CN110870912A

CN110870912A - Application of methylene blue dye as vaccine adjuvant, vaccine containing adjuvant and application of vaccine

Info

Publication number: CN110870912A
Application number: CN201811008795.5A
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: 2018-08-31
Filing date: 2018-08-31
Publication date: 2020-03-10

Abstract

The invention relates to application of methylene blue dye as a vaccine adjuvant, a vaccine containing the methylene blue dye adjuvant, and a method for preventing and treating diseases related to local lesions, in particular solid tumors by using the vaccine.

Description

Application of methylene blue dye as vaccine adjuvant, vaccine containing adjuvant and application of vaccine

Technical Field

Background

The treatment of localized lesions, particularly intractable localized lesion diseases (e.g., solid tumors, intractable microbial infections, intractable skin diseases, etc.), has been a difficult scientific problem. Due to the support of a great deal of research work, solid tumors are often used as research models for locally diseased diseases, especially refractory locally diseased diseases. The solid tumor is a tumor disease with tumor symptoms, the tumor body is a characteristic pathological change tissue containing tumor cells, the tumor cells are characteristic pathological change cells of tumor origin, and the tumor-related molecules are molecular level substances which are related to the tumor origin and have certain specificity. In the case of pancreatic cancer tumor bodies, pancreatic cancer cells account for only about 30% by volume of the tumor bodies. It can be seen that in addition to tumor cells and tumor-associated molecules therein, there are often still a greater number of other components (sometimes also referred to as the microenvironment of the tumor cells) present in tumor body tissues, including other various cells, various intercellular substances, various ducts, etc.

Drug therapy is a major treatment for solid tumors, especially advanced malignant solid tumors. Antineoplastic drugs can be classified into tumor cell-targeted drugs (commonly referred to as cytotoxic drugs), tumor tissue-targeted drugs (commonly referred to as topical drugs), and (tumor-associated) molecular-targeted drugs according to different action targets. In the last half century since the 50 s of the 20 th century, the mainstream of development of antitumor drugs is a cytotoxic drug (cancer cell proliferation inhibitor). Until today, such drugs still hold an important position. In the 20 th century and the 80 th century, Japanese scientists introduced anhydrous ethanol into hepatoma bodies percutaneously to cause protein denaturation and cell dehydration, so that the tumor bodies are subjected to coagulation necrosis and chemical ablation. Since then, more effective chemical ablative agents (high acid, base concentrations) were developed by multi-national researchers. The target area of the small molecule targeting drug is mainly the following tumor-related molecules: membrane receptors, components of cell signal transduction pathways, cell cycle regulatory proteins, and important proteins or factors involved in angiogenesis, and the like.

Immunotherapy is in the spotlight. Monoclonal antibodies are primarily therapeutic by targeting the following tumor-associated molecules: certain key molecules of tumor survival (e.g., CD20, VEGF/VEGFR2, HER2, EGFR, CD19, CD25, SLAMF7, CD38, etc.), tumor immunity-inherent and adaptation-related molecules (e.g., inhibition of certain immune cytokines), and in particular tumor immune suppression/activation modulating molecules (e.g., targeted immune suppression molecules such as CTLA-4, PD-1, and PD-L1, targeted immune activation molecules such as CD137/OX40, etc.). Despite their limited indications and efficacy, immunotherapy is unprecedented in expectation.

Anti-tumor vaccines are another hot direction. It uses tumor cell containing tumor antigen (tumor specific antigen or tumor related antigen), ectosome, polypeptide and nucleic acid sequence to induce patient self-immune response, and aims at overcoming immunosuppression state to inhibit tumor growth and even eliminate tumor. However, tumor antigens are often less antigenic and the addition of vaccine adjuvants seems to be a good strategy. However, in the prior art, the number of alternative vaccine adjuvants is small, and the development of more vaccines is limited. Thus, there is still a need to develop new anti-tumor vaccine adjuvants and new vaccines to meet various clinical needs that the prior art has not yet met. In fact, there is an urgent need for the prevention and treatment of other localized conditions, particularly intractable localized conditions.

Disclosure of Invention

The invention aims to provide application of methylene blue dye as a vaccine adjuvant, a vaccine containing the methylene blue dye as the adjuvant, and a method and application for treating solid tumors by using the vaccine.

According to one aspect of the present disclosure, there is provided the use of a methylene blue-based dye as a vaccine adjuvant. According to one embodiment, the vaccine further comprises one or more antigens and a pharmaceutically acceptable liquid carrier.

According to another aspect of the present disclosure, there is provided a vaccine comprising a methylene blue-based dye as an adjuvant, one or more antigens, and a pharmaceutically acceptable liquid carrier.

According to a further aspect of the present disclosure there is provided a method of preventing and treating solid tumors comprising topically administering to an individual in need thereof a therapeutically effective amount of a vaccine according to the present disclosure.

According to one embodiment, the methylene blue type dye includes the following compounds and derivatives thereof: methylene blue, patent blue, isothio blue, preferably methylene blue.

According to one embodiment, the concentration (w/v) of the methylene blue stain in the vaccine is 2% or less, preferably 0.3 to 2%, more preferably 0.5 to 1.5%.

Compared with the application of methylene blue dye in the prior art, the technical scheme of the application of the methylene blue dye has the following advantages: it can be used as adjuvant to participate in specific immunity; compared with the existing vaccine adjuvant, the vaccine adjuvant has the following advantages: the immune effect can be better enhanced under the animal experiment condition.

The vaccine comprising the methylene blue dye adjuvant according to the invention has the following advantages compared with other vaccines of the prior art: shows faster and higher curative effect.

Embodiments according to the present invention have the following advantages over the prior art for the treatment of localized disease conditions: compared with the existing cytotoxic drugs, the compound has almost non-toxic systemic safety and obviously higher local lesion curative effect; compared with the existing molecular targeted drugs, the compound has less rigorous screening of indications and great potential for rapidly growing tumor bodies, large tumor bodies and blood-poor tumor donors; compared with the existing chemical ablation agent, the chemical ablation agent has higher specificity, namely, the chemical ablation agent has higher effectiveness on local lesion and obviously lower local irritation on non-lesion tissues, so that the chemical ablation agent has larger intervention adaptation range and higher application volume; compared with the existing hardening agent, the compound has higher curative effect and safety. The application and the vaccine of the invention are not affected by the drug resistance problem of the existing cytotoxic drugs and the existing molecular targeted drugs. In addition, the application and the vaccine are convenient to prepare and low in cost, and are particularly beneficial to leading the vast population who is difficult to bear high expense to enjoy safe and effective treatment.

Detailed Description

The inventor of the invention finds that the methylene blue dye can be used as an adjuvant in a tumor-bearing animal experiment and applied to a vaccine for treating solid tumors, and then further prepares a vaccine containing the methylene blue dye adjuvant.

In the context of the present invention, the term "vaccine" refers to any drug capable of specifically inducing an immune response in a body against a target antigen, thereby effectively preventing or treating a disease associated with the target antigen. The term "vaccine adjuvant" (adjuvant for short) refers to a substance that enhances the specific immune function of a vaccine. Vaccine adjuvants differ from immunopotentiators in the general sense that they tend to enhance the non-specific immune function of the body by absorption.

Within the scope of the present invention, the term "therapeutically effective amount" refers to an amount of a drug that is used to treat a disease (e.g., a tumor) and achieve an effective effect (e.g., reduce or/and alleviate symptoms of the disease).

In the present disclosure, the term "methylene blue-based dye" is used to refer to methylene blue and its analogs, especially the in vivo stain analogs. The term "analog" refers to a substance that is not identical to, but structurally or/and qualitatively similar to the specified compound, and includes, for example, complexes, derivatives, hydrates, salts (including acid or base compounds) and the like of the specified compound. The term "derivative" refers to a more complex compound resulting from the replacement of an atom or group of atoms contained in a given compound with another atom or group of atoms. In the prior art, methylene blue dyes have various applications in the preparation of drugs for treating local pathological diseases, for example, they can be used as antidotes, chemical ablation agents and the like. As an antidote, it is administered intravenously at very low concentrations (less than 0.05%). As a chemoablative agent, it is administered topically at high concentrations (greater than 2%). In the examples of the present invention, the inventors of the present invention found that when the local administration concentration (w/v) of methylene blue is in a certain interval (e.g. 0.3% -2%), it significantly enhances the effect of the vaccine on inhibiting the growth of tumor tissue. This local administration concentration may be that critical concentration that would allow the methylene blue-like dye to be converted from a chemoablative agent to an adjuvant for the destruction of tumor tissue by the vaccine. According to the application of the invention, the vaccine should specify the local administration concentration of the methylene blue dye adjuvant in the instruction of using the vaccine so as to ensure that the vaccine is used as an adjuvant instead of a chemical ablation agent so as to avoid risks.

According to one embodiment, the methylene blue-based dye may include, for example, the following compounds and derivatives thereof: methylene blue, patent blue, isothio blue, preferably methylene blue.

In the present disclosure, the antigen comprised in the vaccine is selected from antigens directed against solid tumors. Such solid tumors include, for example, secretory gland tumors and skin tumors.

Within the scope of the present invention, the term "secretory gland tumor" refers to a malignant or non-malignant tumor in a secretory gland or a tissue or organ comprising a secretory gland; the term "secretory gland" refers to a structure composed of gland cells or gland cell groups that performs a secretory function (secretion), and includes exocrine glands and endocrine glands. Exocrine glands are ducted secretory glands including pancreas (excluding islets of langerhans), small intestine, sweat glands, sebaceous glands, large intestine, apocrine glands, parotid gland, submandibular glands, mammary glands, stomach glands, liver, etc. Endocrine glands are some of the human body's secretory glands without efferent ducts, including thyroid, parathyroid, adrenal gland, pituitary, pineal, pancreatic islet, thymus, gonad, etc. Secretory adenocarcinomas include, for example, breast cancer, liver cancer, colon cancer, pancreatic cancer, thyroid cancer, prostate cancer, lung cancer, head and neck cancer, nasopharyngeal cancer, and are typically represented by breast cancer, liver cancer, colon cancer.

Within the scope of the present invention, the term "skin tumor" refers to a malignant or non-malignant tumor in a skin tissue or organ, which is typically represented by melanoma.

In the present disclosure, the vaccine may further comprise amino acid nutrients as an additional vaccine adjuvant to enhance the efficacy of the vaccine.

In the present disclosure, the term "amino acid nutrient" refers to an amino acid compound with nutrition and health effects, preferably selected from amino acids, amino acid polymers and amino acid derivatives with nutrition and health effects, more preferably selected from amino acid nutriceuticals and amino acid adjuvants with nutrition and health effects, which are carried in the chinese, us or european official pharmacopoeia or guidelines. In the context of the present invention, the term "nutraceutical effect" is used to refer to an in vivo effect resulting from the action of one or more of the following organisms: providing energy, participating in the synthesis of bioactive substances (e.g., proteins), participating in part of the metabolism, maintaining the intestinal micro-ecological balance of animals, and participating in other physiological regulation beneficial to the health of the body (e.g., regulating protein synthesis, regulating immune response).

In the scope of the present invention, the amino acid polymer, and the amino acid derivative as the amino acid nutrient are preferably amino acids selected from the group consisting of: proteinogenic amino acids and nonproteinogenic amino acids.

In the context of the present invention, the term "proteinogenic amino acids" refers to the main amino acids that make up a protein; the term "non-proteinogenic amino acids" refers to amino acids other than proteinogenic amino acids that may also be used as nutraceutical functional components in nutraceuticals, traditional diets and functional diets (e.g., health diets).

Specifically, in the present disclosure, the proteinogenic amino acids include amino acids selected from the group consisting of nonpolar amino acids (e.g., alanine, valine, leucine, isoleucine, phenylalanine, proline), polar neutral amino acids (e.g., tryptophan, tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine), basic amino acids (e.g., lysine, arginine, histidine), acidic amino acids (e.g., aspartic acid, glutamic acid), the above are L-type α -amino acids except glycine.

The amino acid polymer may be selected from oligopeptides and polypeptides comprising amino acids as described above.

In the present disclosure, the term "oligopeptide" is used to refer to an amino acid polymer comprising 2 to 10 identical or different amino acids linked by peptide bonds, while the term "polypeptide" is used to refer to an amino acid polymer comprising 11 to 100 identical or different amino acids linked by peptide bonds, for the amino acids making up the oligopeptide or polypeptide, all of one or more of the above amino acids may be present, as well as additional amino acids.

The amino acid derivative may, for example, be selected from amino acid salts comprising the amino acids as described above. In the context of the present invention, the term "amino acid salt" is used to indicate salts of amino acids with acids or bases as described above, for example sodium, calcium, potassium, iron, magnesium, zinc, magnesium salts with bases and the like, as well as salts with acids, for example hydrochloride, hydrobromide, sulphate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulphonate, p-toluenesulphonate, amino acid salts and the like. More specifically, for example, lysine hydrochloride, histidine hydrochloride, glutamic acid hydrochloride, cysteine hydrochloride, arginine hydrochloride, glycine sulfate, lysine hydrochloride, aspartic acid hydrochloride, etc.

In the present disclosure, the amino acid nutrient includes one or more of the following amino acid compounds having a nutraceutical effect: amino acids, amino acid salts, oligopeptides, and polypeptides. In the pharmaceutical composition of the present invention, the amino acid nutrient may be one or more of amino acids, amino acid salts, oligopeptides and polypeptides, for example, 2, 3, 4 or 5 or more.

In the present disclosure, the amino acids, amino acid salts, oligopeptides and polypeptides as the amino acid nutrients are preferably amino acids 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, β -alanine, taurine, gamma-aminobutyric acid (GABA), theanine, citrulline and ornithine or salts thereof or oligopeptides and polypeptides comprising or consisting of amino acids selected from the group consisting of arginine, lysine, glycine, cysteine, alanine, serine, aspartic acid and glutamic acid.

In one embodiment, the amino acid nutrient is selected from arginine, an arginine salt, or an oligopeptide or polypeptide containing arginine.

In one embodiment, the amino acid nutrient is selected from lysine, a lysine salt, or an oligopeptide or polypeptide containing lysine.

In one embodiment, the amino acid nutrient is selected from glycine, a glycine salt, or an oligopeptide or polypeptide containing glycine.

In one embodiment, the amino acid nutrient is selected from glutamic acid, a glutamate, or an oligopeptide or polypeptide containing glutamic acid.

In one embodiment, the concentration (w/v) of the amino acid nutrient in the vaccine is 2-40%, 5-40%, 7.5-30%, 10-25% or 18-25%, preferably 15% -25% or 20% -25%.

In one embodiment, the amino acid based nutrient is selected from amino acids or amino acid salts having a nutraceutical effect and the concentration (w/v) of the amino acid or amino acid salt in the vaccine is greater than or equal to 5%, preferably greater than or equal to 7.5%, 10-25% or 18-25%, more preferably 15% -25% or 20% -25%.

In one embodiment, the amino acid based nutrients are selected from oligopeptides and polypeptides having nutraceutical effects and the concentration (w/v) of said oligopeptides and polypeptides in the vaccine is greater than or equal to 5%, preferably 7.5-25%, more preferably 10% -25%.

In one embodiment, the amino acid nutrient is a combination of the amino acid and/or amino acid salt and the oligopeptide and/or polypeptide and the concentration (w/v) of the combination in the vaccine is greater than or equal to 5%, preferably 7.5% -25%, more preferably 10-25%.

In the context of the present invention, unless otherwise indicated, the term "concentration" refers to the weight/volume percent concentration% (w/v) of the specified component in the topical pharmaceutical composition. The term "local administration concentration" refers to the concentration of a specified component at the time the drug is administered locally, which may be the concentration of the specified component at the site where the drug contacts the target area (e.g., injection needle hole or infusion tube outlet).

In the vaccine according to the present disclosure, it may further optionally comprise an analgesic. The analgesic is used to reduce the pain experienced by the patient and may be any suitable one known to those skilled in the art, such as benzyl alcohol, procaine hydrochloride, chlorobutanol, lidocaine hydrochloride, and the like. The concentration of the analgesic in the vaccine may be, for example, 0.1-4% by weight. For example, the concentration of benzyl alcohol may be 1 to 4%, and the concentrations of procaine hydrochloride, chlorobutanol and lidocaine hydrochloride may be 1 to 3%, respectively.

The vaccine according to the present disclosure may further optionally comprise a slow release carrier. The sustained release carrier may be any suitable one known to those skilled in the art, including, for example, a gel matrix, a microparticle carrier, a micelle matrix, and the like. The concentration (w/v) of the slow release carrier in the vaccine may be, for example, 0.5-13%, preferably 1-12% or 1-15%.

The vaccine according to the present invention may further optionally comprise a polyvalent metal compound. The polyvalent metal compound may be any suitable one known to those skilled in the art, and may be, for example, a metal salt (e.g., iron salt, copper salt, magnesium salt, zinc salt, manganese salt, aluminum salt, calcium salt, etc.) or a polyvalent metal compound (e.g., iron-containing heme, water-soluble acid-complexed iron, etc.). The concentration (w/v) of the polyvalent metal compound in the vaccine is, for example, 0.02 to 2%, which is preferably 0.05 to 1.5%, more preferably 0.1 to 1.2% or 0.1 to 1.6%.

The vaccine according to the invention may further optionally comprise a decoupling agent, such as Dinitrophenol (DNP), which may be present in the vaccine at a concentration (w/v) of, for example, 0.1-15%.

The vaccine according to the present invention may further optionally comprise a biological extract and analogues thereof, which may be any suitable one known to the skilled person, including for example: polysaccharides (e.g., algal polysaccharides, medicinal plant polysaccharides, fungal polysaccharides), glycosides (e.g., ginsenosides, panax notoginseng saponins, polygonum cuspidatum saponins, holothurian saponins, etc.), polyphenols (e.g., flavonoids, hydrolyzed tannins, procyanidins, tea polyphenols, resveratrol, apple polyphenols, etc.), terpenes (e.g., artemisinin, inula flower lactone, sesquiterpene lactone, etc.), flavones (e.g., puerarin, luteolin, silibinin, chrysin, amino acid lignin, nobiletin, licorice total flavonoids, etc.), alkaloids (e.g., camptothecin alkaloids, alline alkaloids, vinca alkaloids, petroseline alkaloids, berberine alkaloids, etc.). The concentration of the biological extract and analogues thereof in the vaccine may be, for example, 0.1-15% w/v.

The vaccine according to the present disclosure may be any topically administrable dosage form, preferably an injectable, more preferably a topical injectable, which may comprise the active ingredient (the methylene blue-based dye, the antigen (s)) and a liquid carrier.

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

Therefore, the vaccine disclosed by the application can be in the dosage form of injection, and the injection comprises liquid injection and powder injection for injection.

The powder injection for injection can comprise sterile dry powder and a solvent, one of the methylene blue dye and the one or more antigens is partially or completely contained in the sterile dry powder, the liquid carrier is contained in the solvent, and the concentrations of the methylene blue dye and the one or more antigens are respectively the concentrations of the methylene blue dye and the one or more antigens in a mixture of the sterile dry powder and the solvent.

According to another aspect of the present disclosure, there is provided a vaccine for treating solid tumors in lyophilized or semi-lyophilized form obtained by lyophilizing or semi-lyophilizing one of the whole or a part of the vaccine comprising the methylene blue dye as a vaccine adjuvant, one or more antigens and a pharmaceutically acceptable carrier according to the present disclosure.

The vaccine according to the present disclosure may further optionally comprise an excipient. The excipient may be any suitable one known to those skilled in the art and may include, for example, one or more of the following: dispersion media, preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, viscosity-increasing agents, and the like. The viscosity-increasing agent is, for example, sodium carboxymethylcellulose, polyvinylpyrrolidone or gelatin. Such as an antioxidant (e.g., ascorbic acid).

It will be appreciated by those skilled in the art that the vaccine according to the present disclosure should be formulated for local administration to the target area, preferably as an injection, more preferably as a local injection.

Vaccines according to the present disclosure can be prepared as follows: preparing a liquid medicament containing the methylene blue dye as the vaccine adjuvant, one or more antigens, a solvent (a pharmaceutically acceptable liquid carrier) and other substances (such as amino acid nutrients) optionally existing. The liquid medicament may be a solution (e.g. a solution in a hydrophilic vehicle, preferably an aqueous solution), a suspension, or an emulsion comprising the topically active ingredient. When the liquid drug is a suspension, the dispersion medium may be any suitable medium known to those skilled in the art, such as a micro-material or a nano-material. When the liquid drug is an emulsion, the dispersion medium may be any suitable one known to those skilled in the art, such as a vegetable oil, a synthetic oil or a semi-synthetic oil which may be used for injection. The vegetable oil may be, for example, cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and peanut oil.

According to the preparation method of the invention, the concentration of the methylene blue dye, one or more antigens and optionally other components used as vaccine adjuvants is greater than or equal to the local administration concentration of the methylene blue dye, one or more antigens and other components in the vaccine. When the concentration is more than that in the pharmaceutical composition of the present invention, it can be further diluted for use.

According to one embodiment disclosed herein, the vaccine liquid injection of the present invention can be prepared by a method comprising the steps of: 1) adding the methylene blue dye and one or more antigens which are used as vaccine adjuvants and are required according to the local administration concentration into a solvent to prepare liquid; 2) adding other optional components (such as amino acid nutrients, analgesics and the like) in required amounts according to the local administration concentration to the liquid prepared in the step 1) and mixing uniformly to obtain a liquid medicine; 3) sterilizing the liquid medicine prepared in the step 2) and preparing the liquid injection. When in use, the bacteria-removing liquid medicine in the liquid injection can be directly used as a local administration liquid medicine or used as a diluted liquid medicine.

According to one embodiment disclosed herein, the vaccine liquid injection of the present invention can be prepared by a method comprising the steps of: 1) adding the methylene blue dye used as the vaccine adjuvant, one or more antigens and optionally other components which are required according to the local administration concentration into a solvent (or a pharmaceutically acceptable liquid carrier), uniformly mixing, and sterilizing to prepare a sterilization liquid I; 2) the required amount of other optional components (such as amino acid nutrients) according to the local administration concentration is added into a solvent (or a pharmaceutically acceptable liquid carrier) to be uniformly mixed and sterilized to prepare the sterilization liquid II. When the liquid medicine is used, the sterilization liquid I and the sterilization liquid II form a mixed liquid before or after entering a local administration instrument, and the mixed liquid can be directly used as a local administration liquid medicine or diluted liquid medicine.

According to one embodiment disclosed herein, the powder for injection of the vaccine of the present invention may be prepared by a method comprising the steps of: preparing a sterile dry powder containing the methylene blue as a vaccine adjuvant and an antigen in an amount required according to a local administration concentration; and preparing a sterile vehicle containing the required amounts of the other components (e.g., amino acid nutrients, analgesics) according to the concentration to be topically administered. The sterile dry powder is preferably sterile freeze-dried powder, and the preparation method comprises the following steps: 1) preparing a solution comprising the methylene blue-based dye as vaccine adjuvant, one or more antigens, and optionally other components; 2) sterilizing, filtering and packaging; 3) freeze drying; 4) and (5) plugging and capping. The freeze-drying process conditions include, for example: the pre-freezing condition is that the temperature is kept at minus 45 ℃ for 4 hours; sublimation drying condition is that the heating rate is 0.1 ℃/min, and the heating is kept for at least 10 hours when the temperature is raised to-15 ℃; the desorption drying conditions were 30 ℃ for 6 hours. When in use, the sterile dry powder of the powder injection for injection is redissolved in a sterile solvent to form a redissolved liquid medicine which can be directly used as a local administration liquid medicine or diluted.

In the present disclosure, the vaccine is primarily intended as a therapeutic vaccine for the prevention or treatment of solid tumors. According to one embodiment, the solid tumors include secretory gland tumors, skin tumors, and sarcomas.

According to one embodiment, the secretory adenoma comprises a breast tumor, a liver tumor, a colon tumor, a pancreas tumor, a thyroid tumor, a prostate tumor, a lung tumor, a head and neck tumor, a nasopharyngeal tumor, a testicular tumor, a vaginal tumor, a uterine tumor, an ovarian tumor.

According to one embodiment, the skin tumor comprises melanoma.

According to another aspect of the invention there is also provided the use of a vaccine according to the invention in the manufacture of a medicament for the immunoprophylaxis and immunotherapy of a locally diseased condition.

The skilled person will understand that the technical scheme of the vaccine according to the present invention and its effectiveness and safety can be applied to the preparation of immunotherapeutic drugs for solid tumors, as well as other local lesions, especially refractory local lesions, which can be locally administered to different target regions of local lesions.

The use according to the invention, which is the use of a vaccine according to the invention for the preparation of an immunotherapeutic drug for the treatment of solid tumors.

Within the scope of the present invention, the term "tumor body" refers to a mass formed by abnormal proliferation of cells or mutated cells; the term "solid tumor" refers to a tumor having a tumor body, which may be due to any pathology (malignant and non-malignant) and at any stage of the tumor, including for example the following groups classified by tumor cell type: epithelial cell tumors, sarcomas, lymphomas, germ cell tumors, blastomas; and tumors named as the organ or tissue in which the tumor cell foci are located, including, for example, tumors named as the following organs or tissues: skin, bone, muscle, breast, kidney, liver, lung, gall bladder, pancreas, brain, esophagus, muscle of the shoulder, large intestine, small intestine, spleen, stomach, prostate, testis, ovary, or uterus.

The malignant tumor includes, for example, breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, prostate cancer, liver cancer, lung cancer, intestinal cancer, oral cancer, esophageal cancer, gastric cancer, laryngeal cancer, testicular cancer, vaginal cancer, uterine cancer, ovarian cancer, etc. The non-malignant tumor includes, for example, breast tumor, pancreatic tumor, thyroid tumor, prostate tumor, liver tumor, lung tumor, intestinal tumor, oral tumor, esophageal tumor, stomach tumor, nasopharyngeal tumor, laryngeal tumor, testicular tumor, vaginal tumor, uterine tumor, fallopian tube tumor, ovarian tumor, etc.

In the present disclosure, the vaccine according to the invention can also be used as an immunotherapeutic drug for the treatment of non-neoplastic tumours.

In the context of the present invention, the term "non-neoplastic enlargement" refers to enlargement other than a tumor, and includes, for example, hyperplasia (e.g., hyperplasia of the breast, pancreas, thyroid, parathyroid, prostate, etc.), cyst (e.g., cyst of the breast, thyroid, parathyroid, etc.), nodule (e.g., nodule of the breast, thyroid, parathyroid, etc.), abnormal vein mass (e.g., hemorrhoid, etc.), local inflammatory swelling, microbial infection swelling, etc. The hemorrhoid includes internal hemorrhoid, external hemorrhoid, and mixed hemorrhoid.

In the present disclosure, the vaccine according to the invention can also be used as an immunotherapeutic drug for the treatment of local inflammation, in particular refractory inflammation.

Within the scope of the present invention, the term "local inflammation" refers to a non-neoplastic inflammation at a local site, including for example, inflammatory inflammation (inflammatory inflammation), exudative inflammation (inflammatory inflammation) and proliferative inflammation, which may be any suitable one known to the skilled person, and may for example include one or more of the following: arthritis, mastitis, pancreatitis, thyroiditis, prostatitis, hepatitis, pneumonia, enteritis, stomatitis, pharyngitis, periodontitis, esophagitis, gastritis, gastric ulcer, rhinitis, sinusitis, laryngitis, tracheitis, bronchitis, vaginitis, metritis, salpingitis, and oophoritis.

In the present disclosure, the vaccine according to the invention can also be used as an immunotherapeutic drug for the treatment of skin diseases, in particular intractable skin diseases.

Within the scope of the present invention, the term "skin disease" refers to a lesion native or secondary to the skin or skin appendages, which may be any suitable one known to a person skilled in the art, and may for example include one or more of the following: skin cancer, non-malignant tumors of the skin, viral skin diseases (e.g., herpes, warts, rubella, hand-foot-and-mouth disease), bacterial skin diseases (e.g., impetigo, furuncle, leprosy), fungal skin diseases (e.g., various ringworm), sexually transmitted diseases (e.g., syphilis, gonorrhea, and condyloma acuminatum), allergic and autoimmune skin diseases (e.g., contact dermatitis, eczema, urticaria), physical skin diseases (e.g., solar skin diseases, chilblain, corns, rhagades of hands and feet, pressure sores), connective tissue diseases (e.g., lupus erythematosus), skin disorders (e.g., freckles, pigmented nevi, various plaques), skin appendages diseases (e.g., acne, rosacea, seborrheic dermatitis, alopecia areata, alopecia, hyperhidrosis, and bromidrosis).

In the present disclosure, the vaccine according to the present invention can also be used as an immunotherapeutic drug for the treatment of secretory dysfunction of secretory glands.

Within the scope of the present invention, the term "secretory gland" refers to a structure composed of gland cells or gland cell groups that performs a secretory function (secretion), which includes exocrine glands and endocrine glands. The secretory gland secretory dysfunction includes secretory gland hyperfunction (such as hyperthyroidism) and secretory gland hypofunction (such as hypothyroidism, islet hypofunction (one of diabetes) and the like).

In the present disclosure, the vaccine according to the invention can also be used as an immunotherapeutic drug for the interventional treatment of cardiovascular diseases. Interventional therapy has become an important treatment for cardiovascular diseases. Such cardiovascular diseases include, for example, hemangiomas, hypertrophic obstructive cardiomyopathy, atrial fibrillation, cardiac arrhythmias, arterial emboli, and the like.

The vaccine of the present invention and medical preparations comprising the same, when used for the prevention and treatment of local lesion diseases, can also be administered in combination with other interventional therapies, systemic chemotherapy, immunotherapy, photodynamic therapy, sonodynamic therapy, surgical intervention or a combination of such therapies to further improve the therapeutic effect.

The present disclosure includes the following items:

item 1, use of methylene blue dyes as vaccine adjuvants.

Item 2, the use according to item 1, wherein the vaccine further comprises one or more antigens and a pharmaceutically acceptable liquid carrier.

Item 3, a vaccine comprising a methylene blue-based dye as an adjuvant, one or more antigens, and a pharmaceutically acceptable liquid carrier.

Item 4, the use or vaccine according to one of items 1 to 3, wherein the methylene blue type dye comprises the following compounds and derivatives thereof: methylene blue, patent blue, isothio blue, preferably methylene blue.

Item 5, the use or the vaccine according to item 4, wherein the concentration (w/v) of the methylene blue stain in the vaccine is ≦ 2%, preferably 0.3-2%, more preferably 0.5-1.5%.

Item 6, the use or vaccine according to item 2 or 3, wherein the antigen is selected from antigens directed against solid tumors.

Item 7, the use or vaccine according to item 6, wherein the solid tumor comprises a secretory gland tumor and a skin tumor.

Item 8, the use or vaccine of item 7, wherein the secretory gland tumor comprises breast cancer, liver cancer, colon cancer, pancreatic cancer, thyroid cancer, prostate cancer, lung cancer, head and neck cancer, nasopharyngeal cancer.

Item 9, the use or vaccine according to item 7, wherein the skin tumor is melanoma.

Item 10, the use or vaccine according to one of items 2 to 9, wherein the vaccine further comprises an amino acid nutrient as a further adjuvant.

Item 11, the use or the vaccine according to item 10, wherein the amino acid based nutrient comprises one or more of the amino acid compounds, amino acid salts, oligopeptides and polypeptides having a nutraceutical effect, preferably amino acids or salts thereof selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, tyrosine, serine, cysteine, methionine, asparagine, glutamine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, β -alanine, taurine, gamma aminobutyric acid (GABA), tea polyphenols (theanine), cucurbita amino acid (3-amino-3-carboxypyranoic acid), glutamine, citrulline, ornithine, more preferably amino acids or salts thereof selected from the group or oligopeptides and polypeptides comprising or consisting of arginine, lysine, glycine, cysteine, alanine, serine, glutamic acid.

Item 12, the use or the vaccine according to item 11, wherein the amino acid based nutrient is selected from the group consisting of amino acids or amino acid salts with nutraceutical effect and the concentration (w/v) of the amino acid or amino acid salt in the vaccine is more than or equal to 5%, preferably 10-25% or 18-25%.

Item 13, the use or the vaccine according to item 11, wherein the amino acid based nutrients are selected from oligopeptides and polypeptides with nutraceutical effect and the concentration (w/v) of the oligopeptides and polypeptides in the vaccine is more than or equal to 5%, preferably 7.5-25%.

Item 14, the use or the vaccine according to item 11, wherein the amino acid based nutrient is a combination of the amino acid and/or amino acid salt and the oligopeptide and/or polypeptide and the concentration (w/v) of the combination in the topical pharmaceutical composition is more than or equal to 5%, preferably 10-25%.

Item 15, the pharmaceutical composition or use according to item 11, wherein the oligopeptide is one or more selected from the group consisting of glycyl-L-tyrosine, glycylalanine, glycylglycine, lysine-glycine dipeptide, glutamine dipeptide, carnosine (β -alanine histidine copolymer), glutathione, collagen oligopeptides, casein hydrolysate peptides, soy oligopeptides, oligoarginine, oligoglycine, oligolysine, and the polypeptide is one or more selected from the group consisting of polyaspartic acid, polyglutamic acid, polylysine.

Item 16, a vaccine prepared by lyophilization or semi-lyophilization of a vaccine according to one of items 3-15, in part or in whole.

Item 17, the vaccine according to one of items 1 to 16, which is a therapeutic vaccine.

Item 18, use or vaccine according to one of items 1 to 17, wherein the vaccine is for the prevention or treatment of a solid tumor.

Item 19, a method for the prevention and treatment of solid tumors comprising the local administration of a vaccine according to one of items 3 to 18 to an individual in need thereof.

Item 20, the use, vaccine or method according to item 18 or 19, wherein the solid tumor comprises a secretory gland tumor, a skin tumor and a sarcoma.

Item 21, the vaccine, use or method according to item 20, wherein the secretory gland tumor comprises a breast tumor, a liver tumor, a colon tumor, a pancreatic tumor, a thyroid tumor, a prostate tumor, a lung tumor, a head and neck tumor, a nasopharyngeal tumor, a testicular tumor, a vaginal tumor, a uterine tumor, an ovarian tumor.

Item 22, the vaccine, use or method according to item 20, wherein the skin tumor comprises melanoma.

Based on the studies described in more detail below, the vaccine of the present invention, although the specific mechanism remains to be further studied, has been shown to promote effective destruction of the relevant structures of the tissues in which the local lesion is located (e.g., the lesion tissue, the lesion cells, and any structures involved in constituting them), while minimizing damage to the normal tissues of the patient, thereby achieving a pharmaceutical effect of safely and effectively treating the local lesion disease.

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 methylene blue-based dyes and amino acid-based nutrients used in the following examples are listed in table 1.

TABLE 1

In the present invention, L-amino acids are abbreviated as amino acids (for example, L-arginine is abbreviated as arginine), reduced glutathione is abbreviated as glutathione, and alanyl-glutamine dipeptide is abbreviated as glutamine dipeptide.

In the following examples, tests of subcutaneous transplanted tumor animals were performed according to the test guidelines issued by the drug administration, unless otherwise indicated. The test animal is Balb/c nude mouse or mouse with age of 6-8 weeks and weight of 17.5-20.5 g. The subcutaneous transplantation is carried out according to the conventional subcutaneous inoculation method of tumor cells. Unless otherwise stated, the tumor is grown to a desired volume (e.g., 75-500 mm)³) This was randomly divided into several groups of 6 animals each using PEMS 3.2 software (compiled by the national institute of public health, western Sichuan university). Items for experimental observation, measurement and analysis include general state, body weight, food intake, tumor volume, tumor weight, thymus weight, spleen weight, and the like.

The tumor volume calculation formula is as follows:

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

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

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

In the following examples, the tests were each statistically tested for group differences in mean index using Repeated measures analysis of variance (Repeated Meas μ res ANOVA). When the group difference has statistical significance (P is less than or equal to 0.05), the difference between each group and the negative control group is compared by adopting a minimum significant difference method. Quantitative indices are described as mean ± standard error (X ± SEM). When the LEVENE homogeneity test indicates that the variance is not uniform (P is less than 0.05), the difference between the groups is compared by using Mann-Whitney M rank sum test (M-W method). All statistical analyses were performed under SPSS for Windows 13.0 software.

Example 1: vaccine preparation

Some of the vaccines of the present invention containing methylene blue dye adjuvants prepared in this example are listed in table 2.

TABLE 2

Several examples of preparative assays of the present invention are set forth below.

1. Preparation of adjuvants

1) Preparation of methylene blue dye adjuvant

The methylene blue dye (such as 2.35g of methylene blue), other optional components and injection water (such as 76ml) with the total volume of 50-80% are measured according to the required concentration for dissolving, and then the dissolved solution is added with water to be constant volume (such as 100ml) and is uniformly mixed for later use (such as 2.35% methylene blue aqueous solution).

2) Preparation of methylene blue dye/amino acid nutrient composition adjuvant

Methylene blue type dye (for example, 2.36g of methylene blue), amino acid type nutrient (for example, 23.5g of lysine hydrochloride), optionally other components, and water for injection which is fixed to a total volume (for example, 100ml) are measured according to the required concentration, mixed and dissolved at 20 ℃ to 90 ℃, and the preparation (for example, 2.35% methylene blue/23.5% lysine hydrochloride aqueous solution) is stored at room temperature for later use.

2. Preparation of vaccine antigens

Tumor cells (e.g., melanoma B16-f10 fine cell)Cell) suspensions were inoculated subcutaneously into the left forelimb axilla of 6 BALB/c mice, each at 2X 10⁶One cell, mice were sacrificed 16 days later, tumor blocks were stripped, necrotic tissue was removed, cell suspensions were collected after grinding with a 200 mesh sieve and were measured and diluted to the desired cell number, 1/10 volumes of DNase (0.01mg/mL) were added, and repeated freeze thawing (-20 ℃ for 30min and then 37 ℃ for 10min) was performed until tumor cells were completely lysed (microscopic observation after trypan blue staining), and it was verified (inoculated BALB/c mice) that they had lost tumorigenicity. In the present invention, this preparation is designated as cell lysate (2X 10)⁷Individual cells/ml) can be used as a specific antigen for vaccine preparation.

3. Preparation of vaccines

Mixing the vaccine antigen with the required adjuvant to obtain the vaccine. The preparation method comprises 3 methods: dispersing the antigen dry powder into an adjuvant solution (e.g., the adjuvant solution prepared in 1 above); dispersing the adjuvant dry powder into an antigen-containing liquid (e.g., the antigen-containing liquid prepared in 2 above); or mixing an antigen-containing liquid (e.g., the antigen-containing liquid prepared in 2 above) with an adjuvant solution (e.g., the adjuvant solution prepared in 1 above).

For example, in 2.97ml or 2.7ml cell lysate (2X 10)⁷One cell/ml) with 30mg of methylene blue, or 30mg of methylene blue and 300mg of arginine to obtain a cell lysate (2X 10)⁷Individual cells/ml)/1% methylene blue vaccine, or cell lysate (1X 10)⁷Individual cells/ml)/1% methylene blue/10% arginine vaccine.

Example 2: adjuvant Effect study

1. Adjuvant effect

Mice were randomly divided into 13 groups of 6 mice each, of which 2 negative control groups and 11 study groups. The model cell was melanoma B16-f10 cell, as described above (2X 10)⁶Individual cells/0.1 ml) for transplantation tumor modeling. The negative control was physiological saline, which was injected intraperitoneally and subcutaneously, respectively. The 6 study drugs and their administration are shown in the table below. The drugs were all aqueous solutions and were prepared according to the preparation method of example 1. Each group was injected 1 time at day 2, 8, 15 after tumor inoculation, 100. mu.l/mouse. After 5 days, the patient moves relativelyThe drugs were euthanized, tumor weights were determined after dissection, and tumor inhibition rates were calculated from negative controls for each administration regimen, and the results are shown in table 3.

TABLE 3

In each group injected intraperitoneally in the above table, the positive control group met the following expectations: the antitumor cell medicament can target tumor cells so as to inhibit the growth of tumor bodies, and the methylene blue group, the arginine group, the melanoma cell lysate/methylene blue group and the melanoma cell lysate/methylene blue/arginine group do not show the effect of inhibiting the growth of tumor bodies. In each group of subcutaneous injections, there was no significant difference (P >0.05) among the methylene blue group, the arginine group, and the melanoma cell lysate group compared to the negative control group, whereas the melanoma cell lysate/methylene blue group, and the melanoma cell lysate/methylene blue/arginine group showed significant differences (P < 0.05). According to this result, methylene blue/arginine showed a significant adjuvant effect.

2. Further application examples of adjuvants

The following experiments investigated the use of methylene blue dye adjuvants in a variety of vaccines. In the following experiments, the negative control was saline and the drug was aqueous solution, all prepared according to the preparation method of example 1. In the experiment, animals are euthanized 5 days after the administration, the tumor weight is determined after the dissection, and the tumor inhibition rate is calculated from respective negative control groups.

1) Application of the same in breast tumor resisting vaccine

Mice were randomly divided into 6 groups of 6 mice each, of which 1 negative control group and 5 study groups (A, B, C, D, E groups). The model cell was a mammary tumor 4T1 cell, as described above (0.1X 10)⁶Individual cells/0.1 ml) for transplantation tumor modeling. The negative control was physiological saline, and the 5 study drugs were: 1% methylene blue, 20% lysine, 4T1 cell lysate (2X 10)⁷Individual cells/ml), 4T1 cell lysate (2X 10)⁷Cell/ml)/1% methylene blue,4T1 cell lysate (2X 10)⁷Individual cells/ml)/1% methylene blue/20% lysine. The drugs were all aqueous solutions and were prepared according to the preparation method of example 1. Each group was injected subcutaneously 1 time at day 2, 8, 15 after tumor inoculation, 100. mu.l/mouse. 5 days after the administration, the animals were euthanized, and the tumor weight and tumor inhibition rate were calculated after dissection.

A. The inhibition rates of B, C, D, E groups were 3%, 1%, 8%, 26% and 34%, respectively. Compared with the negative control group, A, B, C group has no significant difference (P >0.05), D, E group shows significant difference (P <0.05), and methylene blue, methylene blue/lysine show obvious adjuvant effect.

2) Application of the same in anti-liver tumor vaccines

Mice were randomly divided into 6 groups of 6 mice each, of which 1 negative control group and 5 study groups (A, B, C, D, E groups). The model cell was murine hepatoma H22 cell, as described above (2X 10)⁶Individual cells/0.1 ml) for transplantation tumor modeling. The negative control was physiological saline, and the 5 study drugs were: 1% methylene blue, 20% lysine, H22 cell lysate (2X 10)⁷Individual cells/ml), H22 cell lysate (2X 10)⁷Individual cells/ml)/1% methylene blue, H22 cell lysate (2X 10)⁷Individual cells/ml)/1% methylene blue/20% lysine. The drugs were all aqueous solutions and were prepared according to the preparation method of example 1. Each group was injected subcutaneously 1 time at day 2, 8, 15 after tumor inoculation, 100. mu.l/mouse. 5 days after the administration, the animals were euthanized, and the tumor weight and tumor inhibition rate were calculated after dissection.

A. The inhibition rates of B, C, D, E groups were 4%, 5%, 13%, 27%, and 33%, respectively. Compared with the negative control group, A, B, C group has no significant difference (P >0.05), D, E group shows significant difference (P <0.05), and methylene blue, methylene blue/lysine show obvious adjuvant effect.

3) Application of the same in colon tumor resisting vaccine

Mice were randomly divided into 6 groups of 6 mice each, of which 1 negative control group and 5 study groups (A, B, C, D, E groups). Making model cell of mouse colon cancer CT26 cells, according to the method described above (1X 10)⁶Individual cells/0.1 ml) for transplantation tumor modeling. The negative control was physiological saline, and the 5 study drugs were: 1% methylene blue, 20% arginine, mastadenoma cell lysate (2X 10)⁷Individual cells/ml), breast tumour cell lysate (2X 10)⁷Individual cells/ml)/1% methylene blue, mammary tumour cell lysate (2X 10)⁷Individual cells/ml)/1% methylene blue/20% arginine. The drugs were all aqueous solutions and were prepared according to the preparation method of example 1. Each group was injected subcutaneously 1 time at day 2, 8, 15 after tumor inoculation, 100. mu.l/mouse. 5 days after the administration, the animals were euthanized, and the tumor weight and tumor inhibition rate were calculated after dissection.

A. The inhibition rates of B, C, D, E groups were 1%, 3%, 10%, 23%, and 31%, respectively. Compared with the negative control group, A, B, C group has no significant difference (P >0.05), D, E group shows significant difference (P <0.05), and methylene blue/arginine show obvious adjuvant effect.

Similar vaccine immunization effects can also be observed using other vaccines prepared using the method of example 1 (e.g., the vaccines in table 2).

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. The methylene blue dye is used as vaccine adjuvant.

2. Use according to claim 1, wherein the vaccine further comprises one or more antigens and a pharmaceutically acceptable liquid carrier.

3. A vaccine comprising a methylene blue-based dye as an adjuvant, one or more antigens and a pharmaceutically acceptable liquid carrier.

4. Use or vaccine according to one of claims 1 to 3, wherein the methylene blue type dye comprises the following compounds and derivatives thereof: methylene blue, patent blue, isothio blue, preferably methylene blue.

5. Use or vaccine according to claim 4, wherein the concentration (w/v) of the methylene blue stain in the vaccine is 2% or less, preferably 0.3 to 2%, more preferably 0.5 to 1.5%.

6. The use or vaccine according to claim 2 or 3, wherein the antigen is selected from antigens directed against solid tumors.

7. The use or vaccine according to claim 6, wherein the solid tumors include secretory tumors and cutaneous tumors.

8. Use or vaccine according to claim 7, wherein the secretory gland tumour comprises breast, liver, colon, pancreatic, thyroid, prostate, lung, head and neck, nasopharyngeal carcinoma.

9. Use or vaccine according to claim 7, wherein the skin tumour is melanoma.

10. A vaccine prepared by lyophilisation or semi-lyophilisation of a part or all of a vaccine according to one of claims 3 to 9.