CN114306392A

CN114306392A - Use of probiotic components and pharmaceutical compositions comprising probiotic components

Info

Publication number: CN114306392A
Application number: CN202011064448.1A
Authority: CN
Inventors: 邹方霖; 邹礼常; 王建霞; 王艺羲
Original assignee: Chengdu Kuachang Aopu Medical Technology Co ltd
Current assignee: Chengdu Kuachang Aopu Medical Technology Co ltd
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2022-04-12

Abstract

The present disclosure relates to the treatment of localized disease conditions by topical administration. In particular, the present disclosure relates to the use of probiotic components as active ingredients that provide a local effect or a local synergistic effect for the treatment of local pathological conditions, pharmaceutical compositions comprising the probiotic components, and pharmaceutical kits.

Description

Use of probiotic components and pharmaceutical compositions comprising probiotic components

Technical Field

The present disclosure relates to the treatment of localized disease conditions by topical administration. In particular, the present disclosure relates to the use of probiotic components as active ingredients that provide a local effect or a local synergistic effect for the treatment of topical pathologies, pharmaceutical compositions comprising such probiotic components, and a method for the treatment of topical pathologies.

Background

From the perspective of diseased tissue, a representative model of localized disease is solid tumors. A solid tumor is a neoplastic disease with neoplastic symptoms, the neoplastic body comprising neoplastic body tissues, which tissues comprise tumor cells. In the case of pancreatic cancer tumor bodies, pancreatic cancer cells account for only about 30% by volume of the tumor bodies. It can be seen that in addition to tumor cells, there are often a greater number of other components in tumor body tissue (sometimes also referred to as the microenvironment of the tumor cells), including other various cells, various intercellular substances, various ducts, etc.

Cytotoxic drugs have been the leading drugs used to treat solid tumors for nearly a century. Intratumoral administration can increase its intratumoral concentration, however its pharmaceutical effect is only slightly increased within the expected range of cancer cell inhibition kinetics (usually less than 200%), and thus still almost for a more compliant systemic administration (oral or intravenous). The pharmacological effects of some drugs administered intratumorally are much greater than their expected range of cancer cell inhibition kinetics, showing a distinct pharmacology from systemic administration. For example, classical chemical ablative agents (high purity ethanol, high concentration acidifying or basifying agents) are not pharmacologically characterized by tumor cell destruction, but by tumor body tissue destruction. However, classical chemical ablative agents are very irritating, often have low long-term efficacy, and can be used in practice with very limited intervention volumes (e.g., acid-base doses not exceeding 0.2ml/kg) and sites of intervention (e.g., restrictions on the organs in which the tumor resides, limited ablation at the margins of the tumor, etc.). Thus, classical chemical ablative agents have been clinically faded out of solid tumors over the last decade.

In recent years, there have been some advances in tumor immunotherapy approaches, represented by tumor vaccines, cell adoptive therapies, and immune checkpoint inhibitors. For example, tumor therapy vaccines have undergone a progression from non-specific vaccines to specific vaccines, wherein the vaccine antigens have undergone a progression of non-specific antigens, tumor cell antigens, tumor associated antigens, and tumor neoantigens. The immunotherapy drugs are very different, and the common points show that the gene specificity and the tumor specificity of patients are very high, the obvious difference between the tumor body residual weight (such as tumor inhibition rate) of an administration group and a negative control group can hardly be observed in common animal experiments, and the clinical indication range is very narrow.

The definition of probiotics by the world health organization is: a live non-pathogenic bacterium which, when ingested in sufficient quantities, imparts a health benefit to the host. Studies have shown that probiotics may have 3 mechanisms of action to support their benefits: (1) binding to intestinal epithelial cells to compete for pathogen attachment sites or consume their nutrients to inhibit the growth of pathogenic bacteria; (2) improving the intestinal barrier function; (3) improving the immunity of the organism. Among them, the study of probiotics on improving the immune function of the body is very interesting. Yeast has been studied as a feed additive for enhancing the immune function of swine (e.g., patent publication No. CN 106387398A). Inactivated yeast has also been studied for enhancing immune function, antibacterial and antiviral (e.g., patent publication No. CN 108524925A).

In the prior art, the anti-tumor effect of probiotics is believed to be primarily related to their bacterial immunogenicity. However, the current use of immunogenicity does not show the therapeutic effect of therapeutic vaccine antigens and is therefore mainly used for immune enhancement in the body of a patient. Immune heterogeneity is one of the characteristics of tumor heterogeneity, and the pharmacology enables immune enhancement of probiotics to show strong tumor specificity. Some probiotics have been reported to provide this immune enhancement against very specific tumors (e.g. colorectal cancer), while no meaningful findings have been reported for the majority of the rest of the tumors. Compared to the above-mentioned drugs that can provide therapeutic effects (e.g., the above-mentioned cytotoxic drugs or chemoablative agents, even the above-mentioned tumor vaccines, adoptive cell drugs and immune checkpoint inhibitors), these antitumor immune-enhancing effects provided by probiotics remain negligible in the industry, and it is almost impossible to show meaningful tumor growth inhibition (tumor inhibition rate ≦ 15% or relative tumor proliferation rate ≧ 85%) in animal experiments, for example. In summary, the probiotics of the prior art provide at most an adjuvant therapeutic effect, and far from a therapeutic effect.

Thus, there is still a need to develop new pharmacological effects of probiotics to break through expectations of the probiotics for immune function enhancement and the like in the prior art, thereby providing new pharmaceutical compositions for clinical use, and using them for new medical uses, such as treatment of localized disease (rather than previous adjuvant therapy).

Disclosure of Invention

The invention aims to provide a local medicament for treating local pathological diseases. More specifically, the present invention aims to provide a therapeutic agent for a localized disease which can provide immunotherapy involving local chemotherapy and optionally other chemotherapy, or/and involving secondary immunization and optionally other immunization in the administered region.

According to one aspect of the present disclosure there is provided the use of a probiotic component as an active ingredient providing a therapeutic effect in the manufacture of a topically administrable pharmaceutical composition for the treatment of a locally pathological condition. In one embodiment, the pharmaceutical composition further comprises a chemically active ingredient capable of producing a synergistic effect with the probiotic component, and wherein the ratio of the amount of the probiotic component to the chemically active ingredient (probiotic component weight concentration/co-agent weight concentration) is (1-110)/(1-100).

According to another aspect of the present disclosure there is provided a topically administrable pharmaceutical composition for the treatment of a localized disease condition, comprising a probiotic component which provides a therapeutic effect, and a pharmaceutically acceptable suitable carrier. In one embodiment, the pharmaceutical composition further optionally comprises a chemically active ingredient capable of producing a synergistic effect with the probiotic component.

According to still another aspect of the present disclosure, there is provided a topically administrable pharmaceutical composition for treating a localized disease, comprising a probiotic component capable of providing a therapeutic effect, a chemically active ingredient capable of producing a synergistic effect with the probiotic component, and a pharmaceutically acceptable suitable carrier, wherein the ratio of the amount of the probiotic component to the chemically active ingredient (probiotic component weight concentration/co-agent weight concentration) is (1-110)/(1-100).

In the present disclosure, the probiotic component is selected from those which minimize bacterial immunogenicity, preferably selected from one or more of the group comprising: water-soluble components of probiotics, semi-fluid components of probiotics, water-insoluble particles of probiotics and inactivated probiotics.

According to yet another aspect of the present disclosure, there is provided a method of treating a localized disease condition, comprising the steps of: topically administering a therapeutically effective amount of a pharmaceutical composition according to the present disclosure, within or/and outside of a localized lesion, to an individual in need thereof.

According to yet another aspect of the present disclosure, there is provided a pharmaceutical kit comprising one or more containers filled with a pharmaceutical composition according to the present disclosure.

Embodiments according to the present invention have the following advantages over the prior art of compositions comprising probiotic components for the treatment of locally diseased diseases: provides a novel pharmacology (local effect or local synergy and secondary immunity thereof), so that local treatment comprising chemoablation and/or immunotherapy comprising secondary immunity, which cannot be carried out by the existing probiotic composition, can produce curative effect (such as tumor inhibition rate more than 3 times) and indications (such as breakthrough of tumor specificity limitation in the prior art and dependence limitation on the immune function of a patient), and can also produce immune curative effect (such as production or enhancement of secondary immunity in a lesion area or/and outside the lesion area greatly exceeds the expectation of immune enhancement of a body) and indications which are far beyond the prior art scheme. Furthermore, embodiments of the present invention also have a greatly reduced safety risk of prior art solutions that do not define the mode of administration.

Embodiments according to the present invention have the following advantages over the prior art of other compositions for treating locally diseased conditions: compared with the existing cytotoxic drugs, the compound has almost non-toxic systemic safety and obviously higher long-term curative effect; compared with the existing molecular targeted drugs, the compound has less rigorous screening of indications and great potential for rapidly growing tumor bodies, large tumor bodies and blood-poor tumor donors; compared with the existing chemical ablation agents, the utility model has the advantages of obviously lower local irritation and better long-term effect. The applications and compositions of the present invention are also not plagued by the problem of drug resistance encountered with existing cytotoxic drugs and existing molecular targeted drugs. In addition, the application and the composition are convenient to prepare and low in cost, and are particularly beneficial to leading the vast population who is difficult to bear high expense to enjoy safe and effective treatment.

Detailed Description

It is known that a substance is used as an active ingredient in the treatment of a locally diseased condition because it can exhibit the therapeutic activity under specific conditions. The same substance may exhibit different activities under different conditions, or different pharmacologies. The development of new pharmacologies for existing substances is intended to introduce therapeutic effects more beneficial to the patient, as are the development of new active substances. For example, ethanol has a long history of use as a bactericide in the preparation of pharmaceuticals. However, until the 1980 s, scientists did not find that high concentrations of ethanol could show chemoablative pharmacology against tumor tissue under specific conditions (intratumoral administration). Therefore, in about 20 years thereafter, high-concentration ethanol is widely used for chemoablation treatment of local lesions such as tumor bodies.

The inventors of the present invention have surprisingly found in a tumor-bearing nude mouse experiment, which is commonly used as a model for chemotherapy, that inactivated probiotics, which are commonly used as immunopotentiators, can naturally produce meaningful local chemical effects, and possibly even chemical ablation-like, under certain specific conditions. Other probiotic components, whose composition and structure are very different from them, inherently also exhibit this new pharmacological profile. These specific conditions are not conditions for the application of the probiotic composition of the prior art, but are as defined below.

In one aspect, the present disclosure provides the use of a probiotic component as an active ingredient providing a therapeutic effect in the preparation of a topically administrable pharmaceutical composition for the treatment of a locally pathological condition. The pharmaceutical composition may be a pharmaceutical composition according to the present disclosure.

In the context of the present disclosure, the term "probiotic" (probiotics) refers to non-pathogenic living microorganisms capable of producing beneficial effects on the health of the host, the probiotic comprising cells and the yeast more unicellular microorganisms. The term "probiotic component" refers to a preparation derived from a natural probiotic or its engineered bacteria (e.g., a murein polysaccharide) or an engineered analog of the preparation (e.g., a synthetic or other source of a polysaccharide similar to murein polysaccharide).

In the context of the present disclosure, the term "therapeutic effect" is distinguished from an auxiliary (therapeutic) effect, the former meaning a primary pharmacological effect (e.g. local treatment of a localized disease, or/and immunotherapy of a localized disease) that allows effective remission, amelioration or recovery of a disease, and the latter meaning a secondary pharmacological effect (e.g. immune enhancement of the body) that is not effective in remission, amelioration or recovery of a disease but is also beneficial to the patient. Pharmaceutical compositions comprising an active ingredient that provides a therapeutic effect are generally referred to as therapeutic agents, while pharmaceutical compositions in which the active ingredient provides only an adjunctive therapeutic effect are generally referred to as adjunctive therapeutic agents.

In the context of the present disclosure, the term "pharmaceutical composition" refers to a substance that defines a pharmacological property and gives pharmacological characteristics such as a pharmacological method, a pharmacological composition, and a pharmacological environment necessary for achieving the pharmacological property in a patient. The term "pharmacological means" refers to the administration of a pharmaceutical composition necessary for the achievement of a particular pharmacological property, e.g. oral administration is the pharmacological means necessary for the improvement of the intestinal barrier of the probiotic, while intralesional administration is the pharmacological means necessary for chemical ablation of ethanol. The term "pharmacological composition" refers to the composition of a pharmaceutical composition necessary to achieve a particular pharmacological property, and different pharmacological properties may often have very different pharmacological compositions, e.g., conventional synergy requires only synergistic amounts of the active ingredients, while local conventional synergy also requires synergistic concentrations and the like of the synergistic composition. The term "pharmacological environment" refers to the minimization of exogenous interference within a target area of a pharmaceutical composition necessary to achieve a particular pharmacological agent, e.g., adverse effects that additives (e.g., osmolytes) necessary for other reasons may have on the particular pharmacological agent. Pharmaceutical compositions without specific pharmacological context limitations often necessarily contain inactive ingredients that enhance administration compliance (e.g., flavoring agents for oral drugs) and administration safety (e.g., osmolytes contained in injections), while local-action pharmacology must necessarily require a defined pharmacological context to reduce possible pharmacological interference with the inactive ingredients. Pharmacology of a pharmaceutical composition without specific pharmacologic approach limitations (e.g., intravenous, intramuscular, or other local injections) means that it is possible to achieve in both of these approaches (e.g., immune enhancement of probiotic injections, cell growth inhibition of cytotoxic drugs, intoxication-like response to ethanol), and pharmacology of a pharmaceutical composition with specific pharmacologic approach limitations means that it is only possible to achieve in this approach (e.g., intestinal barrier improvement of oral probiotics, chemical ablation of topically administered ethanol). Pharmacology of a pharmaceutical composition without a specific pharmacological composition definition (e.g., formulation concentration without pharmacological concentration, or dosing dose without pharmacological volume) means that it is possible to achieve a given active ingredient, active ingredient amount ratio, and composition dose (e.g., immune enhancement of probiotic injections, cell growth inhibition of cytotoxic drugs, intoxication-like reaction of ethanol), and pharmacology of a pharmaceutical composition with a specific pharmacological composition definition means that it is possible to achieve pharmacologic chemical ablation only under the conditions of that composition, e.g., ethanol is administered in its pharmacologic approach (intra-lesion), pharmacologic concentration (e.g., > 70% high concentration), pharmacologic volume (e.g., > 0.25 administration volume/target volume ratio).

In the context of the present disclosure, the term "local administration" is to be distinguished from conventional administration (systemic administration), which refers to administration of a drug either intralesionally (e.g., intratumoral administration) or/and extrapathological locally (e.g., subcutaneous injection, intramuscular injection, local implantation, local smearing, local spraying, local instillation, local insertion, or otherwise), whereas conventional administration refers to delivery of a drug to a target site via blood following administration via the alimentary tract (e.g., oral) or blood vessels (e.g., intravenous, intraperitoneal injection). The term "intralesional administration" refers to any administration that provides direct access to the drug, rather than indirect access to the local lesion in the form of a medicated blood stream, such as percutaneous intralesional injection or/and catheter perfusion through a blood vessel into the lesion, and the like.

In one embodiment, the probiotic component may also provide a synergistic effect, and the pharmaceutical composition further comprises a chemically active ingredient capable of producing a synergistic effect with the probiotic component (hereinafter also referred to as synergistic ingredient), and the pharmaceutical composition is prepared such that the administration ratio of the probiotic component to the co-agent (probiotic component weight concentration/co-agent weight concentration) is (1-110)/(1-100). Within the scope of the present invention, the probiotic component is administered topically together with the chemically active ingredient capable of producing a synergistic effect with the probiotic component. In one embodiment, the probiotic component is contained in the same medicament as the chemically active ingredient.

In the context of the present disclosure, the term "synergistic effect" refers to a particular pharmacological property of a particular active ingredient (e.g., a probiotic component) under particular conditions such that the combined effect of the particular active ingredient and the other active ingredients exceeds the additive expectation of their respective individual effects. The term "synergistic ingredient" refers to a co-agent that enables a particular active ingredient (e.g., a probiotic component) to exhibit a synergistic effect under particular conditions.

In yet another aspect of the present disclosure, there is provided a topically administrable pharmaceutical composition for treating a localized disease condition, comprising a probiotic component that provides a therapeutic effect, and a pharmaceutically acceptable suitable carrier. In one embodiment, the pharmaceutical composition further optionally comprises a chemically active ingredient capable of producing a synergistic effect with the probiotic component.

In yet another aspect of the present disclosure, there is provided a topically administrable pharmaceutical composition for treating a localized disease, comprising a probiotic component capable of providing a therapeutic effect including a synergistic effect, a chemically active ingredient capable of producing a synergistic effect with the probiotic component, and a pharmaceutically acceptable suitable carrier, and the ratio of the amount of the probiotic component to the chemically active ingredient (probiotic component weight concentration/co-agent weight concentration) is (1-110)/(1-100).

In one embodiment, the pharmaceutical composition has a pharmacological composition such that the probiotic component provides the therapeutic effect. In one embodiment, the pharmaceutical composition comprises a probiotic component and a pharmacological composition required such that the probiotic component provides a local effect (or/and a local synergistic effect) and optionally other effects, for example one or more of the following groups: preferred pharmacologically active ingredients, pharmacological content (pharmacological concentration, or/and pharmacological volume) of the active ingredient, and local pharmacological environment of the active ingredient. In one embodiment, the probiotic component is as an active ingredient in a use as defined according to the present disclosure. In one embodiment, the probiotic component is contained in the same medicament as the co-agent. In one embodiment, the probiotic component and co-formulation act as active ingredients in the uses, compositions and methods defined according to the disclosure of the present application. In one embodiment, the suitable carrier comprises a liquid carrier. In one embodiment, the liquid carrier comprises water for injection.

In yet another aspect of the present disclosure, there is provided a method of treating a localized disease condition, comprising the steps of: topically administering to an individual in need thereof, intra-and/or extra-lesional, a therapeutically effective amount of a pharmaceutical composition according to the present disclosure. In one embodiment, the method of treating a localized disease condition comprises the steps of: administering to an individual in need thereof, a therapeutically effective amount of the pharmaceutical composition either intralesionally, or both intralesionally and extracesionally. In one embodiment, the method of treating a localized disease condition comprises the steps of: administering a therapeutically effective amount of the drug a and the drug B, either simultaneously or non-simultaneously, respectively, into a lesion in an individual in need thereof. In one embodiment, the method of treating a localized disease condition comprises the steps of: administering to an individual in need thereof a therapeutically effective amount of the drug B within the lesion and a therapeutically effective amount of the drug A outside the lesion, simultaneously or non-simultaneously, respectively. In one embodiment, the medicament a comprises a probiotic component, which provides a therapeutic effect, and a pharmaceutically acceptable suitable carrier; the medicament B comprises a probiotic component which can provide a therapeutic effect including a synergistic effect, a co-agent of the synergistic effect and a pharmaceutically acceptable suitable carrier, and the amount ratio of the probiotic component to the co-agent (probiotic component weight concentration/co-agent weight concentration) is (1-110)/(1-100). In one embodiment, the probiotic component is contained in the same medicament as the co-agent.

In one embodiment, the therapeutic effect preferably comprises a local treatment involving a local effect (or a local synergistic effect) or/and an immunotherapy. In one embodiment, the local effect (or local synergy) comprises a local chemical effect (or local chemical synergy) and optionally other effects. In one embodiment, the local treatment comprises a chemical-like ablation of one or more local lesions and optionally other chemotherapies. In one embodiment, the immunotherapy comprises a secondary immunization and optionally additional immunization of the local effect (or local synergy) within the lesion or/and outside the lesion. In one embodiment, the local effect (or local synergy) is preferably independent of the pathogen (cell, virus or bacteria) proliferation inhibition of the local lesion.

In the context of the present disclosure, the term "local treatment" is to be distinguished from "systemic treatment," which refers to treatment of the patient's body (e.g., the tumor body, the region of tumor body communication, tumor cells contained in other parts of the body), and which refers to treatment of the patient's local region where the local lesion is located (e.g., the administration of the local lesion and other lesion regions in communication therewith). The term "local chemotherapy" refers to local treatment where local effects include local chemical effects (or local chemosynergy).

In the context of the present disclosure, the term "local effect" is distinguished from conventional effects and refers to the pharmacological effect of a drug within the local area of interstitial penetration (e.g., tumor or/and extratumor local area) after local administration, and conventional effects refers to the pharmacological effect of a drug delivered to the target area in the form of blood after administration through the digestive tract or blood vessels after conventional administration. The term "local chemical effect" is meant to include a local effect of a chemical effect. The term "local synergy" is meant to include a synergistic local effect. The term "local chemical synergy" refers to a local chemical effect that includes synergy.

Local chemical action (or local chemical synergy) in the context of the present disclosure includes pharmacologically general local chemical action, chemical ablation, and chemical-like ablation. The term "general local chemical effect" refers to a local chemical effect for which the drug effect does not exceed the maximum expected (e.g., within 200%) kinetic difference for conventional chemical effects of the same drug, e.g., chemotherapy effects produced by conventional administration of cytotoxic drugs. The term "chemical ablation" refers to a local chemical effect where the effect of the drug exceeds the maximum expected (e.g., greater than 200%, preferably greater than 400%) kinetic difference of conventional chemical effects of the same drug (e.g., chemotherapy effects from conventional administration of high concentrations of ethanol), and traditionally refers to the local chemical effect exhibited by classical chemical ablative agents (e.g., high concentrations of ethanol, high concentrations of acid, high concentrations of base). The term "chemical ablation-like" refers to a local effect that includes chemical ablation. Although not caused by classical chemical ablation agents, the chemical ablation is pharmacologically distinct from the common local chemical action.

In the context of the present disclosure, the term "immunotherapy" is distinguished from the term "immunopotentiation", the former referring to an immune effect that can be used alone to achieve a therapeutic effect (e.g., the immune effect of a therapeutic vaccine, specific antibodies, etc.), and the latter referring to an immune effect that cannot be used alone to achieve a therapeutic effect, but still has an adjuvant effect (e.g., an immunopotentiator has the effect of enhancing the immune function of the body). The term "secondary immunization" is to be distinguished from the term "drug-antigenic effect", the latter referring to the antigenic effect of the drug itself (e.g., the antigenic effect caused by any drug entering the body as a foreign body), and the former referring to the immunization associated with administration but distinguished from the antigenic effect of the drug, e.g., the in situ vaccine effect caused by the local chemical effect of the drug). The term "secondary immunity in situ" is distinguished from the term "secondary immunity ex situ", the former referring to the secondary immunity within the lesion (e.g., tumor mass) and the latter referring to the secondary immunity outside the lesion (e.g., axillary intradermal). The term "secondary immunological substance" refers to an immunological substance formed by local administration in the administration area and different from the administered substance itself, such as antigens, adjuvants, or/and other immunological molecules that are released, generated, activated, or/and sequestered for any reason after administration. The term "in situ secondary immune substance" is distinguished from the term "ex situ secondary immune substance," the former referring to secondary immune substances within a lesion (e.g., tumor mass) (e.g., in situ antigens, in situ adjuvants, or/and other immune molecules released, generated, activated, or/and draped within the lesion), and the latter referring to secondary immune substances outside the lesion (e.g., axillary intradermally) (e.g., nodular immune substances formed at the site of administration, other immune molecules released, generated, activated, or/and draped due to the nodule or/and other pharmacological effects at the site of administration, etc.).

In the context of the present disclosure, the term "vaccine antigen" is distinguished from the term "antigen", the latter refers to any substance that is capable of inducing an immune response in a body, and the former refers to an antigen that is capable of inducing an immune response in a body against a particular disease and that is therapeutically effective, e.g., the same substance may be used as a vaccine antigen and an immunopotentiator antigen by a wide variety of techniques. The term "adjuvant" refers to a substance that enhances the immunotherapeutic effect of its antigen in a vaccine. The term "in situ antigen" refers to an in situ secondary immune substance that can act as an antigen. The term "vaccine-like drug" or "vaccine-like" refers to a therapeutic drug that provides a secondary immune effect (as distinguished from immunopotentiators and conventional vaccines) similar to the vaccine effect, and optionally an exogenous antigenic effect.

In the present disclosure, the chemical ablation-like of the compositions of the present invention is highly consistent with that of classical chemical ablation agents, and thus largely inconsistent with the conventional action of prior art compositions that may contain probiotic components, leading to the following differences in composition characteristics: 1) the pharmacological approaches of the compositions of the present invention are strictly limited to topical administration, whereas the pharmacological approaches of the prior art compositions are not limited to topical administration; 2) the pharmacological composition of the present invention exceeds the expectations of the prior art compositions based on their conventional pharmacological composition, such as completely different pharmacologically active ingredient preference principles, completely different pharmacological contents (pharmacological concentration, or/and pharmacological volume), different pharmacological environments (e.g., rejection of salt-type osmo-regulators); 3) thus, the pharmacological effect of the composition of the invention greatly exceeds that expected most based on the kinetic differences of the prior art compositions upon their conventional administration (closer to the conditions of the cellular experiment), for example by a much higher efficacy (tumor inhibition rate greater than 200%, preferably greater than 400%) and new indications (for example local treatment of said local lesions but not of said local lesions) and new applicable patients, which further shows that the difference between the two compositions is not a kinetic difference but a pharmacological difference (for example local effects vs conventional effects).

In one embodiment, the patient for whom the treatment is applicable is selected from one or more of the group comprising: immunosuppressed patients, patients with drug administration within a localized lesion, patients with ablation of a similar type of localized lesion tissue, patients with secondary immune material production within a localized lesion, patients with secondary immune material production within the region administered outside of a lesion.

In the context of the present invention, the term "immunosuppressed patient" refers to any patient represented by a tumor-bearing nude mouse model, for example, a patient whose immune function is at a low level for any reason and who is otherwise unable to achieve normal levels (e.g., immune enhancement) within the time period over which the composition of the present invention provides local effects (e.g., within 7 days of the first administration in example 2 below), for example, a patient who is refractory to radiation therapy or conventional chemotherapy because of low immunity.

In one embodiment, the therapeutic effect is a local treatment involving the local effect (or local synergy) or/and the immunotherapy. In one embodiment, the patient for whom the treatment is applicable is selected from one or more of the group comprising: immunosuppressed patients, patients who can be administered in a local lesion and the local lesion tissue can be ablated in a similar way or/and patients who can produce secondary immunity in the local lesion, patients who can produce secondary immunity in the administration area outside the lesion. In one embodiment, the composition is a chemoablation-like in situ and ex situ immunotherapeutic agent.

In one embodiment, said therapeutic effect is a treatment comprising said immunotherapy involving said local effect (or local synergy), and said immunotherapy comprises a secondary immunization of said local effect (or local synergy) within or/and outside said lesion and optionally other immunizations. In one embodiment, the patient for whom the treatment is applicable is selected from one or more of the group comprising: can be locally administered in lesion, and the locally diseased tissue can be ablated in a chemical way or/and patients with secondary immunity in the locally diseased tissue and patients with secondary immunity in the administration area outside the lesion. In one embodiment, the composition is an in situ or/and ex situ immunotherapeutic drug.

In one embodiment, the therapeutic effect is a combination of the local therapy or/and immunotherapy involving the local effect (or local synergy), and wherein the local therapy includes chemical-like ablation of one or more local lesions and optionally other chemotherapies; the immunotherapy comprises a secondary immunization of the local effect (or local synergy) and optionally other immunizations within the lesion. In one embodiment, the patient for whom the treatment is applicable is selected from one or more of the group comprising: immunosuppressed patients, patients who can be administered within a localized lesion, patients who can be ablated by a similar chemical technique in a localized lesion tissue, patients who can develop secondary immunity in a localized lesion. In one embodiment, the composition is a chemoablation-in situ immunotherapy drug.

In one embodiment, the therapeutic effect is a local treatment involving a local effect (or local synergy), and wherein the local treatment includes an anabolic ablation of one or more local lesions and optionally other chemotherapies. In one embodiment, the patient for whom the treatment is applicable is selected from one or more of the group comprising: immunosuppressed patients, patients who can be administered within a localized lesion, patients who can be similarly chemically ablated in a localized lesion tissue. In one embodiment, the composition is a chemoablative-like drug.

In one embodiment, the therapeutic effect is an immunotherapy comprising a local effect (or local synergy), and wherein the immunotherapy comprises a secondary immunity of the local effect (or local synergy) and optionally other immunizations within the lesion. In one embodiment, the patient for whom the treatment is suitable is selected from patients who can be administered intralesionally and who can develop secondary immunity from a local lesion. In one embodiment, the composition is an in situ immunotherapeutic drug, e.g., one that can provide in situ vaccine activation and optionally other immunization at the target site within the lesion.

In one embodiment, the therapeutic effect is an immunotherapy comprising a local effect (or local synergy), and wherein the immunotherapy comprises a secondary effect of the local effect (or local synergy) outside the lesion and optionally other immune effects. In one embodiment, the patient for whom the treatment is suitable is selected from patients who have developed a secondary immune effect in the area of the administration outside the lesion. In one embodiment, the composition is an immunotherapeutic agent that can provide extra-lesional secondary immunity and optionally other immunity. In one embodiment, the secondary immune effect outside the lesion comprises an immune effect of an abnormal structure (e.g., a nodule) secondary to the local effect (or local synergy). In one embodiment, the immunotherapeutic agent comprises, for example, a vaccine-like agent.

In one embodiment, the probiotic component is selected from one or more that provide an effect comprising one or more of the following groups: said local effect, said secondary effect of said local effect, said immunization of said local administration. In one embodiment, the secondary effect of the local effect comprises an immunological effect involved by an immunological agent secondary to the administration area.

In one embodiment, the probiotic component is preferably selected from those which minimise bacterial immunogenicity, more preferably from one or more selected from the group comprising: water-soluble components of probiotics, semi-fluid components of probiotics, water-insoluble particles of probiotics and inactivated probiotics. In one embodiment, the probiotic component is selected from one or more of a probiotic water-soluble component and engineered analogues thereof, and the composition is a solution composition. In one embodiment, the probiotic component is selected from water insoluble particles of the bacterial component, or/and inactivated probiotics, and the composition is a suspension composition. In one embodiment, the probiotic component is selected from probiotic semi-fluid based components, and the composition is a semi-fluid based composition. In one embodiment, the composition is preferably a semi-fluid type composition or an aqueous solution composition, more preferably a semi-fluid type composition.

In the context of the present disclosure, the term "bacterial immunogenicity" refers to the ability of a bacterium to generate an immune response in a recipient as an intact foreign body, with different bacteria having different bacterial immunogenicity. Live probiotics have the strongest bacterial immunogenicity, but direct entry into the body also carries a strong safety risk. The term "inactivated probiotic" refers to a preparation obtained after a bacterial inactivation engineering treatment, wherein the bacterial inactivation is, for example, one or more of: high-temperature inactivation, high-temperature and high-pressure inactivation, ultraviolet inactivation, chemical reagent inactivation and radiation inactivation. The term "probiotic water-insoluble component" refers to any component obtained from a probiotic with a water solubility of < 0.1%. The term "probiotic water-soluble component" refers to any component obtained from a probiotic with a water solubility of 0.1% or more. The term "disrupted probiotic" refers to a mixture obtained after disruption engineering of the probiotic. The term "disrupted probiotic pellet fraction" refers to water-insoluble pellets separated (e.g. filtered or/and centrifuged) from the disrupted probiotic and further separated fractions thereof and their analogues. The term "disrupted probiotic supernatant component" refers to the supernatant separated (e.g. filtered or/and centrifuged) from the disrupted probiotic and its further separated components and their analogues. The term "probiotic cell wall polysaccharides" refers to polysaccharides and their analogues comprised by the cell wall, such as beta-glucans. The term "probiotic semi-fluid like component" refers to a probiotic component that provides the composition of the present invention with a semi-fluid like morphology.

In the context of the present disclosure, the term "semi-fluid-like" refers to a class of physical forms between liquids and semi-solids, which includes semi-fluids and the like. The term "semi-fluid" refers to an object that flows without external pressure but without macroscopic flow within a time frame at room temperature (e.g., 20 seconds), but that can flow and cause irreversible deformation under clinically (administered) acceptable external pressure (e.g., external pressure that can be applied to a syringe pusher), as distinguished from liquids (which also have fluidity without external pressure) and semisolids (which only reversibly deform under clinically acceptable external pressure). The term "semi-fluid analog" refers to a physical form between a liquid (suspension) and a semi-fluid, close to the semi-fluid, that does not appear when resting at room temperature for about 1 minute, but that does appear with significant delamination of the suspension; which can occur in less than about 1 minute without external pressure at room temperature without macroscopic flow of the semifluid. The suspension may be transformed into a semi-fluid type object. For example, a 5% β -glucan suspension mixed with water can be prepared as a semi-fluid analog by heating a 5% β -glucan aqueous suspension, and a 5% β -glucan/1% methylene blue/1% 5-fluorouracil aqueous suspension can be prepared as a semi-fluid by heating.

In one embodiment, the probiotic water-soluble component is selected from the group comprising one or more of the following groups and derivatives thereof: crushing the probiotic supernatant component, the probiotic extract and the probiotic intracellular water-soluble component. In one embodiment, the probiotic semi-fluid composition is preferably one or more probiotic components selected from probiotic components whose aqueous mixture may form (e.g. by cooling after heating, basification etc.) a semi-fluid composition, e.g. a probioticOne or more of bacteriophagous polysaccharide and its analogues. In one embodiment, the probiotic water-insoluble particles are selected from one or more of the group comprising: breaking the probiotic precipitation component, the probiotic cell wall polysaccharide particles and the probiotic cell wall polysaccharide nanoparticles. In one embodiment, the inactivated probiotic is preferably selected from, but not limited to, inactivated probiotics, all of which are intact bacteria. In one embodiment, the ratio of the amount of incomplete bacteria in the inactivated probiotic is>20% or>30% or the number of intact cells per ml of the composition<10⁵Or even<0.5×10⁵And (4) respectively.

In one embodiment, the probiotic is selected from one or more of the group consisting of natural or engineered bacteria: probiotic bacillus, probiotic lactobacillus, probiotic bifidobacterium and probiotic fungi.

In one embodiment, the probiotic comprises one or more selected from the group consisting of lactobacillus plantarum. In one embodiment, the probiotic comprises one or more selected from lactobacillus probiotic. In one embodiment, the probiotic comprises one or more selected from the group consisting of probiotic bifidobacteria. In one embodiment, the probiotic comprises one or more selected from probiotic fungi.

In one embodiment, the M.gemmuliformis is selected from the group comprising one or more of: comprises Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus firmus, Bacillus coagulans, Bacillus lentus, Bacillus pumilus, and Bacillus natto. In one embodiment, the M.gemmuliformis is preferably selected from the group consisting of one or more of: bacillus licheniformis, Bacillus subtilis, and Bacillus pumilus.

In one embodiment, the lactic acid bacteria comprise one or more selected from the group consisting of lactobacilli or/and bifidobacteria. In one embodiment, the lactobacillus comprises one or more selected from the group consisting of: lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus fermentum. In one embodiment, the lactobacillus is preferably selected from the group comprising one or more of: lactobacillus casei, lactobacillus plantarum, lactobacillus brevis and lactobacillus fermentum. In one embodiment, the bifidobacteria comprise one or more selected from the group consisting of: bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium infantis, Lactobacillus blodgensis, Lactobacillus helveticus, Lactobacillus thermophilus, enterococcus faecium, and Streptococcus faecalis. In one embodiment, the bifidobacteria are preferably selected from the group comprising one or more of: bifidobacterium longum, Bifidobacterium adolescentis, enterococcus faecium, and Streptococcus faecalis.

In one embodiment, the fungus comprises one or more of a yeast or/and a gambling yeast, wherein the yeast comprises one or more of the group consisting of: saccharomyces cerevisiae, Deerbu Trichosporon, Candida, Vecker Han yeast, Pichia yeast, Trichosporon, Schlemm Sphaerotheca, Rhodotorula rubra, Schizosaccharomyces pombe, Bauscerella yeast, and Candida utilis. In one embodiment, the probiotic is one or more of yeast selected from the group consisting of yeast and/or wine yeast.

In one embodiment, the probiotic is preferably selected from the group of probiotics that do not contain a mycolic acid cell wall. In one embodiment, the probiotic is preferably selected from the group consisting of Kluyveromyces. In one embodiment, the probiotic is preferably selected from saccharomyces cerevisiae.

In one embodiment, in the use, pharmaceutical composition or method as defined according to the present disclosure, the technical solution for providing the desired pharmacological composition of the therapeutic effect by the probiotic component is characterized by: the composition of the composition must meet the conditions of pharmacological concentration required for the probiotic component to provide the effect, wherein the pharmacological concentration (local administration concentration) is > 0.1%, ≧ 0.25%, 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 5-15%.

In one embodiment, the probiotic component comprises the inactivated probiotic bacteria, and the amount of said inactivated probiotic bacteria in the pharmaceutical composition must be such that it is administered locally in a concentration > 0.3%, ≧ 0.75%, 0.75-15, preferably 1.5-15% or 5-15%. In one embodiment, the probiotic component comprises the probiotic water-soluble component and the amount of the probiotic water-soluble component in the pharmaceutical composition must be such that its concentration for topical administration is >0.1, or 0.15-25%, preferably 0.35-15% or 5-15%. In one embodiment, the probiotic component comprises particles of said probiotic water-insoluble component, and the content of said particles of probiotic water-insoluble component in the pharmaceutical composition must be such that its concentration for topical administration is >0.5, or 0.5-15%, preferably 1.5-15% or 5-15%. In one embodiment, the probiotic component comprises the probiotic semi-fluid like component and the concentration of the probiotic semi-fluid like component in the pharmaceutical composition is > 2.5%, 2.6-25%, preferably 5-15%.

In the context of the present disclosure, unless otherwise indicated, the term "concentration" refers to the weight percent concentration (w/w) of a specified component in a drug (or composition). The term "formulation concentration" refers to the concentration of a specified component in a pharmaceutical formulation form (e.g., injection or infusion solution). The term "administration concentration" refers to the concentration of a specified component in an administration form (e.g., a dilution of a formulation) of a pharmaceutical formulation. The term "initial intralesional concentration" refers to the concentration of a given component in a drug-containing medium (e.g., drug-containing blood) as the drug enters the lesion.

Even though the administration concentration of the probiotic component of the composition of the present invention is the same as that of the conventional injection, the intra-pathological initial concentrations necessary for their respective pharmacologies (local chemical action vs. immune enhancement) may be greatly different. One of the technical features of the use, composition and method of the present invention is to ensure the local initial concentration (local administration concentration) required for the action, especially for the local chemical action. The topical administration concentration of the probiotic component in the pharmaceutical composition according to the present disclosure is typically the concentration of the probiotic component in the drug at the end point (e.g. needle hole, catheter outlet, etc.) of the administration device (syringe, piercer, infusion catheter, etc.). For injectable powder injections, the administration concentration is the concentration of the probiotic component in the mixture (e.g., suspension) of dry powder and liquid carrier.

In accordance with the present disclosure, the ratio (v/v) of the amount of the pharmaceutical composition administered to the target volume within the localized lesion is >0.1, 0.15-1.5, preferably 0.23-1.5 or 0.5-1.5. Or the application amount of the medicine composition is more than or equal to 1ml, or the application amount in the local lesion is 10-150ml or/and the application amount outside the local lesion is 1.5-50 ml.

In the context of the present disclosure, the term "target" refers to a pharmacologically primary target, such as a cytotoxic drug directed against tumor cells, an immunomodulatory drug directed against a regulatory factor of the immune system, a chemoablative agent directed against tumor tissue, and the like. The term "target region" refers to the spatial extent (e.g., tumor or a portion thereof) of the target for which the present administration is designed. For example, the target region may refer to a tumor body (when the tumor body diameter is small, the required one-time administration dose is clinically feasible) or a part of the tumor body (when the tumor body diameter is large, the required one-time administration dose is clinically infeasible) which is the target of the treatment.

In one embodiment, the composition must be such that the probiotic component provides the local pharmacological environment required for the effect, which requires the presence of other components in addition to the synergistic component to be minimized, preferably in the absence of inactive ingredients required for pharmacology or/and safety of administration in conventional compositions, such as solid excipients and flavoring agents in oral formulations, and osmotic pressure enhancers in conventional injections.

In one embodiment, the pharmaceutical composition, wherein the suitable carrier is water; the co-product is soluble in water and the probiotic component is selected from the probiotic water-soluble component. In one embodiment, the pharmaceutical composition, wherein the suitable carrier is water; the co-product is soluble in water, while the probiotic component is water-insoluble and is selected from one or more of the group comprising: inactivated probiotics, water insoluble particles of probiotic components, and semi-fluid components of probiotics. In one embodiment, the pharmaceutical composition, wherein the suitable carrier is water; the co-ingredient is soluble or/and poorly soluble in water, and the probiotic component is one or more selected from the group consisting of probiotic semi-fluid type components.

In the present disclosure, the chemically active ingredient is preferably one or more selected from the group consisting of weak topically acting compounds and/or cytotoxic drugs. In one embodiment, the co-ingredient is preferably a plurality selected from 2 or more than 2 of the chemically active ingredients. In one embodiment, the co-agent is preferably selected from the group consisting of the weak topically acting compounds and or cytotoxic drugs. In one embodiment, when the chemically active compound comprises a cytotoxic drug, the ratio of the amounts of the probiotic component and the cytotoxic drug (W) is_{Probiotic compositions}/W_{Cytotoxic drug}) Is (1-110)/(1-100). In one embodiment, when the chemically active compound comprises a weak topically acting compound, the ratio of the amounts of the probiotic component and the weak topically acting compound (V) is_{Probiotic compositions}/W_{Compounds with weak local action}) Is (1-90)/(1-100).

In the context of the present disclosure, the term "chemically active ingredient" refers to any active ingredient that provides a chemical action. The term "topical chemical composition" refers to any active ingredient that provides a topical chemical effect. The term "weak topically acting compound" refers to a chemically active ingredient that is less topically acting than a classical chemical ablative agent under shared conditions.

In one embodiment, the weak topically acting compound is selected from one or more of the group comprising: amino acid nutrients, vital dyes, quinines, low-concentration acidifying agents, low-concentration basifying agents, pH buffer systems comprising acidifying or/and basifying agents. In one embodiment, the co-agent is preferably selected from the group consisting of the vital dye and 1 or more other of the chemically active ingredients. In one embodiment, the co-agent is preferably selected from the group consisting of the vital dye and the cytotoxic drug. In one embodiment, the co-ingredient is preferably selected from the group consisting of the vital dyes and amino acid based nutrients. In one embodiment, said co-agent is preferably selected from said cytotoxic drug and 1 or more other of said chemically active ingredients.

In one embodiment, when the weak topically-acting compound includes an amino acid nutrient, the ratio of the amounts of the probiotic component and the amino acid nutrient (W) is greater than the ratio of the amounts of the probiotic component and the amino acid nutrient (W)_{Probiotic compositions}/W_{Amino acid nutrient}) Is (1-20)/(1-100). In one embodiment, when the weak topically-acting compound includes a vital dye, the ratio of the amounts of the probiotic component and the vital dye (W) is such that the probiotic component and the vital dye are in the same amount_{Probiotic compositions}/W_{Vital dyes}) (7-90)/(1-100) in one embodiment, when the weak topical effect compound comprises a quinine drug, the probiotic component and the quinine drug are in a quantitative ratio (W)_{Probiotic compositions}/W_{Quinine medicine}) Is (2-90)/(1-100). In one embodiment, when the weak topical-action compound comprises an acidifying or/and alkalising agent, the probiotic component and the acidifying or/and alkalising agent are in a ratio of amounts (W)_{Probiotic compositions}/W_{Acidifying or/and alkalifying agents}) Is (2-60)/(1-100).

In the present disclosure, the term "amino acid nutrient" refers to an amino acid compound having a nutritional and health-care effect, preferably selected from amino acid nutriceuticals and amino acid adjuvants having a nutritional and health-care effect, which are carried in official pharmacopoeias or guidelines of various countries.

In one embodiment, the amino acid based nutrient is one or more selected from the group comprising: amino acids, amino acid salts, oligopeptides, and polypeptides. In one embodiment, the amino acid based nutrient is preferably an amino acid selected from the group comprising or a salt thereof or an oligopeptide and polypeptide comprising or consisting of: alanine, valine, leucine, isoleucine, 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 nutrient is more preferably an amino acid selected from the group comprising or a salt thereof or oligo-and polypeptides comprising or consisting of: arginine, lysine, glycine, cysteine, alanine, serine, aspartic acid, glutamic acid. In one embodiment, the concentration of the amino acid based nutrient is > 2.5%, or 5-30%, preferably 5-25%.

In one embodiment, the chemically active ingredient comprises one or more selected from vital dyes. In the present disclosure, the term "vital dye" refers to an aromatic compound dye that, upon entry into the living tissue of an animal, is capable of imparting color to the tissue, cells, subcellular units, etc., without unacceptable harm to the animal as a whole.

In one embodiment, the vital dye may be any suitable one known to those skilled in the art, and may be, for example, one or more organic dyes selected from the group consisting of: methylene blue, patent blue, isothio blue, bengal red, toluidine blue, trypan blue, basic blue, eosin, basic fuchsin, crystal violet, gentian violet, neutral red, janus green B, safranin. In one embodiment, the dye is selected from the methylene blue class of dyes. In one embodiment, the methylene blue-based dye may be, for example, a compound selected from the group consisting of: methylene blue, patent blue, isothio blue, new methylene blue. In one embodiment, the methylene blue-based dye is preferably selected from the group consisting of methylene blue and hydrates and derivatives thereof.

In one embodiment, the vital dye concentration is 0.25% or more, or 0.25 to 10%, preferably 0.25 to 1.5% or 2.5% to 10%. In one embodiment, the concentration (w/v) of the methylene blue dye is not less than 0.35%, preferably 0.35-2.5%, more preferably 0.35-1.5% or 0.5-1%. In one embodiment, the concentration (w/v) of the vital dye other than methylene blue dye (e.g. Bengal red) is 1-10%.

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

In one embodiment, the chemically active ingredient comprises one or more selected from the group consisting of the acidulants. In the context of the present invention, the term "acidifying agent" means an acid which is used in the pharmaceutical preparation primarily as an adjuvant, more particularly as a pH adjustment, which does not generally introduce a particular biological activity in addition to providing acidity when used. In the compositions disclosed herein, the acidulant comprises any acidulant that is pharmaceutically acceptable, preferably selected from the group consisting of acidulants approved by the official administrative department of each country, or loaded via the official pharmacopoeia or guidelines of each country.

In one embodiment, the acidifying agent is selected from strong or/and weak acids. In one embodiment, the strong acid is, for example, hydrochloric acid, sulfuric acid, nitric acid, perchloric acid, selenic acid, hydrobromic acid, hydroiodic acid, chloric acid, and the like, preferably hydrochloric acid. In one embodiment, the weak acid is, for example, carbonic acid, boric acid, acetic acid, phosphoric acid, sulfurous acid, pyruvic acid, oxalic acid, tartaric acid, nitrous acid, and the like, preferably acetic acid.

In one embodiment, the ratio of the amount of probiotic component to the acidifying agent (probiotic component weight concentration/acidifying agent weight concentration) is (1-20)/(0.5-50) in one embodiment, the acidifying agent concentration is ≥ 0.5%, 0.5-2% (strong acid), or/and 2-15% (weak acid).

In one embodiment, the chemically active ingredient comprises one or more selected from the group consisting of the alkalizing agents. In one embodiment, the alkalizing agent comprises a strong base or/and a weak base. In one embodiment, the ratio of the amount of probiotic component to the alkalizer (probiotic component weight concentration/alkalizer weight concentration) is (1-20)/(0.5-50). In one embodiment, the concentration of the alkalizing agent is 0.5% or more, 0.5 to 5% (strong base), or/and 2 to 15% (weak base).

In the context of the present invention, the term "alkalinizing agent" refers to a basic compound which is used primarily as an adjuvant, more specifically for pH adjustment, in the preparation of a medicament. In the compositions disclosed herein, the alkalizing agent comprises any alkalizing agent that is pharmaceutically acceptable, preferably selected from the group consisting of those approved by the official administrative authorities of each country, or those loaded via the official pharmacopoeias or guidelines of each country. In the present disclosure, the alkalizing agent includes strong bases and weak bases.

In one embodiment, the strong base is an alkali metal hydroxide or/and an organic strong base, preferably an alkali metal hydroxide. In one embodiment, the alkali metal hydroxide is, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, preferably sodium hydroxide. In one embodiment, the concentration (w/v) of the strong base in the pharmaceutical composition is not less than 0.5%, preferably 0.5-7.5% or 0.75-7.5%.

In one embodiment, the weak base is selected from one or more of the group comprising: polybasic weak acid type inorganic salt, polybasic weak acid type basic inorganic salt and nitrogenous weak base.

In one embodiment, the polybasic weak acid acidic inorganic salt includes, for example: sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydrogen carbonate, potassium hydrogen carbonate, calcium hydrogen carbonate, sodium hydrogen sulfate, preferably sodium hydrogen carbonate. In one embodiment, the concentration (w/v) of the polybasic acid acidic inorganic salt in the pharmaceutical composition is not less than 1%, preferably 2-10% or 3-10%.

In one embodiment, the polybasic weak acid basic inorganic salts include, for example: sodium phosphate, sodium carbonate, potassium carbonate and borax, and sodium carbonate is preferred. In one embodiment, the concentration (w/v) of the polybasic weak acid basic inorganic salt in the pharmaceutical composition is not less than 1%, preferably 2-10% or 3-10%.

In one embodiment, the weak nitrogen-containing base is selected from the group including, for example: ammonia water, ammonia chloride, 2-aminoethanol, tromethamine, triethanolamine, tris (hydroxymethyl) aminomethane, 2-aminoethanol, tromethamine, triethanolamine, meglumine and gluglucosylamine. In one embodiment, the concentration (w/v) of the nitrogen-containing weak base in the pharmaceutical composition is ≥ 2%, preferably 2-35% or 3-35%.

In a specific embodiment, the alkalizing agent is sodium hydroxide and sodium bicarbonate, and in the pharmaceutical composition, the concentration (w/v) of the sodium hydroxide is 2-5%; the concentration (w/v) of the sodium bicarbonate is 3-10%.

In a particular embodiment, the alkalizing agent is sodium carbonate and sodium bicarbonate, and in the pharmaceutical composition, the concentration (w/v) of the sodium carbonate is 3-10%; the concentration (w/v) of the sodium bicarbonate is 3-10%.

In one embodiment, the pharmaceutical composition comprises an acidifying agent or/and an alkalinizing agent and has pH buffering capacity.

In one embodiment, the chemically active ingredient comprises one or more selected from cytotoxic drugs, wherein the cytotoxic drugs comprise active ingredients selected from anti-neoplastic drugs. In one embodiment, the concentration of the antitumor drug active ingredient is more than or equal to 0.1 percent and 0.1-15 percent.

Within the scope of the present disclosure, the term "cytotoxic drug" refers to an active ingredient that achieves its pharmaceutical effect primarily targeted to the diseased cells or structures within the diseased cells, e.g. a substance that shows anti-cancer cell proliferation in cell experiments or in tumor bearing animals, preferably selected from conventional chemotherapeutic drugs. The term "conventional chemotherapeutic agent" refers to an agent that is effective in treating a locally diseased condition by conventional administration at a safe dose, and is selected from any conventional chemotherapeutic agent that is pharmaceutically acceptable, preferably from conventional chemotherapeutic agents that are well known in the art, and more preferably from conventional chemotherapeutic agents (e.g., antineoplastic agents) that have been or will be approved by or loaded into the administrative department of the official authorities of various countries (e.g., FDA or national drug administration).

In one embodiment, the antitumor drug active ingredient may be one or more selected from the group consisting of: drugs that disrupt the structure and function of DNA [ e.g., alkylating agents (e.g., cyclophosphamide, carmustine, etc.), metal platinum complexes (e.g., cisplatin, carboplatin, etc.), DNA topoisomerase inhibitors (e.g., doxorubicin, topotecan, irinotecan, etc.), etc. ], drugs that intercalate into DNA and interfere with transcribed RNA [ e.g., antitumor antibiotics, such as actinomycin, daunorubicin, doxorubicin, etc. ], drugs that interfere with DNA synthesis [ e.g., pyrimidine antagonists (e.g., uracil derivative 5-fluorouracil, furazauracil, bifuran fluorouracil, cytosine arabinoside derivative, cytidine, 5-azacytidine, etc.), purine antagonists (e.g., oncolysin, thioguanine, etc.), folic acid antagonists (e.g., methotrexate, etc.), etc. ], drugs that affect protein synthesis [ e.g., colchicines, vinblastines, taxanes (e.g., paclitaxel, etc. ], docetaxel, etc.).

In the pharmaceutical composition according to the present disclosure, the antitumor drug active ingredient may be one or more selected from the group consisting of: uracil derivatives, cyclophosphamide, gemcitabine, epirubicin, antitumor antibiotics, teniposide, metal platinum complex, and taxanes; preferably one or more selected from the group consisting of: 5-fluorouracil, cyclophosphamide, gemcitabine, epirubicin, antitumor antibiotics, teniposide, metal platinum complex, paclitaxel.

In a particular embodiment, the concentration (w/v) of the antineoplastic drug active ingredient (e.g. cyclophosphamide, carmustine, etc.) selected from said alkylating agents in said pharmaceutical composition is between 0.5-6%, preferably between 0.75-1.5%.

In a specific embodiment, the concentration (w/v) of the antitumor pharmaceutical active ingredient (e.g., cisplatin, carboplatin, etc.) selected from the metal platinum complex in the pharmaceutical composition is 0.03 to 0.15%, preferably 0.05 to 0.15%.

In a particular embodiment, the concentration (w/v) of the antineoplastic drug active ingredient (e.g. doxorubicin, topotecan, irinotecan, etc.) selected from the DNA topoisomerase inhibitors is 0.05-0.20%, preferably 0.075-0.15% in the pharmaceutical composition.

In a particular embodiment, the concentration (w/v) of the antineoplastic drug active ingredient (e.g. actinomycins, daunorubicin, etc.) selected from said antineoplastic antibiotics in said pharmaceutical composition is between 1-4%, preferably between 1-2%.

In a specific embodiment, the concentration (w/v) of the antitumor drug active ingredient selected from the pyrimidine antagonists (e.g., uracil derivative 5-fluorouracil, fururofluorouracil, difurofluorouracil, cytosine derivative cytarabine, cyclocytidine, 5-azacytidine, etc.) in the pharmaceutical composition is 0.5 to 2%, preferably 0.75 to 1.5%.

In one embodiment, the pharmaceutical composition further optionally comprises a biologically active ingredient, wherein the biologically active ingredient is selected from the group comprising one or more of: antigen, immunoregulation antibody, cell factor, adjuvant.

In one embodiment, the biologically active ingredient comprises one or more selected from antigens, wherein the antigens are selected from microbial antigens or tumor antigens, and wherein the microbial antigens are selected from antigens derived from one or more of the following groups of microorganisms: non-probiotic bacteria, such as Streptococcus pyogenes, Serratia marcescens, Bacillus Calmette-Guerin, Clostridium tetani, Clostridium butyricum, Lactobacillus acidophilus; viruses, such as hepatitis B virus, adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, polio virus, measles virus, West Nick valley virus, Coxsackie virus, reovirus; parasites, such as plasmodium; the tumor antigen is one or more selected from the following groups: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, gastric cancer, colorectal cancer, bronchial cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant melanoma, brain tumor, renal cell carcinoma, astrocytoma, and glioblastoma.

In one embodiment, the biologically active ingredient comprises one or more selected from immunomodulatory antibodies, wherein the immunomodulatory antibodies are selected from one or more comprising: antibody blocking agents against inhibitory receptors, such as blocking antibodies against CTLA-4 molecules and PD-1 molecules; antibody blockers against ligands for inhibitory receptors, activating antibodies against immune response cell surface stimulatory molecules, such as anti-OX 40 antibodies, anti-CD 137 antibodies, anti-4-1 BB antibodies; neutralizing antibodies against immunosuppressive molecules in the microenvironment of the localized disease, such as anti-TGF-p 1 antibodies.

In one embodiment, the bioactive component comprises one or more selected from immunomodulatory antibodies, wherein the cytokine is selected from the group consisting of one or more of: tumor necrosis factor, interferon, interleukin.

The dosage form of the pharmaceutical composition according to the present disclosure is a topical dosage form. The pharmaceutical composition according to the present disclosure may be any topically administered dosage form that may contain a probiotic component and that satisfies the necessary conditions for the probiotic component to provide the desired effect. In one embodiment, the topical dosage form comprises an injection, a spread, or an ointment.

In the context of the present invention, the term "injectable formulation" refers to a sterile formulation containing an active ingredient and a liquid carrier and intended for in vivo administration. The injection is classified into a local injection, an intravenous injection, etc. according to the administration mode, and the local injection can be used as the local injection only after the given local administration concentration. The injection is divided into liquid injection, semi-liquid injection, powder injection for injection, etc. according to the commodity form. The powder injection for injection comprises sterile dry powder and a solvent, wherein the sterile dry powder contains part or all of active ingredients, and the solvent contains all of liquid carriers. The concentration of the active ingredient in an injection is the concentration of the active ingredient in its mixture with the entire liquid carrier, usually in the liquid drug at the end point (e.g. needle hole, catheter outlet, etc.) of the topical administration device (syringe, piercer, infusion catheter, etc.). For injectable powder injections, the concentration of the active ingredient is the concentration of the active ingredient in a mixture of sterile dry powder and vehicle (e.g., a reconstituted solution, or the pharmaceutically acceptable liquid carrier).

The pharmaceutical composition according to the present disclosure may further optionally comprise an excipient. 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 emulsifying agents, solubilizers, viscosity-increasing agents, and the like. The viscosity-increasing agent is, for example, sodium carboxymethylcellulose, polyvinylpyrrolidone or gelatin. Such as an antioxidant (e.g., ascorbic acid).

The pharmaceutical composition according to the present disclosure may further optionally comprise a tracer. The tracer may be any suitable one known to those skilled in the art and may include, for example, iodized oil.

The present disclosure also provides a pharmaceutical kit comprising one or more separate containers containing a pharmaceutical composition according to the present disclosure. The separate container may for example comprise an ampoule, a vial or the like.

In one embodiment, the pharmaceutical kit may further comprise instructions or labeling for how to administer the pharmaceutical composition to an individual in need thereof. In one embodiment, said administering comprises administering within said localized lesion, or both within and outside of said localized lesion, wherein said topical administration comprises, for example, subcutaneous injection under the axilla of said individual. At the time of administration, the ratio of the amount of the pharmaceutical composition administered to the target volume within the localized lesion is >0.1, 0.15-1.5, preferably 0.23-1.5 or 0.5-1.5. Or, according to the specific situation of the local lesion, the application amount of the pharmaceutical composition is more than or equal to 1ml, or the application amount in the local lesion is 10-150ml or/and the application amount outside the local lesion is 1.5-50 ml.

According to the preparation method of the present invention, the preparation of the pharmaceutical composition of the present invention comprises the steps of: preparing a topically administrable pharmaceutical formulation containing the probiotic component and optionally other substances, or preparing a topically administrable pharmaceutical formulation containing the probiotic component, the co-product and optionally other substances. Wherein the drug may be a liquid drug or a semi-fluid drug and the liquid drug may be a solution (e.g. a solution in a hydrophilic vehicle, preferably an aqueous solution), a suspension, an emulsion. In one embodiment, wherein the drug may be an in vivo drug delivery agent or a topical application.

In one embodiment, the inactivated probiotic of the invention may be prepared by subjecting the live probiotic to a process comprising one or more of the following steps of inactivation treatment: high-temperature inactivation, high-temperature and high-pressure inactivation, ultraviolet inactivation, chemical reagent inactivation and radiation inactivation. In one embodiment, the broken probiotic component of the present invention may be prepared by a process comprising the steps of: 1) and subjecting the live probiotic bacteria to a process comprising one or more of the following steps of disruption treatment to produce disrupted probiotic bacteria: high-pressure homogenization crushing, oscillating bead crushing, high-speed stirring bead grinding crushing, ultrasonic crushing, impact crushing, osmotic pressure impact crushing, freeze-thaw crushing, enzymatic lysis crushing, chemical crushing, detergent crushing, and the like; if necessary, 2) preparing the preparation of 1) into a broken probiotic isolated component (e.g. broken probiotic water-insoluble component particles, broken probiotic water-soluble component) by separation engineering (e.g. filtration or/and centrifugation). In one embodiment, the water-soluble component of the probiotic of the present invention may be prepared by a method comprising, for example, the preparation of the above-described disrupted probiotic water-soluble component (e.g., the disrupted probiotic supernatant component), or the preparation of a prior art probiotic water-soluble component (e.g., a probiotic extract). In one embodiment, the probiotic water-insoluble component particles of the present invention may be prepared by a process comprising, for example, the preparation of the above-described crushed probiotic water-insoluble component particles (e.g., crushed probiotic precipitated component), or the preparation of the prior art probiotic water-insoluble component particles (e.g., murein polysaccharide). In one embodiment, the probiotic semi-fluid based composition of the present invention may be prepared by a process comprising a warming step of a mixture of, for example, a probiotic polysaccharide or analogue thereof and water. In one embodiment, the warming temperature of the probiotic polysaccharide/water mixture is preferably from 50 to 110 ℃, which warming may be by any suitable method of the prior art (e.g., microwave heating, electric furnace warming, steam warming, etc.).

In one embodiment, the preparation of the pharmaceutical composition of the invention comprising the probiotic semi-fluid like component and the co-formulation comprises the steps of: and uniformly mixing the probiotic polysaccharide or the analogue thereof, the common substance and water, and then heating.

In one embodiment, the preparation of the pharmaceutical composition of the invention further comprises the steps of: such that the concentration of the probiotic component, the co-agent and optionally other substances in the pharmaceutical formulation is greater than or equal to the dosing concentration required in the present technical solution. When it is more than the administration concentration in the pharmaceutical composition of the present invention, it may be further diluted for use.

In one embodiment, the preparation of the pharmaceutical composition of the invention further comprises the steps of: the contents of the probiotic component, the co-product and optionally other substances in the pharmaceutical preparation are adapted to meet the required drug volume/target volume ratio in the solution according to the invention, e.g. drug dispensing and capping according to the drug volume/target volume ratio.

In one embodiment, the preparation of the pharmaceutical composition of the invention further comprises the steps of: subjecting the preparation to a sterilization process, wherein the sterilization comprises one or more of: high temperature sterilization, high temperature and high pressure sterilization, ultraviolet sterilization, chemical reagent sterilization and radiation sterilization.

In accordance with the principles of these methods described above, one skilled in the art can prepare a variety of specific dosage forms comprising the compositions of the present invention by any suitable specific method. For example, variations in the pharmaceutical compositions of the invention include: the composition comprises different types and concentrations of the pharmaceutical composition, different types and concentrations of other drugs, and different types and concentrations of other additives (such as analgesic, activating agent, etc.).

In the present disclosure, the localized disease includes solid tumors, non-neoplastic tumors (e.g., non-neoplastic nodules), localized inflammation (e.g., cervical erosion), secretory gland dysfunction, and skin disorders.

In the context of the present disclosure, the term "locally diseased disease" refers to a disease having symptoms of a local lesion. The term "localized disease (also simply referred to as" lesion ") refers to an abnormal local area of an animal (preferably a human) body, either native or secondary, that includes a structurally (e.g., diseased tissue), morphologically or functionally symptomatic zone and an abnormal region in communication therewith. For example, when the locally diseased disease is a solid tumor, the local disease is the tumor body and its tissue where the tumor cells are located, and the abnormal region in communication with the tumor body is the adjacent region in communication with the tumor body (e.g., in communication with lymphatic or blood vessels) and having or suspected of having tumor cells; when the locally diseased disease is a non-neoplastic mass, the local disease becomes an abnormal non-neoplastic mass, such as a hyperplasia, cyst, nodule, or the like; when the local disease is local inflammation, the local disease becomes an inflammation region, such as an inflammation surface or an inflammation body; when the local lesion disease is abnormal secretion, the local lesion becomes an abnormal source or a secretory gland where the local lesion disease is located. For another example, when the disease is abnormal insulin secretion, the abnormality is caused in the islets of langerhans, and the local tissue is the islets of langerhans or the pancreas in which the islets of langerhans are located; when the condition is a skin condition, the localized tissue is the diseased skin or an appendage 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 having a tumor body, which may be due to any pathology (malignant and non-malignant) and at any stage of the tumor, including for example the following groups classified by tumor cell type: epithelial cell tumors, sarcomas, lymphomas, germ cell tumors, blastomas; and tumors named as the organ or tissue in which the tumor cell foci are located, including, for example, tumors named as the following organs or tissues: brain, skin, bone, muscle, breast, kidney, liver, lung, gall bladder, pancreas, brain, esophagus, muscle, large intestine, small intestine, spleen, stomach, prostate, emerald, ovary, or uterus.

Specifically, the tumor includes malignant tumor and non-malignant tumor, and the malignant tumor includes, for example, breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, esophageal cancer, stomach cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, malignant lymphoma, malignant cerebroma, etc.; non-malignant tumors include, for example, breast tumor, pancreatic tumor, thyroid tumor, prostate tumor, hepatoma, lung tumor, intestinal tumor, oral tumor, esophageal tumor, stomach tumor, nasopharyngeal tumor, laryngeal tumor, testicular tumor, vaginal tumor, uterine tumor, fallopian tube tumor, ovarian tumor, lymphoma, brain tumor, and the like.

In one embodiment, the localized disease condition is selected from a non-neoplastic lesion (e.g., a non-neoplastic nodule). In one embodiment, the non-neoplastic tumor comprises, for example, a secretory gland non-neoplastic nodule. In one embodiment, the secretory glands include, for example, thyroid, breast, liver, lung, intestine (e.g., polyps), and the like.

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

In one embodiment, the localized disease condition is selected from a skin condition. In one embodiment, the skin disease includes, for example, chronic mucocutaneous candidiasis, various ringworms.

The pharmaceutical composition disclosed herein is a therapeutic agent which, when used to treat a disease, may also be administered in combination with other interventional therapies, systemic chemotherapy, immunotherapy, photodynamic therapy, sonodynamic therapy, surgical intervention or a combination of such therapies to further enhance the therapeutic effect.

The present application includes the following embodiments:

1. use of a probiotic composition as an active ingredient providing a therapeutic effect in the manufacture of a topically administrable pharmaceutical composition for the treatment of a locally pathological condition.

2. The use according to item 1, wherein the pharmaceutical composition further comprises a chemically active ingredient capable of producing a synergistic effect with the probiotic component, and wherein the ratio of the amounts of the probiotic component and the chemically active ingredient (probiotic component weight concentration/co-agent weight concentration) is (1-110)/(1-100).

3. A topically administrable pharmaceutical composition for the treatment of a localized disease comprising a probiotic component providing a therapeutic effect, and a pharmaceutically acceptable suitable carrier.

4. The pharmaceutical composition according to item 3, which optionally further comprises a chemically active ingredient capable of producing a synergistic effect with the probiotic component.

5. A topically administrable pharmaceutical composition for the treatment of a locally pathological condition, comprising a therapeutically active probiotic component, a chemically active ingredient capable of exerting a synergistic effect with the probiotic component, and a pharmaceutically acceptable suitable carrier, and the ratio of the amounts of the probiotic component and the chemically active ingredient (probiotic component weight concentration/co-agent weight concentration) is (1-110)/(1-100).

6. The use or pharmaceutical composition according to one of items 1 to 5, wherein the probiotic component is selected from those which minimize bacterial immunogenicity, preferably selected from one or more of the group comprising: water-soluble components of probiotics, semi-fluid components of probiotics, water-insoluble particles of probiotics and inactivated probiotics.

7. The use or pharmaceutical composition according to clause 6, wherein the probiotic water-soluble component is selected from the group comprising one or more of the following groups and derivatives thereof: crushing a probiotic supernatant component, a probiotic extract, a probiotic intracellular water-soluble component and a water-soluble derivative of a probiotic cell wall component; the probiotic semi-fluid like component is preferably selected from one or more of the probiotic components whose aqueous mixture forms a semi-fluid like composition, for example one or more of the probiotic polysaccharides and analogues thereof: the water insoluble particles of the probiotic component are selected from one or more of the group comprising: breaking the probiotic precipitated components, the probiotic cell wall polysaccharide particles and the probiotic cell wall polysaccharide nanoparticles; the inactivated probiotic bacteria are preferably selected from, but not limited to, inactivated probiotic bacteria, all of which are intact bacteria.

8. Use or pharmaceutical composition according to one of items 1 to 7, wherein the probiotic bacteria are selected from one or more of natural or/and engineered bacteria comprising the following group: probiotic bacillus, probiotic lactobacillus, probiotic bifidobacterium and probiotic fungi.

9. The use or pharmaceutical composition according to item 8, wherein the M.pullulans comprises one or more selected from the group consisting of: bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, and Bacillus natto; the lactobacillus comprises one or more selected from the group consisting of: lactobacillus acidophilus, lactobacillus casei, lactobacillus plantarum, lactobacillus brevis, and lactobacillus fermentum; the bifidobacteria comprise one or more selected from the group consisting of: bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, enterococcus faecium, and Streptococcus faecalis; the fungi comprise one or more selected from the following group: a yeast, a diamond saw yeast, and wherein the yeast comprises one or more of: saccharomyces cerevisiae, Delbert/Buerger's yeast, Wilkh's yeast, Pichia yeast, Candida utilis, and lactalbumin yeast.

10. A use or a pharmaceutical composition according to any of items 1 to 9, wherein the probiotic comprises a yeast selected from Saccharomyces cerevisiae and/or Saccharomyces bougii.

11. The use or pharmaceutical composition according to one of clauses 2 or 4 to 10, wherein the suitable carrier is water; the chemically active ingredient is soluble in water, and the probiotic component is water-insoluble and is selected from one or more of the group comprising: inactivated probiotics, water insoluble particles of probiotic components, and semi-fluid components of probiotics.

12. The use or the pharmaceutical composition according to one of items 1 to 11, wherein the concentration of the probiotic component in the pharmaceutical composition is > 0.1%, ≧ 0.25%, 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 5-15%.

13. The use or pharmaceutical composition according to item 12, wherein when the probiotic component comprises the inactivated probiotic, the concentration of said inactivated probiotic in the pharmaceutical composition is > 0.3%, > 0.75%, 0.75-15, preferably 1.5-15% or 5-15%; when the probiotic component comprises the probiotic water-soluble component, the concentration of the probiotic water-soluble component in the pharmaceutical composition is >0.1, or 0.15-25%, preferably 0.35-15% or 5-15%; when the probiotic component comprises said probiotic water-insoluble component particles, the concentration of said probiotic water-insoluble component particles in the pharmaceutical composition is >0.5, or 0.5-15%, preferably 1.5-15% or 5-15%; when the pharmaceutical composition is a semi-fluid composition, the concentration of the probiotic semi-fluid component in the pharmaceutical composition is > 2.5%, 2.6-25%, preferably 5-15%.

14. The use or pharmaceutical composition according to one of items 2 or 4 to 13, wherein the chemically active ingredient is a composition comprising one or more selected from the group consisting of a weak topically acting compound and/or a cytotoxic drug, and wherein when the chemically active compound comprises a cytotoxic drug, the ratio of the amounts of the probiotic component and the cytotoxic drug (W ™)_{Probiotic compositions}/W_{Cytotoxic drug}) Is (1-110)/(1-100); when the chemically active compound comprises a weak topically acting compound, the ratio of the amounts of the probiotic component and the weak topically acting compound (V)_{Probiotic compositions}/W_{Compounds with weak local action}) Is (1-90)/(1-100).

15. The use or pharmaceutical composition according to item 14, wherein the weak topically acting compound is selected from the group comprising one or more of: amino acid based nutrients, vital dyes, quinines, low concentrations of acidifying agents, low concentrations of basifying agents, pH buffering systems comprising acidifying or/basifying agents, and wherein when the weak topically-acting compound includes the amino acid nutrient, the ratio of the amounts of the probiotic component and amino acid nutrient (W) is such that the weak topically-acting compound comprises the amino acid nutrient_{Probiotic compositions}/W_{Amino acid nutrient}) Is (1-20)/(1-100); when the weak topically-acting compound includes the vital dye, the ratio of the amounts of the probiotic component and the vital dye (W)_{Probiotic compositions}/W_{Vital dyes}) Is (7-90)/(1-100); when the weak topically-acting compound comprises the quinine drug, the ratio of the amounts of the probiotic component and the quinine drug (W) is_{Probiotic compositions}/W_{Quinine medicine}) Is (2-90)/(1-100); when the weak topical effect compound comprises the acidifying or/and alkalinizing agent, the probiotic component and the acidifying or/and alkalinizing agent are present in a ratio of amounts (W)_{Probiotic compositions}/W_{Acidifying or/and alkalifying agents}) Is (2-60)/(1-100).

16. The use or pharmaceutical composition according to clause 14 or 15, wherein the chemically active ingredient is selected from the group consisting of vital dyes and 1 or more other of said chemically active ingredients.

17. The use or pharmaceutical composition according to clause 14 or 15, wherein the chemically active ingredient is selected from the group consisting of the cytotoxic drug and a vital dye.

18. The use or pharmaceutical composition according to item 15, wherein the amino acid based nutrient is an amino acid selected from the group comprising or consisting of an oligopeptide and a polypeptide comprising or consisting of: arginine, lysine, glycine, cysteine, alanine, serine, aspartic acid, glutamic acid, and the amino acid nutrient concentration is > 2.5%, or 5-30%, preferably 5-25%.

19. The use, pharmaceutical composition according to clause 15, wherein the vital dyes are selected from the group consisting of bengal red and/or one or more of the following methylene blue dyes: methylene blue, patent blue, isothio blue, new methylene blue, and wherein the concentration of said bengal red is 2.5% -20%; the concentration of the methylene blue dye is more than or equal to 0.25 percent, or 0.25 to 2.5 percent, and preferably 0.5 to 2.5 percent.

20. The use or pharmaceutical composition according to clause 15, wherein the acidifying agent is selected from one or more of strong acids or/and weak acids, and the amount ratio of the probiotic component to the acidifying agent (probiotic component weight concentration/acidifying agent weight concentration) is (1-20)/(0.5-50), and the concentration of the acidifying agent is ≥ 0.5%, 0.5-2% (strong acid), or 2-15% (weak acid), wherein the strong acid is, for example, hydrochloric acid; the weak acid is, for example, oxalic acid, acetic acid, lactic acid, citric acid, malic acid; the alkalizer is selected from one or more of strong alkali and/or weak alkali, and the amount ratio of the probiotic component to the alkalizer (probiotic component weight concentration/alkalizer weight concentration) is (1-20)/(0.5-50), and the alkalizer has a concentration of 0.5% or more, 0.5-5% (strong alkali), or 2-15% (weak alkali), wherein the strong alkali is sodium hydroxide or potassium hydroxide; the weak base is, for example, sodium dihydrogen phosphate, sodium hydrogen carbonate, sodium carbonate.

21. The use or pharmaceutical composition according to item 14, wherein the cytotoxic drug comprises one or more selected from the group consisting of: drugs that disrupt DNA structure and function, such as cyclophosphamide, carmustine, platinum complexes, doxorubicin, topotecan, irinotecan; drugs that interfere with the transcription of RNA embedded in DNA, such as anti-tumor antibiotic drugs; drugs that interfere with DNA synthesis such as 5-fluorouracil (5-Fu), furfluorouracil, difurofluorouracil, cytarabine, cyclocytidine, 5-azacytidine; drugs influencing protein synthesis, such as colchicine drugs, vinblastine drugs and taxane drugs, and the concentrations of the cytotoxic drugs are more than or equal to 0.1% and 0.1-15%.

22. The use or pharmaceutical composition according to one of items 1 to 21, wherein the pharmaceutical composition further optionally comprises a biologically active ingredient, wherein the biologically active ingredient is selected from one or more of the group comprising: antigen, immunoregulation antibody, cell factor, adjuvant.

23. A method of treating a localized disease condition comprising the steps of: topically administering a therapeutically effective amount of a pharmaceutical composition according to one of items 3-23 into or/and outside of a localized lesion in an individual in need thereof.

24. The method of item 23, comprising the steps of: administering a therapeutically effective amount of the pharmaceutical composition to the individual within a localized lesion, or both within and outside of a localized lesion.

25. The method according to item 23 or 24, further comprising optionally further performing one or more other treatments, such as chemotherapy, immunotherapy, radiotherapy, surgery, chemical ablation, physical ablation, before, during or after administration of the pharmaceutical composition.

26. The method according to one of clauses 23 to 25, wherein the probiotic component is present in the pharmaceutical composition in an amount such that its topical administration concentration is > 0.1%,. gtoreq.0.25%, 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 5-15%.

27. The method according to item 26, wherein when said probiotic component comprises said inactivated probiotic bacteria, then said inactivated probiotic bacteria must be present in the pharmaceutical composition in an amount such that its local administration concentration is > 0.3%, > 0.75%, 0.75-15, preferably 1.5-15% or 5-15%; when the probiotic component comprises the probiotic water-soluble component, the content of the probiotic water-soluble component in the pharmaceutical composition must be such that its concentration for topical administration is >0.1, or 0.15-25%, preferably 0.35-15% or 5-15%; when the probiotic component comprises the probiotic water-insoluble component, the content of the probiotic water-insoluble component in the pharmaceutical composition must be such that its concentration for topical administration is >0.5, or 0.5-15%, preferably 1.5-15% or 3.5-15%.

28. The use, pharmaceutical composition or method according to one of items 1 to 27, wherein the ratio (v/v) of the amount of the pharmaceutical composition administered to the target volume within the local lesion is >0.1, 0.15 to 1.5, preferably 0.23 to 1.5 or 0.5 to 1.5.

29. Use, pharmaceutical composition or method according to one of items 1 to 298, wherein alternatively the pharmaceutical composition is administered in an amount of ≥ 1ml, or in a local lesion in an amount of 10-150ml and/or in an amount outside the local lesion in an amount of 1.5-50 ml.

30. The use, pharmaceutical composition or method according to one of items 1-30, wherein the localized disease condition comprises a tumor, a non-neoplastic enlargement, a localized inflammation, a secretory gland dysfunction and a skin disease, wherein the tumor comprises a malignant and a non-malignant solid tumor.

31. The use, pharmaceutical composition or method according to item 31, wherein said solid tumor comprises one or more of the following tumors and secondary tumors thereof: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, esophageal cancer, gastric cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, cerebroma, and lymphoma.

32. Use, pharmaceutical composition or method according to one of items 1 to 32, wherein the therapeutic effect preferably comprises a local treatment or/and immunotherapy involving a local effect (or local synergy), and wherein the local effect (or local synergy) comprises a local chemical effect (or local chemical synergy) and optionally further effects; the local treatment comprises a chemical-like ablation of one or more local lesions and optionally other chemotherapies; said immunotherapy comprises a secondary immunization of said local effect (or local synergy) within or/and outside said lesion and optionally other immunizations.

33. The use, pharmaceutical composition or method according to one of items 1 to 33, wherein the patient for whom the treatment is suitable is selected from the group comprising one or more of: immunosuppressed patients, patients with drug administration within a localized lesion, patients with ablation of a similar type of localized lesion tissue, patients with secondary immune material production within a localized lesion, patients with secondary immune material production within the region administered outside of a lesion.

34. A pharmaceutical composition comprising a probiotic composition together with a pharmaceutically acceptable suitable carrier for use in the treatment of a localized disease condition, wherein said localized disease condition comprises a solid tumour.

35. A pharmaceutical kit comprising one or more containers filled with a pharmaceutical composition according to one of items 3-22.

36. The pharmaceutical kit of clause 35, further comprising instructions or labeling for how to administer the pharmaceutical composition to an individual in need thereof.

37. The pharmaceutical kit of item 36, wherein said administering comprises administering within said localized lesion, or both within and outside of said localized lesion, wherein said outside of said localized lesion comprises, for example, subcutaneous injection under the axilla of said individual.

Based on the studies described in more detail below, the pharmaceutical composition of the present invention, although the specific mechanism remains to be further studied, exhibits pharmaceutical effects of promoting effective destruction of tumor body tissues while minimizing damage to normal tissues of patients, thereby achieving safe and effective treatment of locally diseased diseases.

Examples

The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention thereto.

The probiotics used in the following specific examples are all commercially available. Specifically, the strains of the probiotics are selected from saccharomyces cerevisiae approved by food, drug authorities or loaded in official pharmacopoeia and strains used in probiotic preparations. For example, the following strains contained in probiotic preparations approved by the chinese drug administration: clostridium butyricum strain in live Bacillus coagulans tablets, Bacillus licheniformis strain in live Bacillus licheniformis granules (capsules), Bacillus cereus strain in live oral live Bacillus cereus preparations, Clostridium butyricum strain in live Clostridium butyricum capsules, Saccharomyces boulardii strain in Bruna powder (capsules), Lactobacillus acidophilus strain in Lactobacillus acidophilus capsules, Clostridium butyricum strain in live Clostridium butyricum preparations for oral administration, enterococcus faecium strain and Bacillus subtilis strain in dual live bacterial granules, Lactobacillus plantarum strain, Lactobacillus casei strain and Lactobacillus casei strain in triple live Clostridium butyricum tablets, Lactobacillus casei strain and saccharomycete strain in live vaginal lactobacillus capsules, Bifidobacterium adolescentis strain in live Bacillus bifidus powder, Bifidobacterium longum strain in triple live Bacillus bifidus capsules, Lactobacillus strain, Lactobacillus casei strain, Bacillus bifidus strain in live Bacillus bifidus capsules, Bifidobacterium longum strain in live Bifidobacterium bifidus capsules, Bifidobacterium longum strain, Lactobacillus strain in live Bacillus bifidus capsules, Lactobacillus strain in Bacillus bifidus strain, Lactobacillus strain in Bacillus bifidus triple viable bacteria tablets, Lactobacillus strain in oral live Bacillus bifidus strain, Lactobacillus strain in Bacillus bifidus strain, Lactobacillus strain in Bacillus bifidus strain in the form of Bacillus bifidus strain in the same strain, Lactobacillus strain, and Bacillus bifidus strain in the same strain, and/or strain, Enterococcus faecalis strains, and the like.

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

TABLE 1

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

The experimental animals used in the following examples were all purchased from professional laboratory animals company and were all SPF (Specific Pathogen Free) grade animals. The mice are healthy females with the age of 6-8 weeks and the body weight of 17.5-20.5 g.

In the following examples, unless otherwise indicated, subcutaneous transplantation of tumor animals was performed according to the general practice of subcutaneous inoculation of solid tumor cells according to the guidelines issued by the drug administration. Unless otherwise indicated, examplesThe body tumor grows to a desired volume (e.g., 50-500mm tumor bearing in mice)³) Then for successful modeling, the model was randomly divided into experimental groups of 6 animals each using PEMS 3.2 software (compiled by the national institutes of public health, western, university, Sichuan). Items for experimental observation, measurement and analysis include general state, body weight, food intake, animal graft versus host disease, solid tumor volume, tumor weight, survival time, and the like.

In the following examples, 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 × b²Wherein a represents the tumor length and b represents the tumor width.

Relative tumor proliferation rate R (%) ═ T_RTV/C_RTVX 100, wherein T_RTVAnd C_RTVRelative tumor volumes of study group and negative control group, respectively, the relative tumor volume ═ V_t/V₀In which V is₀Mean tumor volume measured for the day of group administration (i.e., day one); v_tMean tumor volume measured t days after group administration.

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 potency of drug i is denoted as E_iIn which E_iCan be (100-R)_i) % or r_i％。

In the following examples, the experimental results (e.g. tumor weight, tumor volume) are expressed as mean ± standard deviation (x ± s), the differences between the two experimental animal groups and the group mean are compared by significance test using statistical software SPSS 13.0 or SPSS 19.0, the test is performed using statistic t, the test level α is 0.05, P <0.05 indicates that the difference is statistically significant, and P >0.05 indicates that it is not statistically significant.

In the following examples, the type of action (pharmacology) of a drug is studied by drug effect, in particular by comparing the drug effect of the same study drug in different technical protocols. For example, the difference in drug effect between regimen X and Y when drug i is not unusually large (e.g., E_iX/E_iY<200%), it is likely to be the result of different kinetic conditions (effect concentrations) in the substantially same type of drug action (pharmacology); when the drug effect is unusually large (e.g. E)_iX/E_iY>200%), the drug effect of drug i in regimen X should be greater than its kinetic expectation for the type of drug action (pharmacology) in regimen Y, and thus likely to involve a different type of drug action (pharmacology) than regimen Y. When two drugs show distinctly different E_iX/E_iYRelatedly, they are likely to be involved in apparently different pharmacologies; and when both drugs show similar E_iX/E_iYIn relation, they are likely to be involved in the same pharmacology, at least in similar pharmacology (e.g., chemoablative pharmacology and chemoablative pharmacology).

In the following examples, the efficacy of study drugs was evaluated by comparing their efficacy differences with positive controls in the same protocol. For example, when tumor weight difference or volume difference between the study drug group and the positive control group is not statistically significant (P)>0.05), and the efficacy of the study drug group is not less than 50% of the positive control group (e.g., E)_{Positive control group}/E_{Study drug group}200%) or less, the efficacy of the study drug is evaluated as having a similar efficacy (or therapeutically significant efficacy) as the positive control; when the difference in tumor weight or volume between the study drug group and the positive control group was statistically significant (P)<0.05) and the efficacy of the study drug group is greater than 200% of the positive control (e.g., E)_{Study drug group}/E_{Positive control group}>200%), the efficacy of the study drug is evaluated as having efficacy exceeding that expected by similar pharmacology for the positive control, which also tends to indicate the development of a new pharmacology.

In the examples below, chemotherapy positive controls included a classical cytotoxic drug (e.g., 0.5-1% 5-fluorouracil, which has a tumor inhibition rate of 30% or greater under the conditions of the examples below) and a classical chemoablative agent (e.g., 75-99% ethanol, which has a tumor inhibition rate of 15% or greater under the conditions of the examples below). Immunopotentiating positive controls include classical immunopotentiators (e.g., interleukin-12, which has a tumor suppression rate of ≦ 10% under the conditions of the examples below).

In the following examples, the judgment of the effect shared by the compositions is made by the q judgment. Within the scope of the present invention, the combination of drug A and drug B is designated as B/A. A. The single drug effect of B is respectively marked as E_AAnd E_BThe actual shared drug effect of A/B is recorded as E_A+B。

The effect of drug combinations is highly uncertain and is often judged in the industry by the following q:

q is the actual shared effect/theoretical purely additive expected effect.

The q calculation formula has many calculation methods for theoretically and simply adding expected drug effects, and most methods aim at cell experimental effects. It is generally believed that when q is 1, the actual sharing effect is in accordance with theoretical expectations, showing an additive effect; when q <1, the actual sharing effect is weaker than the theoretical expectation, showing antagonism; when q >1, the actual co-action is over the theoretical expectation and shows a synergistic effect.

One method for determining the effect of concomitant medication in animal experiments is the Burgi method (Burgi Y. Pharmacology; Drug actions and reactions. cancer res.1978, 38 (2); 284. 285). The Burgi method was modified for just-in-gold (just-in-gold, equi-probability and curve and "Q50", second college of medicine, Shanghai, 1981, 1, 75-86) by the Q-calculation formula:

q＝E_A+B/(E_A+E_B-E_A·E_B)，

wherein (E)_A+E_B-E_A·E_B) The expected effect is simply added for the pharmacology theory of A drug and B drug. It is generally believed that q ═ 1.00 reflects the purely additive expected effect.

On the basis of the Q calculation of the above-mentioned Jingzheng (Zhang Xiao Wen, and Jingzheng (Zhang Xiao Wen, a new method for estimating the effect of combined medication by Q value, Shuang30 Fang, Shanghai second college of medicine, 1985, 5, p353-354) further makes the Q judgment more suitable for the practical experiment. They reflect the purely additive expected effect with q ═ 0.85 to 1.15. The following examples of the present invention are judged for the shared effect of the combined administration according to the improved cases of the Jinzhengyu (average of the two drugs):

when the combination group does not show a therapeutically significant effect, the combined administration thereof does not show a therapeutically significant co-effect. When the composition group shows a therapeutic effect, the combination of the compositions is additive if q is between 0.85 and 1.15 (in line with theoretical purely additive expectations), significant synergy if q >1.15 (in line with theoretical purely additive expectations), significant antagonism if q <0.85 (not in line with theoretical purely additive expectations); the difference in tumor weight between the composition group and the negative control group was not statistically significant, and the composition showed no significant synergy.

Example 1: preparation of the composition

Many different compositions of the invention can be formulated according to the above-described method of preparation of the compositions of the invention. The composition of the part of the composition of the present invention prepared in this example is listed in table 2 (probiotic component as active ingredient) and table 3 (probiotic component as synergistic active ingredient).

TABLE 2

Examples of the preparation tests for several of the preparations in Table 2 are listed below.

Example 1 a: probiotic bacteria (e.g. 2.5g of dry saccharomyces cerevisiae powder), optionally other components, and a liquid carrier (e.g. water for injection) in a volume up to a total volume (e.g. 100ml) are measured at the desired (e.g. required for topical chemistry), mixed slowly and homogenized and broken up using a homogenizer. The crushed probiotics with different crushing degrees (preferably 100 percent crushing) can be obtained by adjusting the homogenizing process parameters (such as 10000-. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The liquid mixture is dispensed (e.g., 10 ml/bottle) and capped to obtain a dosage form and specification of the pharmaceutical composition that provides intratumoral local action.The resulting preparation was a 1. Using the same method as for the preparation of a1, the preparation of different compositions (e.g. preparations a2-A8 in the above table) can be performed separately from different probiotics.

Further, using the method for preparing disrupted probiotic bacteria in the above-described preparation, a high concentration disrupted probiotic bacteria suspension (e.g., 10% disrupted Saccharomyces cerevisiae suspension) can be prepared from probiotic bacteria (e.g., 10g of Saccharomyces cerevisiae dry powder and water for injection up to 100ml), the suspension can be added to a centrifuge bottle to be centrifuged at a centrifuge, the disrupted probiotic bacteria supernatant component and the disrupted probiotic bacteria sediment component can be obtained in various degrees of centrifugation by adjusting the rotation speed of the centrifuge (e.g., 1000-25000 rpm), the centrifugation time (e.g., 0.5-30 minutes) and the number of times of centrifugation (e.g., 2-4 times), the supernatant can be separated after centrifugation, the remaining sediment component can be dried (e.g., 125 ℃, 90 minutes) to prepare a disrupted probiotic bacteria sediment component dry powder, the difference between the raw material probiotic bacteria (e.g., 10 g., Saccharomyces cerevisiae) and the disrupted probiotic bacteria sediment component dry powder (e.g., 4 g.) prepared therefrom can be used as the disrupted probiotic bacteria supernatant Dry weight of ingredients (e.g., 6 g).

Example 1 b: probiotic water-insoluble component particles (e.g. 1.4g of the crushed dry saccharomyces cerevisiae powder prepared according to the method of example 1 a), optionally other components, and a liquid carrier (e.g. water for injection) in a volume up to a total volume (e.g. 100ml) are measured at the desired (e.g. desired for topical chemical action) concentration and mixed slowly and homogeneously. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The liquid mixture is dispensed (e.g., 10 ml/bottle) and capped to obtain a dosage form and specification of the pharmaceutical composition that provides intratumoral local action. The resulting preparation was a 9. Using the same method as for the preparation of a9, the preparation of different broken probiotic pellet fractions (e.g. preparations a10 and a11 in the above table) can be performed separately from different probiotics.

Example 1 c: measuring water soluble component of probiotic bacteria (such as 1.1g of the disrupted Saccharomyces cerevisiae supernatant prepared according to the method of example 1a or 20ml of the same amount of supernatant) at desired concentration (such as required for topical chemical action), optionally other components, and adding to total volume (for exampleE.g., 100ml) of a liquid carrier (e.g., water for injection), and mixing them slowly and uniformly. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The liquid mixture is dispensed (e.g., 10 ml/bottle) and capped to obtain a dosage form and specification of the pharmaceutical composition that provides intratumoral local action. The resulting preparation was a 12. Using the same method as for the preparation of a12, the preparation of different compositions (e.g. preparations a13-a15 in the above table) can be performed separately from different probiotics.

Example 1 d: the probiotic semi-fluid component (e.g. 10g beta-glucan), optional other components, and a liquid carrier (e.g. water for injection) are measured at the desired (e.g. desired for topical chemistry) concentration, mixed slowly and homogeneously to form a suspension, which is then heated (e.g. at 50-110 ℃ for 0.5-24 hours) and allowed to cool to form a semi-fluid. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The semifluid is dispensed (e.g., 10 ml/vial) and capped to provide a pharmaceutical composition dosage form and format that provides intratumoral local action. The resulting preparation was a 16. Using the same method as for the preparation of a16, the preparation of different semi-fluid compositions can be performed from different probiotic semi-fluidizable components, respectively. Experiments have shown that only when the concentration of the semi-fluidizable component of the probiotic is above a certain threshold (e.g.. beta. -glucan. gtoreq.2.5%) is it possible to convert the liquid in which it is contained into a semi-fluid class upon warming.

Example 1 e: probiotic bacteria (e.g. 2.5g of dry saccharomyces cerevisiae powder), optionally other components, and a liquid carrier (e.g. water for injection) in a constant volume (e.g. 100ml) are measured at the required (e.g. required for topical chemistry) concentration and slowly mixed to obtain a probiotic mixed solution. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The liquid mixture is dispensed (e.g., 10 ml/bottle) and capped to obtain a dosage form and specification of the pharmaceutical composition that provides intratumoral local action. Subjecting the preparation (e.g. to2.5% saccharomyces cerevisiae) was put into a pasteur inactivation type machine for pasteur inactivation (60 ℃, 48 hours), thus obtaining a 17. The preparation of different inactivated probiotics (e.g. preparations a18-a24 in the above table) can be performed separately from different probiotics using the same method as the preparation of a 17.

TABLE 3

Examples of the preparation tests for several of the preparations in Table 3 are listed below.

Example 1 f: probiotic bacteria (e.g. 1.5g saccharomyces cerevisiae), synergistic ingredients (e.g. 20g of resistant ammonia acid), optionally other ingredients, and water for injection to a total volume of 100ml are measured in synergistic ratios and synergistic concentrations as required (e.g. for local synergy) and slowly mixed homogeneously using the same method as for preparation of a1 and disrupted using a homogenizer to obtain a disrupted probiotic/synergistic ingredient suspension. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The suspension is packaged (e.g. 10 ml/bottle) and capped to obtain a pharmaceutical composition dosage form and specification providing intratumoral local effects. The resulting preparation was B1. Using the same method as for the preparation of B1, the preparation of different compositions (e.g. preparations B2-B5 in the above table) can be carried out from different probiotics and/or different synergistic ingredients, respectively.

Example 1 g: the probiotic water-insoluble component particles (e.g. 2g of the crushed dry saccharomyces cerevisiae powder prepared according to the method of example 1 a), the synergistic components (e.g. 10g of reduced glutathione), optionally other components, and water for injection to a total volume of 100ml are measured in synergistic ratios and synergistic concentrations as required (e.g. required for local synergy) and slowly mixed until homogeneous. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) Packaging (e.g. 10 ml/bottle) and sealing to obtain a pharmaceutical composition with local intratumoral effect. The resulting preparation was B6. Using the same method as for the preparation of B6, the preparation of different compositions (e.g. preparations B7 and B8 in the above table) can be performed from different broken probiotic pellet fractions or/and different synergistic ingredients, respectively.

Example 1 h: the probiotic water-soluble component (e.g. 1g of the disrupted Saccharomyces cerevisiae supernatant component prepared as in example 1a or 20ml of the equivalent amount of the supernatant) and the synergistic amount are measured in the desired (e.g. required for local synergy) synergistic ratio and synergistic concentration, the synergistic component (e.g. 20g of the resistant acid), optionally other components, and water for injection to a total volume of 100ml, and they are slowly mixed well. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The liquid mixture is dispensed (e.g., 10 ml/bottle) and capped to obtain a dosage form and specification of the pharmaceutical composition that provides intratumoral local action. The resulting preparation was B9. Using the same method as for the preparation of B9, the preparation of different compositions (e.g., preparations B10-B16 in the above table) can be performed from different disrupted probiotic supernatant components and their synergistic ingredients, respectively.

Example 1 h: the probiotic semi-fluidable component (e.g. 7.5g β -glucan), the synergistic component (e.g. 1g 5-fluorouracil), optionally other components, and a liquid carrier (e.g. water for injection) are measured in synergistic ratios and concentrations as required (e.g. for local synergy), mixed slowly and homogenously to form a suspension, which is then heated (e.g. at a temperature of 50-110 ℃ for 0.5-24 hours) and allowed to cool to form a semi-fluid. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The semifluid is dispensed (e.g., 10 ml/vial) and capped to provide a pharmaceutical composition dosage form and format that provides intratumoral local action. The resulting preparation was B17. The preparation of different semi-fluid compositions (e.g. B18 and B19 in the above table) can be carried out using the same method as the preparation of B17.

Example 1 i: probiotic bacteria (e.g. 2.5g dry powder of saccharomyces cerevisiae) are measured in synergistic ratios and concentrations as required (e.g. required for local synergy)The probiotics/synergistic components mixed solution is obtained by slowly and uniformly mixing the components (such as 1g of methylene blue and 1g of 5-fluorouracil) and injection water with the volume being 100 ml. If the ratio of the volume of the drug to the volume of the target region is determined as desired (e.g., the average volume of a clinically common solid tumor is 30 cm)³) The liquid mixture is dispensed (e.g., 10 ml/bottle) and capped to obtain a dosage form and specification of the pharmaceutical composition that provides intratumoral local action. The preparation (e.g. 2.5% Saccharomyces cerevisiae) is pasteurized in a pasteurized machine (60 deg.C, 48 hours) to obtain B20. Using the same method as for the preparation of B20, the preparation of different inactivated probiotics (e.g., preparations B21-B27 in the above table) can be performed separately from different probiotics.

If the inactivated preparation (such as A1-16, B1-B19) is pasteurized (60 deg.C, 48 hr) in a pasteurized inactivation machine, sterile liquid injection can be obtained.

If the liquid preparation is freeze-dried, a freeze-dried powder bottle for injection can be obtained. The freeze-drying process conditions include, for example: the pre-freezing condition is that the temperature is kept at minus 45 ℃ for 4 hours; sublimation drying condition is that the heating rate is 0.1 ℃/min, and the heating is kept for at least 10 hours when the temperature is raised to-15 ℃; the desorption drying conditions were 30 ℃ for 6 hours. The liquid carrier (e.g. water for injection) is dispensed (e.g. 7.5 ml/bottle) and capped according to the desired concentration of probiotic components to obtain a vehicle bottle for injection. When in use, the sterile solvent in the bottle is pumped into the freeze-dried powder bottle for injection and is uniformly mixed to form liquid medicine (for example, 1.5 percent of saccharomyces cerevisiae crushed component/20 percent of amino acid), and the liquid medicine can be used as injection medicine.

Examples 2-8 below pharmacological studies were conducted on the therapeutic effects that the probiotic composition can provide using an animal model of immunosuppression (nude mice). The nude mouse is a congenital athymic nude mouse, wherein a recessive mutant gene 'nu' positioned on the 11 th chromosome pair is introduced into a BALB/c mouse. The thymus of the patient only has remnant or abnormal epithelium, can not make T cells normally differentiate, and is lack of the auxiliary, inhibiting and killing functions of mature T cells, and low in immunity. In the prior art, tumor-bearing nude mice are widely used for drug studies with chemotherapy rather than immunotherapy.

Example 2: pharmacological research in immunosuppressive animal models and optimization of technical scheme

In one experiment, the test animal was a nude mouse, and the modeled cell was a human liver cancer HepG2 cell at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (mean tumor volume 161.3mm³) The groups were randomly divided into 17 groups as shown in the following table, and these groups were further divided into series 1 (negative control group 01, study drug groups 1 to 6) and series 2 (negative control group 02, drug study groups 7 to 15). The negative control substance is physiological saline, the study drugs are as shown in the following table, and the study drugs are prepared according to the preparation method of the embodiment 1, wherein the live probiotics are saccharomyces cerevisiae, the inactivated probiotics are heat-inactivated saccharomyces cerevisiae, the water-insoluble component particles of the probiotics are beta-glucan particles, the water-soluble component of the probiotics is a crushed saccharomyces cerevisiae supernatant component, the semifluid component of the probiotics is a semifluid formed by heating and cooling 5% beta-glucan, and the mixture of the water-insoluble component and the water-soluble component (abbreviated as probiotic component mixture) of the probiotics is crushed saccharomyces cerevisiae. The administration modes of the animals in the series 1 are intravenous injection, and the administration modes of the animals in the series 2 are intratumoral injection. Each group was administered 1 time with an injection volume of 150. mu.l/tube. 7 days after administration, animals were euthanized, tumor tissue was dissected and removed to determine tumor weight, and tumor inhibition rates were calculated from each series of negative control groups, and the results are shown in the following table.

TABLE 4

Group i	Medicine i	Mode of administration	Tumor weight (x + -s _ g)	Tumor inhibition rate
					01	Physiological saline	Intravenous injection	0.241±0.113	-
1	1% 5-Fluorouracil	Intravenous injection	0.139±0.094	42.3％
					2	Anhydrous ethanol	Intravenous injection	0.226±0.113	6.1％
3	4×10⁴U/ml interleukin-12	Intravenous injection	0.230±0.102	4.7％
					4	5% inactivated probiotic	Intravenous injection	0.233±0.107	3.5％
5	5% probiotic water-insoluble component particles	Intravenous injection	0.228±0.114	5.2％
					6	5% probiotic Water-soluble component	Intravenous injection	0.238±0.093	1.3％
02	Physiological saline	Intratumoral injection	0.263±0.129	-
					7	1% 5-Fluorouracil	Intratumoral injection	0.133±0.094	49.5％
8	Anhydrous ethanol	Intratumoral injection	0.122±0.085	53.6％
					9	4×10⁴U/ml interleukin-12	Intratumoral injection	0.241±0.111	8.1％
10	5% inactivated probiotic	Intratumoral injection	0.165±0.063	37.2％
					11	5% probiotic water-insoluble component particles	Intratumoral injection	0.183±0.097	30.4％
12	5% probiotic Water-soluble component	Intratumoral injection	0.180±0.079	31.7％
					13	5% probiotic semifluid component	Intratumoral injection	0.157±0.081	40.2％
14	5% probiotic component mixture	Intratumoral injection	0.168±0.081	36.2％
					15	5% live probiotic	Intratumoral injection	0.201±0.101	23.6％

It is generally believed that 5-fluorouracil provides conventional chemical effects under conventional dosing conditions (study group 1) and general local chemical effects under local dosing conditions (study group 8), and that the difference in potency between the two is primarily a difference in cytotoxicity kinetics (low concentration vs high concentration of drug acting on tumor cells within the tumor). In the above table, the tumor inhibition rate of study group 7 did not increase more than the expected limit of the improvement based on the kinetics of the latter, as compared with study group 1 (E)₈/E₁<200%). These results also indicate that the same substance (5-fluorouracil) can be used as the same active ingredient (cytotoxic drug) under different conditions of action (systemic vs topical application).

It is generally believed that high concentrations (75-99%) of ethanol provide a chemical effect in the form of an intoxication-like response under conventional dosing conditions (study group 2) and in the form of local dosing conditions (study group 8) that provide necrosis of tumorigenic tissues and thus tumor-inhibiting chemical ablation. In the above table, an intoxication-like reaction was observed within 5 minutes after injection in more than 50% of the animals in study group 2, while no cytotoxic effect expected based on the results of the cell experiment was observed during the experimental period. Compared with the study group 2, the tumor inhibition rate of the study group 8 is improved by far exceeding the expected limit of the improvement based on the kinetics of the latter (E)₉/E₂>200%) showed pharmacological differences. In fact, these results also indicate that the same substance (ethanol) can be used as different active ingredients (intoxicating agents vs chemoablation agents) under different conditions of action (systemic vs topical application).

The interleukin-12 is clinically used as an immunopotentiator. It is known that even in tumor-bearing animal models with normal immune background, no significant tumor body efficacy is observed with the use of immunopotentiators alone. The results of study groups 3 and 9 in the above table are in anticipation of immunopotentiators perhaps having adjuvant but no therapeutic effect.

In the prior art, the anti-tumor effect of inactivated probiotics is based on the immunity enhancing effect, and the application symptoms of the effect do not comprise tumor treatment of tumor-bearing nude mice, particularly effective destruction of tumor bodies of the tumor-bearing nude mice. In the above table, study group 4 did not show significant tumor body efficacy as study group 3, and appeared to be in line with its prior art expectations. Surprisingly, the difference in tumor inhibition rates between study group 10 and study group 4 using the same drug far exceeded the expected limit of kinetic improvement for the same drug (E)₁₁/E₄>200%). In addition, the tumor inhibition rate of study group 10 was not less than 50% of study groups 7 and 8, and the difference in tumor weights between groups was not statistically significant (both were P)>0.05)。

In the prior art, the pharmaceutical effect of the water-insoluble component particles of probiotics is considered to be lower than that of inactivated probiotics containing intact thallus of the probiotics, whereas the water-soluble component of the probiotics should be much lower than the water-insoluble component particles of the probiotics. In the above table, study groups 5 and 6 showed the same drug effect as study group 4; study groups 11 and 12 showed the same drug effect as study group 10. Furthermore, one animal died shortly after injection in each of study groups 4 and 5, showing that intravenous injection of a 5% concentration suspension of the probiotic component may have a non-negligible safety risk.

In the prior art, the pharmaceutical effect of semi-fluid compositions of probiotic components has not been reported. In the above table, study group 13 showed significantly higher tumor inhibition than study group 11, despite the same chemical composition (5% β -glucan) and only different physical form of the drug used (semi-fluid vs suspension). Study group 14, like study groups 11-13, showed the same drug effect as study group 10.

In the prior art, live probiotics have a stronger immunological effect than the probiotic composition described above. However, the tumor inhibition rate shown by study group 15 was not significantly higher than that shown by study groups 11-14. Indeed, in study groups 11-15, the tumor weights were not statistically significant between any two groups (both P > 0.05).

Indeed, the probiotic component can only be applied if administered enterallyProvide intestinal flora regulation; and the immunity enhancing effect provided by the probiotic component can be applied to both internal and external intestinal administration. In parenteral administration, the prior art preferably uses, or at least does not exclude, administration by intravenous injection. The above studies indicate that the conventional administration protocols (oral, intravenous, etc.) must be excluded and that it is only possible to use a topical, in particular intralesional, administration protocol to allow the probiotic composition to provide the above mentioned tumor-bearing patients with new functions which it would not provide in the prior art (e.g. E in the above table)₁₁/E₄>200%). The following examples further investigate this new function.

In one experiment, the test animals were nude mice randomly divided into 2 study groups (1 and 2), 6 per study group. Study drugs for study groups 1 and 2 were chemoablative agent (75% aqueous ethanol) and probiotic composition (5% inactivated probiotic in the previous experiment), respectively. Each group was administered 1 time, and the administration was made into the muscle mass on the outer side of the right leg, and the injection amount was 100. mu.l/tube. 7 days after administration, the animals were euthanized, and nude mice were dissected to remove a lateral muscle mass specimen from the right leg, and after slicing and washing , the area of abnormal (e.g., necrosis, nodules, etc.) regions distinguished from normal muscles was measured.

The abnormal region areas of study groups 1 and 2 were 35.14 + -15.92 mm2 and 34.12 + -15.81 mm2, respectively, and the difference between the two was not statistically significant (P > 0.05). These results demonstrate that the local effect provided by the probiotic composition is similar to ethanol, which mainly, or at least non-negligibly, involves local chemical effects, in particular chemoablation, and that this chemoablation is independent of conventional effects (immune effects, cytotoxic effects, or tumor body vessel destructive effects), which mainly, or at least non-negligibly, involves tissue destructive effects of drug-permeable regions (tissue toxic effects).

The pharmacological function is the fundamental characteristic of the medicine, any substance lacking the pharmacological function cannot be applied to the medicine, and the old medicine can creatively generate new application as the new medicine by finding the new pharmacological function. According to the results of the above studies and more similar studies, the probiotic composition of the present invention may provide the above-mentioned topical effects. The pharmacological function of this local effect is such that the composition of the invention provides a pharmaceutical effect which greatly exceeds that expected from the current tumor technology of the probiotic component, at least in the area of administration, and thus is a therapeutic agent rather than an immunopotentiator of the prior art; this pharmacological profile also allows the compositions of the present invention to provide a medical use well beyond that contemplated by the current tumor technology for probiotic components, for example for chemo-like ablation of locally diseased tissue to treat any locally diseased disease involving the diseased tissue. The achievement of this local effect also requires that the compositions of the invention have pharmacological properties which differ from those of the prior art. For example, in addition to the above-mentioned specific pharmacological methods, specific pharmacological compositions are also required.

First, the preferred embodiment of the probiotic component of the present composition differs from prior art embodiments. For example, in the prior art, the expectation that the probiotic component provides a pharmacological function (immune enhancing effect) is: live probiotics are stronger than dead probiotics, dead probiotics which completely retain intact bacteria are stronger than dead probiotics which do not completely retain intact bacteria, and water-insoluble component particles (such as mural polymeric polysaccharide particles) of probiotics are stronger than water-soluble water components of probiotics, while no semifluid probiotic component has been reported to have an immunological effect. However, as shown in the above studies and more similar studies, the selection of probiotic components in the compositions of the present invention that provide the above-described topical effect exceeds the above-described selection sequence expectations of the prior art: the latter is preferred between live and dead probiotics, the latter being preferred between dead probiotics, which remain completely intact and dead probiotics, which do not remain completely intact (e.g. the ratio of the amount of incomplete bacteria>20% or>30% or the number of intact cells per ml of the composition<10⁵Or even<0.5×10⁵Inactivated probiotics) or even between a suspension comprising particles of the water-insoluble component of the probiotic and a semi-fluid comprising the semi-fluid type component of the probiotic. Thus, its concentration in a composition can be characterized by its concentration by weight rather than by the number of intact cells per volume.

Secondly, the selection principle of other essential components of the composition of the present invention is also substantially different from the prior art. It is well known that different pharmaceutical dosage forms generally require different pharmaceutical compositions. In the prior art, compositions comprising probiotic components are usually in oral form or in conventional injectable form, the former usually necessarily comprising solid excipients, while the latter usually necessarily comprising osmolytes (salts or monosaccharides) of the probiotic components to ensure that their liquid medicine is at or near the same osmotic pressure as blood. While the composition of the present invention must meet, preferably also only meet, the requirements to provide such a topical effect, its preferred embodiment does not necessarily comprise a solid excipient or an osmotic pressure enhancing agent to avoid reducing said topical effect of the probiotic component. More similar studies have also shown that the probiotic semi-fluid based composition, in addition to being an active ingredient that provides the above mentioned topical effect, may optionally also be present as a slow release carrier to produce a slow release synergistic effect with a wide range of drugs, including almost all chemotherapeutic and biological drugs.

Example 3: pharmacological research of local action and optimization of pharmacological concentration

In one experiment, the test animals were nude mice, and the modeled cells were breast cancer cells (MDA-MB231) at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (tumor volume mean 158.2 mm)³) The groups were randomized into 4 control groups (0, 01-03) and 12 study groups (1-12) as shown in the following table. The negative control was physiological saline, the positive control was ethanol, and the study drugs are shown in the table below. The medicaments are liquid preparations, wherein the inactivated probiotics is heat inactivated saccharomyces boulardii, the water-insoluble component particles of the probiotics are beta-glucan particles, the water-soluble components of the probiotics are crushed saccharomyces boulardii supernatant components, the water-insoluble component particles of the probiotics and the water-soluble component mixture (abbreviated as probiotic component mixture) are crushed saccharomyces boulardii, and the liquid preparations are prepared according to the preparation method of the example 1. Each group was administered 1 time with the injection amounts shown in the following table. 7 days after administration, animals were euthanized, tumor tissue was dissected and removed to determine tumor weight, and tumor inhibition was calculated from the negative control group, and the results are shown in the following table.

TABLE 5

It is known that ethanol can only be used as a chemical ablative agent if it exceeds a threshold concentration (e.g., 70%). In the above table, the results of study group 01 demonstrate that ethanol in this embodiment is similar to intravenous ethanol and fails to serve as an active ingredient for providing chemical ablation. The tumor inhibition rates of study group 02 and study group 01 differed greatly (E)₀₂/E₀₁>200%), and no difference from 03 (E)₀₃/E₀₂(< 200%) showing the expected chemical ablation.

Study groups 1-3 injected the same dose of probiotic composition at different concentrations into the same volume of tumor. In the above table, the tumor inhibition rates between study group 2 and study group 02 did not differ much (E)₃/E₀₂79%), tumor weight difference not statistically significant (P)>0.05) showed similar local effects. Study groups 5, 8, 11 also obtained similar local effects as study group 02. The results of study groups 3, 6, 9, 12 further showed the dosing concentration dependence of the local effect.

It is generally considered that the same drug (e.g., the same drug as a cancer cell inhibitory drug, a tumor angiogenesis inhibitory drug, or an immunological drug) is administered at the same dose, and the drug effects are the same. However, the probiotic component may in different technical solutions obtain different pharmacological effects by providing different pharmacological effects. For example, in the prior art, the probiotic composition may be administered orally or intravenously for its pharmacological function (e.g. immune enhancement), as it can be seen that the pharmacological concentration is not the administered concentration but a blood concentration (usually very low, e.g. 0.25X 10)^-5%) can provide the pharmacological effect only by meeting the administration dosage. The composition thereof must be limited only by the essential components thereof (e.g., probiotics, essential excipients, essential osmotic pressure enhancers), and the formulation thereof must be formulated only by the formulation (e.g., high concentration formulation can save transportationTransportation, storage costs, injection of appropriately high concentrations can reduce injection volume and thus time). It is well known that the concentration of a formulation can be relatively broad, but it is inherently different from the concentration required for administration of a pharmacological effect (and thus an indication). For example, intravenous administration is often done by pre-dilution to give administration concentrations much less (e.g., more than 5-fold dilution) than the formulation concentration to avoid safety risks associated with concentrated rapid entry of the drug into the blood.

According to the above and more similar studies, even with intratumoral administration, the difference in the drug effect of the probiotic components at the same dose and different administration concentrations may even exceed the kinetic difference expectation (e.g. E in the above table)₂/E₁>200%). Thus, the concentration at which the probiotic component is administered in the present composition is not required for other effects (e.g. an enhanced synergy of rabbit disease), but rather for said local effect, and thus a pharmacological concentration. One of the requirements of the probiotic component to provide a therapeutic effect including said local effect is that it must be present in the composition in an amount such that its pharmacological concentration (local administration concentration) is>0.1%,. gtoreq.0.25%, 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 5-15%. Wherein when said probiotic composition comprises said inactivated probiotic, said inactivated probiotic has a pharmacological concentration of>0.3 percent, more than or equal to 0.75 percent, 0.75 to 15 percent, preferably 1.5 to 15 percent or 5 to 15 percent; when the probiotic composition comprises the probiotic water-soluble composition, the pharmacological concentration of the probiotic water-soluble composition is>0.1, or 0.15-25%, preferably 0.35-15% or 5-15%; when the probiotic composition comprises the probiotic water-insoluble composition particles, the pharmacological concentration of the probiotic water-insoluble composition particles is>0.5, or 0.5-15%, preferably 1.5-15% or 5-15%; when the probiotic component comprises the probiotic semi-fluid component, the pharmacological concentration of the probiotic semi-fluid component is>2.5%, 2.6-25%, preferably 5-15%. The pharmacological concentration necessary for the composition of the present invention not only limits its composition as a feature, but it must also appear as a pharmacological condition in the approval of a new drug, and also as an application condition in the instructions for use of the drug. In fact, the therapeutic effect is saidAs necessary for this dosing regimen, the compositions of the present invention are more similar to chemoablative agents than to others (e.g., cancer cell inhibiting drugs, tumor angiogenesis inhibiting drugs, or immunological drugs).

Example 4: pharmacological research of local action and preferred pharmacological volume

It is well known that the component content of pharmaceutical compositions of different pharmacological properties often needs to be defined with different characteristics. Although conventional pharmaceutical compositions have no lesion target volume-dependent pharmacological volume beyond the administered dose, the following experiments have addressed this study.

In one experiment, the test animal was a nude mouse, the modeled cell was a human pancreatic cancer cell (PANC-1) at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (tumor volume mean 213.1 mm)³) The groups were randomized into 16 experimental groups (4 control groups and 12 study groups). The negative control was physiological saline, the positive control was ethanol, and the study drugs are shown in the table below. The drugs 1 to 12 were prepared according to the preparation method of example 1, wherein the inactivated probiotic was heat-inactivated saccharomyces boulardii, the probiotic water-insoluble fraction particles were β -glucan particles, the probiotic water-soluble fraction was a disrupted saccharomyces boulardii supernatant fraction, the probiotic water-insoluble fraction particles and the water-soluble fraction mixture (abbreviated as probiotic fraction mixture) were disrupted saccharomyces boulardii. Drugs 1, 4, 7, 10 were prepared according to the preparation method of example 1, drugs 2, 5, 8, 11 were 2.5-fold aqueous dilutions of drugs 1, 4, 7, 8, and drugs 3, 6, 9, 12 were 5-fold aqueous dilutions of drugs 1, 4, 7, 8, respectively. Each group was dosed 1 time, 100. mu.l per injection of the negative control group, 20, 50, 100. mu.l per injection of the high (10%), medium (4%), low (2%) group of 3 identical fractions (01-03, 1-3, 4-6, 7-9) in the study group. 7 days after administration, animals were euthanized, tumor tissue was dissected and removed to determine tumor weight, and tumor inhibition was calculated from the negative control group, and the results are shown in the following table.

TABLE 6

Study groups 01-03 injected ethanol into the same volume of tumor at different dose volume/target volume ratios. It is known that ethanol can only be used as a chemical ablative agent if it exceeds a threshold dose volume/tumor volume ratio (e.g., 0.15). In the above table, the results of study group 01 demonstrate that ethanol in this embodiment is similar to intravenous ethanol and fails to serve as an active ingredient for providing chemical ablation. The tumor inhibition rates of study group 02 and study group 01 differed greatly (E)₀₂/E₀₁>200%), and no difference from 03 (E)₀₃/E₀₂(< 200%) showing the expected chemical ablation.

Study groups 1-3 injected the same dose of probiotic composition at different dose volume/target volume ratios into the same volume of tumor. In the above table, the tumor inhibition rates between study group 2 and study group 02 did not differ much (E)₃/E₀₂105%), tumor weight difference not statistically significant (P)>0.05) showed similar local effects. Study groups 5, 8, 11 also obtained similar local effects as study group 02. The results of study groups 3, 6, 9, 12 further show the correlation of this local effect on the target volume and the dependence on the administered volume.

Generally, the same drug (e.g., the same cancer cell inhibitory drug, tumor angiogenesis inhibitory drug, and immunological drug) is administered at the same dose, and the drug effects are the same. However, according to the above and more similar studies, even with intratumoral administration of higher concentrations of the composition, the difference in the drug effect of the probiotic component at the same dose and different administration volume/target volume ratio may even exceed the kinetic difference expected (e.g. E in the above table)₂/E₁>200%). Thus, the administration volume (administration volume/target volume ratio) of the probiotic component in the present invention is no longer a pharmacodynamic issue, but a pharmacological issue.

In the prior art, the antitumor effect of probiotic compositions can be measured by their blood concentration (usually very low, e.g. 0.25X 10)^-5%) is administered in a volume dependent only on the dose required for the blood concentration and on the doseThe volume of the target area within the tumor is irrelevant. Thus, the composition can be limited only by the necessary components (e.g., probiotics, necessary excipients, necessary tonicity enhancers) and the formulation is limited only by the formulation (e.g., high concentration formulations can save transportation and storage costs, injection at appropriately high concentration can reduce injection volume and thus time), and there is no limitation in the volume to volume ratio of administration to target volume. It is well known that the injection time is often reduced as much as possible clinically to improve patient compliance.

However, the above studies and more similar studies show that one of the requirements for the probiotic component to provide said therapeutic effect is that it must be present in the composition in an amount such that its administration volume/target volume ratio is >0.09, 0.1-1.5, preferably 0.23-1.5 or 0.5-1.5. The pharmacological volume defined by this administration volume/target volume ratio not only limits its composition as a feature (as it is its content), but this feature must also appear in the new drug approval as a pharmacological condition for the composition, and also in the instructions for its use. Indeed, the compositions of the present invention are more similar to chemoablative agents than others (e.g., cancer cell inhibiting drugs, tumor angiogenesis inhibiting drugs, or immunological drugs) in terms of the pharmacological volume as a component content profile.

Clinically, although the volume of a large number of tumor bodies is more than or equal to 30cm³However, the volume of drugs (e.g., certain cytokines) administered in a tumor area based on immunopotentiating pharmacology is generally low (e.g.,. ltoreq.2 ml). Thus, the standard volume of such pharmaceutical unit preparations (e.g., solution or powder injection bottles) is generally not large. However, the composition of the present invention can be administered only under the conditions satisfying the above-mentioned administration volume (or administration volume/target volume ratio). For example, the volume of the target area of the tumor is more than or equal to 30cm³And the ratio of the administration volume/the target volume is selected to be 0.2, the required administration volume of the pharmaceutical composition of the present invention is 6.0cm or more³The volume of the unit formulation may be 6ml and multiples thereof. It is well known that a pharmaceutical specification may also be in the form of one of the usual desired amounts of active ingredient to achieve the desired pharmacological effect. For example, each slice comprisesDifferent aspirin contents of "bai' apirin" can have different indication ranges.

Example 5: common pharmacological research and technical scheme optimization in immunosuppressive animal models

In one experiment, the test animals were nude mice, the modeled cells were lung tumor cells (A549) at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 153.7 mm)³) The groups were randomly divided into 18 groups as shown in the following table, and these groups were further divided into series 1 (negative control group 01, study drug groups 1 to 7) and series 2 (negative control group 02, study drug groups 8 to 16). The negative control was physiological saline and study drugs were prepared as shown in the table below, all prepared according to the preparation method of example 1. The probiotic component is inactivated probiotics (heat-inactivated saccharomyces boulardii), and the administration modes of animals in the liquid preparation series 1 are intravenous injection; the animals in series 2 were administered intratumorally, with study group 16 administering 10% arginine about 2 hours after probiotic injection. Each group was dosed 1 time with each drug, at an injection rate of 150. mu.l/drug. 7 days after administration, animals were euthanized, tumor tissue was dissected and removed to determine tumor weight, and tumor inhibition rates were calculated from each series of negative control groups, and the results are shown in the following table.

TABLE 7

In the above table, composition study groups 5 and 7 did not show a therapeutically significant co-effect when injected intravenously, these compositions were then not therapeutic pharmaceutical compositions. Composition study 6 showed similar efficacy to study 2 but did not show significant synergy (q ═ 1.07). These results indicate that where the probiotic component is unable to provide topical effects, including chemical effects, its co-use with the chemically active substance does not appear to produce a local synergistic effect.

When injected intratumorally, the composition study groups 12-16 all showed therapeutic efficacy compared to the positive control group 9. The results of the composition study group 12 show that the co-use of a probiotic component (reference example 2) and a chemoablative agent as chemoablative-like drugs does not show a significant synergistic effect as expected (q ═ 0.95). Composition study group 13 results show that the co-administration of the probiotic component and the cytotoxic drug shows a clear synergistic effect (q ═ 1.17). The results of study group 14 show that the co-use of a probiotic component and a weak topically acting compound shows a clear synergistic effect (q ═ 1.41). In particular, the difference in tumor weight between study group 15 and the negative control group is statistically significant, and the use of this probiotic component in combination with the three components of the two different chemically active compounds (the weak topically acting compound and the cytotoxic drug) may inherently also result in further synergy based on the synergy of their two components (e.g. q ═ 1.21). Furthermore, q is >1.15 calculated for the probiotic component/cytotoxic drug composition (E13) in combination with the weak topically acting compound (E10), or the probiotic component/weak topically acting compound composition (E14) in combination with the cytotoxic drug (E9), respectively. In addition, the synergistic component of study group 16, although identical to the drug component of study group 14, was not in the same agent and showed no significant synergy (q ═ 0.57).

In addition, the study groups 11 and 12 animals produced a more intense response after injection in the majority, indicating that the probiotic component, either as a single drug or in a non-synergistic pharmaceutical composition, should be used under analgesic conditions to avoid safety risks. However, this strong stimulatory effect was unexplained from the study groups 13-15, showing that synergistic pharmaceutical compositions of probiotic components with weak topically acting compounds and/or cytotoxic drugs have the safety expected for a super single effect.

In other experiments, the use of other probiotic components (e.g., probiotic semi-fluid-like components, probiotic water-soluble components, probiotic water-insoluble component particles) in combination with the chemically active compound also gave results consistent with the above table. The following experiments have conducted more studies on the composition of probiotic components, in particular three-component compositions.

In one experiment, the test animals were nude mice, the model cells were gastric tumor cells (BGC823), and the number of cells was 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. In the study, successfully modeled test animals (tumor volume mean 164.4 mm)³) The groups were randomly divided into 14 test groups (1 negative control group and 13 study groups). The negative control was physiological saline and study drugs 1-13 were formulated as described in the preparation of example 1, as shown in the following table, wherein: the inactivated probiotics is heat inactivated saccharomyces boulardii, the probiotics semi-fluid component is 7.5% beta-glucan in the semi-fluid composition, the probiotics water-soluble component is a crushed saccharomyces boulardii supernatant component, the probiotics water-insoluble component particles and the water-soluble component mixture (abbreviated as probiotics component mixture) are crushed saccharomyces boulardii, the synergistic component 1 is 1% methylene blue, the synergistic component 2 is 1% 5-fluorouracil, and the synergistic component 3 is a 1% methylene blue/1% 5-fluorouracil composition. The composition of beta-glucan and synergistic component is semi-fluid, and other medicines are all liquid. Each group was administered 1 time with an injection volume of 150. mu.l/tube. 7 days after administration, animals were euthanized, tumor tissue was dissected and removed to determine tumor weight, and tumor inhibition was calculated from the negative control group, and the results are shown in the following table.

TABLE 8

Both two-component study groups 8 and 9 showed significant synergy (q 1.41 and 1.17, respectively) upon intratumoral injection, and three-component study groups 10-13 showed significant synergy (q 1.23, 1.21, 1.17 and 1.25, respectively) when viewed as a combination of probiotic components with chemically active compound 1/chemically active compound 2 combination (synergistic component 3). They likewise show a clear synergistic effect when viewed as a combination of a synergistic combination of probiotic component/chemically active compound 1 (synergistic component 1) and chemically active compound 2 (synergistic component 2), or a combination of a synergistic combination of probiotic component/chemically active compound 2 (synergistic component 2) and chemically active compound 1 (synergistic component 1), respectively (q both > 1.15).

According to the results of the above studies and more similar studies, the co-action of the drugs that the probiotic component can provide is closely related to the administration protocol in which it is used: conventional dosing regimens (oral, intravenous, etc.) must be excluded and only intratumoral dosing regimens are used to make it possible for the probiotic bacteria to provide the above-mentioned tumor-bearing patients with a new drug co-usage not provided by the prior art. The following examples further investigate the pharmacological effects of this new function.

In one experiment, the test animals were nude mice randomly divided into 3 study groups (1, 2 and 3), 6 per study group. The study drugs of study groups 1, 2 and 3 were chemoablation agent (75% ethanol aqueous solution), probiotic component/weak topical compound (5% inactivated probiotic/synergistic component 1 in the previous experiment), probiotic component/weak topical compound/cytotoxic drug composition (7.5% probiotic semi-fluid type component/synergistic component 3 in the previous experiment), respectively. Each group was administered 1 time, and the administration was made into the muscle mass on the outer side of the right leg, and the injection amount was 100. mu.l/tube. 7 days after administration, the animals were euthanized, and a nude mouse right lateral leg muscle mass specimen was dissected out and subjected to gross pathological analysis, and the area of abnormal (e.g., necrosis, tubercle, etc.) regions distinguished from normal muscles was measured in sections.

The abnormal area areas of study groups 1, 2 and 3 were 32.24. + -. 13.71mm, respectively²、46.78±13.64mm²And 72.35. + -. 23.71mm². This result demonstrates that the probiotic composition provides a local synergy similar to, but stronger than ethanol, which is primarily, or at least non-negligibly, inclusive of local chemical synergy. This local chemical synergy appears to be chemical-like ablation, which primarily, or at least non-negligibly, involves tissue destruction of the subcutaneous drug-permeable zone (tissue toxicity) independent of conventional effects.

According to the results of the above studies and more similar studies, the probiotic composition of the present invention provides the above-mentioned local synergy. The pharmacological function of the local synergistic effect enables the composition to provide a drug effect greatly exceeding the expected drug effect of the existing tumor technology of the single probiotic component at least in the administration area, and also provide a drug effect greatly exceeding the expected drug effect of the addition effect of the single probiotic component and the single common medicine, thereby becoming a therapeutic drug capable of providing chemical ablation; this pharmacological function also allows the composition of the invention to provide a medical use that greatly exceeds the prior art expectation of single or probiotic component/co-use of probiotic components, for example for chemo-like ablation of locally diseased tissue to treat any locally diseased disease involving the diseased tissue. The achievement of this local effect also requires that the compositions of the invention have pharmacological properties which differ from those of the prior art. For example, in addition to the above-mentioned specific pharmacological methods, specific pharmacological compositions are also required.

First, the preferred embodiment of the probiotic component in the composition of the invention goes beyond the expectation of the prior art, namely the use of the selection principle of the probiotic component of the invention in example 2 which provides said topical effect.

Secondly, the preferred embodiment of the co-product of the probiotic component of the present composition to provide the above-mentioned synergistic effect is also beyond the expectation of the prior art co-product embodiments. The above studies and more similar studies have also shown that even limiting the synergistic composition to a range of chemically active compounds, even those that provide local chemical effects, the choice of co-drug (synergistic composition) for such synergistic effects is still difficult to predict. Surprisingly, the chemoablative-like providing probiotic composition does not provide a local synergistic effect to the chemoablative-providing ethanol, but to the poorly locally acting compound or/and the pharmacologically different cytotoxic drug. Wherein the weak topically acting compound comprises, for example, an amino acid based nutritional agent, a methylene blue based dye at a concentration < 3%, a quinine based drug at a concentration < 3%, a low concentration of an acidifying agent, a low concentration of an alkalinizing agent, a pH buffering system comprising an acidifying or/and alkalinizing agent. Furthermore, it is only possible to administer the probiotic composition in combination with its synergistic components in one medicament to provide said synergistic effect.

Still further, the preferred embodiment of the above-described synergistic co-formulation provided by the probiotic component of the present composition is beyond the expectations of the prior art co-formulations. A synergistic composition (probiotic component/chemically active compound composition) is obtained in the field of solid tumors to exceed expectations. It is rare that the synergistic composition may also form a further synergistic composition with the chemically active compound, i.e. a probiotic component/chemically active compound 1 (e.g. a weak topically acting compound)/chemically active compound 2 (e.g. a cytotoxic drug) composition. This is also a preferred embodiment of the composition of the invention. A more preferred embodiment of the composition of the invention is a probiotic component/methylene blue type dye/other chemically active compound, wherein the concentration of the probiotic component is 1-15%, the concentration of the methylene blue type dye is 0.5-1.5% and the concentration of the other chemically active compound is 0.1-35%.

More similar studies have also shown that the probiotic semifluid composition, in addition to being an active ingredient providing the above-mentioned local synergy, may optionally also be present as a slow release carrier to produce a slow release synergy with a wide range of drugs, including almost all chemotherapeutic and biological drugs.

Example 6: pharmacological research of local synergistic effect and preferred pharmacological dose ratio thereof

In one experiment, the test animal was a nude mouse, and the modeled cell was a human liver cancer cell (HepG2) at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. In the study, successfully modeled test animals (mean tumor volume 155.7 mm)³) The groups were randomized into negative control and 12 study groups. The negative control was physiological saline and the composition of the study drugs included as shown in the table below: 4 probiotics component single medicines with variable concentrations, 4 pH buffer system single medicines with variable concentrations, and a composition of 4 probiotics component with variable concentrations and 4 pH buffer systems with variable concentrations, wherein the probiotics component is a crushed low blakeslea yeast supernatant component, and the pH buffer system is a sodium bicarbonate-sodium hydroxide buffer system. The drugs were all aqueous liquids and were prepared according to the preparation method of example 1. The medicine is applied to each group for 1 time, and the injection amount is 150 mu l/unit. Animals were euthanized 7 days after dosing, tumor tissue was dissected and tumor mass was determined and tumor inhibition was calculated from negative controls. The tumor inhibition rates for each study drug group are shown in the table below.

TABLE 9

The sodium bicarbonate-sodium hydroxide buffer system reduces the pH of sodium hydroxide from greater than 12.5 to around 11, generally considered to be a decrease in alkalinity, which appears to be detrimental to its local chemistry. In the above table, composition groups 9 and 12 showed no significant synergy (q 0.96 and 1.10, respectively), whereas composition groups 10 and 11 showed significant synergy (q 1.18 and 1.32, respectively), with a synergistic amount ratio (W probiotic component/W co-formulation) between 0.2/14.3 and 20/0.29.

In one experiment, the test animal was a nude mouse, and the modeled cell was a human liver cancer cell (HepG2) at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. In the study, successfully modeled test animals (tumor mean volume 163.2 mm)³) The groups were randomized into negative control and 12 study groups. The negative control was physiological saline and the composition of the study drugs included as shown in the table below: 4 probiotic component single drugs with variable concentrations, 4 weak local acting compound single drugs with variable concentrations, and a composition of 4 probiotic component with variable concentrations and 4 weak local acting compounds with variable concentrations, wherein the probiotic component is inactivated 37312312312and the saccharomyces cerevisiae and the weak local acting compound is methylene blue. The drugs were all aqueous liquids and were prepared according to the preparation method of example 1. Each group was administered 2 times, and the 2 nd dose was administered 7 days after the 1 st dose, and the injection amount was 150. mu.l/dose. Animals were euthanized 7 days after the end of the 2 nd dose, tumor tissue was dissected out to determine tumor weight, and tumor inhibition rate was calculated from the negative control group. The drugs for each studyThe group tumor inhibition rates are shown in the following table.

Watch 10

Group number	Research medicine	Tumor weight (x + -s _ g)	Tumor inhibition rate
				01	Physiological saline	0.253±0.117	-
1	0.2% probiotic composition	0.240±0.109	5.3％
				2	0.5% probiotic composition	0.207±0.105	18.1％
3	10% probiotic composition	0.155±0.070	38.6％
				4	20% of probiotic component	0.144±0.081	43.1％
5	3% methylene blue	0.073±0.033	71.1％
				6	1% methylene blue	0.186±0.096	26.4％
7	0.50% methylene blue	0.208±0.115	17.7％
				8	0.15% methylene blue	0.227±0.107	10.1％
9	0.2% probiotic composition/3% methylene blue	0.063±0.026	75.2％
				10	0.5% probiotic composition/1% methylene blue	0.112±0.094	55.9％
11	10% probiotic composition/0.50% methylene blue	0.071±0.031	71.8％
				12	20% probiotic component/0.15% methylene blue	0.111±0.062	56.1％

In the above table, composition groups 9 and 12 showed no significant synergy (q 1.04 and 1.14 respectively), while composition groups 10 and 11 showed significant synergy (q 1.41 and 1.45 respectively), with a synergistic amount ratio (W probiotic component/W co-formulation) between 0.2/3 and 20/0.15. These results indicate that the probiotic component does not appear to provide a local chemical synergy, subject to the weakness of the local chemical provided by the weak locally acting compound.

In one experiment, the test animal was a nude mouse, and the modeled cell was a human liver cancer cell (HepG2) at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. In the study, successfully modeled test animals (mean tumor volume 168.7 mm)³) The groups were randomized into negative control and 12 study groups. The negative control was physiological saline and the composition of the study drugs included as shown in the table below: 4 probiotic component single drugs with variable concentrations, 4 weak local acting compound single drugs with variable concentrations, and a composition of 4 probiotic component with variable concentrations and 4 weak local acting compounds with variable concentrations, wherein the probiotic component is cracked 37312312312312and the Saccharomyces cerevisiae supernatant component is lysine. The drugs were all aqueous liquids and were prepared according to the preparation method of example 1. Each group was administered 2 times, and the 2 nd dose was administered 7 days after the 1 st dose, and the injection amount was 150. mu.l/dose. Animals were euthanized 7 days after the end of the 2 nd dose, tumor tissue was dissected out to determine tumor weight, and tumor inhibition rate was calculated from the negative control group. The tumor inhibition rates for each study drug group are shown in the table below.

TABLE 11

Group number	Research medicine	Tumor weight (x + -s _ g)	Tumor inhibition rate
				01	(physiological saline)	0.238±0.191	-
1	0.2% probiotic composition	0.230±0.106	3.2％
				2	0.5% probiotic composition	0.199±0.099	16.4％
3	10% probiotic composition	0.126±0.061	47.1％
				4	20% of probiotic component	0.111±0.065	53.4％
5	25% lysine	0.109±0.074	54.1％
				6	20% lysine	0.134±0.073	43.6％
7	5% lysine	0.211±0.091	11.3％
				8	1% lysine	0.226±0.112	5.2％
9	0.2% probiotic composition/25% lysine	0.093±0.020	61.1％
				10	0.5% probiotic composition/20% lysine	0.076±0.038	67.9％
11	10% probiotic composition/5% lysine	0.065±0.026	72.6％
				12	20% probiotic composition/1% lysine	0.094±0.055	60.3％

In the above table, composition groups 9 and 12 showed no significant synergy (q 1.11 and 1.09, respectively), whereas composition groups 10 and 11 showed significant synergy (q 1.29 and 1.36, respectively), with a synergistic amount ratio (W probiotic component/W co-formulation) between 0.2/25 and 20/1. These results further illustrate that the probiotic component does not appear to provide a local chemical synergy, subject to the weakness of the local chemical provided by the weak locally acting compound.

In one experiment, the test animal was a nude mouse, the modeled cell was a human pancreatic cancer cell (PANC-1) at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. In the study, successfully modeled test animals (mean tumor volume 157.3 mm)³) The groups were randomized into negative control and 12 study groups. The negative control was physiological saline and the composition of the study drugs included as shown in the table below: 4 probiotics component single drugs with variable concentrations, 4 chemotherapy drug single drugs with variable concentrations, and a composition of 4 probiotics components with variable concentrations and 4 chemotherapy drugs with variable concentrations, wherein the probiotics component is broken 37312312, and the saccharomyces cerevisiae supernatant component and the chemotherapy drug are gemcitabine. The drugs were all aqueous liquids and were prepared according to the preparation method of example 1. Each group was administered 1 time with an injection volume of 100. mu.l/dose. Animals were euthanized 7 days after dosing, tumor tissue was dissected and tumor mass was determined and tumor inhibition was calculated from negative controls. The tumor inhibition rates for each study drug group are shown in the table below.

TABLE 12

Group number	Research medicine	Tumor weight (x + -s _ g)	Tumor inhibition rate
				01	(physiological saline)	0.294±0.189	-
1	0.2% probiotic composition	0.284±0.115	3.3％
				2	0.5% probiotic composition	0.278±0.109	5.6％
3	10% probiotic composition	0.161±0.080	45.1％
				4	20% of probiotic component	0.140±0.096	52.4％
5	5% Jixitabine	0.191±0.077	35.1％
				6	2.5% Jixitabine	0.204±0.103	30.4％
7	0.1% of Jixitabine	0.271±0.114	7.8％
				8	0.05% of Jixitabine	0.278±0.101	5.6％
9	0.2% probiotic composition/5% gemcitabine	0.172±0.062	41.4％
				10	0.5% probiotic composition/2.5% gemcitabine	0.107±0.069	63.7％
11	10% probiotic composition/0.1% gemcitabine	0.101±0.070	65.6％
				12	20% probiotic composition/0.05% gemcitabine	0.117±0.073	60.2％

In the above table, composition groups 9 and 12 showed no significant synergy (q is 1.11 and 1.09, respectively), while composition groups 10 and 11 showed significant synergy (q is 1.88 and 1.33, respectively), the synergistic amount ratio (W)_{Probiotic compositions}/W_{Shared article}) Between 0.2/5 and 20/0.05. These results indicate that the probiotic component does not appear to provide a local chemosynergistic effect subject to the intensity of the local chemical effect provided by the cytotoxic drug.

The probiotic component may provide different pharmacological effects, e.g. different synergistic effects, in different technical solutions as described previously. According to the above studies and more similar studies, the synergistic amount ratio of the probiotic components in the present composition is not required for other synergistic effects (e.g. enhanced synergy of rabbit plague), but for said local synergistic effect, and thus is a pharmacological amount ratio. In the use, composition, or method of the present invention, one prerequisite for a topical synergistic regimen of probiotic components is: a pharmacological quantity ratio (W) of the probiotic component to the synergistic component of the chemically active compound_{Probiotic compositions}/W_{Chemically active compounds}) Is (1-110)/(1-100), wherein: when the chemically active compound comprises a cytotoxic drug, the ratio of the pharmacological amounts of the probiotic component and the cytotoxic drug (W)_{Probiotic compositions}/W_{Cytotoxic drug}) Preferably (1-110)/(1-100); when the chemically active compound comprises a weak topically acting compound, the ratio of the pharmacological amounts of the probiotic component and the weak topically acting compound (W)_{Probiotic compositions}/W_{Compounds with weak local action}) Preferably (1-90)/(1-100), wherein the pharmacological amount ratio is (W) when the weak topically acting compound is an amino acid nutrient_{Probiotic compositions}/W_{Amino acid nutrient}) Preferably (1-20)/(1-100); when the weak topically acting compound is a vital dye, the pharmacological dose ratio (W)_{Probiotic compositions}/W_{Vital dyes}) Preferably (7-90)/(1-100); when the weak topically acting compound is an acidifying or/and alkalifying agent, the pharmacological dose ratio (W)_{Probiotic compositions}/W_{Acidifying or/and alkalifying agents}) Preferably (2-60)/(1-100).

Example 7: pharmacological research of local synergistic effect and pharmacological concentration optimization thereof

In one experiment, the test animal was a nude mouse, the modeled cell was a human pancreatic cancer cell (PANC-1) at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 152.8 mm)³) Randomized into 1 control group (0) and 9 study groups (1-9) as shown in the following table. The negative control was physiological saline and study drugs are shown in the table below. The medicaments are all liquid preparations, wherein the probiotic component is a crushed saccharomyces cerevisiae supernatant component, and the chemical active compound is arginine, and the preparation method is prepared according to the preparation method of the embodiment 1. Each group was administered 1 time, the negative control group was injected in an amount of 100. mu.l per injection, and the study group was administered in an amount of 100, 200 and 500. mu.l per injection for each of 3 high, medium and low concentration groups of the same fraction (01-03, 1-3, 4-6 and 7-9). .7 days after administration, animals were euthanized, tumor tissue was dissected and removed to determine tumor weight, and tumor inhibition was calculated from the negative control group, and the results are shown in the following table.

Watch 13

Group of	Medicine	Tumor weight (x + -s _ g)	Tumor inhibition rate
				0	Physiological saline	0.246±0.123	-
1	0.1% probiotic composition	0.218±0.096	11.3％
				2	0.25% probiotic composition	0.189±0.069	23.1％
3	0.5% probiotic composition	0.169±0.088	31.4％
				4	2% arginine	0.226±0.104	8.1％
5	5% arginine	0.210±0.113	14.7％
				6	10% arginine	0.193±0.075	21.4％
7	0.1% probiotic composition/2% arginine	0.206±0.082	16.3％
				8	0.25% probiotic composition/5% arginine	0.146±0.091	40.7％
9	0.5% probiotic composition/10% arginine	0.089±0.020	63.9％

In the above table, study groups 7-9 injected the same composition, the same ratio of amounts and the same dose but different concentrations of probiotic composition/chemically active compound into the same volume of tumor. Of these, composition group 7 showed no significant synergy (q ═ 0.88), whereas composition group 8 showed significant synergy (q ═ 1.18), while the results of study group 9 (q ═ 1.39) further showed dosing concentration dependence of this synergy.

It is generally considered that the same drug composition (e.g., the same pharmaceutical composition of a cancer cell inhibitory drug, a tumor angiogenesis inhibitory drug, and an immunological drug) has the same drug-sharing effect when the amount ratio of the active ingredients is the same and the dose to be administered is the same. However, it is possible that the probiotic components may in different technical solutions obtain different shared pharmacological effects by providing different shared pharmacological effects. In the prior art, the co-administration of the probiotic composition with other drugs, if any, can be carried out by oral or intravenous administration of the probiotic composition, it being seen that what is required is a blood level thereof (which is generally very low, e.g. 0.25X 10^-5%) the composition can provide the drug only by satisfying the administration dosage and the dosage ratioThe functions are shared. Thus, the composition thereof must be composed only by its essential components (e.g., probiotics, synergistic components, essential excipients, essential osmotic pressure enhancers) and the necessary quantitative ratio. As described in example 3, the formulation composition is then limited only by formulation (e.g., high concentration formulations can save transportation, storage costs, and injection at appropriately high concentrations can reduce injection volume and thus time).

According to the above and more similar studies, even with intratumoral administration, the difference in the drug effect of the probiotic components at the same dose and different administration concentrations may even exceed the kinetic difference expectation (e.g. E in the above table)₂/E₁>200%). Thus, the concentration at which the probiotic component is administered in the present composition is not required for other effects (e.g. for rabbit enhancement), but rather for said local synergistic effect, and thus is a pharmacological concentration. One of the requirements of the probiotic component to provide a therapeutic effect including said local synergy is that it must be present in the composition in an amount such that it is administered locally at a concentration>0.1%,. gtoreq.0.25%, 0.25-25%, preferably 0.5-15%, more preferably 1-15% or 1-3.5%. Wherein, when the probiotic component is selected from the group consisting of inactivated probiotic bacteria, the concentration of inactivated probiotic bacteria administered is>0.3 percent, more than or equal to 0.75 percent, 0.75 to 15 percent, preferably 1.5 to 10 percent or 1.5 to 3.5 percent; when the probiotic component is selected from the probiotic water-soluble component, the probiotic water-soluble component is administered at a concentration of>0.1, or 0.15-25%, preferably 0.25-15%; when the probiotic component is selected from the probiotic water-insoluble component particles, the probiotic water-insoluble component particles are administered at a concentration of>0.5, or 0.5-15%, preferably 1.5-15% or 1.5-5.5%; when the probiotic component is selected from the group consisting of probiotic semi-fluid components, the probiotic semi-fluid component is administered at a concentration of>2.5%, 2.6-25%, preferably 5-15%; when the probiotic component is selected from a mixture of inactivated probiotics and other probiotic components (e.g., probiotic water-soluble components), the mixture is administered at a concentration of>0.25%, 0.35-15%, preferably 0.5-25% or 1.5-15%. The concentration of probiotic component administered necessary for the composition of the invention not only limits its composition as a feature, but also must be taken as a featureThe pharmacological conditions appear in the approval of new drugs and must also appear in the instruction manual of the drugs as the application conditions.

Furthermore, more studies have shown that similar to what is described in example 4, even in the dosing regimen according to the invention, even in addition to the dose ratio regimen and dosing concentration regimen according to the invention, different administration volume/target volume ratios of the probiotic components of the same dose can provide different shared pharmacological effects. In the use, composition, or method of the present invention, one condition of the topical synergistic regimen of the probiotic component is: the composition of the composition comprising it must be such that its administration volume/target volume ratio is >0.09, 0.1-1.5, preferably 0.23-1.5 or 0.50-1.5. The pharmacological volume defined by this administration volume/target volume ratio not only limits its composition as a feature (as it is its content), but this feature must also appear in the new drug approval as a pharmacological condition for the composition, and also in the instructions for its use. Indeed, the compositions of the present invention are more similar to chemoablative agents than others (e.g., cancer cell inhibiting drugs, tumor angiogenesis inhibiting drugs, or immunological drugs) in terms of the pharmacological volume as a component content profile.

Example 8: short-term single-use pharmacology/shared pharmacology research and technical scheme optimization of lesion region administration in common animal model

In one experiment, the experimental animals were BALB/c mice, and the modeled cells were breast cancer 4T1 cells at 0.5X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 160.1 mm)³) The groups were randomly divided into 21 groups as shown in the following table, and these groups were further divided into series 1 (negative control group 01, study drug groups 1 to 6) and series 2 (negative control group 02, drug study groups 7 to 19). The negative control was physiological saline, and the study drugs, all liquid formulations, were prepared according to the preparation method of example 1, as shown in the following table, wherein: the probiotic component 1 is inactivated probiotics (heat inactivated Saccharomyces boulardii), the probiotic component 2 is water insoluble probiotic granule (beta-glucan), and the probiotic component 3 is probiotic waterThe soluble component (the crushed saccharomyces boulardii supernatant component) and the probiotic component 4 are a mixture of water-insoluble component particles of probiotics and water-soluble component (the crushed saccharomyces boulardii). The administration modes of the animals in the series 1 are intravenous injection, and the administration modes of the animals in the series 2 are intratumoral injection. Each group was administered 2 times, and the 2 nd dose was administered 7 days after the 1 st dose, and the injection amount was 150. mu.l/dose. 7 days after the end of the 2 nd dose, the animals were euthanized, dissected and tumor tissue was removed to determine tumor weight, and the tumor inhibition rate was calculated from each series of negative control groups, and the results are shown in the following table.

TABLE 14

Group of	Medicine	Tumor weight (x + -s _ g)	Tumor inhibition rate
				01	Physiological saline	0.358±0.231	_
1	1% 5-Fluorouracil	0.222±0.080	38.1％
				2	75% ethanol	0.348±0.114	2.8％
3	4×10⁴U/ml interleukin-12	0.339±0.112	5.3％
				4	1% methylene blue	0.345±0.109	3.7％
5	10% probiotic component 1	0.352±0.115	1.6％
				6	10% probiotic component 1/1% methylene blue	0.349±0.107	2.4％
02	Physiological saline	0.374±0.233	0
				7	1% 5-Fluorouracil	0.209±0.095	44.1％
8	75% ethanol	0.294±0.087	21.3％
				9	4×10⁴U/ml interleukin-12	0.357±0.099	4.6％
10	1% methylene blue	0.324±0.104	13.4％
				11	10% probiotic component 1	0.280±0.086	25.1％
12	10% probiotic component 2	0.272±0.083	27.3％
				13	10% probiotic component 3	0.286±0.089	23.5％
14	10% probiotic component 4	0.271±0.090	26.7％
				15	10% probiotic component 1/1% methylene blue	0.143±0.060	61.8％
16	10% probiotic composition 2/1% methylene blue	0.129±0.065	65.4％
				17	10% probiotic composition 3/1% methylene blue	0.148±0.077	60.3％
18	10% probiotic composition 4/1% methylene blue	0.142±0.064	62.1％
				19	10% probiotic composition 1/1% methylene blue/1% 5-fluorouracil	0.005±0.012	98.6％

It is known that in the drug treatment of solid tumors, only a chemotherapeutic drug can attack its target as soon as it enters the target region, thereby exhibiting the effect of the chemotherapeutic drug in a short period of time. In the above table, study groups 1-3 and 7-9 are in full agreement with their therapeutic results in immunosuppressive animal models (examples 2 and 5), in line with the expectations of cytotoxic, chemoablative and immunopotentiating effects, respectively.

In the above table, the tumor inhibition rates of study groups 5 and 6 are consistent with study group 3 and appear to be consistent with their prior art (non-therapeutic, especially non-chemo-therapeutic) expectations. Study group 11 and studyThe difference in tumor inhibition rates between groups 5 far exceeded the expected limit of kinetic improvement for the same drug (E)₁₁/E₅>200%) and the tumor inhibition rate was not much different from that of study group 8 (classical chemoablative agent group). Study groups 12-14 were the same as study group 11. The tumor inhibition rates of the composition research groups 15-18 are significantly greater than those of their respective probiotic component single drug groups 11-14, and they all show significant synergistic effects (q is 1.76, 1.77, 1.78 and 1.70, respectively). A three component composition (study group 19) formed with the probiotic component of a synergistic composition (study group 15) with one chemically active compound and further with another chemically active compound (study group 7) also showed significant synergy (q ═ 1.25). These results are in full agreement with the treatment results in example 5.

According to the results of the above examples 2, 5 and the study of this example and more similar studies, the probiotic composition, as well as in the animal model of immunosuppression, provides a short-term drug effect closely related to the dosing regimen with it: conventional dosing regimens (oral, intravenous, etc.) must be excluded and only intratumoral dosing regimens are used to make it possible to have the probiotic composition provide the tumor-bearing patient with new functions that it does not provide in the prior art (e.g. E)₁₁/E₅>200%). The following examples further investigate this new function.

In one experiment, the test animals were BALB/c mice, randomly divided into 4 study groups (1, 2, 3 and 4), 6 per study group. Study drugs for study groups 1, 2, 3 and 4 were chemoablation agent (75% ethanol aqueous solution), probiotic component aqueous solution (10% probiotic component 1 in the previous experiment), probiotic component/chemically active substance (10% probiotic component 1/1% methylene blue in the previous experiment), and probiotic component/chemically active substance 1/chemically active substance 2 (10% probiotic component 1/1% methylene blue/1% 5-fluorouracil in the previous experiment), respectively. Each group was administered 1 time, and the administration was made into the muscle mass on the outer side of the right leg, and the injection amount was 100. mu.l/tube. 7 days after administration, the animals were euthanized, and a mouse lateral hamus muscle mass specimen was dissected out and subjected to gross pathological analysis, and the area of abnormal (e.g., necrosis, tubercle, etc.) regions distinguished from normal muscles was measured in sections.

The abnormal area areas of study groups 1, 2 and 3 were 33.16. + -. 13.44mm, respectively²、34.01±14.24mm²And 49.61 + -23.27 mm²、78±4.31mm². These results are highly consistent with the results of similar experiments in examples 2 and 5.

According to the results of the above studies of examples 2-8 and further similar studies, the probiotic composition of the present invention is similar to high concentration ethanol, and the short term drug effect exhibited after administration is primarily, or at least non-negligibly, local effect (or local synergy) in either immunosuppressed or general patients, and the local effect (or local synergy) is primarily, or at least non-negligibly, local chemical effect (or local chemical synergy), especially similar to chemical ablation, which is primarily, or at least non-negligibly, tissue destruction in the drug penetration zone independent of conventional effects. The pharmacological function of this local effect is such that the composition of the invention provides a pharmaceutical effect which greatly exceeds that expected from the prior art of probiotic compositions, at least in any region of the patient to be administered, and thus is a therapeutic agent rather than an immunopotentiator of the prior art; this pharmacological function also allows the composition of the invention to provide a medical use that greatly exceeds that contemplated in the prior art for probiotic components, for example for chemo-like ablation of locally diseased tissue to treat any locally diseased disease involving the diseased tissue. Furthermore, the composition of the present invention can be used for the treatment of local lesions (e.g. tumor body) which is difficult to treat by the anti-local lesion disease drugs (e.g. cytotoxic drugs, antiviral drugs, antibacterial drugs, angiostatic drugs, immunological drugs, etc.) in the prior art. For example, in contrast to these anti-focal disease drugs, which are primarily based on anti-pathogens, the compositions of the present invention may be used as a cathartic drug for the treatment of the above indications.

The achievement of this local effect also requires that the compositions of the invention have pharmacological properties which differ from those of the prior art. In addition to the above-mentioned specific pharmacological methods, specific pharmacological compositions are required, for example, preferred technical solutions of the pharmacological compositions in examples 2 to 7 (for example, selection of probiotic components, selection of other essential components, selection of co-ingredients, pharmacological quantity ratio, pharmacological concentration, pharmacological volume, etc.).

Example 9: medium-and-long-term single-use pharmacology/shared pharmacology research and technical scheme optimization of lesion region administration in common animal model

In one experiment, the experimental animals were BALB/c mice, and the modeled cells were breast cancer 4T1 cells at 0.5X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 107.4 mm)³) The groups were randomly divided into 1 negative control group (01) and 16 drug study groups (1-16). The negative control was physiological saline and study drugs are shown in the table below, but the drugs used in study groups 6, 9, 12, and 19 were semi-fluid compositions and were all liquid compositions prepared according to the preparation method of example 1. Wherein the probiotic component 1 is inactivated probiotic (heat-inactivated 37312; wine yeast), the probiotic component 2 is a semi-fluid component (beta-glucan) of probiotic forming a semi-fluid composition, the probiotic component 3 is a mixture of water-insoluble component particles of probiotic and water-soluble component (crushed 37312; wine yeast), and the dilution of each drug is 3 times of the dilution of 1 volume of drug added with 2 volumes of water for injection. Each group was administered 2 times by injection, and the 2 nd administration was performed before the tumor volume measurement on the 7 th day from the first administration. Study groups 13 and 14 were administered at 300. mu.l/case and the other groups were administered at 100. mu.l/case. Study group 15 was administered to the right forelimb axilla, and each of the other groups was administered intratumorally. Study group 16 was administered intratumorally a first time of 1/1% probiotic composition and 5-fluorouracil/1% methylene blue 1.5% and intratumorally a second time of 1.5% probiotic composition 1, with the other groups administered twice with the same drug. Tumor volume (V) was measured for each group on days 7 and 21 of the 2 nd administration₇、V₂₁) And the relative tumor proliferation rate (R) was calculated from the negative control group, and the results are shown in the following table.

Watch 15

In the above table, on day 7, the drug effects of study groups 1-12 are in full agreement with study groups 7-18 of the previous example table, further illustrating that the probiotic composition of the present invention regimen inevitably, even possibly primarily, provides the said local effect (study groups 5-7) or local synergy (study groups 8-12) upon administration. The study groups 13 and 14 were not administered at concentrations that satisfy their pharmacologically preferred concentrations, so that the pharmacological effects of the study groups 5 and 6, respectively, were very different (both of them were the same dose as the administration component)>200%). The administration mode of study group 15 did not satisfy the administration mode required for local synergy, so that it was very different in drug effect (E) from study group 8 (administered drug was identical)₈/E₁₅>200%). Study group 16, which was the same drug as study group 11 at the first administration, showed similar local synergy to study group 11.

It is well known that the kinetics of chemotherapeutic drug effects is characterized by the fact that the drug is not attacked any more after the drug has been metabolised. The results on day 21 in the table above show a greater increase in relative tumor proliferation rates (e.g., greater than 1.50 for both 21R/7R days) for study groups 1 and 2, compared to day 7, indicating a significant reduction in the effect of short-term drug effects. The relative tumor proliferation rates of study group 3 were still high, consistent with the expectation that the immunopotentiator alone did not exhibit significant intermediate-term efficacy. The relative tumor proliferation rates in study groups 5-7 were less elevated (e.g., 21 day R/7 day R were less than 1.25), significantly different from study groups 1-3. The relative tumor proliferation rates in study groups 8-12 were also less elevated (e.g., 21 day R/7 day R were each less than 1.25), similar to study groups 5-7. The relative tumor proliferation rates of study groups 13-15 were still high, similar to study group 3. The relative tumor proliferation rate increase was also small in study group 16, similar to study group 11.

The above results show that the composition of the present invention, study groups 5 to 12 and 16, showed novel pharmacology different from the chemical or immunopotentiation effect, which was not shown in study groups 1 to 3, for the middle and long term effects. In light of the significant differences between study groups 5-12, 16 and study groups 13-15, this new pharmacological profile is clearly related to the local effect or local synergy provided by the probiotic component of the present invention.

The results of the above and more similar studies indicate that the short-term effects following intralesional administration of the compositions of the present invention are primarily, or at least non-negligibly, local effects (or local synergy), wherein the long-term efficacy is primarily, or at least non-negligibly, secondary effects resulting from the local effects (or local synergy), and the secondary effects are primarily, or at least non-negligibly, immune effects (e.g., vaccine effects). Thus, the compositions of the invention are completely different from classical immunological drugs (e.g. vaccines) in that the substance acting as an antigen is likely to be predominantly, or at least non-negligibly, comprised of an in situ immunological substance (e.g. an in situ antigen) produced after administration. In addition, the probiotic components, like other foreign substances, may also have a non-specific antigenic effect, which may also strengthen the efficacy of the in situ vaccine.

According to the above studies, the chemo/immunopharmacological function comprising this local effect (or local synergy) and its secondary immune effect makes it possible for the composition of the present invention to provide a pharmaceutical effect that greatly exceeds the prior art expectation of the probiotic component, thus being a therapeutic agent rather than the prior art immunopotentiators; this pharmacological profile also allows the compositions of the invention to provide a medical use that is far superior to that contemplated in the prior art for probiotic compositions, for example for any localized disease, preferably one that is treatable by the stimulation of body immunity associated with chemoablation of the localized diseased tissue and its secondary immune effects. Furthermore, the composition of the present invention can be used for the treatment of local lesion diseases (e.g., solid tumors) which are difficult to treat by the anti-local lesion disease drugs (e.g., cytotoxic drugs, antiviral drugs, antibacterial drugs, angiostatic drugs, immunological drugs, etc.) in the prior art. For example, in contrast to these anti-focal disease drugs, which are primarily based on anti-pathogens, the compositions of the present invention may be used as tissue poison/immune drugs for the treatment of the above indications.

The achievement of this local effect (or local synergy) and its secondary immune effect also requires that the composition of the invention must have pharmacological properties different from those of the prior art. In addition to the specific pharmacological approaches described above, specific pharmacological compositions are required, such as their preferred technical solutions in examples 2-8 (e.g., selection of probiotic components, selection of co-ingredients, pharmacological dose ratios, pharmacological concentrations, pharmacological volumes, etc.).

Example 10: single pharmacology/shared pharmacology research and technical scheme optimization of local drug delivery outside lesion area in common animal model

Although the efficacy of the drug administered extratumorally was not evident under the conditions of example 9, the study of extratumoral administration was not abandoned. In one experiment, the test animals were BALB/c mice，The cells modeled were breast cancer 4T1 cells at 0.25X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. On day 5 after modeling, the test animals with visually apparent tumors were randomly assigned to 1 negative control group (01) and 11 drug study groups (1-11), each of 10 animals. The negative control was physiological saline and the study drugs of drug study groups 1-11 were prepared as described in example 1, below. Wherein the probiotic component 1 is heat-inactivated saccharomyces boulardii, the probiotic component 2 is beta-glucan particles, and the composition of the probiotic component and the shard is prepared by adding dry powder of the shard into the probiotic component and uniformly stirring. Except study groups 10 and 11, each of the other groups was administered 2 times by injection, and the 2 nd administration was performed before tumor volume measurement on day 7 from the first administration. Study groups 8 and 9 were administered at 10. mu.l/case, and the other groups were administered at 120. mu.l/case. Study groups 10 and 11 the probiotic component/co-agent composition was administered intradermally to the left underarm of the animal on the same day as the first intratumoral administration of the probiotic component, and the other groups were administered the same agent for both administrations. Tumor volume (V) was measured on days 7 and 21 of the 2 nd dose from study groups 1-10₇、V₂₁) And the relative tumor proliferation rate (R) was calculated from the negative control group, and the results are shown in the following table.

TABLE 16

In the above table, on day 7, the results of study groups 1 and 2 were consistent with the pharmacological expectations of cytotoxic drugs and chemoablative agents, respectively, the results of study group 3 were consistent with the expectation that immune enhancing agents would not provide a therapeutically significant effect even when introduced into the body, the results of study groups 5-9 were similar to study groups 2 or 3, and the results of study groups 10 and 11 were consistent with the expectation that local intratumoral administration of chemoablative-like drugs would be therapeutically effective. These results further illustrate that the primary cause of the short-term efficacy of the compositions of the present invention (groups 5-7) is a local effect (or local synergy) and not other effects (e.g., immune effects). This local effect (or local synergy) may show short term efficacy when intratumoral (study groups 10 and 11) and not when extratumoral (study groups 5-7) as with compositions not complying with the composition protocol (pharmacological volume) of the invention (study groups 8 and 9).

In the above table, the results on day 21 were significantly less effective in study group 1 compared to day 7, no therapeutic effect was observed in study groups 2 and 3, and study groups 8 and 9 were still similar to study groups 2 and 3. Surprisingly, the relative tumor proliferation rates of study groups 5-7 all decreased significantly (e.g., study group 5 decreased the relative tumor proliferation rate from 94% to 71%), indicating a new pharmacological effect different from study groups 1-3, 8, and 9. The relative tumor proliferation rates of study groups 10 and 11 remain at a therapeutically superior level, showing the intra-tumoral local synergy provided by the probiotic composition and its secondary effects (e.g. in situ vaccine effect) combined with the pharmacological effects of the new pharmacologic effect described above.

Furthermore, the study groups 5 and 6 mostly developed strong responses after injection, indicating that the probiotic component single drug should be used under analgesic conditions to avoid safety risks. However, this strong stimulatory effect was unexplained and not observed in study group 7, indicating that the synergistic pharmaceutical composition of probiotic components with weak topically acting compounds and/or cytotoxic drugs has the safety expected for a super single effect.

In the above table, the difference in the administration between study groups 5 and 8, 6 and 9 is the administration volume, which is again the pharmacological condition of the short-term effect including the local effect (or local synergistic effect) in examples 2 to 10. The new pharmacological effects exhibited by the intermediate drug effects of study groups 5-7 should then include, perhaps even be primarily, secondary effects of this short-term effect. The following examples further investigate this short-term effect.

In one experiment, the test animals were BALB/c mice, randomized into 2 study groups (1 and 2), 6 per study group. Study drugs for study groups 1 and 2 were 10% probiotic composition 1 and 10% probiotic composition 1/20% resistance to alanine, respectively, from the previous experiment. Each group was administered 1 time, and administered into the axilla of the right anterior leg with an injection amount of 100. mu.l/tube. 7 days after administration, the animals were euthanized, and then the subcutaneous nodule specimens of the right anterior leg axilla of the mice were dissected out and the area of abnormal (e.g., necrosis, nodule, etc.) regions (e.g., nodule cross-section) different from normal muscles was measured after the section washing.

The abnormal region areas of study groups 1 and 2 were 30.15. + -. 11.31mm2 and 45.74. + -. 13.71mm2, respectively. These results are highly consistent with the results of similar experiments in example 9. These results demonstrate that the probiotic composition of the present invention, similar to high concentrations of ethanol, exhibits primarily a local effect (or local synergy) upon topical administration, and that the local effect (or local synergy) primarily, or at least non-negligibly, includes a local chemical effect (or local chemical synergy), especially a local chemical effect (or local chemical synergy) similar to chemical ablation, which primarily, or at least non-negligibly, includes subcutaneous drug-permeable tissue disruption independent of these effects. These local effects (or local synergy) are completely consistent with the local effects (or local synergy) exhibited by intratumoral administration in examples 2, 5 and 9.

The results of the above and further similar studies indicate that the short-term effects following topical (preferably immunologically beneficial topical) administration of the compositions of the present invention are primarily, or at least non-negligibly, local effects (or local synergy), with long-term efficacy primarily, or at least non-negligibly, including secondary effects resulting from the local effects (or local synergy), and secondary effects primarily, or at least non-negligibly, including vaccine effects. Thus, the compositions of the invention which provide an extratumoral vaccine effect are entirely different from classical vaccines in that the substance acting as an antigen is likely to consist primarily, or at least non-negligibly, of the nodular antigen produced after administration. In addition, the probiotic components, like other foreign substances, may also have a non-specific antigenic effect, which may also strengthen the efficacy of the vaccine.

According to the above studies, the immunopharmacological function comprising this secondary immunization allows the compositions of the present invention to provide a pharmacological effect that greatly exceeds that expected from the prior art of probiotic compositions, thus being therapeutic agents (vaccinoids) rather than prior art immunopotentiators; this pharmacological function also allows the composition of the invention to provide a medical use that greatly exceeds that envisaged in the prior art for probiotic components, for example for any localised disease, preferably one that is treatable by immunostimulation of the body in association with a locally acting (or locally synergistic) secondary immune effect. Furthermore, the composition of the present invention can be used for the treatment of local lesion diseases (e.g., solid tumors) which are difficult to treat by the anti-local lesion disease drugs (e.g., cytotoxic drugs, antiviral drugs, antibacterial drugs, angiostatic drugs, immunological drugs, etc.) in the prior art. For example, in contrast to these anti-focal disease drugs, which are based primarily on anti-pathogens, the compositions of the present invention can be used as immuno-drugs (vaccinoids) for the treatment of the above indications.

The achievement of this secondary immunological effect also requires that the compositions of the invention have pharmacological properties which differ from those of the prior art. In addition to the specific pharmacological approaches described above, specific pharmacological compositions are required, such as their local effects (or local synergy) in examples 2-8, pharmacological composition preferences (e.g., selection of probiotic components, selection of co-agents, pharmacological dose ratios, pharmacological concentrations, pharmacological volumes, etc.). For example, although no relationship between the volume of the drug administered and the volume of the target region within the tumor has been found, a requirement for the volume of its administration is apparently to be formed as an antigenThe desired nodules, for example: when the total volume of the tumor body is more than or equal to 2.85cm³The administration of a dose of more than 0.01ml/kg of human, 0.015-0.25ml/kg of human, preferably 0.020-0.25ml/kg of human, requires a white administration volume of the probiotic component (or of the pharmaceutical composition comprising it) for extratumoral local injection according to the invention>2 times, or 3 to 100 times, preferably 5 to 100 times (e.g.. gtoreq.1 ml, or 1.5 to 50ml) in the administration volume of a conventional vaccine.

Similar results were obtained in similar experiments using the examples above using some other compositions of the present invention prepared by the method of example 1.

Various modifications of the invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including all patents, patent applications, journal articles, books, and any other publications, cited in this application is hereby incorporated by reference in its entirety.

Claims

2. Use according to claim 1, wherein the pharmaceutical composition further comprises a chemically active ingredient capable of producing a synergistic effect with the probiotic component, and wherein the ratio of the amount of the probiotic component to the chemically active ingredient (probiotic component weight concentration/co-agent weight concentration) is (1-110)/(1-100).

4. The pharmaceutical composition according to claim 3, further optionally comprising a chemically active ingredient capable of producing a synergistic effect with the probiotic component.

6. Use or pharmaceutical composition according to one of claims 1 to 5, wherein the probiotic component is selected from those which minimize bacterial immunogenicity, preferably from one or more selected from the group comprising: water-soluble components of probiotics, semi-fluid components of probiotics, water-insoluble particles of probiotics and inactivated probiotics.

7. Use or pharmaceutical composition according to one of claims 1 to 6, wherein the probiotic bacteria are selected from one or more of natural or/and engineered bacteria comprising the following group: probiotic bacillus, probiotic lactobacillus, probiotic bifidobacterium and probiotic fungi.

8. A pharmaceutical kit comprising one or more containers filled with a pharmaceutical composition according to any one of claims 3-7.

9. The use, pharmaceutical composition or kit according to one of claims 1 to 7, wherein the locally diseased state comprises tumors, non-tumor enlargement, local inflammation, secretory gland dysfunction and skin diseases, wherein the tumors comprise malignant and non-malignant solid tumors.

10. The use, pharmaceutical composition or kit according to claim 9, wherein said solid tumors comprise one or more of the following tumors and secondary tumors thereof: breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, esophageal cancer, gastric cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, cerebroma, and lymphoma.