CN112316147A

CN112316147A - Pharmaceutical composition comprising a semi-fluid of tissue cells and an active ingredient and process for preparing the composition

Info

Publication number: CN112316147A
Application number: CN201910706687.3A
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: 2019-08-01
Filing date: 2019-08-01
Publication date: 2021-02-05

Abstract

The present application relates to the use of a semi-fluid comprising tissue cells, a pharmaceutical composition comprising an active ingredient and said semi-fluid comprising tissue cells, a process for the preparation of the pharmaceutical composition, and a method of treating or inhibiting a pathogenic disease.

Description

Pharmaceutical composition comprising a semi-fluid of tissue cells and an active ingredient and process for preparing the composition

Technical Field

Background

The treatment of pathogenic diseases, especially intractable pathogenic diseases (e.g. solid tumors, intractable microbial infections, intractable skin diseases, etc.), has been a difficult scientific problem. Solid tumors are often used as a research model for pathogenic diseases, as it is an intractable pathogen disease, as supported by a large amount of research work.

Various substances have been used to produce tumor vaccines. Microbial substances were first applied to the development of solid tumor vaccines. Streptococcus pyogenes, Serratia marcescens, BCG and the like are found to have curative effects on certain tumors in sequence. Live, attenuated or genetically modified obligate or facultative anaerobic bacteria can selectively multiply in tumors (which can be used as colonization carriers) and can also release certain antigens to activate the body's immune system. The cases of obvious remission of tumor diseases also appear after the infection of influenza virus or injection of rabies vaccine. In addition, live, attenuated or genetically modified oncolytic viruses can suppress tumors by two means (selective tumor cell killing and anti-tumor immunity). On the other hand, plasmodium, toxoplasma, etc. have also been found to be resistant to tumor growth. However, microbial vaccines either show some efficacy but higher safety risks or show higher safety but are less effective.

To improve specificity, vaccines comprising tumor antigens are of greater interest. The first appeared to be whole tumor cell vaccines. The whole-cell vaccine has more comprehensive expression, but has complex components and weak specificity. Researchers have therefore sought to screen individual harmless antigenic sites (e.g., certain exosomes, surface proteins, nucleic acids, polypeptides, etc.) from tumor cells to create subunit vaccines. The discovery of so-called tumor-specific antigens and tumor-associated antigens has been encouraging. However, although the components are single, the specificity is strong, the safety is high, and the immunogenicity is low. With the development of the current genetic technology, tumor neoantigens (Neoantigen) are making people a lot again. Tumor neoantigens refer to a class of proteins/epitope polypeptides that are caused by mutations in cancer cell genes, are absent from normal cells, and are recognized by immune cells. However, the polypeptide sequence generally consists of several or tens of amino acids, and has a small molecular weight, a simple chemical structure, and high safety but weak immunogenicity, and it is still difficult to induce immune responses of sufficient strength.

Although microbial (antigen) drugs, tumor cell subunit (antigen) drugs, tumor neogenetic (antigen) drugs, allogeneic lymphocyte (antigen) drugs are numerous and varied, the development of solid tumor drugs seems to be less advanced from a clinical point of view. Big problems are also in old places: how can a solid tumor drug antigen be developed that has the clinically effective desired (anti-tumor) antigenicity while not requiring antigenicity (e.g., GVHD) and is within the clinically safe tolerance? Several other serious pathogenic diseases, jeopardizing that it is a persistent pathogenic disease (e.g., hepatitis b) also have similar problems.

Thus, there is still a need to develop new drugs to meet the urgent clinical needs that the prior art has not yet met.

Disclosure of Invention

The object of the present patent application is to provide a pharmaceutical composition which is more potent (e.g. including immuno-chemo-combination or/and immuno-plus-boost combination) and thus less antigenic (e.g. GVHD), a medicament comprising the pharmaceutical composition, a process for the preparation of the medicament, and a method for combating pathogenic diseases. In particular, the provided pharmaceutical compositions comprise an active ingredient against pathogenic disease and a semi-fluid comprising animal tissue cells as a synergistic ingredient for the active ingredient.

One aspect of the disclosure provides the use of a semifluid comprising animal tissue cells as a synergistic component of an active ingredient against a pathogenic disease in the preparation of a topical medicament for the treatment or inhibition of a pathogenic disease.

Another aspect of the disclosure provides a topical pharmaceutical composition for treating or inhibiting a pathogenic disease comprising an active ingredient against the pathogenic disease and a semi-fluid comprising animal tissue cells, wherein the active ingredient is dispersed in the semi-fluid comprising animal tissue cells.

A further aspect of the present disclosure provides a method for treating or inhibiting a pathogenic disease comprising administering topically to an individual in need thereof by implantation, preferably by injection, a topical pharmaceutical composition comprising an anti-pathogenic disease active ingredient and a semi-fluid comprising animal tissue cells as a synergistic ingredient to the active ingredient.

A further aspect of the present disclosure provides a method for preparing a pharmaceutical composition for combating pathogenic disease, the pharmaceutical composition comprising a semi-fluid of animal tissue cells and an active ingredient, the method comprising the steps of:

a. providing a preparation comprising said cells, which preparation may be one or more of the following groups: natural cell preparations, engineered cells, connective tissue comprising natural cells, organ tissue comprising natural cells, enriched fractions of natural cells,

b. providing the active ingredient in a liquid form, and,

c. mixing said preparation comprising said cells with said active ingredient, if necessary semifluidizing and/or severely damaging the mixture to obtain a semifluid comprising said animal tissue cells and active ingredient; or

c. Subjecting the preparation containing the cells to a semifluid and/or severe damage treatment to obtain a semifluid, adding an active ingredient thereto and mixing to obtain a semifluid containing the animal tissue cells and the active ingredient.

A further aspect of the disclosure provides a pharmaceutical composition prepared according to the above method.

The semi-fluid according to the invention has the following advantages compared to the pathogenic disease drugs of the prior art: more comprehensive pharmacology, higher effectiveness, higher safety, wider indication spectrum and more and better synergistic action selectivity.

The anti-pathogenic disease regimen according to the present invention is more easily combined with other related treatment regimens. In addition, the application and the preparation of the medicine are controllable, the cost is low, the treatment scheme is simple and easy to implement, and the medicine is particularly beneficial to leading the wide population who is difficult to bear high expense to enjoy safe and effective treatment.

Detailed Description

According to one aspect of the present disclosure there is provided the use of a semifluid comprising animal tissue cells as a synergistic component of an active ingredient against a pathogenic disease in the manufacture of a topical medicament for the treatment or inhibition of a pathogenic disease.

According to one aspect of the present disclosure there is provided a topical pharmaceutical composition for treating or inhibiting a pathogenic disease comprising an active ingredient against the pathogenic disease and a semi-fluid comprising animal tissue cells, wherein the active ingredient is dispersed in the semi-fluid comprising animal tissue cells.

According to one aspect of the present disclosure there is provided a method for treating or inhibiting a pathogenic disease comprising topically administering to an individual in need thereof by implantation, preferably by injection, a topical pharmaceutical composition comprising an anti-pathogenic disease active ingredient and a semi-fluid comprising animal tissue cells as a synergistic ingredient to the active ingredient.

In the present disclosure, the term "animal tissue cells" refers to cells derived from animal natural tissues and artificial derivatives thereof.

In the present disclosure, the term "semi-fluid" refers to an object that flows without external pressure within a limited time (e.g., 20 seconds) without visible flow to the naked eye, but that flows and causes irreversible deformation under clinically (applied) acceptable external pressure (e.g., external pressure that may be applied to a syringe pusher), as distinguished from fluids (which flow without external pressure), solids (which are not flowable under clinically acceptable external pressure), and semi-solids (which are only reversibly deformable under clinically acceptable external pressure). For example, the tissues of certain animal organs (e.g., muscle mass, liver, stomach, intestine, heart, lung, pancreas, cartilage, joints, etc.), and certain gels that do not flow under pressure (e.g., fibrin glue) are semi-solid, rather than semi-fluid.

In the present disclosure, the term "active ingredient" refers to a substance that is capable of producing a certain prophylactic or therapeutic effect when topically applied alone under the same conditions.

In the present disclosure, the term "pathogen" refers to a pathogenic organism, including, for example, parasites (e.g., protozoa, worms, etc.), bacteria, fungi, viruses, rickettsia, chlamydia, mycoplasma, spirochetes, or other biological agents, including, for example, microbial recombinants, diseased cells (e.g., solid tumor cells). The term "pathogenic disease" refers to a disease associated with a pathogen.

In one embodiment, the composition is of a composition and form such that it forms a semi-fluid nodule at the site of administration and the semi-fluid comprising animal tissue cells provides a slow release carrier for the active ingredient and is capable of producing a synergistic effect therewith of: immune synergy and/or tissue destruction synergy

Within the scope of the present invention, the synergistic requirements (or basic technical solutions) of said compositions are: for example, by applying conditions which form a semi-fluid nodule at the site of administration which elicits an immune and/or tissue destruction synergy in composition (e.g., quantitative ratio), nature (e.g., softness), morphology/structure, and is capable of sustained release of the active ingredient.

In one embodiment, the active ingredient is selected from one or more of biological products against pathogenic diseases; the semifluid acts as an immunologically synergistic component of the active ingredient and acts as a slow release carrier.

In one embodiment, the active ingredient is selected from one or more anti-pathogenic disease chemotherapeutic agents; the semifluid is used as a chemotherapy synergistic component of the chemotherapy drug and plays a role of a slow release carrier.

In one embodiment, the active ingredient is selected from the group consisting of biologicals against pathogenic diseases and chemotherapeutic agents; the tissue cell semifluid acts as an immune synergistic component and a tissue destruction synergistic component of the active ingredients and acts as a slow release carrier.

Within the scope of the present invention, the pharmaceutical composition comprising an effective amount of an active ingredient and an effective amount of a tissue cell-containing semifluid as a synergistic component of the active ingredient is used as an immunologically active ingredient and/or a tissue-disrupting ingredient for the preparation of a medicament for pathogenic diseases.

In one embodiment, the pharmaceutical composition comprising an effective amount of an active ingredient and an effective amount of a semi-fluid comprising tissue cells as a synergistic component of the active ingredient is used as an immunologically active ingredient for the manufacture of a medicament for a pathogenic disease.

In one embodiment, the pharmaceutical composition comprising an effective amount of an active ingredient and an effective amount of a semi-fluid comprising tissue cells as a synergistic component of the active ingredient is used as a tissue-destructive ingredient in the manufacture of a medicament for a pathogenic disease.

In one embodiment, the semifluid comprising the active ingredient and the tissue cell composition is used in the manufacture of a medicament for a pathogenic disease as an immunologically active ingredient and a tissue-disrupting ingredient.

In one embodiment, the semifluid comprising the active ingredient and the tissue cell composition is used in the preparation of a medicament for a pathogenic disease as a delivery agent for an in situ antigen.

In the present disclosure, the term "in situ antigen" refers to a substance contained in a lesion caused by a pathogen in vivo, which is likely to induce a specific immune response of the body (e.g., intratumoral solid tumor cell material, exosome, polypeptide and nucleic acid sequence containing information of a solid tumor antigen), and actually contains a large amount of antigenic information different from that of a normal body, except that the information is shielded by some pathological factors (e.g., tumor microenvironment) and thus cannot be recognized and reacted by a conventional immune system.

In the scope of the present invention, the requirements (or basic technical solutions) of the tissue cell-containing semifluid as a synergistic carrier for active ingredients against pathogenic diseases are: it forms a semi-fluid nodule at the administration zone similar in composition (e.g. containing cells), nature (e.g. softness), morphology/structure to a gel semi-solid, but more dispersible, to enable controlled release of the active ingredient, thereby producing a therapeutic effect that exceeds the additive effect of the carrier and the active ingredient.

In the scope of the present invention, the requirements (or basic technical solution) for the semi-fluid containing tissue cells as an immune synergistic component for an immune component against pathogenic diseases are as follows: such that it forms an effective antigen at the administration area, activates a pathogen in situ antigen, or/and acts as a carrier for the immunological component to produce an immunotherapeutic effect beyond the additive effect of the semifluid antigen and the immunological component.

In the scope of the present invention, the requirements (or basic technical solutions) for the synergistic effect of the tissue cell-containing semifluid as a chemotherapeutic agent against pathogenic diseases are: such that it forms a more potent antigen at the administration site, a destructive effect on neoplastic tissue, or/and acts as a carrier for the chemotherapeutic agent to produce a therapeutic effect that exceeds the additive effect of the semifluid antigen and the chemotherapeutic agent. For example, the semi-fluid intratumoral drug of the invention may be involved as a tissue-disrupting component in the release of intratumoral in situ antigens.

Within the scope of the present invention, the tissue cells are preferably selected from cells contained in connective tissue and/or semisolid tissue derived from other than connective tissue.

In one embodiment, the tissue cells are one or more selected from cells derived from animal connective tissue.

In one embodiment, the tissue cells are one or more cells selected from the group consisting of cells derived from semi-solid tissue other than animal connective tissue.

In one embodiment, the semi-fluid comprises animal tissue cells selected from one or more of the following group and derivatives thereof: muscle cells, blood cells, immune cells, stem cells such as mesenchymal stem cells, hematopoietic stem cells.

In one embodiment, the immune cell is selected from one or more of the following group and derivatives thereof: dendritic cells, macrophages, leukocytes, wherein the leukocytes are selected from one or more of: granulocytes, monocytes, lymphocytes, wherein said lymphocytes are selected from one or more of the following: t cells, B cells, naked cells.

In one embodiment, the ratio of the amount of cells and composition (v/v) is > 22%, preferably 33% -86%, 45% -86%, or 55% -86%.

In one embodiment, the tissue cells have a hematocrit of>22％(or at a cell concentration of>5.6×10⁹One/ml), preferably 33% to 86% (or a cell concentration of 8.4X 10)⁹-22×10⁹Individual cell/ml), 45% -86% (or cell concentration 11.5X 10)⁹-22×10⁹Individual cells/ml), or 55% -86% (or cell concentration 14.0X 10)⁹-22×10⁹Individual cells/ml).

In one embodiment, the composition comprises a hematocrit of 55% to 86% (or a cell concentration of 14.0 x 10)⁹one/ml-22X 10⁹Pieces/ml).

In one embodiment, the active ingredient is selected from one or more of a biological product against a pathogenic disease or/and a chemotherapeutic drug, and the amount ratio (w/w or v/v) of the active ingredients in the composition is (0.1-30)/100. In one embodiment, the amount ratio (w/w or v/v) of the chemotherapeutic agent in the composition is (0.1-30)/100. In one embodiment, the amount ratio (w/w or v/v) of the biological product in the composition is (0.1-30)/100.

In one embodiment, the requirement for synergy of the composition includes its inclusion in the topical administration of the drug, wherein the topical administration is an intralesional administration and/or a topical administration outside of the lesion. In one embodiment, the composition is contained in an intratumoral administration (e.g., intratumoral administration) of the drug in the diseased area. In one embodiment, the composition is contained in a topical application outside the diseased region of the drug (e.g., a topical application outside a neoplasm). In one embodiment, the composition is comprised in both intralesional administration and ex-situ administration of the drug. In one embodiment, the topical application outside the lesion comprises one or more of: subcutaneous, intramuscular, mucosal administration.

In one embodiment, the essential forms/structures of the composition that are synergistic include: it is a semi-fluid composition and it forms a semi-fluid nodule comprising the active ingredient and tissue cells at the site of administration.

In one embodiment, preferred morphological/structural conditions of the tissue cell-containing semifluid as an immune synergistic component include: the semi-fluid is a highly off-natural state, preferably a severely damaged state, wherein the highly off-natural state comprises viscosifying; the severe injury is selected from one or more injuries including: solidification, mechanical disruption, ultrasonic damage, thermal damage, freeze-thaw damage, irradiation damage, chemical damage. In this embodiment, the tissue cell-containing semifluid is an immune component (e.g., an antigen or adjuvant) that induces an immune response as a highly divergent native cell population, preferably as a severely damaged cell population.

In one embodiment, the tissue cells comprised by the composition are severely damaged cells, wherein the severe damage is selected from the group consisting of one or more of the following: mechanical disruption, coagulation, ultrasonic damage, thermal damage, freeze-thaw damage, irradiation damage, chemical damage.

In the context of the present invention, the thickening means that the amount of tissue cells or/and additives added to the fluid containing tissue cells is so high that the system is no longer fluid but becomes a non-fluid viscous substance. From fluid to semi-fluid, significantly away from its natural state.

In the present disclosure, the term "severe injury" refers to an injury state that not only loses physiological function but is recognized by the body's immune system and is eliminated in response to major trauma.

Within the scope of the present invention, the solidification damage, mechanical disruption, ultrasonic damage, thermal damage, freeze-thaw damage, irradiation damage, chemical damage may be obtained by the following processes, respectively: solidification treatment, mechanical crushing, ultrasonic treatment, heat treatment, freeze thawing treatment, irradiation treatment and chemical treatment. It is well known that these treatments can alter the structure, morphology of the cell contents (e.g. tissue), leading to severe damage. These treatments not only cause damage to the cell contents, but often also alter the cell structure, morphology, causing cell damage (e.g., mechanical damage to cells, ultrasonic damage, thermal damage, freeze-thaw damage, radiation damage, chemical damage). After such damage, the cell contents (e.g., tissue) and even cells lose physiological function (e.g., are no longer available for organ or tissue transplantation, and the cells proliferate poorly), and are more easily recognized and responded to by the body's immune system as a major trauma (e.g., its dominant antigenicity is no longer allogeneic).

In the context of the present invention, the solidification is a process of converting a liquid into a solid or semi-solid, which includes solidification processes selected from any liquid tissue known in the art, such as: self-coagulating (e.g., self-coagulating blood), thermal coagulating (e.g., thermal coagulating blood), coagulant coagulating (e.g., coagulant coagulating blood). Among them, thermal coagulation can be performed by heat treatment, and coagulation by a coagulant is performed by adding a coagulant to a liquid (for example, thrombin and calcium chloride are added to blood).

Within the scope of the present invention, mechanical disruption includes mechanical segmentation (e.g., tissue sampling) and shear disruption. The shear crushing means that an object to be treated (for example, a coagulum) is subjected to shearing at a rotational speed of 10 rpm or more, preferably 10 to 50000 rpm, wherein the shear crushing can be performed by a stirrer, a grinder or a homogenizer . The coagulum can be changed into granules after mechanical crushing.

In the context of the present invention, the ultrasound treatment is carried out by placing the object to be treated (e.g. blood, cell pellet) in an ultrasound apparatus and subjecting it to ultrasound (e.g. at an operating frequency of 2 to 60kHZ) in order to destroy its structure.

Within the scope of the present invention, the heat treatment is selected from the group comprising one or more of: direct heat treatment, steam heat treatment, freeze-drying heat treatment, microwave heat treatment, radio frequency heat treatment and laser heat treatment, wherein the heat treatment temperature is more than or equal to 40 ℃, and preferably 60-115 ℃. Such as heat treatment of blood.

Within the scope of the present invention, the freeze-thaw treatment includes a freezing treatment and a thawing treatment of the frozen matter, wherein the freezing treatment is selected from mechanical refrigeration or/and liquid nitrogen refrigeration, and the freezing temperature is ≦ 60 ℃ and preferably-60 ℃ to-160 ℃. Such as freeze-thaw treatment of blood, cells.

Within the scope of the present invention, the irradiation treatment (e.g. with an intensity of 20-100Gy) is selected from one or more of the following: x-ray irradiation treatment, gamma-ray irradiation treatment, photosensitive drug + ultraviolet irradiation treatment. Such as irradiation treatment of blood and cells.

In the context of the present invention, chemical treatment refers to the addition of a chemical breaker (e.g. a hardening agent such as an acid, base, alcohol, etc.) to the object to be treated to break its structure. Such as chemical treatment of blood, cells.

In one embodiment, the semi-fluid composition comprising an active ingredient and tissue cells comprises a semi-fluidized product of a mixture of the active ingredient and tissue cells, or/and a mixture of the active ingredient and a semi-fluid comprising tissue cells, wherein the semi-fluid comprising tissue cells is a semi-fluidized product of tissue cells or their contents (e.g., tissue components, tissue cell-containing liquid, etc.).

In one embodiment, the semi-fluidization comprises one or more of the following group: semi-fluid thickening of a liquid, semi-fluid solidification of a liquid, disruption of a non-liquid or solidified substance.

In the present disclosure, the term "semi-fluid viscosified" refers to a viscosification of a non-fluid dope to a semi-fluid dope. For example: when the hematocrit of the white blood cells/blood plasma or red blood cells/blood plasma of the engineering tissue reaches more than 70 percent, the system is converted from fluid to semi-fluid; the term "semi-fluid coagulation" refers to a coagulation that converts a liquid to a semi-fluid, such as self-coagulation (e.g., coagulation without heat, without a coagulant), thermal coagulation (e.g., medium-low temperature heat coagulation), etc., of blood or tissue cell/blood mixtures; the term "comminution" means a process of fragmenting non-liquid or solidified matter into pieces by mechanical partitioning or shear-breaking so as to form a semi-fluid, preferably injectable semi-fluid, wherein the shear-breaking can be carried out by means of a blender, mill or homogenizer (e.g. at a rotational speed of > 10 rpm, preferably at a shear of 10-50000 rpm).

In one embodiment, the semi-fluid composition is selected from one or more of the following groups: the composition is one or more selected from the following group: a semi-fluid dope comprising the active ingredient and tissue cells, a semi-fluid coagulate comprising the active ingredient and tissue cells, a semi-fluid homogenate comprising the active ingredient and tissue cells, a coagulate comprising the active ingredient and tissue cells, preferably in the form of: a semi-fluid coagulum comprising the active ingredient and tissue cells, a disrupted semi-fluid product comprising the active ingredient and tissue cells, a disrupted product of a coagulum comprising the active ingredient and tissue cells. In this embodiment, the semifluid is preferably an antigen that induces an immune response as an abnormal nodule in the body, as if it were a severely damaged nodule.

In one embodiment, the semi-fluid composition comprises one or more of the following groups: a semi-fluid coagulum comprising the active ingredient and tissue cells, a disrupted semi-fluid product comprising the active ingredient and tissue cells, a disrupted product of a coagulum comprising the active ingredient and tissue cells.

In one embodiment, the coagulum comprises a self-coagulum, a coagulum-coagulum, a thermal coagulum of a liquid system comprising the active ingredient and cells. In one embodiment, the disruptant comprises a population of particles. In one embodiment, the particles are preferably macroscopic particles distinguishable to the naked eye. In one embodiment, the average diameter of the cross-section of the longest end of the particle is ≥ 100nm, preferably 500nm-1mm or 1 μm-1 mm.

In one embodiment, the composition, properties, morphology/structure and application conditions of the semi-fluid composition are such that it forms a volume at the site of application>100mm³Preferably ≥ 200mm³The semifluid nodule. Under this technical scheme, the semifluid composition is preferably a severely injured tumor body biomimetic antigen that is a semifluid nodule antigen of a larger size or a larger tumor body.

In the present disclosure, the term "antigen of a larger size of a semifluid nodule" refers to an antigen of a specific immunogen caused by the structural (morphological) characteristics of the larger size of the semifluid nodule itself, which is distinguished from antigens of molecular morphology (e.g., microbial antigens, tumor antigens, allogeneic immune cell antigens, etc.), and antigens of semi-solid morphology (e.g., semi-solid graft antigens, etc.). The terms "microbial antigen", "tumor antigen", "allogeneic immune cell antigen", "semi-solid graft antigen" refer to an antigen for which a species-specific molecule of a microorganism, a pathogen-specific molecule of a tumor cell, a xenogeneic immune cell, a species of a semi-solid graft or a xenogeneic specific molecule, respectively, is a specific immunogen. When these antigens are used as vaccine antigens, the corresponding vaccines are referred to as "semifluid nodule antigen vaccines", "microbial vaccines", "tumor antigen vaccines", "allogeneic immune cell vaccines", and the like, respectively. The more complex the structure, the more antigenic determinants (e.g., epitopes) it usually carries. For example, many vaccines used clinically are still primarily live microbial vaccines, although microbial subunit antigens are safer and easier to prepare.

In the present disclosure, the term "severely damaged tumor biomimetic antigen" refers to a semifluid nodular antigen that has multiple tumor-like characteristics (including tumor-like components and morphological features) but is more easily recognized as a foreign body by immunity due to severe damage.

In one embodiment, the semi-fluid is preferably a squeezable semi-fluid. Within the scope of the present invention, the extrudable semi-fluid is defined as a semi-fluid that flows through a syringe at a clinically acceptable pressure.

In one embodiment, the semifluid is an implant, preferably an injection, and its single animal dose is >0.1ml, preferably ≧ 0.2ml or 0.2-25 ml. The requirement for the semifluid to act as an antigen provides multiple oncosomal characteristics, while larger injection volumes make the resulting semifluid nodule more readily immunologically recognizable as a semifluid nodule antigen.

Within the scope of the present invention, the semi-fluid injection is a semi-fluid that can be administered directly by conventional injection systems. Semi-solid implants, in turn, are often surgically implanted or administered in a fluid (liquid) form a semi-solid (e.g., in situ gelling) nodule at the site of administration by conventional injection systems.

In one embodiment, the semi-fluid composition has a subcutaneous half-disappearance of 0.1 days or more, preferably 0.1 to 30 days. In one embodiment, the semi-fluid composition has a subcutaneous half-disappearance of 1 to 30 days.

Within the scope of the present invention, the tissue cell-containing semifluid (sometimes simply referred to as semifluid) comprises both the semifluid product of the mixture of the active ingredient and the tissue cells, or/and the semifluid of the mixture of the active ingredient and the tissue cell-containing semifluid.

In one embodiment, the tissue cells are inactivated tissue cells.

In the context of the present invention, the inactivated tissue cells are tissue cells that have undergone severe injury and lost proliferative activity.

Within the scope of the present invention, the tissue cells contained in the semifluid are derived from a mammal. The mammal is selected from one or more of the following: human, pig, horse, sheep, cattle, rabbit, mouse. These animals may be either fully naturally evolved animals or animals modified by biotechnology (e.g., gene editing technology) (e.g., GAL antigen knockout pigs).

In one embodiment, the tissue cells are selected from one or more of the following groups: natural cells in the connective tissue and/or semi-solid tissue, natural cells in the connective tissue-enriched fraction, natural cell preparations and/or engineered cells derived from the connective tissue and/or semi-solid tissue.

In one embodiment, the composition comprises one or more of the following groups: the connective tissue and/or semi-solid tissue comprising the cells, the connective tissue-enriched component and/or semi-solid tissue-enriched component comprising the cells, a cell preparation and/or engineered cells derived from the enriched tissue of the cells, a blood mixture comprising the cells.

In one embodiment, the composition is selected from one or more of the following groups: a semi-fluid coagulum comprising the active ingredient and the connective tissue, a semi-fluid mixture comprising the active ingredient and the connective tissue, a semi-fluid disruption comprising the active ingredient and the connective tissue and/or semi-solid tissue, a semi-fluid coagulum comprising the active ingredient and the connective tissue-enriched component and/or semi-solid tissue-enriched component, a semi-fluid disruption comprising the active ingredient and the connective tissue-enriched component and/or semi-solid tissue-enriched component, a semi-fluid dope comprising the natural cell preparation and/or engineered cell, a semi-fluid coagulation comprising the natural cell preparation and/or engineered cell, a semi-fluid disruption comprising the natural cell preparation and/or engineered cell.

In one embodiment, the semi-solid tissue is preferably selected from the group comprising one or more of: intestine, stomach, meat, pancreas, spleen, liver, lung, cartilage, joints, skin, placenta, umbilical cord, preferably tissue comprised by one or more organs selected from the group consisting of: meat, spleen, liver, placenta, umbilical cord.

In one embodiment, the connective tissue is preferably selected from the group comprising one or more of: blood, bone marrow, spinal cord, more preferably blood.

In one embodiment, the blood is selected from one or more of the following groups: natural blood, cell-enriched natural blood components, engineered blood comprising blood cell preparations and plasma.

In one embodiment, the semi-fluid is one or more selected from the group consisting of: a semi-fluid coagulum comprising the native blood, a semi-fluid coagulum comprising the blood component, a semi-fluid coagulum comprising the engineered blood, a disruption of a coagulum of the blood.

In one embodiment, the semi-fluid is a semi-fluid coagulum comprising native blood. In one embodiment, the semi-fluid is a semi-fluid coagulum comprising the blood component. In one embodiment, the semifluid is a semifluid clot comprising the engineered blood. In one embodiment, the blood, blood component, or semi-fluid coagulum of engineered blood includes one or more of: self-coagulum, thermal coagulum, coagulant semi-fluid coagulum.

In the present disclosure, the term "self-coagulum" refers to a coagulum formed by the natural coagulation of blood, blood components, or engineered blood. The term "thermal coagulation" refers to a coagulation formed by heat treatment of blood, blood components or engineered blood. The term "coagulant semi-fluid coagulum" refers to a semi-fluid coagulum (rather than a solid or semi-solid coagulum) formed by the addition of a coagulant to blood, blood components, or engineered blood.

In one embodiment, the clotting agent comprises a blood clotting agent, wherein the blood clotting agent is selected from the group consisting of one or more of: thrombin such as bovine thrombin, porcine thrombin, recombinant human thrombin, autologous thrombin, other blood coagulation proteins such as coagulation factors, prothrombin complexes, and their activated forms, calcium ion providers such as calcium chloride, calcium hydroxide, calcium carbonate.

In one embodiment, the semi-fluid is a disruption of a coagulation of the blood. In one embodiment, the disruptant comprises a population of particles. In one embodiment, the particles are preferably macroscopic particles distinguishable to the naked eye. In one embodiment, the average diameter of the cross-section of the longest end of the particle is ≥ 100nm, preferably 500nm-1mm or 1 μm-1 mm.

In one embodiment, the semi-vanishing period of the blood-containing semi-fluid is ≧ 1 day, preferably 2-20 days.

In one embodiment, the semi-fluid is a semi-fluid dope comprising the cells, and wherein the hematocrit is ≧ 70%. In one embodiment, the semi-fluid dope comprises a tackifier. In one embodiment, the tackifier is selected from the group comprising one or more of: polyethylene glycol, starch, sodium carboxymethyl cellulose, polyvinyl pyrrolidone.

The compositions according to the invention comprise a semifluid which, when used as an immunosynergistic component under the conditions described above, is preferably a semifluid whose dominant antigenicity is antigenic against the pathogenic disease and not against the host.

In one embodiment, the semifluid is preferably a semifluid with a tumor inhibition rate of ≥ host-resistant rate, preferably a tumor inhibition rate/host-resistant rate of ≥ 150%.

The complexity of immune recognition and immune response caused by substances as antigens far outweighs the consequences caused by their use as chemotherapeutic drugs. In a multi-level and diversified network pattern of an immune system, one substance may cause various immune reactions and show various antigenicities. In the present disclosure, the term "dominant immune response" refers to the major immune response that is activated. The term "dominant antigenicity" refers to the antigenicity exhibited in a dominant immune response. For example, allogeneic lymphocytes can attack normal cells (graft versus host response) and cancer cells (graft versus tumor response) of the recipient, and can also activate lymphocytes of the recipient to attack cancer cells (host versus tumor response) and allogeneic cells (host versus graft response). Since the dominant immune response is graft-versus-host response followed by graft-versus-tumor response, which is weak, allogeneic lymphocytes are themselves an anti-host antigen and can be used as an anti-tumor cell (e.g., leukemia) antigen, but are difficult to use as a solid tumor vaccine antigen.

In one embodiment, the cells contained in the semifluid may be xenogeneic or xenogeneic antigenically minimized cells.

In the present disclosure, the term "xenoantigen" refers to an antigen derived from a different species of a subject and representative of its species specificity; the term "allogenic antigen" refers to antigens derived from alloallelic differences in a subject, such as major histocompatibility antigens (MHC antigens, e.g., Human Leukocyte Antigens (HLA)), minor histocompatibility antigens (mH antigens), and other histocompatibility antigens (e.g., human blood group antigens, tissue-specific antigens, etc.).

When the semifluid according to the present invention is used as an immune synergistic component under the above-mentioned conditions, cells contained therein can be preferably selected by the following means.

In one embodiment, the cell is one or more selected from the group consisting of: cells derived from allogeneic tissues with the same or similar ABO blood type or HLA, cells derived from allogeneic tissues, and cells derived from autologous tissues.

In one embodiment, the blood cells comprise cells derived from allogeneic tissue of either consistent ABO blood group or HLA-matched.

In one embodiment, the cells comprise cells derived from autologous tissue.

In one embodiment, the cells include cells derived from autologous tissues and allogeneic tissues.

In one embodiment, the biological product is selected from one or more of the following groups: pathogen antigens, cell preparations, immunoregulatory antibodies, cytokines.

In the present disclosure, the term "pathogen antigen" refers to an antigen derived from a pathogen, including, for example, pathogens, pathogen subunits, and the like; the term "pathogen subunit" refers to immunologically active components of pathogens and artificial analogs thereof, such as parasites, bacteria, viruses, tumor cells, their immunologically active components (DNA, proteins, polypeptide fragments, etc.) (e.g., tumor subunit antigens, unmethylated cytosine guanine dinucleotide-deoxyoligonucleotides (CpG ODNs), etc.).

In one embodiment, the semifluid is used in the preparation of a pathogen disease vaccine as an adjuvant to a pathogen antigen, wherein the pathogen antigen is selected from one or more of the group consisting of: microbial antigens, tumor antigens.

In one embodiment, the microbial antigen is selected from antigens derived from one or more of the following groups of microorganisms: bacteria such as one or more of the following: streptococcus pyogenes, Serratia marcescens, Bacillus Calmette-Guerin, Clostridium tetani, Clostridium butyricum, Lactobacillus acidophilus, and Bacillus bifidus; viruses are, for example, one or more of the following: hepatitis B virus, adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, measles virus, West Nick valley virus, Coxsackie virus, reovirus; parasites such as plasmodium.

In one embodiment, the tumor antigen is selected from one or more of the group comprising: 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 the present disclosure, the term "cell preparation" refers to a collection of cells obtained by engineering production methods (e.g., cell purification, cell culture, etc.), such as a collection of lymphocytes obtained by lysing erythrocytes from a cell mixture in the spleen.

In one embodiment, the cell preparation is a preparation of natural or engineered cells selected from the group comprising one or more of: cells rich in tissue such as muscle cells, blood cells; immune cells such as peripheral blood mononuclear cells, T cells, B cells, NK cells, lymphocytes; stem cells such as mesenchymal stem cells, hematopoietic stem cells.

In one embodiment, the cell content is greater than 10⁵Per mm³Preferably 10⁵～10⁹Per mm³. In one embodiment, the semi-fluid comprises autologous blood and cells. In one embodiment, the ratio of the amount of cells to blood cells (v/v) is 10% to 90%. In one embodiment, the cells are preferably selected from allogeneic cells, syngeneic cells and autologous cells, more preferably autologous cells.

In one embodiment, the immunomodulatory antibodies are selected from one or more of the following groups: 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 solid tumor microenvironment, such as anti-TGF-p 1 antibodies. In one embodiment, the immunomodulatory antibody-like drug is present in the pharmaceutical composition in an amount > 0.1%, preferably 0.25-10%.

In one embodiment, the cytokine is selected from one or more of the following: tumor necrosis factor, interferon, interleukin. In one embodiment, the cytokine is present in the pharmaceutical composition in an amount > 0.1%, preferably 0.25-3%.

In one embodiment, the chemotherapeutic agent is selected from one or more of cytotoxic agents and/or conventional ineffective but topically effective compounds. In one embodiment, the ratio of the amount of cytotoxic drug to the amount of semi-fluid (w/w or v/v) is (0.1-15)/100. In one embodiment, the ratio of the amount of said conventional ineffective but topically effective compound to the amount of semi-fluid (w/w or v/v) is (0.5-30)/100.

In the present disclosure, the term "cytotoxic drug" refers to a drug (e.g., an anti-tumor chemotherapeutic) that is effective by absorption at a safe dose, selected from any pharmaceutically acceptable cytotoxic drug, preferably selected from those known in the art, and more preferably selected from those approved or to be approved by, or loaded or to be loaded in, the chinese, U.S. or european official pharmacopoeia (e.g., FDA or chinese drug administration).

In one embodiment, the cytotoxic drug 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 following drugs and their analogous derivatives: 5-fluorouracil, gemcitabine, epirubicin, an anti-solid tumor antibiotic, teniposide, a metal platinum complex and paclitaxel.

In one embodiment, the concentration of the anti-tumor chemotherapeutic in the semi-solid is greater than 50% of its saturation concentration, preferably 50-500% of its saturation concentration, wherein the saturation concentration refers to the saturation concentration of the anti-solid tumor chemotherapeutic in its pharmaceutical vehicle.

In one embodiment, the conventionally ineffective but topically effective compound is selected from one or more of the following groups: amino acid nutrient, ineffective aromatic compound, and plant active ingredient.

In the present disclosure, the term "conventional ineffective but topically effective compound" refers to a drug (e.g., an anti-tumor chemotherapeutic) that is clinically ineffective by absorption at a safe dose, selected from drugs other than the anti-tumor chemotherapeutic already loaded in, for example, the chinese, us, or european official pharmacopoeia.

In the context of the present invention, the amino acids, amino acid salts, oligopeptides as the amino acid nutrient are preferably amino acids or salts thereof selected from the group 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; more preferably an amino acid or a salt thereof selected from the group or an oligopeptide comprising or consisting of: arginine, lysine, glycine, cysteine, alanine, serine, aspartic acid, glutamic acid. In one embodiment, the concentration (w/w) of the amino acid based nutrient in the pharmaceutical composition is greater than or equal to 5%, preferably 10-35% or 18-35%, more preferably 15% -35% or 20% -35%.

In the present disclosure, the term "ineffective aromatic compound" refers to an aromatic compound that is not effective in inhibiting tumors by absorption at a safe dose. The ineffective aromatic compound includes any ineffective aromatic compound which is pharmaceutically acceptable. Within the scope of the present invention, the ineffective aromatic compound is one or more selected from the group consisting of: pigment aromatic compounds, salicylic acid compounds and quinoline compounds. In one embodiment, the concentration (w/v) of the ineffective aromatic compound in the pharmaceutical composition is not less than 0.35%, preferably 0.35-20%.

In the present disclosure, the term "chromoaromatic compound" refers to a pharmaceutically acceptable aromatic compound capable of selectively absorbing or reflecting light of a specific wavelength at a target region, which may include, for example, vital dyes, photosensitizers, and colored chemotherapeutic agents. In one embodiment, the pigment aromatic compound may be one or more selected from the group consisting of: methylene blue (including its hydrates), patent blue, isothio blue, bengal red, mixed porphyrin-based photosensitizers, porphyrin-based compounds (e.g., porphyrins, porphins, purpurins, endogenous porphyrins), nitrophenol compounds. In one embodiment, the concentration (w/v) of the pigment aromatic compound in the pharmaceutical composition is not less than 0.35%, preferably 0.5-10%.

In one embodiment, the salicylic acid-based compound is one or more selected from salicylic acid and one or more of the following compounds and derivatives thereof: acetylsalicylic acid, difluorosalicylic acid, disalicylate, dicumarol and aspirin lysine. In one embodiment, the concentration (w/v) of the salicylic acid-based compound in the pharmaceutical composition is not less than 0.5%, preferably 0.5-2.0%.

In one embodiment, the quinolines are selected from water-soluble quinolines, preferably from one or more of the following: quinine, quinine hydrochloride, quinine dihydrochloride, chloroquine. In one embodiment, the concentration (w/v) of said quinolines in said pharmaceutical composition is ≥ 3%, preferably 3-6%.

In the present disclosure, the term "non-animal bioactive ingredient" refers to pharmaceutically active extracts of plants and fungi and analogs thereof. The term "extract" refers to a particular component (e.g., a purified product based on a particular structure) obtained from a feedstock by separation and other processing. The term "analog" refers to a natural product, derivative, semi-synthetic, or synthetic, although not identical, but similar in structure and/or nature.

Within the scope of the present invention, the plant active ingredient is selected from the group consisting of biological extracts and analogues thereof having one or more of the following structures: glycosides, polyphenols, polysaccharides, terpenes, and flavones.

In one embodiment, the chemotherapeutic agent is selected from one or more of the following groups: anti-pathogenic disease chemotherapeutic drugs such as 5-fluorouracil, gemcitabine, epirubicin, antitumor antibiotics, teniposide, metal platinum complexes, paclitaxel, amino acid nutrients such as arginine, lysine, glycine, cysteine, glutamic acid, or salts thereof, or oligopeptides comprising the same, ineffective aromatic compounds such as methylene blue, acetylsalicylic acid, quinine monohydrochloride, quinine dihydrochloride, non-animal bioactive ingredients such as algal polysaccharides, medicinal plant polysaccharides, fungal polysaccharides, artemisinin.

In one embodiment, the composition further optionally comprises an adjuvant selected from conventional adjuvants. In the present disclosure, the term "adjuvant" refers to the additive disclosed in the present invention, which can enhance the body's ability to respond to the vaccine antigen or change the type of immune response after being mixed with the vaccine antigen. According to this definition, vaccine adjuvants differ from immunopotentiators in the general sense that their action often does not have to be mixed with vaccine antigens.

In the present disclosure, the term "conventional adjuvant" refers to any suitable adjuvant known to those skilled in the art, which may be, for example, an oil/milk adjuvant. In the present disclosure, the term "oil/milk adjuvant" refers to an oil or/and emulsion based adjuvant (e.g. freund's adjuvant, MF 59).

The additive in the semi-fluid according to the present disclosure may further optionally include 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).

In one embodiment, the pathogen disease comprises one or more of the following groups: tumors, microbial infections. In one embodiment, the pathogenic disease is selected from a tumor, preferably from a solid tumor.

In the present disclosure, the term "solid tumor" is used to refer to any malignant tumor having a tumor mass. Such as leukemia, malignant lymphoma, etc., are non-solid tumors.

In one embodiment, the solid tumor is preferably selected from the group consisting of tumor volume>85mm³Preferably ≥ 200mm³More preferably not less than 300mm³The solid tumor of (3).

In one embodiment, the solid tumor comprises: 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 pathogenic disease is selected from a microbial infection. In one embodiment, the microbial infection includes a viral infection and a bacterial infection. In one embodiment, the viral infection includes, for example, hepatitis b virus infection, hepatitis c virus infection, aids virus infection; the bacterial infections include, for example, leprosy, chronic mucocutaneous candidiasis.

The invention also includes the use of the semi-fluid pharmaceutical composition for the preparation of a vaccine or an immuno-chemotherapeutic combination for the local lesion.

The pharmaceutical compositions according to the present disclosure are for the treatment or inhibition of pathogenic diseases and may also be administered in combination with other therapies, such as interventional therapies, systemic chemotherapy, other immunotherapy (e.g., de-immune tolerant immunotherapy), photodynamic therapy, sonodynamic therapy, surgical intervention or a combination of such therapies to further enhance the therapeutic effect.

According to yet another aspect of the present disclosure, there is provided a method of preparing a pharmaceutical composition for treating or inhibiting a pathogenic disease, the pharmaceutical composition comprising a semi-fluid of animal tissue cells and an active ingredient, the method comprising the steps of:

b. providing the active ingredient in a liquid form, and,

According to yet another aspect of the present disclosure, there is provided a pharmaceutical composition prepared according to the above method.

Where relevant, the definitions and descriptions of various pertinent terms in the foregoing aspects of the disclosure herein apply to this aspect as well.

Within the scope of the present invention, the term "liquid tissue" refers to a tissue having fluidity without the limitation of a container, and includes, for example: blood, semen, saliva, fluid engineered tissue, and the like.

Severely damaged semifluids according to the present disclosure can be prepared, for example, as follows: subjecting the provided tissue to a severe damage treatment and obtaining a semi-fluid, or preparing the tissue as a semi-fluid followed by a severe damage treatment, wherein the severe damage treatment is selected from one or more of the following treatments disclosed herein: viscosifying treatment, mechanical disruption, coagulating treatment, heat treatment, freeze-thaw treatment, irradiation treatment, chemical treatment, and adding an active ingredient disclosed herein (e.g., chemotherapeutic agent, biologic, etc.) prior to, or/and during the treatment of severe damage to the tissue.

In one embodiment, said viscosifying comprises concentrating and/or adding an additive, wherein said additive is selected from the group comprising one or more of a cell preparation and/or a viscosifying agent, wherein said viscosifying agent is selected from the group comprising one or more of: polyethylene glycol, starch, sodium carboxymethyl cellulose, polyvinyl pyrrolidone.

In one embodiment, the severe injury treatment comprises mechanical disruption. For example, the bone marrow, muscle mass, liver, lung, heart, solid engineered tissue, and the like are severely damaged by mechanical disruption.

In one embodiment, the severe damage treatment comprises a heat treatment. For example, the bone marrow, muscle mass, liver, lung, heart, solid engineered tissues, etc. are heat inactivated, or the blood or blood/engineered cell composition is heat coagulated and severely damaged.

In one embodiment, the severe insult treatment comprises a freeze-thaw treatment. For example, the bone marrow, muscle mass, liver, lung, heart, blood, engineered cells, and the like are severely damaged by freezing and thawing.

In one embodiment, the severe damage treatment comprises an irradiation treatment. For example, the bone marrow, muscle mass, liver, lung, heart, blood, engineered cells, and the like are severely damaged by irradiation.

In one embodiment, the severe damage treatment comprises a chemical treatment. For example, the bone marrow, muscle mass, mechanical debris such as liver, lung, and heart, and fluid tissues such as blood are severely damaged by addition of an appropriate amount of acid, alkali, or alcohol.

In one embodiment, the severe damage treatment comprises a solidification treatment. For example, blood or blood/engineered cell compositions are severely damaged by self-coagulation, thermal coagulation or coagulation with the addition of coagulants.

In the method for preparing a pharmaceutical composition according to the present disclosure, the semi-fluid pharmaceutical composition prepared as above may be further divided into separate packages, and the separate packages may be prepared as a preparation (preferably, an implant, more preferably, an injection) for clinical use, or further prepared as a lyophilized preparation (e.g., a powder for injection) for clinical use.

In the present disclosure, the term "injection" refers to a drug that is administered through a needle cannula in compliance with the injection standards of the drug administration. The injection includes systemic injection (such as intravenous injection) and topical injection (such as subcutaneous injection, intramuscular injection, and tissue injection).

Within the scope of the invention, the needle cannula comprises, for example: a conventional syringe needle tube, a conventional puncture needle, a conventional implantation needle, and a conventional infusion needle.

The process conditions of the freeze-drying treatment 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 lyophilized preparation can be mixed with a liquid medium (e.g., water or an aqueous solution containing a viscosity-enhancing agent) and then directly administered topically.

Based on the studies described in more detail below, the drug of the present invention shows an organic unity of rapid treatment and follow-up treatment, short and long-lasting effects, and little damage to normal tissues of patients, although specific mechanisms remain to be further studied, thereby achieving a pharmaceutical effect of safely and effectively treating diseases.

Examples

The present invention is further illustrated by the following specific examples, which are not to be construed as limiting the invention thereto. In the following examples, all experimental animals were performed according to the relevant regulations and industry discipline. Unless otherwise specified, all tests were carried out according to the usual methods.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Some of the additives used in the following examples are 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. Taking mice as an example, there are 5 types: BALB/C mice, C57BL/6 mice, CB6F1 mice, CC3HF1 mice, nude mice, wherein: the CB6F1 mouse is a BALB/C X C57BL/6 hybrid F1 generation female mouse (the phenotype is H-2)^b/d) The CC3HF1 mouse is a BALB/C x C3H hybrid F1 generation female mouse (the phenotype is H-2)^d/k) The nude mice are mutant line (BALB/c-nu) mice obtained by introducing nude genes (nu) into BALB/c mice. 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, BALB/C mice are abbreviated as B mice and C57BL/6 mice are abbreviated as C mice, unless otherwise indicated. The organs and tissues of the experimental animals are purchased by experimental animal companies or prepared by the conventional method according to the rules of experimental animals, and comprise the following components: blood, bone marrow, spinal cord, skin, intestine, stomach, meat, pancreas, spleen, liver, lung, cartilage. Human placenta and human umbilical cord are legally obtained from a handling organization specified by the law.

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, solid tumors grow to the desired volume (e.g., mice carry tumors 30-500 mm)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.

The solid tumor volume calculation formula is as follows:

solid tumor volume (V) ═ l/2 × a × b²Wherein a represents a solid tumor length and b represents a solid tumor width.

The solid tumor growth inhibition rate (abbreviated as tumor inhibition rate in the invention) is calculated by the following formula:

tumor inhibition rate Y (%) ═ (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.

The observation items and scores of the animal graft versus host disease are shown in table 2 below.

TABLE 2 graft versus host disease score

Observation index	0 point (min)	1 minute (1)	2 is divided into
				Body mass reduction	<10％	10％～25％	>25％
Ability to move	Is normal	Attenuation of	Standing still
				Fur and fur	Is normal	Mild disorder	Color difference and disorder
Degree of skin integrity	Is normal	Mild molting of toe and tail	Severe hair loss
				Posture of a person	Is normal	Mild degree of bow back	Severe arch with upward

Each animal scored 5 points in total, with a maximum of 10 points and a minimum of 0 points.

The graft-versus-host ratio is calculated as follows:

(iii) anti-host rate Z (%) (TP-CP)/10 × 100%, wherein TP is the mean value of study group graft-versus-host disease scores; CP is the average value of the negative control group graft-versus-host disease score; 10 is the highest possible value of the graft versus host disease score for each animal.

In the following examples, experimental results (e.g. tumor weights) are expressed as means ± standard deviation (x ± s), and differences between two experimental animal groups and group means are compared by significance testing using statistical software SPSS 19.0, the testing being performed using statistic t, test level α being 0.05, P <0.05 indicating that the differences are statistically significant, otherwise they are not statistically significant.

Within the scope of the present invention, the combination of drug A and drug B is designated as B/A. In the following examples, unless otherwise indicated, agent a and agent B are semi-fluid and active components, respectively, comprising animal tissue cells. Unless otherwise stated, the drug effects of A, B, A/B are tumor inhibition. Improving the drug effect of antitumor drugs is always the biggest medical problem in the world. Even a few percent of efficacy is difficult and difficult to improve, so that the theoretical expectation of drug combination is usually not high, and once realized, the significance is great. The drug effect generated by the combined use of the drugs can be theoretically determined according to the judgment of q:

q-the actual combined effect/theoretical purely additive expected effect.

When q is 1, the actual combination effect is in accordance with theoretical expectations, showing additive effects; when q is less than 1, the actual effect is weaker than the theoretical expectation, and antagonism is shown; when q >1, the actual combination effect is more than theoretically expected and shows a synergistic effect.

The q calculation used in the present invention is based on the modified just-in-gold method (just-in-gold, addition in concomitant medication, Chinese Pharmacology 1980; 1(2), 70-76) of the Burgi method (Burgi Y. Pharmacology; Drug actions and reactions. cancer res.1978, 38(2), 284-285):

q＝E_A+B/(E_A+E_B-E_A·E_B)，

wherein E_A+BFor the actual combined effect of the A and B drugs, E_AFor the single-use effect of A, E_BThe effect of the single medicine B is shown, (E)_A+E_B-E_A·E_B) The expected effect is simply added for the pharmacology theory of A drug and B drug. To better fit the practical error range in animal experiments, he further replaced q 1 with q 1 ± 15% as an additive decision formula.

Another method for determining the efficacy of anti-tumor combinations, which is common in the literature, is based on a significance test (e.g., p-test). The pharmacological effects (tumor inhibition rate) of the compositions in both the q-and p-judgment were judged as a clear relationship between the actual combined effect and the theoretical expectation in the following antitumor animal experiments:

1) when q is 0.85. ltoreq. 1.15, and E_A+BAnd E_ABetween or E_A+BAnd E_BIf the difference between the two is not statistically significant (p is more than or equal to 0.05), the result is judged to be additive effect or effect which is not over expected;

2) when q is<0.85, and E_A+BAnd E_AAnd E_A+BAnd E_BThe difference between them is statistically significant (p)<0.05), the antagonism is judged to be obvious;

3) when q is>1.15, and E_A+BAnd E_AAnd E_A+BAnd E_BThe difference between them is statistically significant (p)<0.05), the synergistic effect is judged to be obvious.

Example 1: preparation of pharmaceutical compositions

In the following examples, the animal tissue cells used are selected from one or more of the following groups:

1) natural cells in connective tissue and/or semi-solid tissue. For example, using organs and/or tissues (e.g., muscle, spinal cord, bone marrow, blood, etc.) that contain cells of a desired animal tissue;

2) desired cells enriched in the connective tissue component and/or the semi-solid tissue component. For example, a blood component enriched in cells of a desired animal tissue (e.g., cell-enriched blood) is used;

3) natural cell preparations and/or engineered cells derived from connective and/or semi-solid tissue. Natural cell preparations include purified natural cells and derivatives thereof. For example, cell pellets, white blood cell pellets, red blood cell pellets, platelet pellets isolated from natural blood according to the prior art. For example, hematopoietic stem cells extracted and prepared from tissues such as bone marrow according to the prior art. Also for example, autologous or allogeneic blood cells, such as DC cells, LAK cells, TIL cells, CIK cells, DC-CIK, CTL cells, TCR-T cells, CAR-T cells, NK cells, γ δ stem cells, and the like, are induced, activated, expanded in vitro. For another example, lymphocytes are obtained from the spleen of an animal after a treatment of removal by lysis of erythrocytes according to the prior art: taking spleen after the animal is killed, crushing the spleen gently, adding serum-free DMEM culture solution and erythrocyte lysate (0.0075% ammonium chloride/0.0026% Tris sterile aqueous solution for hydrolysis and diluting to 500mL), stirring gently, standing for 5 minutes, performing centrifugal washing for 3 times (the centrifugal condition is 1000 rpm/min, and the sediment resuspension is serum-free DMEM), and finally obtaining lymphocyte sediment.

1. Preparation of a semi-fluid comprising animal tissue cells

By the disclosed method, a semi-fluid comprising animal tissue cells can be prepared. Table 3 below lists the semi-fluid (preparation number) prepared in this example, part of which contains the animal tissue cells, the preferred tissue from which it was prepared, the major preparation steps, and the effects achieved by the preparation and its nodular nature.

TABLE 3

Hematocrit: the hematocrit of the semi-solid tissue is provided by veterinarians of relevant animal laboratories and the hematocrit of the blood is determined according to routine blood measurements (e.g., using a fully automated hematology analyzer BC 5000). For example, 43% fineThe cell concentration in the blood of the hematocrit mouse was 11X 10⁹One per ml.

The function is as follows: a indicates the presence of semifluidization (+ presence), and B indicates the presence of severe damage (+ presence).

And (3) nodule: indicates whether a semifluid nodule (+ energy) can be formed at the injection site, as follows: 100ul of the preparation in the table above was injected subcutaneously into the left axilla of BALB/c mice to form a nodule, and the nodule was irreversibly deformed by pinching down the index finger and thumb. When the hematocrit of the cell suspension is 77% or more, it turns into a semi-fluid thick matter. The subcutaneous half-disappearance of other semifluid nodules ranged from 1 to 30 days, except for the semifluid dope (X25) ranged from 0.1 to 0.5 days. When the cell suspension has a hematocrit of 70% or more, it is converted to a semi-fluid viscous substance.

Mice: the mice in the table are BALB/c mice.

Several examples of the preparation tests of the semifluids according to the invention are listed below.

Example 1 a: the spinal cord tissue 20g obtained by extracting (puncturing or dissecting and removing the outer membrane) from the spinal cord tissue containing the membrane is the injectable semi-fluid preparation X1 in the upper surface. Preparation X2 in the above table was obtained if X1 was placed in a cup and a gas permeable lid was added and steamed in steam (about 100 ℃ C.) for 30 minutes.

Example 1 b: 20g of a lean meat mass taken from a mouse was subjected to tissue extraction (peeling to remove fat, membrane, tendon and blood vessel), and the obtained muscle tissue of the mouse was a semisolid tissue, which was then crushed in a blender (rotation speed 3000-. The shear-crushing rotation speed and time may be set so that the particles obtained have a cross-sectional average size of 100nm or more, preferably 500nm to 0.8mm or 1 μm to 0.8 mm. Preparation X5 was irreversibly deformed under pressure and was semi-fluid. Preparation X6 in the above table was obtained if X5 was further placed in a cup and a gas permeable lid was added and steamed in steam (about 100 ℃ C.) for 10 minutes.

When X5 was further sealed in a plastic bag, frozen in liquid nitrogen (below-80 ℃ C.) for 30 minutes and thawed at 37 ℃ and this freeze-thaw can be performed one or more times, preparation X7 from the above table was obtained. When X5 was further sealed in a plastic bag and placed in an ultrasonic apparatus (temperature 5-25 deg.C, operating frequency 10-30 kHZ) for 5-10 minutes, preparation X8 in the above table was obtained.

The preparation of preparations X3, X9, X10, X11, X20 and X21 in the above table, respectively, can be carried out using the same method as that for X5. The preparation of preparation X4 in the above table can be carried out using the same method as the preparation of X6.

Example 1 c: 20g of a piece of skin taken from the back of a pig was subjected to tissue extraction (peeling to remove fat), and the obtained skin tissue was a semisolid tissue, which was then placed in a cup with a gas-permeable lid, steamed in steam (about 100 ℃) for 120 minutes, and then placed in a mixer while hot and sheared and crushed (rotation speed 3000 + 10000 rpm, total time 1-3 minutes), so that preparation X12 in the above table was obtained. X12 is semi-fluid when heated (e.g. 60-100 deg.C), can be packaged into injector, and can be used as injectable vaccine when heated (e.g. 60 deg.C).

Example 1 d: one new zealand immune was sacrificed and 15ml of the immune blood was taken and allowed to stand at room temperature for 30 minutes to obtain self-coagulated blood (X13 in the above table).

Example 1 e: one new zealand immune was sacrificed and 15ml of the immune blood was placed in a cup pre-filled with anticoagulant sodium citrate, slowly stirred well, then a gas permeable lid was added to the cup, and then placed in steam (about 100 ℃) to steam for 20-50 minutes to obtain heat coagulated blood (X14 in the above table). The preparation of preparations X15 and X16 in the above table can be carried out separately using the same method as that for X14.

Example 1 f: about 6ml of blood was obtained from each of the orbits of a plurality of mice (with or without anticoagulant), mixed in a cup, treated in an ultrasonic instrument (temperature 5-25 ℃ C., operating frequency 10-30kHZ,;) for 1-30 minutes, then covered with a gas-permeable lid, and steamed in steam (about 100 ℃ C.) for 20 minutes to obtain preparation X17 in the above table.

Example 1 g: separately collecting blood (with or without anticoagulant) from orbit of multiple mice, adding preset coagulant (such as thrombin with final concentration of 10-100U/1ml and calcium chloride with concentration of 5-25 mmol/L) into the cup, mixing uniformly, standing for 30 min to obtain semisolid coagulum, and stirring and crushing (rotation speed of 3000 plus 10000 rpm, total time of 1-3 min) to obtain preparation X18 in the above table.

Example 1 h: preparation X19 in the above table was obtained by separately bleeding the orbit of a plurality of mice, centrifuging to remove 40% serum, adding to the cup a gas permeable lid, and steaming in steam (about 100 ℃ C.) for 20 minutes.

Preparation X20 in the above table was obtained by the same preparation using human blood.

Example 1 i: preparation X23 in the above table was obtained by sampling muscle tissue on the hind leg of a pig using a puncture needle and then drawing the highly disrupted muscle tissue into the syringe barrel.

Example 1 j: approximately 10ml of blood was drawn from the horse ear using a blood drawing needle, and then the syringe containing the blood was heated in a microwave oven for 1 minute with the small needle tip set upright to obtain preparation X24 in the above table.

Example 1 k: preparation X25 in the above table was obtained by separately bleeding the orbit of a plurality of mice and centrifuging to remove 45% of the serum.

Example 1 l: preparation X26 in the above table was obtained from human leukocyte pellets that were filtered off during the production of leukoreduced erythrocytes (blood centrifugation + leukocyte sorting enrichment).

Example 1 m: preparation X27 in the above table was obtained by adding 0.6g of mouse lymphocyte pellet and 0.4g of mouse blood to the cup, gently mixing, adding a gas permeable lid to the cup, and steaming in steam (about 100 ℃ C.) for 20 minutes.

Example 1 n: preparation X28 in the above table was obtained by mixing 4g of human plasma with 6g of human leukocyte pellet taken from a leukocyte-removing filter, and steaming the cup with a gas-permeable lid in steam (about 100 ℃ C.) for 10 minutes.

The preparations are injectable semifluid and can be used after being subpackaged into injectors.

2. The composition of the invention and the preparation of the medicament

The semifluid of the invention comprising animal tissue cells is used as a synergistic component of the active ingredient for the preparation of a medicament. Wherein the active ingredient comprises a biological agent and/or a chemotherapeutic agent.

Chemotherapeutic drugs and most biologicals are commercially available (e.g., table 1).

The tumor antigens used in this example were prepared as follows: tumor cell fluid (10)⁸-10⁹Individual cells/ml) were subjected to conventional freeze-thaw inactivation treatment (by placing in liquid nitrogen at less than-80 ℃ for 30 minutes and then thawing at 37 ℃ for 3 times), and then verified as non-tumorigenic by conventional tumor cell transplantation tumor experiments, i.e., tumor antigens including the following obtained from respective tumor cell fluids: sarcoma antigen (S180 cells), liver cancer antigen (H22 cells), colon cancer antigen (CT26 cells), breast cancer antigen (4T1 cells), melanoma antigen (B16 cells), lung cancer antigen (LLC cells).

The composition containing the semifluid of the animal tissue cells and the active ingredients is prepared by adding the active ingredients before, during and after the semifluid or/and severe damage treatment of the animal tissue cells and appropriately mixing the active ingredients by the preparation method disclosed by the invention. The following table lists a portion of the semi-fluid composition prepared in this example (preparation number), the preferred tissue and preferred active ingredients from which it was prepared, the major preparation steps, and the effects achieved by the preparation and its nodular nature.

TABLE 4

And (3) nodule: indicates whether a semifluid nodule (+ energy) can be formed at the injection site, as follows: 100ul of the preparation in the table above was injected subcutaneously into the left axilla of BALB/c mice to form a nodule, and the nodule was irreversibly deformed by pinching down the index finger and thumb. The subcutaneous half-disappearance period of the above semifluid nodules is 1-30 days.

Mice: the mice in the table are BALB/c mice.

Mixing and stirring: in this example, unless otherwise stated, the mixing agitation is mechanical agitation (rotation speed 10-10000 rpm, total time 1-3 minutes), which can cause mechanical disruption and mechanical damage to the tissue.

Several examples of the preparation of the semi-fluid containing animal tissue cells and active ingredients of the present invention are listed below.

Example 1 o: the spinal cord tissue obtained by extracting (puncture extracting or dissecting and peeling off the outer membrane) from the membrane-containing spinal cord tissue (4.9 g) and 0.1g of docetaxel were mixed in a mixer (rotation speed 1000-. When this Y1 was further placed in a cup with a gas-permeable lid and steamed in steam (about 100 ℃ C.) for 20-50 minutes, Y2 in the above table was obtained.

The preparation of preparation Y3 in the above table can be carried out using the same method as the preparation of Y1.

The preparations of preparations Y4, Y5 in the above table can be carried out separately using the same method as the preparation of Y2.

Example 1 p: separately, blood (with or without anticoagulant) was drawn from each of the orbit of a plurality of mice and mixed, 4.9g of the blood was mixed with 0.1g of 5-Fu in a cup, and then a gas-permeable lid was attached to the cup and steamed in steam (about 100 ℃) for 5-50 minutes to obtain preparation Y6 in the above table.

The preparations of preparations Y7, Y8 and Y9 in the above table can be carried out, respectively, using the same method as the preparation of Y6.

Example 1 q: the spinal cord tissue obtained by extracting (puncturing or dissecting and removing the outer membrane) from the spinal cord tissue containing the membrane (4.95 g) and 0.05g of the CpG ODN were placed in a mixer and stirred (1000 rpm; 2000 rpm; total time 1-3 minutes) to obtain a 1% CpG ODN/99% spinal cord semi-fluid composition (preparation Y10). The heat treated 1% CpG ODN/99% spinal cord semi-fluid composition (preparation Y11) was obtained by further placing the Y10 in a cup with a gas permeable lid and heating in a water bath (about 60 ℃) for 5-50 minutes.

Using mouse muscle tissue and the same method as for Y10 preparation, a 1% CpG ODN/99% mouse muscle semifluid composition (preparation Y12) was prepared.

Using mouse muscle tissue and the same method as for Y11 preparation, a heat-treated 1% CpG ODN/99% mouse muscle semifluid composition (preparation Y13) was prepared.

Example 1 r: separately, blood (with or without anticoagulant) was drawn from each of the orbit of a plurality of mice and mixed, 5g of the blood was mixed with 500 ten thousand units of interferon alpha in a cup, and then a gas-permeable lid was attached to the cup and the cup was steamed in steam (about 100 ℃) for 20 to 15 minutes to obtain 500 ten thousand units of interferon alpha/5 g of a mouse blood semifluid composition (preparation Y14).

Using the same procedure, a cytokine/blood semi-fluid composition (e.g., Y15) can be prepared separately when blood from other animals is used, or when other cytokines are used.

Example 1 s: blood (with or without anticoagulant) was collected from each of the orbit of a plurality of mice and mixed, 4.95g of the blood was mixed with 0.05g of anti-PD-1 antibody in a cup, and then a gas-permeable lid was attached to the cup and steamed in steam (about 100 ℃) for 20-15 minutes to obtain a 1% anti-PD-1 antibody/99% mouse blood semifluid composition (preparation Y16).

The same procedure is used when using blood from other animals, or when using other immunomodulatory antibodies, to prepare immunomodulatory antibody/blood semi-fluid compositions, respectively.

Example 1 t: separately, blood (with or without anticoagulant) was collected from each orbit of a plurality of mice and mixed, and 1.5ml of the blood was mixed with 1ml of freeze-thaw inactivation solution (10) for breast cancer cells⁹Individual cells/ml) was stirred well in the cup, and then a gas-permeable lid was added to the cup, which was put into steam (about 100 ℃) to steam for 5-50 minutes, to obtain preparation Y17 in the above table.

Y19 was prepared using the same method when other cancer cell freeze-thaw inactivation solutions were used.

Example 1 u: 1.5ml human blood and 1ml breast cancer cell freeze-thawing inactivation solution (10)⁹One cell/ml) in the cup, adding a gas-permeable cover to the cup, steaming in steam (about 100 deg.C) for 5-50 minPreparation Y18 was obtained as in the above table.

Example 1 v: separately, blood (with or without anticoagulant) was collected from each orbit of a plurality of mice and mixed, and 1.5ml of the blood was mixed with 1ml of lung cancer cell fluid (10)⁹Individual cells/ml) in a cup, sealed in a plastic bag, frozen in liquid nitrogen (below-80 ℃) for 30 minutes, and thawed at 37 ℃, and this freeze-thaw can be performed one or more times to obtain preparation Y20 in the above table.

Example 1 w: 0.2ml of mouse lymphocyte precipitate (hematocrit 72%), 0.2ml of liver cancer cell sap (10%⁹Individual cells/ml), 0.6ml mouse plasma was added to the cup and gently mixed, and the cup was then covered with a gas permeable lid and placed in steam (about 100 ℃) to steam for 20 minutes, yielding preparation Y21 in the above table.

Example 1 x: preparation Y22 in the above table was obtained by adding 0.6ml of mouse lymphocyte pellet (hematocrit 72%), 10mg of methylene blue liver, 0.39ml of mouse plasma to the cup, gently mixing, adding the cup to a gas permeable lid, and steaming in steam (about 100 ℃) for 20 minutes.

Example 1 y: 0.3g of human leukocyte precipitate (hematocrit 77%), 0.2ml of liver cancer cell sap (10%⁹Individual cells/ml), 0.5ml of human plasma were added to the cup and gently mixed, and the cup was then covered with a gas permeable lid and placed in steam (about 100 ℃) for 20 minutes to obtain preparation Y23 in the above table.

Example 1 z: preparation Y24 in the above table was obtained by adding 0.6g of human leukocyte pellet, 10mg of methylene blue, and 0.39ml of human plasma to the cup, gently mixing, adding a gas-permeable lid to the cup, and steaming in steam (about 100 ℃ C.) for 20 minutes.

The preparations are injectable semifluids and can be used as vaccines after being subpackaged into injectors.

Example 2: synergistic regimen for semifluid compositions comprising animal tissue cells

In the compositions of the present invention, the semi-fluid comprising animal tissue cells may provide the immunogenicity required for immune synergy. The following experiments investigated the requirements in this technical scheme for immunogenicity.

In one of the tests that were carried out,the experimental animals were BALB/c mice, the modeled cells were breast cancer 4T1 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 206 mm)³) The test groups were randomly divided into 6 test groups (as shown in the table below). The test groups were divided into 1 negative control group (group 0) and 5 study groups. The negative controls were all saline and study drugs are shown in the table below. The study drug was prepared as follows:

the human leukocyte precipitate and human plasma are obtained by extracting and obtaining normal blood according to the prior art and then centrifuging. The human leukocyte solution is diluted solution with hematocrit of 35% obtained by adding human leukocyte precipitate (hematocrit of 70.5%) into physiological saline of the same volume. The hematocrit is determined by conventional methods. The 50% human plasma is a mixture of 50% human plasma and an equal volume of 50% normal saline. Human leukocyte/human blood A semi-fluid (prepared as in preparation Y9 of example 1) was formed by adding an equal volume of human plasma (hematocrit 35%) to a human leukocyte pellet and heating to solidify the mixture. The human leukocyte/human blood semisolid is a gel (semisolid) prepared by adding porcine thrombin (final concentration of 100U/1ml) and calcium chloride (final concentration of 20mmol/L) into human leukocyte/human blood , and cutting into a volume of about 400mm³The small blocks are used.

Each experimental group was administered once by subcutaneous injection to the left axilla, 200. mu.l/patient. Study group 5 semisolid was surgically implanted subcutaneously into the left flank of mice. The drugs of the other experimental groups were injected by syringe. Animals were euthanized at day 14 post-dose, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 5.

TABLE 5

Group number	Research medicine	Form of the composition	Node (B)	Tumor weight (x + -s)	Tumor inhibition rate
						0	Physiological saline	Liquid, method for producing the same and use thereof	-	0.92±0.18g	-
1	50% human blood	Liquid, method for producing the same and use thereof	-	0.89±0.11g	3％
						2	Human leukocyte blood/human blood	Liquid, method for producing the same and use thereof	-	0.84±0.19g	8％
3	Human leukocyte liquid	Liquid, method for producing the same and use thereof	-	0.87±0.10g	5％
						4	Human leukocyte/human blood semifluid	Semifluid	+	0.61±0.10g	43％
5	Semi-solid human leukocyte/human blood	Semi-solid	+	0.75±0.26g	19％

Note: + is the formation of nodules, -is the absence of nodules; x is the tumor weight average (g, the same table below).

In the above table, the tumor inhibition rates of the study groups 1, 2, and 3 were all low. While the q-score (q ═ 1.13) for study group 2 showed additive effects between study groups 1, 2, and 3, the tumor weights between study groups 2 and 1 and 2 and 3 had no statistical significance (P ═ 0.0931>0.05 and P ═ 0.3762>0.05, respectively), and the additive effects shown for group 2 were not significant. The results indicate that neither leukocyte (even very high concentrations of xenogenous leukocytes) fluids, human plasma fluids, nor leukocyte/human plasma composition fluids show a significant tumor burden reduction effect.

However, the tumor inhibition rates of study groups 2, 4, and 5 were very different. The drug effect of study group 4 not only exceeded that of study group 2 and study group 5, but even showed drug effects exceeding the additive effect of the two groups (q ═ 1.69>1.15), and the tumor weight differences between study groups 4 and 2, and 4 and 5 were statistically significant (P ═ 0.0271<0.05, P ═ 0.0472<0.05, respectively). This result demonstrates that even with the same composition, concentration and administration, it is possible that the cell-containing material will exhibit significantly different tumor burden reduction effects if only the morphological structure is different (liquid, semi-fluid, semi-solid). This shows that semifluids may have a completely different mechanism of immune action than liquids and semisolids.

The above results and more similar studies indicate that semifluids containing animal tissue cells exhibit a theoretically expected potency that significantly exceeds the sum of the liquid, semisolid, and even liquid and semisolid effects of the same composition, potentially providing immune synergy for biologically active products with immune effects. This was further verified by the following experiment.

In one experiment, the experimental animals were BALB/c mice, the modeled cells were sarcoma S180 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 342 mm)³) The test groups were randomly divided into 13 test groups (as shown in the table below). The test groups were 2 series, the tail vein injection series had 1 negative control group (group 01) and 2 study groups, and the subcutaneous injection series had 1 negative control group (group 02) and 10 study groups. The negative controls were all saline and study drugs are shown in the table below. The study drug was prepared as follows:

the C mice were C57BL/6 mice, and the B mice were BALB/C mice. Animal tissue cells are contained in blood and muscle, respectively. The mouse muscle semifluid was a semifluid prepared from mouse meat by the method of preparation X5 in example 1. Blood from 4 mice was prepared as follows: the natural blood (hematocrit 42%, 4341%) is fresh blood taken from mice to which anticoagulant (sodium citrate) is added. 6ml of the natural blood is separated out and centrifuged, 3ml of serum is taken out, and the rest part in the centrifuge tube is mixed uniformly to obtain the concentrated blood of the mouse (the hematocrit is 68%). 2ml of serum was mixed with 2ml of natural blood to give diluted blood (1) for mice (hematocrit 22%). The remaining 1ml of serum was then mixed with 2ml of native mouse blood to give diluted mouse blood (2) (hematocrit 33%). The hematocrit of each blood was measured according to a conventional method. Each of the mouse blood semifluids was a semifluid obtained by heat coagulation of the corresponding mouse blood (prepared by the methods of preparation X16 and X19 in example 1, respectively). Semisolid mouse blood is prepared by adding pig thrombin (final concentration of 100U/1ml) and calcium chloride into mouse blood(final concentration 20mmol/L) gel formed (semi-solid), which was cut to a volume of about 400mm³The small blocks are used.

Each experimental group was administered 1 time in the following administration mode (subcutaneous injection is administered in the left axilla) and each time 400. mu.l/patient was administered. Study group 10 mice were subcutaneously implanted with semisolid by surgery. The drugs of the other experimental groups were injected by syringe. In this experiment, administration to study groups 1, 3, 5 and 7-11 can be considered an allogeneic administration model (which may represent more than 99% of allogeneic administrations), and administration to other study groups can be considered a fully compatible administration model and an autologous administration model. Animals were euthanized at day 14 post-dose, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 6.

TABLE 6

*: + is for the formation of nodules, -is for the absence of nodules

In the above table, the tumor inhibition rates of study groups 1, 2, 3 and 4 were low, and the difference in tumor weights between them and the respective negative control groups was not statistically significant (P-0.9332 >0.05, P-0.5340 >0.05, P-0.3788 >0.05 and P-0.5458 >0.05, respectively). This result demonstrates that the liquid contents of the cells do not show a significant tumor burden reduction effect whether they are administered intravenously or subcutaneously in blood containing syngeneic or allogeneic cells. More generally, liquid inclusions of cells may be difficult to prefer as antigens capable of significantly reducing tumor mass.

In fact, it is generally believed that allogeneic blood transfusion to a patient with a tumor will result in increased secretion of interleukin-2, interleukin-10 and interleukin-24, inhibition of interleukin-2 secretion by helper T lymphocytes, a reduction in the B lymphocyte stimulatory response and antibody production, and a negative modulation of cellular immunity. Allogeneic blood is thus considered as a potential inhibitor of immune effector cells and also as a stimulator of immunosuppressive cells, which down-regulates the beneficial tumor-suppressing immune function. However, in the above table, study groups 5 and 6 both showed higher tumor inhibition rates. The difference in tumor weights between each of them and the negative control group was statistically significant (P0.0004 <0.05, P0.0002 < 0.05). Furthermore, tumor weights between study groups 5 and 3 (P ═ 0.0005), 5 and 1 (P ═ 0.0004), 6 and 4 (P ═ 0.0001), 6 and 2 (P ═ 0.0002) were statistically significant (P < 0.05). While the tumor weight difference between study groups 5 and 6 was not statistically significant (P ═ 0.1375> 0.05). The above results indicate that whether a cell containing substance (e.g., blood) can be used as an antigen to significantly reduce tumor mass is significantly more correlated with its state (whether liquid or semi-fluid) than is genetically identical.

In the above table, study group 10 had a lower tumor inhibition rate than study group 9, while the difference in tumor weight between study groups 10 and 9 was statistically significant (P ═ 0.0147< 0.05). The results further illustrate that the preferred state of the cell-containing material as an antigen that significantly reduces tumor mass is semi-fluid, not semi-solid. In addition, semi-fluid phases are more semi-solid with greater ease of handling and patient compliance for clinical use.

In addition, in the C mouse blood semifluid research group, the tumor inhibition rates are arranged in the order of magnitude: study group 5, study group 9, study group 8, and study group 7. Among them, the difference in tumor weight between study groups 7 and 3 (P ═ 0.2139) was statistically insignificant (P >0.05), while the differences in tumor weight between study groups 8, 9, 5 and 7, respectively, were statistically significant (P ═ 0.0107<0.05, P ═ 0.0227<0.05, P ═ 0.0008<0.05, respectively). Furthermore, the tumor inhibition rate of study group 11 (mouse muscle tissue hematocrit above 60%) was comparable to study group and 6, respectively, with no statistical significance for the difference in tumor weights between them (P0.5241 > 0.05). This result indicates that the association of the blood semifluid (more generally, the semifluid containing cells) as an antigen that significantly reduces the tumor mass with the concentration (> 22%, preferably ≧ 33%) of the cells it contains is higher than its cellular identity.

According to the above and more similar studies, the requirements for providing immune synergy with a semi-fluid comprising animal tissue cells in the composition of the present invention are: its composition and morphology is such that it forms a semifluid nodule at the site of administration that activates an effective immune response. The composition and morphology of the semifluid containing animal tissue cells is such that it forms a semifluid nodule at the site of administration that activates an effective immune response. The requirement seems to be to form locally in vivo tissues that are physiologically similar in composition and morphology to the lesion (e.g. tumor mass), are more readily recognized by the body (e.g. semifluid highly deviating from the natural state or highly damaged) and elicit an effective specific immune response against it and the lesion (e.g. tumor mass) similar to it. Thus, the basic technical solution for providing immune synergy by a semi-fluid containing animal tissue cells in the composition of the present invention is as follows:

a semi-fluid comprising animal tissue cells is applied as an immuno-synergistic component of the composition;

the animal tissue cells contained in the semifluid are remote from their natural environment, preferably in an environment that is readily recognized by the immune system as severely damaged tissue. In addition, the animal tissue cells contained in the semi-fluid composition of the invention are preferably severely damaged cells (e.g., cells that have lost proliferative activity). Severely damaged cells can often expose cellular contents (e.g., cellular contents released by cell membrane disruption);

the above-mentioned semi-fluid containing animal tissue cells is preferably one or more selected from the group consisting of: a semi-fluid dope comprising said cells, a coagulum comprising said cells, a homogenate of non-fluid tissue comprising said cells, a homogenate of a coagulum comprising said cells, more preferably one or more selected from the group consisting of: a coagulate comprising the cells, a disrupted product of a non-liquid tissue comprising the cells, and a disrupted product of a coagulate comprising the cells. These cell-containing semifluids exhibit a state of high deviation from the natural state or high damage as compared with the natural tissue containing cells;

the cell-containing semifluid is contained in a topical application (e.g., a tumor-area application and/or an extratumoral topical application of the drug);

the semi-fluid containing cells is a semi-fluid implant, preferably a semi-fluid injection. The semi-fluid injection is an injection which can be directly administered by a conventional injection system in a semi-fluid manner, and the semi-solid implant is usually implanted by surgery or forms a semi-solid (e.g. gelated) nodule at the administration site after being administered by a conventional injection system in a fluid (liquid) manner.

Furthermore, the semifluid comprises cells having a hematocrit of>22% (or cell concentration of>5.6×10⁹One/ml), preferably 33% to 86% (or a cell concentration of 8.4X 10)⁹-22×10⁹Individual cell/ml), 45% -86% (or cell concentration 11.5X 10)⁹-22×10⁹Individual cells/ml), or 55% -86% (or cell concentration 14.0X 10)⁹-22×10⁹Individual cells/ml). In which the semi-fluid dope composition, the ratio of the amount of the cells to the amount of the composition (v/v) is 70% or more (or the cell concentration is 17.9X 10 or more)⁹Individual cell/ml), preferably 70% to 86% (or a cell concentration of 17.9X 10)⁹-22×10⁹Individual cells/ml).

The following experiments investigated the combined solution based on the semi-fluid solution described above.

In one experiment, the experimental animals were BALB/c mice, and the modeled cells were breast cancer 4T1 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 236 mm)³) The test groups were randomly divided into 16 test groups (as shown in the table below). The test groups were 2 series, the tail vein injection series had 1 negative control group (group 01) and 3 study groups, and the subcutaneous injection series had 1 negative control group (group 02) and 11 study groups. The negative controls were all saline and study drugs are shown in the table below. The study drug was prepared as follows:

the breast cancer antigen is a freeze-thaw inactivation solution (10) of breast cancer cells prepared by the method of example 1 from the tumor body of a breast cancer-bearing mouse obtained by the same modeling method⁹Individual cells/ml). The cells used are contained in the blood. Blood from 4 mice was prepared as follows: the natural blood (hematocrit 42%, 4143%) was fresh blood taken from mice to which anticoagulant (sodium citrate) was added. Is divided into6ml of the crude blood was centrifuged, 3ml of serum was removed, and the remaining portion of the tube was mixed to give concentrated mouse blood (hematocrit 68%). 2ml of serum was mixed with 2ml of natural blood to give diluted blood (1) for mice (hematocrit 22%). The remaining 1ml of serum was then mixed with 2ml of native mouse blood to give diluted mouse blood (2) (hematocrit 33%). The hematocrit of each blood was measured according to a conventional method. Each breast cancer antigen/mouse blood is 40% breast cancer cell freeze-thaw inactivation solution (10)⁹Individual cells/ml) and 60% of each mouse blood. Each breast cancer antigen/mouse plasma semifluid was a semifluid formed by heat coagulation of each breast cancer antigen/mouse blood (prepared according to the preparation method of preparation Y19 in example 1). The mouse native blood semifluid is a semifluid formed by heating and coagulating mouse blood (prepared by the method of preparation X16 in example 1).

In this experiment, the administration to the study group can be considered an allogeneic administration model (which may represent more than 99% of allogeneic administrations). Each experimental group was administered once in the administration mode shown in the following table (subcutaneous injections were all administered subcutaneously in the left axilla), and each administration was 400. mu.l/tube. Animals were euthanized at day 14 post-dose, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 7.

TABLE 7

And (3) nodule: + is for the formation of nodules, -is for the absence of nodules

*: mouse blood semifluid tumor inhibition rate: according to the related experiments, the tumor inhibition effect of the semifluid of diluted mouse blood is less than that of the semifluid of 100 percent of mouse blood

In the above table, the q of the composition group (q 0.83<0.85) showed antagonism among the study groups 3, 2 and 1 subjected to intravenous injection, whereas the tumor weights of the study groups 3 and 1 and 3 and 2 had no statistical significance (P0.7662 >0.05 and P0.9317 >0.05, respectively), and thus the composition group showed no significant antagonism. The q of the composition group (q 0.79<0.85) showed antagonism among study groups 6, 5 and 4 injected subcutaneously, whereas the tumor weights of study groups 6 and 4 and 6 and 5 had no statistical significance (P0.3274 >0.05 and P0.7771 >0.05, respectively), and thus the composition group showed no significant antagonism. The results demonstrate that the liquid compositions show no synergistic or even no significant additive effect, whether injected intravenously or subcutaneously.

However, between study groups 8, 7, 5 injected subcutaneously, the q-judged (q >1.77>1.15) of the composition group showed synergy, and the tumor weight differences between study groups 8 and 5, and 8 and 7 were all statistically significant (P ═ 0.0006<0.05, P ═ 0.0327<0.05, respectively), so the composition group showed significant synergy. This result demonstrates that there are completely different interactions between the liquid and semi-liquid containing the cells of the animal tissue and the active ingredient.

In the above table, the tumor inhibition rates in the study group of compositions injected subcutaneously are in the order of magnitude: study group 14, study group 8, study group 12, study group 10. Between study groups 10, 9, 5, the q-judged (q ═ 0.95) of the composition group showed additive effects, but the difference in tumor weight between study groups 10 and 5 had statistical significance (P ═ 0.0288<0.05), and the difference in tumor weight between 10 and 9 had no statistical significance (P ═ 0.2850>0.05), so the composition group showed no significant additive effects, more no synergistic effects. As the hematocrit increased, the q-judged (q ═ 1.29>1.15) of the composition group showed synergy among the study groups 12, 11, 5, and the tumor weight differences between the study groups 12 and 5, 12 and 11 were all statistically significant (P ═ 0.0012<0.05, P ═ 0.0274<0.05, respectively), so the composition group showed significant synergy. Further, between study groups 14, 13, and 5, the q of the composition group (q ═ 1.25>1.15) showed synergy, and the tumor weights of study groups 14 and 5 and 14 and 13 were statistically significant (P ═ 0.0001<0.05 and P ═ 0.0283<0.05, respectively), so that the composition group showed significant synergy. The results show that the synergistic effect of the cell-containing semifluid on the biological product is dependent on the hematocrit. The hematocrit may be involved in determining many properties (e.g., softness) of the nodules formed in vivo by the semifluid, and thus in determining its synergistic effects.

Similar results were obtained using other preparations from example 1 (e.g., Y14-Y18, etc.).

In one experiment, the experimental animals were BALB/c mice, and the modeled cells were breast cancer 4T1 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (average tumor volume 215 mm)³) The groups were randomized into 8 experimental groups, including 1 negative control group and 7 study groups. The negative controls were all saline and study drugs are shown in the table below. The study drug was prepared as follows:

the PD- (L)1 antibody used is commercially available, and the cells used are contained in blood. The PD- (L)1 antibody drugs were all aqueous solutions of indicated concentrations, the mouse blood was taken from fresh blood of BALB/c mice to which an anticoagulant (sodium citrate) was added, the mouse blood semifluid was preparation X16 in example 1, and PD- (L)1 antibody/mouse blood semifluid at different weight ratios were prepared according to the preparation method of preparation Y16 in example 1.

In this experiment, the administration to the study group can be considered as an isogenic administration model (which can represent allogenic and autologous administration). Each experimental group was administered once in the form of subcutaneous injection in the left axilla (subcutaneous injection means subcutaneous injection in the left axilla) at a dose of 250. mu.l/dose. Animals were euthanized at day 14 post-dose, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 8.

TABLE 8

In the above table, in the PD- (L)1 antibody/blood semifluid composition study group, the tumor inhibition rates are ranked from large to small: study group 7, study group 6, study group 5. Between study groups 5, 2, 1, the q-judged (q ═ 1.11) of the composition group showed additive effects, but the difference in tumor weight between study groups 5 and 2 had statistical significance (P ═ 0.0019<0.05) while the difference in tumor weight between study groups 5 and 1 had no statistical significance (P ═ 0.2980>0.05), so the composition group showed no significant additive effects. However, between study groups 6, 3, 1, the q of the composition group was judged (q ═ 1.30>1.15) to show synergy, and the tumor weight differences between study groups 6 and 3, and between 6 and 1 were all statistically significant (P ═ 0.0003<0.05, P ═ 0.0060<0.05, respectively), so the composition group showed significant synergy. Further, between study groups 7, 4, and 1, the q of the composition group was judged (q ═ 1.54>1.15) to show synergy, and the tumor weight differences between study groups 7 and 4, and 7 and 1 were statistically significant (P ═ 0.0003<0.05, and P ═ 0.0038<0.05, respectively), so the composition group showed significant synergy. According to this and further results, the synergistic quantitative ratio (w: w) of PD- (L)1 antibodies to the semi-fluid composition (more generally, to their cognate immunomodulatory antibodies to the semi-fluid composition) is >0.05/100, preferably ≧ 0.1/100.

The following further experiments confirm the above ranges of the synergistic amount ratio.

In one experiment, the experimental animals were BALB/c mice, the modeled cells were sarcoma S180, at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animal (Holland sarcoma mouse, average tumor volume 251mm³) The animals were randomized into 8 experimental groups (1 negative control group and 7 study groups) and intratumorally administered to the animals. The negative control was physiological saline and study drugs are shown in the table below. The study drug was prepared as follows:

0.1 percent of BCG vaccine and 50 ten thousand IU/ml of human recombinant interferon are respectively liquid prepared by water for injection. The cells used are contained in the blood. The mouse blood semifluid was X16 of example 1, 0.1% BCG/99.9 mouse blood semifluid, and the human recombinant interferon/mouse blood (50 ten thousand IU/ml) semifluid were thermal coagulates of the indicated amounts of the mixture of the biological product and the mouse blood, respectively (prepared according to the preparation method of Y14 or Y18 of example 1.

Each experimental group was administered intratumorally once, 250. mu.l/patient. Animals were euthanized at day 14 after drug administration, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 9.

TABLE 9

Group number	Research medicine	Ratio of measurement	Tumor weight (x + -s)	Tumor inhibition rate
					0	Physiological saline	-	1.73±0.26g	-
1	Mouse blood semifluid	-	1.28±0.33g	26％
					2	0.1% BCG vaccine	-	1.45±0.31g	16％
3	0.1% BCG/99.9% mouse blood semifluid	0.1/100	0.81±0.22g	53％
					4	Human recombinant interferon 50 ten thousand IU/ml	-	1.51±0.35g	13％
5	Human recombinant interferon/mouse blood (50 ten thousand IU/ml) semifluid	1/100	0.90±0.20g	48％

In the above table, the q-judged (q ═ 1.20>1.15) of the composition group showed a synergistic effect among the study groups 3, 2, and 1, and the tumor weight differences among the study groups 3 and 2 and 3 and 1 were all statistically significant (P ═ 0.0022<0.05, and P ═ 0.0166<0.05, respectively), so that the composition group showed a significant synergistic effect. Between study groups 5, 4 and 1, the q-judged (q ═ 1.33>1.15) of the composition group showed synergy, and the tumor weights between study groups 5 and 4 and between 5 and 1 were statistically significant (P ═ 0.004<0.05 and P ═ 0.0379<0.05, respectively), so that the composition group showed significant synergy.

The above results and more similar studies indicate that a semifluid composition comprising animal tissue cells of the invention and an anti-pathogenic biological product provides synergistic destruction of pathogenic tissue. This synergy is likely to be due to the synergy of the animal tissue cell-containing semifluid of the invention as an immunological component (antigen) or/and as a slow release carrier. The enlarged damage of pathogen-induced pathological tissues is beneficial to the generation of endogenous vaccines.

Example 3: study and optimization of specific immunogens in semi-fluids containing animal tissue cells

The immunogen comprising the semi-fluid of animal tissue cells is the basis for its use as an immunologically synergistic component in the compositions of the invention. This was investigated in the following experiments and the animal tissue cells were preferably selected on the basis thereof.

In one experiment, the experimental animals were CB6F1 mice, and the modeled cells were hepatoma H22 cells at 2X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Of the successfully modeled test animals, one part was used to prepare the tumor particle semifluid, and the other part (tumor mean volume 213 mm)³) The groups were randomly divided into 10 test groups (as shown in the table below, 1 negative control group (group 0) and 9 study groups were included). The negative control was physiological saline and 8 study drugs are shown in the table below. The study drug was prepared as follows:

the cells used are contained in blood or meat. The preparation of CB6F1 mouse concentrated blood and CB6F1 mouse concentrated blood semifluid was the same as in example 2. CB6F1 mouse muscle mass is about 400mm of a volume³The block-shaped mouse meat. The 4 mouse muscle particle semifluids and the CB6F1 mouse tumor tissue particle semifluids were prepared as follows: the meat mass of 4 mice and the tumor body of the liver cancer-bearing CB6F1 mouse are respectively stripped, and then the two mice are respectively placed in a stirrer to be crushed (the rotating speed is 1000-10000 r/min, the total time is 1-3 minutes), so that tissue particles which have the average cross section size of less than 1mm multiplied by 1mm and can be distinguished by naked eyes are respectively obtained (prepared by the preparation method of the preparation X5 in the example 1). The pig muscle particle semifluid was preparation X4 in example 1. Horse blood semifluid was preparation X24 in example 1.

Each experimental group was administered subcutaneously 1 time to the left axilla, 400. mu.l/mouse. Study group 3 mice muscle mass was surgically implanted subcutaneously in the left rib of mice. The drugs of the other experimental groups were injected subcutaneously by syringe into the left flank of CB6F1 mice. In this experiment, administration to study groups 1-4 and 8 can be considered a full-phase transplantation model or an autologous administration model, administration to study group 5 can be considered an allogeneic semi-phase administration model, administration to study group 6 can be considered an allogeneic administration model, and administration to study group 7 can be considered a xenogeneic administration model. Animals were measured for graft versus host disease score on day 14 post-dose, then euthanized, tumor weight determined after dissection, and tumor inhibition rate calculated from the negative control group, with the results shown in table 10.

Watch 10

It is generally believed that antigens in molecular form (e.g., whole tumor cell antigens, heterologous glycoprotein antigens, allogeneic cell antigens, subunit antigens) have distinct structural components (e.g., Pathogen-associated molecular patterns, PAMPs) that are distinct from those of normal bodies, while Pattern-recognition receptors (PRRs) that recognize these structural components (e.g., PAMPs) are present on cells of the natural immune system in the body, which PRRs are stimulated and initiate an adaptive immune response to attack the same or similar structural components (e.g., cross-antigen components in tumor cells). Thus, higher graft-versus-host responses mediated by heterologous or allogeneic grafts through their antigenic molecules (e.g., heterologous glycoproteins, allogeneic cells, etc.) can lead to higher graft-versus-tumor reactivity.

In the above table, the comparison between study groups 2 and 1 shows that the score for the resistance to host disease is not statistically significant (P ═ 0.3016>0.05), indicating that the immunogenicity of transplant rejection (against normal host cells and tumor cells) is comparable, whereas the difference in tumor weight (against tumor bodies in the host) is statistically significant (P ═ 0.0023< 0.05). Comparison between study groups 4 and 3 showed that the anti-host disease score was statistically significant (P0.0034 <0.05), indicating that the immunogenicity of graft rejection (against host cells) was significantly different, whereas the tumor weight difference (against host tumor bodies) was not statistically significant (P0.8408 > 0.05). According to these results, the expression of the cell-containing semifluid as a solid tumor vaccine antigen was not clearly directly correlated with its anti-host response. Thus, the antigenicity of the semifluid comprising cells of the invention, under the conditions disclosed herein, appears to be significantly different from that of the grafts of the prior art (usually liquid or semisolid), the latter often having an antitumor immunogenicity directly correlated with that of its graft rejection.

In the above table, the mouse muscle particle semifluid graft-versus-host disease scores (anti-host rates) were from large to small for study group 7, study group 6, study group 5, and study group 4, roughly reflecting the correlation of graft-versus-host disease with the foreign antigens (heterologous and xenogenic). For example, the anti-host disease score between study groups 7 and 4 was statistically significant (P ═ 0.0040< 0.05). However, the tumor weight difference between study groups 7 and 4 was not statistically significant (P-0.6085 > 0.05). It is generally believed that xenogeneic antigenicity is much higher than isogenic antigenicity in the immune response of conventional grafts. In the above table, the difference in tumor weight between study groups 9 (including horse blood) and 2 (including mouse blood) was not statistically significant (P ═ 0.7295>0.05), although the difference in graft versus host disease scores between them was indeed statistically significant (P ═ 0.0005<0.05), under the requirements of the present disclosure.

In addition, an initial study of the experiment was conducted using the muscle particle semifluid from the CB6F1 mouse as a negative control for the tumor particle semifluid from the CB6F1 mouse. However, in the above table, the difference in tumor weights between study 8 (drug tissue enriched with tumor cells) and study 4 (drug tissue without tumor cells) was not statistically significant (P ═ 0.8375> 0.05).

Similar results were obtained with the other preparations of example 1. For example, in one experiment, the test animals were BALB/c mice, and the modeled cells were breast cancer 4T1 cells at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. 14 days after modeling, successfully modeled Homophorus mice (tumor body mean volume 306 mm)³) The groups were randomized into 8 experimental groups (as shown in the table below, including 1 negative control group and 7 study groups). The negative control was physiological saline and the study drugs are shown in the table below and are all selected from the group consisting of realgarThe preparation of example 1. Each experimental group was administered subcutaneously 1 time to the left axilla at a dose of 400. mu.l/animal, euthanized 10 days after administration, tumor weight was determined after dissection, and tumor inhibition rate was calculated from the negative control group, and the results are shown in Table 11.

TABLE 11

Group number	Research medicine	Tissue of inclusion	Tumor inhibition rate
				0	Physiological saline	-	0
1	X10	Pig lung	23％
				2	X11	Pork liver	25％
3	X12	Pigskin	21％
				4	X20	Human placenta	36％
5	X21	Human umbilical cord	37％
				6	X22	Porcine muscle tissue	33％
7	X23	Pig blood	38％

The above results of this example further demonstrate that the semifluid comprising animal tissue cells in the compositions of the invention have immunogenicity against pathogenic tissues (e.g., tumor bodies) different from conventional grafts (typically fluid, semi-solid or solid), the antigenicity of the latter being generally associated with the anti-host antigenicity of its foreign molecules (e.g., xenogenic antigenic molecules, heterologous antigenic molecules, etc.). The semi-fluid of the invention comprising animal tissue cells, however, appears to have an anti-solid tumor immunogenicity comparable to that of highly damaged pathogen-induced diseased tissue (e.g., tumor body tissue).

According to the above studies and more similar studies, under the requirements and preferred conditions disclosed in the present invention, a semifluid comprising animal tissue cells shows immunological activity against pathogens and their resulting local lesions, and thus can be used as a synergistic immunological component in the composition of the present invention. The technical solution for the semi-fluid application comprising animal tissue cells in the composition of the invention is further preferably as follows: the semifluid of the invention is preferably one whose dominant antigenicity is against said pathogenic diseased tissue (e.g. tumor bodies) and not against host antigenicity, e.g. one whose tumor inhibition rate is ≥ against host, preferably ≥ 150% against host. Thus:

the source of essential animal tissue cells comprised by the semifluid of the invention does not include tumor cell body tissue, preferably selected from non-tumor body tissue;

the semi-fluid of the invention comprises essential animal tissue cells in the semi-fluid, preferably in a state remote from nature, or in a tissue remote from nature or severely damaged.

The essential animal tissue cells comprised by the semifluid according to the invention do not comprise cells with a strong graft versus host response (e.g. enriched with xenoglycoprotein antigens), preferably selected from cells with a low graft versus host response, such as one or more of the following groups: heterologous cells not enriched for heterologous glycoprotein antigens, allogeneic cells not enriched for heterologous gene antigens, more preferably selected from one or more of the following: allogeneic cells, allogeneic cells and autologous cells with the same ABO blood type or similar HLA.

Similarly, where the animal tissue cells are provided by the organ or tissue fraction in which they are present, the essential tissue or tissue fraction organ or tissue comprised by the semifluid of the invention does not include a tissue or tissue fraction organ or tissue in which the graft versus host response is strong (e.g., enriched with xenoglycoprotein antigens), and is preferably selected from a tissue or tissue fraction organ or tissue in which the graft versus host response is not strong, such as one or more of the following groups: a xenogeneic tissue or tissue component organ or tissue not enriched for xenogeneic glycoprotein antigens, a allogeneic tissue or tissue component organ or tissue not enriched for xenogeneic antigens not responsive to the host, more preferably selected from one or more of the following: allogenic allogeneic tissues or tissue components or organs or tissues, allogenic allogeneic tissues or tissue components or organs or tissues, autologous tissues or tissue components or organs or tissues with matched ABO blood types or similar HLA.

Under the conditions disclosed herein and preferred above, the composition of the present invention comprises more preferably one or more cells selected from the group consisting of tissue derived from semi-solid tissue and/or connective tissue. The semi-solid tissue is selected from one or more of the following: intestine, stomach, meat, pancreas, spleen, liver, lung, placenta, umbilical cord, preferably selected from one or more of: meat, spleen, liver, placenta, umbilical cord. The connective tissue is one or more selected from the group consisting of: blood, bone marrow, spinal cord, preferably blood.

Example 4: study of chemotherapeutic drug/cellular semifluid composition synergy protocol

The tissue-disrupting and/or sustained-release properties of the semi-fluid comprising animal tissue cells are the basis for its use as a tissue-disrupting and/or sustained-release synergistic component in the compositions of the invention. This was investigated in the following experiments.

In one experiment, the test animal was a nude mouse, and the modeled cell was sarcoma S180, at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Successfully modeled test animals (Holland sarcoma mice, average tumor volume 218mm³) The test groups were randomly divided into 7 test groups (as shown in the table below). The test groups were 2 series, the intratumoral injection series had 1 negative control group (group 01) and 4 study groups, and the subcutaneous injection series had 1 negative control group (group 02) and 1 study group. The negative controls were all saline and the study drugs were as shown in the table below, respectively: BALB/c mouse fresh blood, absolute ethanol (positive control for ablator), mouse blood semifluid (X16 of example 1), human placenta semifluid (X21 of example 1), which contains muscle cells or blood cells, respectively. Each experimental group was administered once, 200. mu.l/patient, and the administration mode (subcutaneous injection is subcutaneous injection in the left axilla) is shown in the following table.

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 nude mouse only has remnant or abnormal epithelium, which can not lead the T cell to be normally differentiated, lacks the auxiliary, inhibiting and killing functions of mature T cell and has low cell immunity. The animals were euthanized on day 7 after drug administration of the sarcoma nude mice, tumor weights were determined after dissection, and the tumor inhibition rates were calculated from the negative control group, and the results are shown in table 12.

TABLE 12

Group number	Research medicine	Mode of administration	Tumor weight (x + -s)	Tumor inhibition rate
					01	Physiological saline	Intratumoral injection	1.91±0.34g	-
1	Human placental semifluid	Intratumoral injection	0.97±0.21g	49％
					2	Mouse blood semifluid	Intratumoral injection	1.30±0.15g	32％
3	Blood of mouse	Intratumoral injection	1.70±0.22g	11％
					4	Anhydrous ethanol	Intratumoral injection	1.13±0.27g	41％
02	Physiological saline	Subcutaneous injection	1.99±0.21g	-
					5	Mouse blood semifluid	Subcutaneous injection	1.87±0.15g	5％

In the above table, the difference in tumor weight between study groups 5 and 02 was not statistically significant (P ═ 0.437> 0.05). This result and more similar studies indicate that the cell-containing semifluid of the composition of the invention shows immunosuppressive effects mainly as a thymus-dependent antigen in previous subcutaneous injections of tumor-bearing mice.

However, in the above table, the tumor weights were statistically significant between study groups 2 and 3 and between study groups 2 and 01 (P ═ 0.0043<0.05 and P ═ 0.0014<0.05, respectively). The tumor weights between study groups 2 and 4 and between study groups 1 and 4 were not statistically significant (P-0.2096 >0.05, P-0.2783 >0.05, respectively). This result and more similar studies suggest that the semifluid of the present invention can be used in immune synergy as a thymus-dependent antigen, and in chemotherapy synergy as a thymus-independent tissue-disrupting component. It is well known that destruction of tumor body tissue may give secondary release of tumor antigens (in situ) and produce a vaccine effect.

In one experiment, the test animal was a nude mouse, and the modeled cell was sarcoma S180, at 1X 10⁶Individual cells/animal right axillary subcutaneous transplantation tumor modeling. Test animals successfully modeled (Holland sarcoma mice, mean tumor volume 209mm³) The test groups were randomly divided into 10 test groups (1 negative control group and 9 study groups) and intratumorally administered to animals. The negative control was physiological saline and study drugs are shown in the table below. The study drug was prepared as follows:

the cells are contained in the blood. Blood semifluid is blood taken from other nude mice prepared according to the method of preparation X16 in example 1, 0.5% 5-Fu, 0.5% methylene blue, 10% arginine/10% glycine are aqueous solutions of chemotherapeutic agents, respectively, and other drugs are chemotherapeutic agent/autologous blood semifluid compositions of blood taken from other nude mice prepared according to the method of preparation Y6, Y8, Y9 in example 1, respectively. Each experimental group was administered intratumorally once, 200. mu.l/patient. Animals were euthanized at day 14 after drug administration, tumor weights were determined after dissection, and tumor inhibition rates were calculated from the negative control group, and the results are shown in table 13.

Watch 13

Group number	Research medicine	Tumor weight (x + -s)	Tumor inhibition rate
				0	Physiological saline	1.87±0.17g	-
1	Blood semifluid of nude mice	1.35±0.21g	28％
				2	0.5％5-Fu	1.27±0.18g	32％
3	0.5% 5-Fu/99.5% nude mouse blood semifluid	0.71±0.25g	62％
				4	1% 5-Fu/99% nude mouse blood semifluid	0.49±0.08g	74％
5	0.5% methylene blue	1.63±0.28g	13％
				6	0.5% methylene blue/99.5% nude mouse blood semifluid	0.92±0.21g	51％
7	1% methylene blue/99% nude mouse blood semifluid	0.65±0.07g	65％
				8	10% arginine/10% glycine	1.40±0.29g	25％
9	10% arginine/10% glycine/80% nude mouse blood semifluid	0.92±0.12g	51％

Mouse blood semifluid tumor inhibition rate: according to the related experiments, the tumor inhibition effect of the semifluid of diluted mouse blood is less than that of the semifluid of 100 percent of mouse blood

In the above table, the tumor inhibition rate of study group 4 was higher than that of study group 3, and that of study group 7 was higher than that of study group 6. Between study groups 3, 2 and 1, the q of the composition group (q ═ 1.22>1.15) showed synergy, and the tumor weight differences between study groups 3 and 2 and 3 and 1 were all statistically significant (P ═ 0.0012<0.05 and P ═ 0.0007<0.05, respectively), so the composition group showed significant synergy. Between study groups 6, 5 and 1, the q-judged (q ═ 1.38>1.15) of the composition group showed a synergistic effect, and the tumor weight differences between study groups 6 and 5 and between 6 and 1 were all statistically significant (P ═ 0.0006<0.05 and P ═ 0.0054<0.05, respectively), so that the composition group showed a significant synergistic effect. Between study groups 9, 8, 1, the q-judged (q >1.17>1.15) of the composition group showed synergy, and the tumor weight differences between study groups 9 and 8, 9 and 1 were all statistically significant (P ═ 0.0041<0.05, P ═ 0.0015<0.05, respectively), so the composition group showed significant synergy.

The above results and more similar studies indicate that a semi-fluid composition comprising animal tissue cells of the invention and an anti-pathogenic chemotherapeutic agent provides synergistic destruction of pathogenic tissue. This synergy is likely to be due to the animal tissue cell-containing semifluid of the invention acting synergistically as an immunological component (antigen), synergistically as a tissue destruction agent, or/and synergistically as a sustained release carrier. The enlarged damage of pathogen-induced pathological tissues is beneficial to the generation of endogenous vaccines.

According to the studies of the above examples and further similar studies, a semifluid containing animal tissue cells can be used as a synergistic component in the preparation of a medicament against pathogenic diseases. Specifically, the compound can be used as a vaccine antigen, a vaccine adjuvant or/and a carrier vaccine adjuvant to form a composition with other immune medicaments (such as immune biological products) for preparing immune medicaments (such as vaccines against pathogenic diseases), can also be used as an immune medicament and a tissue destruction component or/and a synergistic component of a carrier and other biological products for preparing medicaments against pathogenic diseases, and can also be used as a chemotherapeutic medicament to form a composition for preparing immune medicaments against pathogenic diseases (such as immune-chemotherapeutic compound medicaments against pathogenic diseases) for preparing medicaments against pathogenic diseases.

The requirements of the composition of the invention are: its composition and morphology is such that it can stimulate a more effective immune response than the active ingredient alone, and/or a more effective chemotherapeutic effect. Thus, the basic technical scheme of the composition of the invention is as follows:

the use of a semifluid comprising animal tissue cells as a synergistic component for an active ingredient against pathogenic diseases;

the above-mentioned semi-fluid containing animal tissue cells and active ingredients is preferably one or more selected from the group consisting of: a semi-fluid dope comprising the active ingredient and animal tissue cells, a coagulum comprising the active ingredient and animal tissue cells, a morselate of non-aqueous cement tissue comprising the active ingredient and comprising the cells, a morselate of a coagulum comprising the active ingredient and animal tissue cells, more preferably one or more selected from the group consisting of: a coagulum comprising the active ingredient and animal tissue cells, a morselized product of a non-aqueous liquid tissue comprising the active ingredient and the cells, a morselized product of a coagulum comprising the active ingredient and animal tissue cells;

the semi-fluid comprising animal tissue cells and an active ingredient is contained in the intratumoral and/or extratumoral topical administration of the vaccine;

the semi-fluid containing the animal tissue cells and the active ingredient is a semi-fluid implant, preferably a semi-fluid injection. The semi-fluid injection is an injection which can be directly administered by a conventional injection system in a semi-fluid manner, and the semi-solid implant is usually implanted by surgery or forms a semi-solid (e.g. gelated) nodule at the administration site after being administered by a conventional injection system in a fluid (liquid) manner.

Under more preferred conditions, the semi-fluid composition of the invention comprising animal tissue cells and an active ingredient against a pathogenic disease exhibits an immune synergy, a chemotherapeutic synergy and/or a carrier synergy. The synergistic conditions for the semi-fluid composition are: the ratio of the amount of the active ingredient to the amount of the composition (w/w or v/v) is (0.1-30)/100, and the ratio of the amount of the cell to the amount of the composition (v/v) is>22% (or cell concentration of>5.6×10⁹One/ml), preferably 33% to 86% (or a cell concentration of 8.4X 10)⁹one/ml-22X 10⁹One cell per ml) or 45% -86% (or the cell concentration is 11.5X 10)⁹one/ml-22X 10⁹Pieces/ml). In which the semi-fluid dope composition, the ratio of the amount of the cells to the amount of the composition (v/v) is 70% or more (or the cell concentration is 17.9X 10 or more)⁹One/ml), preferably 70% to 86% (or a cell concentration of 17.9X 10)⁹one/ml-22X 10⁹Pieces/ml).

More preferably, the concentration of the active ingredient is greater than or equal to the concentration at which it acts when administered topically alone, wherein:

the concentration of the biological product is greater than or equal to the concentration at which it acts when administered topically alone, for example: the content of the tumor antigen is more than 10⁵Per mlmm³Preferably 10⁵～10⁹Per mlmm³Tumor antigens contained in individual tumor cells, the concentration of the microbial antigens being>0.1 percent, the content of the immunoregulation antibody medicine is more than or equal to 0.1 percent, preferably 0.25 to 5 percent, and the like; or/and

the concentration of the chemotherapeutic agent is greater than or equal to the concentration at which it acts when administered topically alone, for example: the concentration of the cytotoxic drug is more than 50% of the saturation concentration of the cytotoxic drug, and preferably 50% -500% of the saturation concentration of the cytotoxic drug; the concentration of the conventional ineffective compound is > 0.25%, preferably 0.35-30% (for example, the local administration concentration of the amino acid nutrient is more than 5%, preferably 5-30%, the local administration concentration of the ineffective aromatic compound is more than 0.25%, preferably 0.35-10%, and the local administration concentration of the plant or fungus active ingredient is more than 0.25%, preferably 0.75-15%).

Example 6: further uses of the composition

According to the above results, more applications of the semi-fluid containing animal tissue cells of the present invention as a synergistic component of active ingredients for drugs against pathogenic diseases were investigated.

1. Tumor-bearing nude mouse experiment

In the following experimental series comprising 9 experiments, the test animals were nude mice, the modeled cells were 9 human solid tumor cells as shown below, respectively, and the conventional transplantation tumor modeling was performed subcutaneously in the right axilla of the animals, respectively. The successfully modeled test animals in each series of experiments were randomly divided into 5 experimental groups, 1 negative control group and 4 study groups (A, B, C, D groups). The negative control was physiological saline and the A, B, C, D study drugs were preparations Y1, Y4, Y6, Y9 in example 1, respectively. Each experimental group was administered intratumorally 1 time, 200. mu.l/patient. Animals were euthanized on day 7 after drug administration, tumor weights were determined after dissection, and tumor inhibition rates were calculated from negative control groups of each series, and the results of 9 series of experiments are shown below.

1) Application of the compound in treating breast tumor

Successfully modeled nude mice bearing human breast cancer cells (MDA-MB231) (tumor body average volume 203 mm)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. B, C, D groups have average tumor inhibition rates of 85%, 72%, 68% and 83%, respectively, which meet the standard of effective anti-solid tumor (tumor inhibition rate is more than or equal to 40%).

2) Application of the same in lung cancer treatment

Successfully modeled nude mouse with human lung cancer cells (A549) (average tumor volume 207 mm)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. The average tumor inhibition rates of B, C, D groups were 75%, 63%, 61%, and 79%, respectively, which all meet the standard of effective anti-solid tumor (tumor inhibition rate is more than or equal to 40%).

3) Application of the compound in thyroid cancer treatment

Successfully modeled nude mice bearing human thyroid cancer cells (SW579) (average tumor volume 204 mm)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. The mean tumor inhibition rates of B, C, D groups were 79%, 71%, 66% and 82%, respectively, which all met the standard of effective anti-solid tumor (tumor inhibition rate ≥ 40%).

4) Use in the treatment of prostate cancer

Successfully modeled human prostate cancer cell (LNCaP/AR) bearing nude mice (tumor body average volume 208 mm)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. The mean tumor inhibition rates of B, C, D groups were 85%, 76%, 71%, and 83%, respectively, which all met the standard of effective anti-solid tumor (tumor inhibition rate ≥ 40%).

5) Application of the compound in liver cancer treatment

Successfully modeled nude mice (tumor mean volume 213 mm) with human hepatoma cells (HepG2)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. The mean tumor inhibition rates of B, C, D groups were 83% and 71% respectively65 percent and 81 percent of the total percent of.

6) Use in the treatment of head and neck cancer

Successfully modeled nude mice bearing human head and neck cancer cells (Fmu da) (tumor body average volume 213 mm)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. The mean tumor inhibition rates of B, C, D groups were 86%, 73%, 70% and 87%, respectively, and all met the standard of effective anti-solid tumor (tumor inhibition rate is more than or equal to 40%).

7) Application of the compound in treatment of nasopharyngeal carcinoma

Successfully modeled nude mice with human nasopharyngeal carcinoma cells (CNE1) (tumor mean volume 206 mm)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. The mean tumor inhibition rates of B, C, D groups were 91%, 81%, 79% and 82%, respectively, which all meet the standard of effective anti-solid tumor (tumor inhibition rate is more than or equal to 40%).

8) Application of the compound in treatment of gastric cancer

Successfully modeled nude mouse with human gastric cancer cell (BGC823) (tumor body average volume 204 mm)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. The average tumor inhibition rates of B, C, D groups were 71%, 66%, 62% and 73%, respectively, which all meet the standard of effective anti-solid tumor (tumor inhibition rate is more than or equal to 40%).

9) Use in the treatment of ovarian cancer

Successfully modeled nude mice with human ovarian cancer cells (PA1) (tumor body average volume 201 mm)³) The groups were randomized into a negative control group and 4 study groups (A, B, C, D). A. The mean tumor inhibition rates of B, C, D groups were 86%, 72%, 71%, and 85%, respectively, which all meet the standard of effective anti-solid tumor (tumor inhibition rate ≥ 40%).

Similar results were obtained using the other chemotherapeutic drug containing composition preparations of example 1.

2. Tumor bearing mouse experiment

In the following experimental series, which included 5 experiments, the experimental animals and the modeled cells were each as follows. Modeling cells conventional transplantation tumor modeling was performed subcutaneously in the right axilla of animals, respectively. The successfully modeled test animals in each series of experiments were randomly divided into 4 experimental groups, 1 negative control group and 3 study groups (A, B, C groups). The negative control was physiological saline and the A, B, C study drugs were preparations Y10, Y22, Y24 in example 1, respectively. Each experimental group was administered intratumorally 1 time, 250. mu.l/patient. Animals were euthanized at day 14 after drug administration, tumor weights were determined after dissection, and tumor inhibition rates were calculated from each series of negative control groups, and the experimental results are shown below.

1) Application of the compound in liver cancer treatment

In one experiment, the test animals were BALB/c mice, and the modeled cells were hepatoma H22 cells (2X 10)⁶One cell/one), graft tumor modeling was performed subcutaneously in the right axilla of the animal. Tumor-bearing animals successfully modeled (average tumor volume 215 mm)³) The groups were randomized into 4 experimental groups (1 negative control group and 3 study groups). A. The mean tumor inhibition rates of B, C groups were 43%, 47%, and 51%, respectively.

2) Application of the compound in treatment of intestinal cancer

In one experiment, the experimental animals were BALB/c mice and the modeled cells were colon cancer CT26 cells (1X 10)⁶One cell/one), graft tumor modeling was performed subcutaneously in the right axilla of the animal. Tumor-bearing animals successfully modeled (average tumor volume 206 mm)³) The groups were randomized into 4 experimental groups (1 negative control group and 3 study groups). A. The mean tumor inhibition rates of B, C groups were 41%, 46%, and 43%, respectively.

3) Application of the compound in breast cancer treatment

In one experiment, the experimental animals were BALB/c mice and the modeled cells were breast cancer 4T1 cells (1X 10)⁶One cell/one), graft tumor modeling was performed subcutaneously in the right axilla of the animal. Tumor-bearing animals successfully modeled (average tumor volume 216 mm)³) The groups were randomized into 4 experimental groups (1 negative control group and 3 study groups). A. The mean tumor inhibition rates of B, C groups were 44%, 49%, and 47%, respectively.

4) Application of the compound in treating malignant melanoma

In a testIn the experimental animals, C57BL/6 mice were used, and melanoma B16 cells (1X 10) were used as model cells⁶One cell/one), graft tumor modeling was performed subcutaneously in the right axilla of the animal. Tumor-bearing animals successfully modeled (average tumor volume 218 mm)³) The groups were randomized into 4 experimental groups (1 negative control group and 3 study groups). A. The mean tumor inhibition rates of B, C groups were 41%, 49%, and 51%, respectively.

5) Application of the same in lung cancer treatment

In one experiment, the test animals were C57BL/6 mice, and the modeled cells were lung cancer (LLC cells) (1X 10)⁶One cell/one), graft tumor modeling was performed subcutaneously in the right axilla of the animal. Tumor-bearing animals successfully modeled (average tumor volume 202 mm)³) The groups were randomized into 4 experimental groups (1 negative control group and 3 study groups). A. The mean tumor inhibition rates of B, C groups were 38%, 42%, and 45%, respectively.

Similar results as above were obtained using the other composition preparations of example 1.

Based on the above results and preliminary analysis of the immune synergy, tissue destruction synergy, and/or sustained release synergy strategies leading to these results, the semi-fluid comprising animal tissue cells in the compositions of the present invention, as a synergistic component, exhibits properties of targeting pathogen-causing diseased tissue (e.g., tumor mass) under the presently disclosed technical solutions. For example, the use of a semifluid comprising animal tissue cells in the compositions of the invention as an antigen providing immune synergy is distinguished from prior art antigens of molecular morphology (e.g., pathogen antigens, conventional graft antigens, and single cell antigens) that have been shown to have specific immunogens against it that resemble highly damaged pathogen-causing diseased tissue (e.g., tumor bodies). It is well known that the main characteristic of chemotherapeutic drugs is their composition, while the main characteristic of antigens is their specific immunogen. The immunogen is likely because it forms a pathogen-induced diseased tissue (e.g., tumor) nodule, but is semi-fluid and highly damaging (cells in the semi-fluid are remote from their state in the native organ or tissue) but is more readily recognized by the immune system than the pathogen-induced diseased tissue (e.g., tumor), thereby eliciting an effective immune response against the pathogen-induced diseased tissue (e.g., tumor) it mimics.

According to the above studies and more similar studies, the composition of the present invention can be applied to a wide spectrum of pathogen diseases, such as tumors, microbial infections. Wherein the tumor is preferably a solid tumor, wherein the solid tumor is one or more selected from the group consisting of: 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. Wherein the microbial infection is selected from one or more of the following groups: HIV, hepatitis B, hepatitis C, leprosy, chronic mucocutaneous candidiasis.

The present disclosure includes the following items:

item 1, use of a semifluid comprising animal tissue cells as a synergistic component of an active ingredient against a pathogenic disease for the preparation of a topical medicament for the treatment or inhibition of a pathogenic disease.

Item 2, a topical pharmaceutical composition for treating or inhibiting a pathogenic disease comprising an active ingredient against the pathogenic disease and a semi-fluid comprising animal tissue cells, wherein the active ingredient is dispersed in the semi-fluid.

Item 3, a method of treating or inhibiting a pathogenic disease comprising administering topically to an individual in need thereof by implantation, preferably by injection, a topical pharmaceutical composition comprising an anti-pathogenic disease active ingredient and a semifluid comprising animal tissue cells.

Item 4, use, pharmaceutical composition or method according to one of items 1 to 3, wherein the composition has a composition and morphology such that it forms a semifluid nodule at the site of administration.

Item 5, use, pharmaceutical composition or method according to one of items 1 to 4, wherein the tissue cells comprised by the semi-fluid are one or more selected from the group consisting of cells comprised by a semi-solid tissue derived from an animal connective tissue and/or other than a connective tissue.

Item 6, use, pharmaceutical composition or method according to one of items 1 to 5, wherein the tissue cells comprised by the semi-fluid are one or more selected from the group consisting of: muscle cells, blood cells, immune cells, stem cells such as mesenchymal stem cells, hematopoietic stem cells, wherein said immune cells are selected from one or more of the following cells and derivatives thereof: dendritic cells, macrophages, leukocytes and derivatives thereof, wherein the leukocytes are selected from one or more of: granulocytes, monocytes, lymphocytes, wherein said lymphocytes are selected from one or more of the following: t cells, B cells, naked cells.

Item 7, use, pharmaceutical composition or method according to one of items 1 to 6, wherein the composition comprises the tissue cells in a quantitative ratio (v/v) of>22% (or cell concentration of>5.6×10⁹Individual cells/ml), preferably 33% to 86% (or a cell concentration of 8.4X 10)⁹-22×10⁹Individual cell/ml) or 55% -86% (or cell concentration is 14.0X 10)⁹-22×10⁹Individual cells/ml).

Item 8, the use, pharmaceutical composition or method according to one of items 1 to 7, wherein the composition comprises the active ingredients in a quantitative ratio (w/w or v/v) of (0.1 to 30)/100, and wherein the active ingredients are selected from one or more of biologies against pathogenic diseases or/and chemotherapeutic drugs.

Item 9, the use, the pharmaceutical composition or the method according to one of items 1 to 8, wherein the composition comprises tissue cells in a highly deviated native state, preferably a severely damaged state, wherein the highly deviated native state comprises viscosification; the severe injury is selected from one or more injuries including: solidification, mechanical disruption, ultrasonic damage, thermal damage, freeze-thaw damage, irradiation damage, chemical damage.

Item 10, use, pharmaceutical composition or method according to one of items 1 to 9, wherein the composition is one or more selected from the group consisting of: a semi-fluid dope comprising the active ingredient and tissue cells, a semi-fluid coagulate comprising the active ingredient and tissue cells, a semi-fluid homogenate comprising the active ingredient and tissue cells, a coagulate comprising the active ingredient and tissue cells, preferably in the form of: a semi-fluid coagulum comprising the active ingredient and tissue cells, a disrupted semi-fluid product comprising the active ingredient and tissue cells, a disrupted product of a coagulum comprising the active ingredient and tissue cells.

Item 11, use, pharmaceutical composition or method according to one of items 1 to 10, wherein the animal tissue cells are selected from one or more of the following group: natural cells in the connective tissue and/or semi-solid tissue, natural cells in the connective tissue-enriched fraction, natural cell preparations and/or engineered cells derived from the connective tissue and/or semi-solid tissue.

Item 12, use, pharmaceutical composition or method according to one of items 8 to 11, wherein the composition is selected from one or more of the following group: a semi-fluid coagulum comprising the active ingredient and the connective tissue, a semi-fluid mixture comprising the active ingredient and the connective tissue, a semi-fluid disruption comprising the active ingredient and the connective tissue and/or semi-solid tissue, a semi-fluid coagulum comprising the active ingredient and the connective tissue-enriched component and/or semi-solid tissue-enriched component, a semi-fluid disruption comprising the active ingredient and the connective tissue-enriched component and/or semi-solid tissue-enriched component, a semi-fluid dope comprising the natural cell preparation and/or engineered cell, a semi-fluid coagulation comprising the natural cell preparation and/or engineered cell, a semi-fluid disruption comprising the natural cell preparation and/or engineered cell.

Item 13, a use, a pharmaceutical composition, or a method according to one of items 9 to 12, wherein the connective tissue is selected from the group comprising one or more of: blood, bone marrow, spinal cord, preferably blood.

Item 14, the use, the pharmaceutical composition or the method according to one of items 9 to 12, wherein the semi-solid tissue is comprised by an organ selected from the group consisting of one or more of: intestine, stomach, meat, pancreas, spleen, liver, lung, cartilage, joints, skin, placenta, umbilical cord, preferably tissue comprised by one or more organs selected from the group consisting of: meat, spleen, liver, placenta, umbilical cord.

Item 15, use, pharmaceutical composition or method according to one of items 1 to 14, wherein the animal tissue cells are selected from cells with a mild graft-versus-host response, preferably selected from one or more of the following: xenogeneic cells, allogeneic cells, allogeneic cells and autologous cells, wherein the xenogeneic cells are matched with ABO blood types or similar to HLA, and the allogeneic cells and the autologous cells are not rich in xenogeneic glycoprotein antigens.

Item 16, the use, pharmaceutical composition or method according to one of items 1 to 15, wherein the animal tissue cells are one or more selected from the group consisting of: allogeneic cells, autologous cells.

Item 17, the use, pharmaceutical composition or method according to one of items 1 to 16, wherein the animal tissue cells are autologous cells.

Item 18, a pharmaceutical composition or a method for use according to one of items 5 to 17, wherein the biological product is selected from one or more of the following group: pathogen antigens, immunomodulatory antibodies, cytokines, and the amount ratio (w/w or v/v) of said biological product in said composition is (0.1-30)/100.

Item 19, the use, pharmaceutical composition or method according to item 18, wherein the pathogen antigen is selected from one or more of the following group: microbial antigens, tumor antigens.

Item 20, the pharmaceutical composition or the method for use according to item 19, wherein the microbial antigen is an antigen selected from one or more of the following groups of microorganisms: bacteria, such as Streptococcus pyogenes, Serratia marcescens, Bacillus Calmette-Guerin, Clostridium tetani, Clostridium butyricum, Lactobacillus acidophilus, and Bifidobacterium; 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.

Item 21, the pharmaceutical composition for use according to item 19, or the method, wherein the tumor antigen is one or more selected from the group consisting of: 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.

Item 22, the use, pharmaceutical composition or method according to item 18, wherein the immunomodulatory antibody is selected from one or more of the group consisting of: 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 solid tumor microenvironment, such as anti-TGF-p 1 antibodies.

Item 23, the use, pharmaceutical composition or method according to item 18, wherein the cytokine is selected from one or more of: tumor necrosis factor, interferon, interleukin.

Item 24, use, pharmaceutical composition or method according to one of items 5 to 23, wherein the chemotherapeutic drug is selected from a cytotoxic drug and/or a conventional non-effective but topically effective compound, and wherein the amount ratio (w/w or v/v) of the chemotherapeutic drug and the semi-fluid is (0.1-30)/100.

Item 25, the use, pharmaceutical composition or method according to item 24, wherein the cytotoxic drug is selected from one or more of the following: 5-fluorouracil, gemcitabine, epirubicin, antibiotics against pathogen diseases, teniposide, metal platinum complex, paclitaxel.

Item 26, the use, pharmaceutical composition or method according to item 24, wherein the conventionally ineffective but topically effective compound is selected from the group consisting of one or more of: amino acid nutrients, conventional ineffective local effective aromatic compounds and non-animal bioactive components.

Item 27, the use, pharmaceutical composition or method according to item 26, wherein the conventionally ineffective but topically effective compound is selected from the group consisting of one or more of: amino acid nutrients such as arginine, lysine, glycine, cysteine, glutamic acid, or salts thereof, or oligopeptides comprising the same; conventional ineffective topically effective aromatic compounds such as methylene blue, acetylsalicylic acid, quinine monohydrochloride, quinine dihydrochloride; non-animal bioactive components such as algal polysaccharides, medicinal plant polysaccharides, fungal polysaccharides, artemisinin.

Item 28, the use, the pharmaceutical composition or the method according to one of items 1 to 27, wherein the pathogenic disease is selected from one or more of the following group: tumors, microbial infections.

Item 29, the use, the pharmaceutical composition or the method according to item 28, wherein the tumor is preferably a solid tumor, and wherein the solid tumor is tumor volume>85mm³Preferably ≥ 200mm³More preferably not less than 300mm³The solid tumor of (3).

Item 30, the use, pharmaceutical composition or method according to item 28 or 29, wherein the solid tumor is one or more selected from the group consisting of: 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.

Item 31, the use, pharmaceutical composition or method according to item 28, wherein the microbial infection is selected from one or more of the following group: HIV, hepatitis B, hepatitis C, leprosy, chronic mucocutaneous candidiasis.

Item 32, a method according to one of items 3-31, wherein the method further comprises treatment with one or more of: interventional therapy, chemotherapy, other immunotherapy, photodynamic therapy, sonodynamic therapy, surgical intervention.

Item 33, a method for preparing a pharmaceutical composition for treating or inhibiting a pathogenic disease, the pharmaceutical composition comprising a semi-fluid of animal tissue cells and an active ingredient, the method comprising the steps of:

a. providing a preparation comprising said cells, which preparation may be one or more of the following groups: natural cell preparations, engineered cells, connective tissue comprising natural cells, organ tissue comprising natural cells, enriched fractions of natural cells;

b. providing the active ingredient;

Item 34, the method of item 33, wherein the semifluidizing is selected from one or more of: semi-fluid thickening of a liquid, semi-fluid solidification of a liquid, disruption of a non-liquid or solidified substance.

Item 35, the method of item 33, wherein the severe injury treatment is selected from one or more of: viscosity thickening treatment, mechanical crushing, solidification treatment, heat treatment, freeze thawing treatment, irradiation treatment and chemical treatment.

Item 36, a pharmaceutical composition prepared according to the method of one of items 33-35.

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

1. Use of a semifluid comprising animal tissue cells as a synergistic component of an active ingredient against a pathogenic disease in the manufacture of a topical medicament for the treatment or inhibition of a pathogenic disease.

2. A topical pharmaceutical composition for treating or inhibiting a pathogenic disease comprising an active ingredient against the pathogenic disease and a semi-fluid comprising animal tissue cells, wherein the active ingredient is dispersed in the semi-fluid.

3. The use, pharmaceutical composition or method according to claim 1 or 2, wherein the composition is of a composition and morphology such that it forms a semifluid nodule at the site of administration.

4. Use, pharmaceutical composition or method according to one of claims 1 to 3, wherein the tissue cells comprised by the semi-fluid are one or more selected from the group consisting of cells comprised by animal connective tissue and/or semi-solid tissue other than connective tissue.

5. The use, pharmaceutical composition or method according to any one of claims 1 to 4, wherein the tissue cells comprised by the semi-fluid are selected from one or more of the following cells and derivatives thereof: muscle cells, blood cells, immune cells, stem cells such as mesenchymal stem cells, hematopoietic stem cells, wherein said immune cells are selected from one or more of the following cells and derivatives thereof: dendritic cells, macrophages, leukocytes and derivatives thereof, wherein the leukocytes are selected from one or more of: granulocytes, monocytes, lymphocytes, wherein said lymphocytes are selected from one or more of the following: t cells, B cells, naked cells.

6. Use, pharmaceutical composition or method according to one of claims 1 to 5, wherein the tissue cells comprised by the composition are in a highly deviant, preferably severely damaged state, wherein the highly deviant state comprises viscosification; the severe injury is selected from one or more injuries including: solidification, mechanical disruption, ultrasonic damage, thermal damage, freeze-thaw damage, irradiation damage, chemical damage.

7. A method of preparing a pharmaceutical composition for treating or inhibiting a pathogenic disease, the pharmaceutical composition comprising a semi-fluid of animal tissue cells and an active ingredient, the method comprising the steps of:

b. providing the active ingredient;

8. The method according to claim 7, wherein the semifluidizing is selected from one or more of: semi-fluid thickening of a liquid, semi-fluid solidification of a liquid, disruption of a non-liquid or solidified substance.

9. The method according to claim 7, wherein the severe injury treatment is selected from one or more of the following: viscosity thickening treatment, mechanical crushing, solidification treatment, heat treatment, freeze thawing treatment, irradiation treatment and chemical treatment.

10. A pharmaceutical composition prepared according to the process of any one of claims 7-9.