Pharmaceutical composition for treating tumors

Abstract

This invention provides a pharmaceutical composition for treating tumors, which, when formulated for intratumoral injection, exhibits excellent tumor inhibition and elimination effects, and can safely, rapidly, and potently achieve antitumor efficacy against a variety of different tumors. This invention also provides a method for preparing the composition and its applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a pharmaceutical composition for treating tumors. The composition, when formulated for intratumoral injection, exhibits excellent tumor inhibition and elimination effects, and can safely, rapidly, and effectively achieve anti-tumor efficacy against a variety of different tumors. This invention also relates to a method for preparing the composition and its applications. Background Technology

[0002] Proliferative diseases pose a serious threat to modern society. The growth of tumors, due to their unique characteristics including uncontrolled cell proliferation, the ability to invade local and even distant tissues, lack of differentiation, lack of detectable symptoms, and lack of effective treatment and prevention, presents a severe challenge to modern medicine. More than 10 million people worldwide are diagnosed with cancer each year, and cancer / tumor causes 6 million deaths annually, accounting for 12% of all deaths worldwide.

[0003] Currently, commonly used anti-tumor methods in clinical practice include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Although these treatments have achieved certain clinical success, they suffer from problems such as poor selectivity, poor safety, significant toxic side effects (e.g., bone marrow suppression, cardiotoxicity, neurotoxicity), easy development of drug resistance, and high incidence of tumor recurrence. Furthermore, these treatments generally only achieve a certain degree of slowing or control of tumor growth, and cannot completely reverse tumor growth, let alone achieve complete elimination and radical cure of the tumor.

[0004] Ascorbic acid, commonly known as vitamin C, is an essential water-soluble vitamin and a powerful antioxidant. Its main known functions include: 1) Antioxidant: scavenging free radicals and protecting cells from oxidative damage; 2) Collagen synthesis: acting as a coenzyme for proline hydroxylase and lysine hydroxylase, promoting collagen synthesis and wound healing; 3) Promoting iron absorption: reducing poorly absorbed ferric iron (Fe3+) to more easily absorbed ferrous iron (Fe2+); 4) Enhancing immune function: supporting the activity and function of white blood cells. Furthermore, a few studies have reported the use of ascorbic acid injections as adjuvant therapy for tumors. For example, Chinese patent CN111939153A mentions using ascorbic acid as a prodrug in the preparation of drugs for treating liver cancer, with administration methods including intravenous or intramuscular injection. However, this patent only involves early studies at the cellular level and has not verified the actual antitumor efficacy of ascorbic acid in animals or humans. Furthermore, based on the inventors' earlier research, ascorbic acid alone has limited efficacy in treating tumors, cannot fully reverse tumor growth or completely eliminate tumors, and has the drawback of easy tumor recurrence.

[0005] Therefore, developing novel anti-tumor drugs with safe and potent tumor-suppressing effects, especially those capable of reversing tumor growth and even achieving complete elimination and cure of tumors, is of great clinical significance and has an urgent market demand. Summary of the Invention

[0006] In one aspect, the present invention provides a pharmaceutical composition for treating tumors, comprising component A and component B as active ingredients, and optionally a pharmaceutically acceptable carrier or excipient. In another aspect, the present invention provides a pharmaceutical composition for treating tumors, prepared by mixing component A, component B, and optionally a pharmaceutically acceptable carrier or excipient. In yet another aspect, the present invention provides an intratumoral injection composition for treating tumors, comprising component A and component B as active ingredients, and optionally a pharmaceutically acceptable carrier or excipient. The above compositions are collectively referred to as "compositions of the present invention" throughout this invention.

[0007] In another aspect, the present invention provides a method for preparing the composition of the invention, comprising the step of mixing component A, component B, and optionally a pharmaceutically acceptable carrier or excipient.

[0008] In another aspect, the present invention provides the use of the compositions of the present invention in the preparation of a medicament for treating tumors.

[0009] In another aspect, the present invention provides a method for treating tumors, comprising administering the composition of the present invention.

[0010] Other embodiments and advantages of the invention will be set forth in part in the description which follows, and will be apparent from the description, or may be learned by practice of the invention. The embodiments and advantages of the invention will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims.

[0011] It should be understood that the foregoing description of the invention and the following detailed description are merely exemplary and illustrative, and not intended to limit the scope of the claimed invention. Attached Figure Description

[0012] Figure 1 These are the tumor growth curves of samples 1-2 and the graph showing the change in mouse body weight after intratumoral injection.

[0013] Figure 2 These are the tumor growth curves for samples 1-4 and the changes in mouse body weight after intratumoral injection.

[0014] Figure 3 These are comparative photographs of the tumor sites in mice with lung squamous cell carcinoma samples 1-5 before administration, on day 3, day 7, and day 21 after administration.

[0015] Figure 4 The curves show the inhibition of tumor growth in tumor-bearing mice by components A, B, and the A / B combination. The horizontal axis represents the number of days, and the vertical axis represents the tumor volume.

[0016] Figures 5A-5C These are the specific morphological observation results of mice in the A / B composition experimental group at different stages. Figure 5A The A / B combination is administered immediately after administration. Figure 5B : 2 hours after administration of the A / B composition. Figure 5C Day 21 after administration of the A / B combination.

[0017] Figures 6A-6C Figure 6 shows the specific morphological observation results of mice in the component B experimental group at different stages. Figure 6 A: Immediately after administration of 22% unit volume component B in the A / B composition. Figure 6 B: 2 hours after administration of 22% unit volume component B in the A / B composition. Figure 6 C: 4 hours after administration of 22% unit volume component B in the A / B composition.

[0018] Figures 7A-7E These are the specific morphological observation results of mice in the A / B combination administration experimental group at different stages. Figure 7A Samples 1-5 before administration. Figure 7B Samples 1-5 were administered on day 3. Figure 7C : 7 days after taking the medication for samples 1-5. Figure 7D : Day 21 after administration of samples 1-5. Figure 7E Long-term observation of samples 1-5 after administration.

[0019] Figures 8A-8B These are the pH values ​​measured at three locations on the left, right, and center of the tumor at different time points. Figure 8A ); and pH values ​​measured at different distances from the tumor at different time points ( Figure 8B ).

[0020] Figure 9A-9I These are pathological slide photographs of the experimental and control groups at 24 hours, 48 ​​hours, 72 hours, 7 days, and 21 days after drug administration.

[0021] Figures 10A-10E These are pathological slide photographs of various tissues and organs 24 hours to 21 days after drug administration. Detailed Implementation

[0022] This invention provides a novel anti-tumor drug composition with safe and potent tumor-suppressing effects, particularly capable of reversing tumor growth and even achieving complete elimination and radical cure of tumors.

[0023] Unless otherwise defined below, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. References to technology as used herein are intended to refer to technology as commonly understood in the art, including obvious modifications or equivalent substitutions by those skilled in the art. While it is believed that the following terms are fully understood by those skilled in the art, the following definitions are provided to better explain this invention.

[0024] definition As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “comprising,” and other variations thereof, are inclusive or open-ended and do not exclude other elements or method steps not listed.

[0025] In the context of describing this invention (especially in the context of the claims), the use of the terms “a,” “an,” “the,” and similar designations should be understood to cover both singular and plural forms unless otherwise stated. Unless otherwise stated in this invention, the enumeration of numerical ranges in this invention is intended only as a shorthand method of individually referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were individually described in this invention. Unless otherwise required, the use of any and all example or exemplary language (e.g., “such as”) provided in this invention is intended to better illustrate the invention and not to limit the scope of the invention. The language in the specification should not be construed as indicating that any unclaimed element is necessary for the practice of this invention.

[0026] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the patient is a human. In some embodiments, the subject has experienced and / or exhibited at least one symptom of a disease or condition to be treated and / or prevented.

[0027] As used herein, the term "treatment" means the elimination, reduction, or improvement of a disease or condition and / or symptoms associated with that disease or condition. While not excluded, treatment of a disease or condition does not require the complete elimination of the disease, condition, or symptoms associated with it. The term "treatment" and its synonyms encompass the administration of a therapeutically effective amount of the compound of the present invention to a subject who requires such treatment. Treatment may be directed at symptoms, for example, to suppress symptoms. Treatment may be effective in the short term, directed at the intermediate term, or may be long-term treatment, for example, in the context of maintenance therapy.

[0028] As used in this invention, the term "prevention" refers to a method of preventing the onset of a disease or condition and / or its accompanying symptoms, or preventing a subject from becoming ill. As used in this invention, "prevention" also includes delaying the onset of a disease and / or its accompanying symptoms and reducing the risk of a subject becoming ill. The term "prevention" may include "preventive treatment," which refers to reducing the likelihood of a subject who does not have, but has a risk of recurrence of, a disease or condition, or a relapse of such disease or condition, or who is prone to recurrence of such disease or condition, or who has previously controlled a disease or condition, re-occurring with such disease or condition.

[0029] As used herein, the term "therapeutic effective amount" or "effective dose" refers to the amount of an active ingredient, when administered by the method of the present invention, sufficient to effectively deliver an active ingredient for treating a target condition or disease to a subject in need. In the case of cancer, tumors, or other proliferative conditions, a therapeutically effective amount of an agent may reduce (i.e., to some extent delay or terminate) unwanted cell proliferation; reduce the number of tumor cells; reduce tumor size; inhibit (i.e., to some extent delay or terminate) tumor cell infiltration into peripheral organs; inhibit (i.e., to some extent delay or terminate) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer to some extent. The administered composition may be cytotoxic and / or cytosolic in terms of the degree to which it inhibits the growth of existing cancer cells and / or kills existing cancer cells.

[0030] In this invention, the term "alkyl" when used alone or as part of another group refers to a straight-chain or branched aliphatic hydrocarbon (i.e., C1-C1) containing one to eighteen carbon atoms. 18 Alkyl groups or straight-chain or branched aliphatic hydrocarbons containing a specified number of carbon atoms (e.g., C1 alkyl such as methyl, C2 alkyl such as ethyl, etc.). In one embodiment, the alkyl group is C1-C2. 10 Alkyl group. In another preferred embodiment, the alkyl group is C1-C6 alkyl. In another embodiment, the alkyl group is C1-C4 alkyl. In yet another embodiment, the alkyl group is C1-C3 alkyl, i.e., methyl, ethyl, propyl, or isopropyl. Non-limiting exemplary C1-C 18 Alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, isobutyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, and decyl. In another embodiment, one or more of the hydrogen atoms of the alkyl group are replaced by deuterium atoms, i.e., the alkyl group is labeled with a deuterium isotope. A non-limiting exemplary deuterated alkyl group is -CD3.

[0031] In this invention, the term "alkoxy" when used alone or as part of another group refers to an alkyl group attached to a terminal oxygen atom. In one embodiment, the alkyl group is a C1-C6 alkyl group, and therefore the resulting alkoxy group is referred to as "C1-C6 alkoxy". In another embodiment, the alkyl group is a C1-C4 alkyl group, and therefore the resulting alkoxy group is referred to as C1-C4 alkoxy. Non-limiting exemplary alkoxy groups include methoxy, ethoxy, and tert-butoxy groups.

[0032] This invention covers any compound in the compositions of the invention that is isotopically labeled (i.e., radioactively labeled) by replacing one or more atoms with atoms having different atomic masses or mass numbers. Examples of isotopes that can be incorporated into the compositions of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, and chlorine, such as... 2 H (or deuterium (D)), 3 H, 11 C 13 C 14 C 15 N、 18 O、 17 O、 31 P, 32 P, 35 S, 18 F and 36 Cl, for example 3 H, 11 C and 14 C. In one embodiment, a compound is provided in a composition wherein substantially all atoms at a certain position within the compound are replaced by atoms with different atomic masses or mass numbers. In another embodiment, a compound is provided wherein substantially all atoms at a certain position within the compound are replaced by deuterium atoms, for example, all hydrogen atoms of a -CH3 group are replaced by deuterium atoms to obtain a -CD3 group. In another embodiment, a compound is provided wherein a portion of the atoms at a certain position within the compound are replaced, i.e., a certain position of the compound of the present invention is enriched with atoms with different atomic masses or mass numbers. In another embodiment, a compound is provided wherein none of the atoms of the compound are replaced by atoms with different atomic masses or mass numbers. The isotopically labeled compounds of the present invention can be prepared by methods known in the art.

[0033] The compounds in the compositions of this invention contain one or more asymmetric centers, thus producing enantiomers, diastereomers, and other stereoisomers. This invention covers all such possible forms, as well as their racemic and resolved forms and the use of mixtures thereof. In view of this invention, individual enantiomers can be isolated according to methods known in the art.

[0034] As used in this invention, the term "stereoisomer" is a general term for all isomers of a single molecule that differ only in the spatial orientation of their atoms. Stereoisomers include enantiomers and isomers of compounds having more than one chiral center that are not mirror images of each other (diastereomers).

[0035] Unless otherwise stated, the stereochemical terminology and conventions used in this specification are consistent with those in [the original text]. Pure&Appl.Chem , No. 68 Those consistent with those described in Volume 2193 (1996).

[0036] As used in this invention, the term "about" includes ±10% of the referenced number. Thus, "about 10" refers to 9 to 11.

[0037] As used in this invention, the term "pharmaceutically acceptable salt" includes acid addition salts and base addition salts of compounds.

[0038] Suitable acid addition salts are formed from acids that form non-toxic salts. Examples include acetates, adipates, aspartates, benzoates, benzenesulfonates, bicarbonates / carbonates, hydrogen sulfates / sulfates, borates, camphorsulfonates, citrates, cyclohexylaminosulfonates, ethanedisulfonates, ethanesulfonates, formates, fumarates, glucohepanoates, glucuronates, hexafluorochlorophosphates, hydrochlorides, hydrobromide / bromine, hydroiodide / iodide, hydroxyethanesulfonates, lactates, malates, maleates, malonates, methanesulfonates, methyl sulfate, naphthates, 2-naphthalenesulfonates, nicotinates, nitrates, orotates, oxalates, palmitates, pamoates, phosphates / hydrogen phosphates, dihydropyrophosphate aldonicates, stearates, succinates, tannic acid, tartrates, toluenesulfonates, trifluoroacetates, and neofolates.

[0039] Suitable base addition salts are formed from bases that form non-toxic salts. Examples include aluminum salts, arginine salts, benzathine penicillin salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts. In a preferred embodiment, a suitable base addition salt is a sodium or potassium salt.

[0040] For a review of suitable salts, see “Handbook of Medicinal Salts: Properties, Selection and Use” by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds in the compositions of this invention are known to those skilled in the art.

[0041] As used herein, the term "solvent" refers to a substance formed by a combination, physical bonding, and / or solvation of the compounds of the present invention with solvent molecules, such as disolvents, monosolvents, or hemisolvents, wherein the ratio of solvent molecules to the compounds of the present invention is about 2:1, about 1:1, or about 1:2, respectively. This physical bonding involves, to varying degrees, ionization and covalent bonding (including hydrogen bonding). In some cases (e.g., when one or more solvent molecules are bound to the lattice of a crystalline solid), the solvate can be separated. Therefore, a solvate includes a solution phase and a separable solvate. The compounds of the present invention can be in a solvated form with pharmaceutically acceptable solvents (e.g., water, methanol, and ethanol), and the present invention is intended to include both solvated and unsolvated forms of the compounds.

[0042] One type of solvate is a hydrate. The term "hydrate" refers to a specific subset of solvates in which the solvent molecule is water. Solvates typically function as pharmacologically equivalents. The preparation of solvates is known in the art. Representative and non-limiting methods for preparing solvates involve dissolving the compound in a desired solvent (organic solvent, water, or a mixture thereof) at a temperature above 20°C to about 25°C, then cooling the solution at a rate sufficient to form crystals, and separating the crystals by known methods such as filtration. Analytical techniques such as infrared spectroscopy can be used to confirm the presence of the solvent in the solvate crystals.

[0043] In the context of this invention, "pharmaceutically acceptable carrier" means a diluent, adjuvant, excipient, or medium that is administered with a therapeutically active ingredient and is suitable for exposure to human and / or other animal tissues within a reasonable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications corresponding to a reasonable benefit / risk ratio.

[0044] Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions or cartridges of this invention include, but are not limited to, sterile liquids such as water and oils, including those derived from petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. The pharmaceutical composition may also contain small amounts of stabilizers, protectants, isotonic agents, preservatives, wetting agents, emulsifiers, or pH buffers, as needed. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

[0045] As used in this invention, "cancer / tumor metastasis" refers to cancer or tumor that has spread (metastasized) from its original site to another area of ​​the body. Almost all cancers or tumors have the potential to metastasize. Whether metastasis occurs depends on a complex interplay of various tumor cellular factors, including cancer type, the degree of maturity (differentiation) of tumor cells, the location and age of the cancer, and other factors that are not yet fully understood. There are three pathways of metastasis: spread from the local tumor to surrounding tissues, reaching distant sites via the bloodstream, or reaching adjacent or distant lymph nodes via the lymphatic system. Each type of cancer can have a representative pathway of spread. Tumors are named according to their primary site (e.g., breast cancer that has metastasized to the brain is called metastatic breast cancer that has metastasized to the brain).

[0046] About this invention In one aspect, the present invention provides a pharmaceutical composition for treating tumors, comprising, as an active ingredient: 1) Component A, which is selected from Compounds of formula (I) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (I) Among them, R1, R2, and R3 are each independently selected from H, C1-C 10 Alkyl, C1-C 18 Alkyl group, -P(=O)(OH)2, -S(=O)2(OH) and glucoside; R4 and R5 are each independently selected from H, OH, C1-C6 alkoxy groups and C1-C6 alkoxy groups. 18Alkyloxy group; or R4 and R5 together with the C atom attached to them to form an oxo group (-C(=O)); and / or Compounds of formula (II) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (II) Among them, R6, R7, R8, and R9 are each independently selected from H, C1-C. 10 Alkyl, C1-C 18 Alkyl groups, -P(=O)(OH)2, -S(=O)2(OH) and glucosides; R 10 Selected from H and OH; and 2) Component B is a compound selected from the following: sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, sodium ethoxide, and potassium ethoxide; Based on the total weight of the composition, the mass percentage of component A is 0.5-50%; and / or the mass percentage of component B is 0.5-50%; And optional pharmaceutically acceptable carriers or excipients.

[0047] In one embodiment, the compound of formula (I) is selected from L-ascorbic acid, L-ascorbyl glucoside, L-ascorbyl palmitate, L-ascorbyl-2-phosphate, L-ascorbyl-2-sulfate, D-ascorbic acid, 5-O-acetyl-L-ascorbic acid, 5-keto-L-ascorbic acid, and 5-diether-L-ascorbic acid. In a preferred embodiment, the compound of formula (I) is selected from L-ascorbic acid. Without wishing to be limited by any particular theory, the present invention has found that the above-preferred component A compound can achieve a superior tumor-suppressive effect.

[0048] In one embodiment, the compound of formula (II) is selected from citric acid.

[0049] In one embodiment, component A is selected from compounds of formula (I). In one embodiment, component A is selected from compounds of formula (II). In one embodiment, component A is selected from a combination of compounds of formula (I) and compounds of formula (II).

[0050] In one embodiment, component B is selected from sodium hydroxide or potassium hydroxide. In a preferred embodiment, component B is selected from sodium hydroxide. Not wishing to be limited by any particular theory, the present invention has found that the above-mentioned preferred component B can achieve superior tumor inhibition and elimination effects.

[0051] In one embodiment, the molar ratio between component A and component B is any value or range between 20:1 and 1:20. In a preferred embodiment, the molar ratio between component A and component B is 10:1 to 1:10. In a preferred embodiment, the molar ratio between component A and component B is 5:1 to 1:5. In a preferred embodiment, the molar ratio between component A and component B is 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, or any value or range therebetween, such as 1:1 to 1:5. In a preferred embodiment, the molar ratio between component A and component B is 2:1 to 1:2.

[0052] In one embodiment, the mass percentage of component A is 5-50% based on the total weight of the composition. In one embodiment, the mass percentage of component A is 10-30% based on the total weight of the composition. In one embodiment, the mass percentage of component A is 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, or any value or range therebetween, such as 10%-25%, based on the total weight of the composition.

[0053] In one embodiment, the mass percentage of component B is 5-50% based on the total weight of the composition. In one embodiment, the mass percentage of component B is 10-30% based on the total weight of the composition. In one embodiment, the mass percentage of component B is 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, or any value or range therebetween, such as 10%-25%, based on the total weight of the composition.

[0054] In one embodiment, the composition comprises component A, component B, and optionally a pharmaceutically acceptable carrier or excipient, i.e., no other components not listed. In another embodiment, the composition comprises component A, component B, and optionally a solvent.

[0055] In one embodiment, the composition is an injectable preparation.

[0056] In one embodiment, the composition is an intratumoral injection.

[0057] In one embodiment, the composition is an injection solution or a lyophilized powder for injection. In a preferred embodiment, the composition is an injection solution. In one embodiment, the solvent of the injection solution is water, ethanol, or other pharmaceutically available solvent types, preferably water, such as sterile water for injection.

[0058] In one embodiment, the mass-volume concentration of component A in the injection solution is 0.5-50%. In another embodiment, the mass-volume concentration of component A in the injection solution is 5-30%. In yet another embodiment, the mass-volume concentration of component A in the injection solution is 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, or any value or range therebetween, such as 10%-25%.

[0059] In one embodiment, the mass-volume concentration of component B in the injection solution is 0.5-50%. In another embodiment, the mass-volume concentration of component B in the injection solution is 5-30%. In yet another embodiment, the mass-volume concentration of component B in the injection solution is 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, or any value or range therebetween, such as 10%-25%.

[0060] In one embodiment, the pH of the injection solution is 9-14. In another embodiment, the pH of the injection solution is 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or any value or range therebetween, such as 10-13.5. In a preferred embodiment, the pH of the injection solution is 11-14. In a more preferred embodiment, the pH of the injection solution is 12-14, such as 12.5-13.5. In a most preferred embodiment, the pH of the injection solution is 13-13.5, such as about 13, 13.1, 13.2, 13.3, 13.4, or 13.5. Not wishing to be limited by any particular theory, the present invention has found that the above-mentioned preferred pH ranges can achieve superior tumor inhibition and elimination effects.

[0061] In one embodiment, the pharmaceutically acceptable carrier is, for example, but not limited to, sterile liquids such as water and oils, including oils derived from petroleum, animal, plant, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is an exemplary carrier when the pharmaceutical composition is administered by injection. Physiological saline and aqueous solutions of glucose and glycerol can also be used as liquid carriers, particularly for injection. Suitable pharmaceutical excipients include mannitol, glycerol, propylene glycol, water, ethanol, etc. As needed, the pharmaceutical composition may also contain a pharmaceutically acceptable carrier selected from stabilizers, protectants, isotonic agents, preservatives, wetting agents, emulsifiers, or pH buffers.

[0062] In another aspect, the present invention provides a pharmaceutical composition for treating tumors, which is prepared by mixing the following components: 1) Component A, which is selected from Compounds of formula (I) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (I) Among them, R1, R2, and R3 are each independently selected from H, C1-C 10 Alkyl, C1-C 18 Alkyl group, -P(=O)(OH)2, -S(=O)2(OH) and glucoside; R4 and R5 are each independently selected from H, OH, C1-C6 alkoxy groups and C1-C6 alkoxy groups. 18 Alkyloxy group; or R4 and R5 together with the C atom attached to them to form an oxo group (-C(=O)); and / or Compounds of formula (II) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (II) Among them, R6, R7, R8, and R9 are each independently selected from H, C1-C. 10 Alkyl, C1-C 18 Alkyl groups, -P(=O)(OH)2, -S(=O)2(OH) and glucosides; R 10 Selected from H and OH; and 2) Component B, which is a compound selected from the following: sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, sodium ethoxide, potassium ethoxide; wherein, based on the total weight of the composition, the mass percentage of component A is 0.5-50%; and / or the mass percentage of component B is 0.5-50%; And 3) optional pharmaceutically acceptable carriers or excipients.

[0063] In one embodiment, preferred options for the characteristics of the pharmaceutical compositions described above are as defined throughout the present invention.

[0064] In another aspect, the present invention provides an intratumoral injection composition for treating tumors, comprising, as an active ingredient: 1) Component A, which is selected from Compounds of formula (I) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (I) Among them, R1, R2, and R3 are each independently selected from H, C1-C 10 Alkyl, C1-C 18Alkyl group, -P(=O)(OH)2, -S(=O)2(OH) and glucoside; R4 and R5 are each independently selected from H, OH, C1-C6 alkoxy groups and C1-C6 alkoxy groups. 18 Alkyloxy group; or R4 and R5 together with the C atom attached to them to form an oxo group (-C(=O)); and / or Compounds of formula (II) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (II) Among them, R6, R7, R8 and R9 are each independently selected from H, C1-C6 alkyl, C1-C 18 Alkyl groups, -P(=O)(OH)2, -S(=O)2(OH) and glucosides; R 10 Selected from H and OH; and 2) Component B is a compound selected from the following: sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, sodium ethoxide, and potassium ethoxide; And optionally a pharmaceutically acceptable carrier or excipient, wherein the pH of the injection solution is 9-14.

[0065] In a preferred embodiment, the pH of the injection solution is 12-14.

[0066] In one embodiment, the mass-volume concentration of component A in the injection solution is 0.5-50%.

[0067] In one embodiment, the mass-volume concentration of component B in the injection solution is 0.5-50%.

[0068] In one embodiment, the molar ratio between component A and component B is 20:1 to 1:20.

[0069] In one embodiment, preferred options for the above-described composition features are as defined throughout the invention.

[0070] In another aspect, the present invention provides a method for preparing the composition of the present invention, which includes the step of mixing component A, component B and optionally a pharmaceutically acceptable carrier or excipient.

[0071] In one embodiment, the mixing includes preparing solutions of combination A and component B separately, and then uniformly mixing them. In one embodiment, the mixing includes preparing a solution of combination A or component B, and then adding another component in solid form to the solution. In one embodiment, the mixing is carried out at ambient temperature. In one embodiment, the mixing is carried out at room temperature. In one embodiment, the mixing includes stirring or sonication. In one embodiment, the preparation method further includes the step of lyophilizing or drying the resulting mixture.

[0072] In another aspect, the present invention provides the use of the compositions of the present invention in a medicament for treating tumors.

[0073] In one implementation, the tumor is a solid tumor.

[0074] In one embodiment, the tumor is selected from: carcinoma, sarcoma, Kaposi's sarcoma, malignant glioma, meningioma, astrocytoma, melanoma, myoblastoma, brain cancer, skin cancer, adenocarcinoma, malignant epithelial tumor, urinary system tumor, prostate cancer, urothelial carcinoma, bladder urothelial carcinoma, bladder cancer, ovarian cancer, breast cancer, uterine cancer, pancreatic cancer, liver cancer, colon cancer, lung adenocarcinoma, lung cancer, small cell lung cancer, bone cancer, neuroblastoma, intestinal cancer, colorectal cancer, rectal cancer, colon cancer, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, medullary thyroid carcinoma, papillary thyroid carcinoma, kidney cancer, renal parietal cell carcinoma, cervical cancer, cervical squamous cell carcinoma and adenocarcinoma, uterine corpus cancer, endometrial cancer, choriocarcinoma, testicular cancer, invasive breast cancer, urethral cancer, melanoma, brain tumor. Glioma, astrocytoma, meningioma, medulloblastoma, peripheral neuroectodermal tumors, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, hepatocellular carcinoma, gallbladder cancer, bronchogenic carcinoma, small cell lung cancer, non-small cell lung cancer, head and neck tumors, basal cell carcinoma, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, craniopharynx, thyroid tumors, osteosarcoma, chondrosarcoma, myoma, liposarcoma, fibrosarcoma, Ewing's sarcoma, and plasmacytoma.

[0075] In one embodiment, the tumor is selected from: squamous cell carcinoma of the lung, adenocarcinoma of the lung, adenosquamous carcinoma of the lung, gastric adenocarcinoma, rectal adenocarcinoma, colonic adenocarcinoma, esophageal squamous cell carcinoma, greater omentum adenocarcinoma, ovarian adenocarcinoma, esophageal adenocarcinoma, small intestinal adenocarcinoma, small cell neuroendocrine gastric cancer, gastric squamous cell carcinoma, pelvic adenocarcinoma, pancreatic cancer, glioma, pelvic adenocarcinoma, and liver cancer.

[0076] In one embodiment, the drug further comprises one or more adjunctive therapeutic agents, or is administered co-administered therewith. In one embodiment, the one or more adjunctive therapeutic agents are selected from anticancer agents, such as chemotherapeutic agents, antibody preparations, ADCs, nucleic acid drugs, etc.

[0077] In another aspect, the present invention provides a method for treating tumors, comprising administering the composition described herein.

[0078] In one embodiment, the administration is an intratumoral injection.

[0079] The therapeutically effective amount of the composition of the invention required for use in treatment varies depending on the nature of the condition being treated, the duration of activity required, and the age and condition of the subject, and is ultimately determined by the attending physician. Dosage and intervals can be individually adjusted to provide intratumoral levels of the composition of the invention sufficient to maintain the desired therapeutic effect. The desired dose can be administered as a single dose or as multiple doses at appropriate intervals.

[0080] In one embodiment, the composition of the present invention achieves significantly improved antitumor activity and safety compared to the individual application of the components therein (e.g., sequential application).

[0081] In another aspect, the present invention provides a kit comprising the compositions of the present invention, the compositions being packaged in a manner that facilitates their use in practicing the methods of the present invention. In one embodiment, the kit comprises the compositions of the present invention, and instructions for administering the compositions to a subject suffering from a tumor. In one embodiment, the compounds or compositions are packaged in unit dosage forms. The kit may also include means adapted to administer the compositions according to the intended route of administration.

[0082] This invention combines ascorbic acid or its analogues and / or citric acid or its analogues with sodium hydroxide or its analogues, and administers the mixture via intratumoral injection in an alkaline environment to achieve safe and effective antitumor efficacy. It is not intended to be limited by specific theories, but it is believed that the above components in the composition of this invention can achieve synergistic antitumor effects, while also achieving improved safety and improved efficacy.

[0083] The composition of the present invention has the following beneficial effects: 1) Excellent tumor treatment efficacy: The composition of the present invention can not only completely inhibit the growth of tumors, but also significantly reduce the tumor volume, and even achieve complete elimination of tumors at the administration site, thereby achieving the purpose of completely treating tumors.

[0084] 2) Excellent in vivo safety: When the composition of the present invention is administered intratumorally, it can kill tumor cells in situ without having a substantial effect on normal cells, tissues, nerves and blood vessels outside the tumor of the subject, and it will not have a substantial adverse effect on other organs and their functions of the patient, thus having excellent in vivo safety.

[0085] 3) Single-dose administration / rapid onset of action: The composition of the present invention can achieve excellent tumor treatment effect with only a single dose, without the need for multiple or long-term administration; animal experimental results show that it can kill tumors within hours after administration, significantly reduce tumor volume within 3 days after administration, and completely eliminate tumors within 21 days after administration, with a rapid onset of action.

[0086] 4) Excellent intratumoral drug diffusion performance: After local injection, the composition of the present invention can rapidly diffuse and take effect within the tumor, reaching the edge of the tumor and achieving effective inactivation of the entire tumor within the "visible" range. 5) Long-term inhibition of tumor recurrence: Long-term animal experimental studies have shown that the composition of the present invention can achieve no tumor recurrence for an observation period of more than one year after a single administration, and truly achieve complete cure of tumor.

[0087] Example To make the objectives and technical solutions of this invention clearer, the invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are not intended to limit the scope of the invention. Furthermore, specific experimental methods not mentioned in the following embodiments are performed according to conventional experimental methods.

[0088] The embodiments of the present invention employ the following equipment: 1. Leica-APS300S Advanced Intelligent Dehydrator 2. Leica-HistoCore Arcadia H Paraffin Embedding Machine 3. Leica-HistoCore Arcadia C paraffin-embedded cold stage 4. Leica-HistoCoreBIOCUT Manual Rotary Paraffin Slicer 5. PHY-III Pathological Tissue Drying Instrument 6. Anhui Jiawen DHG-9075A forced-air drying oven 7. Thermo Scientific-Gemini AS Fully Automatic Slide Staining Machine 8. Leica CV5030 Automatic Cover Slicer 9. Jiangfeng KF-PRO-005-EX Fully Automated Digital Pathology Slide Scanner 10. Waters ACQUITY I-Class UPLC (Ultra-High Performance Liquid Chromatography) 11. Waters SYNAPT XS High Resolution Mass Spectrometer 12. Agilent 1260 HPLC Infinity II 12. Sartorius BCE series precision analytical balance 13. POLYTRON PT2500E Tissue Grinder 14. Bruker Avance NEO 800 NMR Spectrometer 15. SevenDirect SD20 pH meter, Mettler Toledo pH meter 16. Vevo LAZRTIGHT Vevo 3100 High-Resolution Small Animal Ultrasound Imaging System 17. Vevo LAZR-X High-Resolution Small Animal Ultrasonic Photoacoustic Imaging Chamber.

[0089] Preparation Examples All raw materials were commercially available and had a purity of ≥99%; the water used was sterile distilled water or sterile water for injection; pH was measured using a Mettler SevenDirect SD20 pH meter.

[0090] pH adjustment solution preparation Prepare 1 mmol / ml and 0.1 mmol / ml NaOH solutions for pH adjustment: Dilute 4 g of NaOH with sterile distilled water to 100 ml to obtain 100 ml of 1 mmol / ml NaOH solution, then pour out 10 ml and dilute to 100 ml to obtain 0.1 mmol / ml NaOH solution; Prepare 1 mmol / ml and 0.1 mmol / ml HCl solutions for pH adjustment: Dilute 8.3 ml of concentrated hydrochloric acid with sterile distilled water to 100 ml to obtain 100 ml of 1 mmol / ml HCl solution. Pour out 10 ml of the HCl solution and dilute it to 100 ml to obtain 0.1 mmol / ml HCl solution.

[0091] Preparation of control samples Control sample 1-1 (1% NaOH solution alone (pH=13.40)): Under argon atmosphere and at room temperature, 0.15 mol (6 g) of solid sodium hydroxide was first dissolved in a small amount of water, stirred slowly, and water was added to 600 g to prepare 600 g of 1% sodium hydroxide solution. The pH of the resulting solution was measured to be 13.40. The solution was then sealed in 2 ml ampoules and stored at low temperature.

[0092] Control samples 1-2 (isolated saturated NaOH solutions (used to prepare the NaOH stock solution for samples 1-5) (pH=13.857)): Under argon atmosphere and at room temperature, 1.5 mol (60 g) of solid sodium hydroxide was first dissolved in a small amount of water, stirred slowly, and then 100 ml of water was added to prepare the saturated sodium hydroxide solution.

[0093] Control sample 2-1 (ascorbic acid solution alone (pH=2.013)): Under argon atmosphere and at room temperature, 0.1 mol (17.6 g) of ascorbic acid was dissolved in sterile distilled water and the solution was adjusted to 100 ml; the pH was measured to be 2.013; 20 ml of the solution was taken out, purged with argon gas and sealed into 2 ml ampoules, and stored in the dark and at low temperature. Control sample 2-2 (single saturated ascorbic acid solution (used to prepare the stock solution of ascorbic acid for samples 1-5 (pH=1.65))): Under argon atmosphere and at room temperature, accurately weigh 0.142 mol (25 g) of L-ascorbic acid solid using a balance, first add a small amount of water to dissolve it, stir slowly, add 100 ml of water to prepare the saturated L-ascorbic acid solution; Control sample 3-1 (hydrochloric acid and sodium hydroxide solution (pH=7.00)): Take 4.17 ml of concentrated hydrochloric acid (12 mol / L) and mix it with 2.64 ml of saturated NaOH (19 mol / L) solution. While stirring slowly, add sterile distilled water to the mixed solution system to 100 ml; measure pH=7.00 (25℃); take 20 ml of the solution, seal it in a 2 ml ampoule, and store it at low temperature.

[0094] Control sample 4-1 (L-ascorbic acid and berberine solution (pH=10.05)): Under argon atmosphere and at room temperature, 50 ml of sterile distilled water solution containing 0.1 mol (37.18 g) berberine hydrochloride was added dropwise to 30 ml of sterile distilled water solution containing 0.1 mol (17.61 g) L-ascorbic acid, forming a paste. 20 ml of water was added, and the mixture was heated to 35°C. 10 ml of 0.1 mol (4 g) NaOH solid solution was added dropwise, forming a brown liquid. After standing for 2 hours, the pH was measured to be 10.21 (25.0°C). 0.7 ml of 0.1 mmol / ml HCl solution was added dropwise using a syringe needle to adjust the pH, and the resulting brown solution had a pH of 10.05 (25.0°C).

[0095] Control sample 4-2 (L-ascorbic acid and berberine solution (pH=13.50)): Take 20 ml of the above pH=10.05 solution, add 0.3 g of solid NaOH under argon atmosphere, and then add 1 mmol / ml NaOH solution to adjust; the pH=13.50 (25.0℃) brownish-black solution was measured.

[0096] Control sample 5-1 (solution of L-ascorbic acid and matrine (pH=6.27)): Under argon atmosphere and at room temperature, 50 ml of sterile distilled water containing 0.1 mol (24.84 g) matrine was added dropwise to 30 ml of sterile distilled water suspension containing 0.1 mol (17.61 g) L-ascorbic acid. The mixture was stirred for 0.5 h and then diluted to 100 ml of clear solution. The pH was measured to be 6.27 (25.0℃).

[0097] Control sample 6-1 (a solution of citric acid and matrine (pH=3.67)): Under an argon atmosphere and at room temperature, 30 ml of sterile distilled aqueous solution of 0.1 mol (19.21 g) citric acid was added dropwise to 50 ml of sterile distilled aqueous solution of 0.1 mol (24.84 g) matrine, stirred for 0.5 h, and then diluted to 100 ml; the pH was measured to be 3.67 (25.0℃). Control sample 6-2 (a solution of citric acid and matrine (pH=7.216)): Take 20 ml (0.02 mmol) of the above-prepared solution with pH=3.67, stir under nitrogen, and add 9.92 g (0.04 mmol) of matrine in 4 portions; heat slightly to 30 °C, and measure the pH of the solution to be measured as 7.216 (25.0 °C).

[0098] Control sample 7-1 (a solution of citric acid and berberine (pH=6.105)): Under an argon atmosphere and at room temperature, 1.921 g of 0.01 mol citric acid was added to 10 ml of sterile distilled water, and 3.718 g of 0.01 mol berberine hydrochloride was added to the above citric acid solution; the volume was prepared to 20 ml; the sample was a red liquid, and the pH was 6.105 (25.0℃).

[0099] Invention Sample Preparation 1. Solution of L-ascorbic acid and sodium hydroxide Under an argon atmosphere and at room temperature, 1.5 mol (60 g) of solid sodium hydroxide was first dissolved in a small amount of water, stirred slowly, and then 100 ml of water was added to prepare a saturated sodium hydroxide solution.

[0100] Under an argon atmosphere and at room temperature, accurately weigh 0.142 mol (25 g) of L-ascorbic acid solid using a balance, dissolve it in a small amount of water, stir slowly, add 100 ml of water, and prepare a saturated L-ascorbic acid solution.

[0101] Sample 1-1 (pH=11.00): Under argon atmosphere and at room temperature, take 39.16 g of the above saturated solution containing 44.49 mmol of L-ascorbic acid, add 7.11 g of the above saturated solution containing 66.66 mmol of sodium hydroxide dropwise with a syringe, mix and stir to obtain a clear solution sample, and measure pH=11.00 (25℃); seal in a 2 ml ampoule under nitrogen atmosphere, protect from light, and store at low temperature.

[0102] Samples 1-2 (pH=12.00): Under argon atmosphere and at room temperature, take 30.0 g of the above saturated solution containing 34.08 mmol of L-ascorbic acid, add 6.897 g of the above saturated solution containing 64.66 mmol of sodium hydroxide dropwise with a syringe needle, mix and stir to obtain a clear solution sample, and measure pH=12.00 (25℃); seal in a 2 ml ampoule under nitrogen atmosphere, protect from light, and store at low temperature.

[0103] Samples 1-3 (pH=13.00): Under argon atmosphere and at room temperature, take 30.0 g of the above saturated solution containing 34.08 mmol of L-ascorbic acid, add 8.554 g of the above saturated solution containing 80.19 mmol of sodium hydroxide dropwise with a syringe, mix and stir to obtain a clear solution sample, and measure pH=13.00 (25℃); seal in 2 ml ampoules under nitrogen atmosphere, protect from light, and store at low temperature.

[0104] Samples 1-4 (pH=13.92): Under argon atmosphere and at room temperature, take 15.046 g of the above saturated solution containing 17.09 mmol of L-ascorbic acid, add 22.543 g of the above saturated solution containing 211.34 mmol of sodium hydroxide dropwise with a syringe, mix and stir to obtain a clear solution sample, and measure pH=13.92 (25℃); seal in 2 ml ampoules under nitrogen atmosphere, protect from light, and store at low temperature.

[0105] Samples 1-5 (pH=13.30): Under argon atmosphere and at room temperature, take 590g of the above saturated solution containing 670.15mmol of L-ascorbic acid, add 175.08g of the above saturated solution containing 1641.05mmol of sodium hydroxide dropwise with a syringe, mix and stir to obtain a clear solution sample, and measure pH=13.30 (25℃); seal in 2ml ampoules under nitrogen atmosphere, protect from light, and store at low temperature.

[0106] Samples 1-6 (pH=7.00): Under argon atmosphere and at room temperature, 0.1 mol (4.0 g) NaOH was added to 15 ml of sterile distilled water, and then 10 ml of sterile distilled water was added dropwise until dissolved. The solution was cooled to 5°C. 0.1 mol (17.6 g) L-ascorbic acid was weighed and dissolved in 30 ml of sterile distilled water. The NaOH solution was added dropwise to the L-ascorbic acid suspension and stirred for 0.5 h. The volume of the aqueous solution was then adjusted to 100 ml. The pH was measured to be 5.89. 20 ml of the solution was taken out and adjusted to pH=7.00 (25.0°C) with 1.5 ml of 0.1 mmol / ml NaOH solution. The solution was then sealed in 2 ml ampoules under argon atmosphere and stored in the dark and at low temperature.

[0107] Samples 1-7 (pH=10.00): Take 20 ml of the pH=5.89 solution prepared above, and adjust the pH to 10.00 (25.0℃) with 1.1 ml of 1 mmol / ml NaOH solution under argon atmosphere; seal the solution in 2 ml ampoules under argon atmosphere and store in the dark and at low temperature.

[0108] Samples 1-8 (pH=11.80): Take 20 ml of the pH=5.89 solution prepared above, and adjust the pH to 11.80 (25.0℃) with 0.75 g solid NaOH under argon gas; seal the solution in 2 ml ampoules with argon gas, and store in the dark and at low temperature.

[0109] Samples 1-9 (pH=13.50): Take 20 ml of the pH=5.89 solution prepared above, and adjust the pH to 13.50 (25.0℃) with 2.52 g of solid NaOH under argon gas; seal the solution in 2 ml ampoules with argon gas, and store in the dark and at low temperature.

[0110] Samples 1-10 (NaOH concentration is 10% of that in Samples 1-3): Under argon atmosphere and at room temperature, take 30.0 g of a saturated solution containing 34.08 mmol of L-ascorbic acid, add 0.8554 g of a saturated solution containing 8.019 mmol of sodium hydroxide dropwise using a syringe, mix and stir, then add 7.699 g of water to obtain a clear solution sample; seal it in a 2 ml ampoule under nitrogen atmosphere, protect it from light, and store it at low temperature.

[0111] Sample 1-11 (NaOH concentration is 30% of that in Sample 1-3): Under argon atmosphere and at room temperature, take 30.0 g of a saturated solution containing 34.08 mmol of L-ascorbic acid, add 2.566 g of a saturated solution containing 24.057 mmol of sodium hydroxide dropwise using a syringe, mix and stir, then add 5.988 g of water to obtain a clear solution sample; seal it in a 2 ml ampoule under nitrogen atmosphere, protect it from light, and store it at low temperature.

[0112] Samples 1-12 (NaOH concentration is 80% of that in Samples 1-3): Under argon atmosphere and at room temperature, take 30.0 g of a saturated solution containing 34.08 mmol of L-ascorbic acid, add 6.843 g of a saturated solution containing 64.152 mmol of sodium hydroxide dropwise using a syringe, mix and stir, then add 1.711 g of water to obtain a clear solution sample; seal it in a 2 ml ampoule under nitrogen atmosphere, protect it from light, and store it at low temperature.

[0113] 2. Solution of D-ascorbic acid and sodium hydroxide Sample 2-1 (pH=7.00): Under argon atmosphere and at room temperature, take 0.1 mol (4.0 g) NaOH, add 50 ml of sterile distilled water, cool to 5 °C, and add dropwise to 0.1 mol (17.61 g) D-ascorbic acid. Stir until clear and dilute to 100 ml. The pH was measured to be 5.75 (25.2 °C), and a precipitate appeared. Adjust the pH to 7.00 (25.0 °C) with 1.5 ml of 1 mmol / ml NaOH solution. Take 20 ml of the solution, fill it with argon gas, seal it in a 2 ml ampoule, and store it in the dark and at low temperature.

[0114] Sample 2-2 (pH=10.05): Take 20 ml of the above solution with pH=7.00, and under argon atmosphere, add 2.2 ml of 1 mmol / ml NaOH solution dropwise with a syringe needle; the pH was measured to be 10.05 (25.0℃); seal the solution in a 2 ml ampoule under argon atmosphere and store it in the dark and at low temperature.

[0115] Sample 2-3 (pH=11.80): Take 20 ml of the above solution with pH=7.00, add 0.64 g of solid NaOH under argon gas, and then add 0.7 ml of 1 mmol / ml NaOH solution. The pH was measured to be 11.80 (25.3℃). The solution was then sealed in 2 ml ampoules under argon gas and stored in the dark and at low temperature.

[0116] Samples 2-4 (pH=13.50): Take 20 ml of the above solution with pH=7.00, add 3.03 g of solid NaOH under argon gas, and measure the pH=13.50 (25.0℃); seal the solution in 2 ml ampoules with argon gas, and store in the dark and at low temperature.

[0117] 3. Solution of sodium D-ascorbate and sodium hydroxide Sample 3-1 (pH=10.00): Under argon atmosphere and at room temperature, 0.1 mol (19.81 g) of sodium D-ascorbate was added to 70 ml of sterile distilled water, and then sterile distilled water was added to a final volume of 100 ml. The solution was heated until completely dissolved, and the pH was measured to be 6.90 (25.0℃). The solution was colorless and clear. 20 ml of the above sodium D-ascorbate solution with pH=6.90 was taken, and under argon atmosphere, 2.0 ml of 1 mmol / ml NaOH solution was added dropwise using a syringe needle. The pH was measured to be 10.00 (25.1℃), and the solution was pale yellow. The solution was then sealed in 2 ml ampoules under argon atmosphere and stored in the dark and at low temperature.

[0118] Sample 3-2 (pH=11.80): Take 20 ml of the above D-ascorbic acid sodium solution with pH=6.90, add 0.75 g of solid NaOH, and then add 0.75 ml of 1 mmol / ml NaOH solution dropwise with a syringe. The pH was measured to be 11.80 (24.5℃), which is a light yellow solution. Seal the solution in 2 ml ampoules with argon gas and store it in the dark and at low temperature.

[0119] Sample 3-3 (pH=13.114): Take 20 ml of the above D-ascorbic acid sodium solution with pH=6.90, add 3.6 g of solid NaOH under argon gas; the pH was measured to be 13.114 (25.0℃); it is a yellow-red liquid; seal it in a 2 ml ampoule filled with argon gas, and store it in the dark and at low temperature.

[0120] 4. Solution of L-ascorbic acid-2-glucoside and sodium hydroxide Sample 4-1 (pH=10.01): Under argon atmosphere, 0.1 mol (33.83 g) of L-ascorbic acid glucoside was dissolved in 50 ml of sterile distilled water, and then sterile distilled water was added to a final volume of 100 ml; the pH was measured to be 6.20 (25.0℃). 20 ml of the above pH=6.20 solution was taken, and under argon atmosphere, 0.5 ml of 1 mmol / ml NaOH solution was added; the pH was measured to be 10.01 (25.0℃). The solution was then sealed in argon atmosphere into 2 ml ampoules and stored in a dark, low-temperature environment.

[0121] Sample 4-2 (pH=11.80): Take 20 ml of the above pH=6.20 solution, add 0.06 g of solid NaOH under argon gas, and measure the pH=11.80 (25.0℃); seal it in a 2 ml ampoule under argon gas and store it in the dark and at low temperature.

[0122] Sample 4-3 (pH=13.50): Take 20 ml of the above pH=6.20 solution, add 0.5 g of solid NaOH under argon gas, and measure the pH=13.50 (25.0℃); seal it in a 2 ml ampoule under argon gas and store it in the dark and at low temperature.

[0123] 5. Solution of L-ascorbic acid-2-phosphate trisodium salt and sodium hydroxide Sample 5-1 (pH=10.09): Under argon atmosphere and at room temperature, 0.1 mol (32.21 g) of L-ascorbic acid-2-phosphate trisodium salt was added to 50 ml of sterile distilled water to dissolve it, and then sterile distilled water was added to 100 ml; the pH was measured to be 8.62 (25.0℃). 20 ml of the above pH=8.62 solution was taken, and 0.18 ml of 1 mmol / ml NaOH solution was added under argon atmosphere, and the pH was measured to be 10.09 (25.1℃), which was a colorless and clear liquid; it was then sealed in 2 ml ampoules under argon atmosphere and stored in the dark and at low temperature.

[0124] Sample 5-2 (pH=11.86): Take 20 ml of the above pH=8.62 solution, add 0.7 ml of 1 mmol / ml NaOH solution under argon gas, and measure the pH=11.86 (25.1℃). It is a colorless and clear liquid. Seal it into a 2 ml ampoule with argon gas and store it in the dark and at low temperature.

[0125] Sample 5-3 (pH=13.50): Take 20 ml of the above solution with pH=8.62, add 0.8 g of solid NaOH under argon gas, and measure the pH=13.50 (25.3℃); seal it in a 2 ml ampoule with argon gas, and store it in the dark and at low temperature.

[0126] 6. Solution of L-ascorbic acid sodium and sodium hydroxide Sample 6-1 (pH=10.00): Under nitrogen atmosphere and at room temperature, 19.8 g of L-ascorbic acid sodium was added to sterile distilled water to a final volume of 100 ml; the color was slightly yellow; the pH was measured to be 7.28 (25.0℃). 20 ml of the above L-ascorbic acid sodium solution (pH=7.28) was taken, and 0.8 ml of 1 mmol / ml NaOH solution was added dropwise using a syringe under nitrogen atmosphere, resulting in a pH of 10.00 (25.0℃); the solution was then sealed in a 2 ml ampoule under nitrogen atmosphere and stored in a dark, low-temperature environment.

[0127] Sample 6-2 (pH=11.80): Take 20 ml of the above L-ascorbic acid sodium solution with pH=7.28, add 0.55 g of solid NaOH under nitrogen atmosphere, and measure pH=11.80 (25.0℃); seal it in a 2 ml ampoule under nitrogen atmosphere and store it in the dark and at low temperature.

[0128] Sample 6-3 (pH=13.50): Take 20 ml of the above L-ascorbic acid sodium solution with pH=7.28, add 2.18 g of solid NaOH under nitrogen atmosphere, and measure the pH=13.50 (25.0℃); seal it in a 2 ml ampoule under nitrogen atmosphere and store it in the dark and at low temperature.

[0129] 7. Solution of sodium D-isoascorbate and sodium hydroxide Sample 7-1 (pH=10.09): Under nitrogen atmosphere and at room temperature, 0.1 mol (19.81 g) of D-isoascorbic acid sodium was added to 70 ml of sterile distilled water, and then sterile distilled water was added to 100 ml to obtain a saturated solution. The pH was measured to be 7.05 (25.0℃). 20 ml of the above D-isoascorbic acid sodium solution with pH=7.05 was taken, and 2.7 ml of 1 mmol / ml NaOH solution was added dropwise with a syringe under nitrogen atmosphere. Then, 0.03 g of solid NaOH was added, and the pH was measured to be 10.09 (25.0℃). The solution was then sealed in a 2 ml ampoule under nitrogen atmosphere and stored in the dark and at low temperature.

[0130] Sample 7-2 (pH=11.81): Take 20 ml of the above-mentioned sodium D-isoascorbate solution with pH=7.05, add 0.42 g of solid NaOH under nitrogen atmosphere, and then add 0.65 ml of 1 mmol / ml NaOH solution dropwise using a 2.5 ml syringe needle. The pH was measured to be 11.81 (25.0℃). The solution was then sealed in a 2 ml ampoule under nitrogen atmosphere and stored in a dark and low-temperature environment.

[0131] Sample 7-3 (pH=13.50): Take 20 ml of the above-mentioned sodium D-isoascorbate solution with pH=7.05, add 2.02 g of solid NaOH under nitrogen atmosphere; measure pH=13.50 (25.1℃); seal with nitrogen atmosphere into 2 ml ampoules, and store in the dark and at low temperature.

[0132] 8. Solution of L-calcium ascorbate and sodium hydroxide Sample 8-1 (pH=10.04): Under nitrogen atmosphere and at room temperature, 0.1 mol (42.62 g) of calcium ascorbate dihydrate was added to 50 ml of sterile distilled water and stirred to dissolve. The volume of the aqueous solution was then adjusted to 100 ml, resulting in a pale yellow solution with a pH of 6.38 (25.1℃). 20 ml of the pH=6.38 solution was taken, and 0.98 g of solid NaOH was added under nitrogen atmosphere, resulting in a pH of 10.04 (25.0℃). The solution was then sealed in 2 ml ampoules under nitrogen atmosphere and stored in the dark and at low temperature.

[0133] Sample 8-2 (pH=11.80): Take 20 ml of the above solution with pH=6.38, add 1.6 g of solid NaOH under nitrogen atmosphere, and measure the pH to be 11.80 (25.0℃); it is a deep yellow liquid. Seal it in 2 ml ampoules under nitrogen atmosphere and store it in the dark and at low temperature.

[0134] Sample 8-3 (pH=13.50): Take 20 ml of the above pH=6.38 solution, add 3.85 g of solid NaOH under nitrogen atmosphere; the pH was measured to be 13.50 (25.1℃); the liquid was wine-red. Seal the solution in 2 ml ampoules under nitrogen atmosphere and store in a dark and low-temperature environment.

[0135] 9. Solution of L-ascorbic acid potassium and sodium hydroxide Sample 9-1 (pH=10.00): Under nitrogen atmosphere and at room temperature, 21.4 g of L-ascorbic acid potassium was taken and diluted with distilled water to 100 ml, and the pH was measured to be 6.317 (25.0℃). 20 ml of the pH=6.317 L-ascorbic acid potassium solution was taken, and 2.25 ml of 1 mmol / ml NaOH was added dropwise using a syringe under nitrogen atmosphere, and the pH was measured to be 10.00 (25.0℃). The solution was then sealed in a 2 ml ampoule under nitrogen atmosphere and stored in a dark, low-temperature environment.

[0136] Sample 9-2 (pH=11.80): Take 20 ml of L-ascorbic acid potassium solution with pH=6.317, add 0.61 g of solid NaOH under nitrogen atmosphere, and measure pH=11.80 (25.2℃); seal with nitrogen atmosphere into 2 ml ampoules, and store in the dark and at low temperature.

[0137] Sample 9-3 (pH=13.50): Take 20 ml of L-ascorbic acid potassium solution with pH=6.317, add 2.41 g of solid NaOH under nitrogen atmosphere, and measure pH=13.50 (25.0℃); seal with nitrogen atmosphere into 2 ml ampoules, and store in the dark and at low temperature.

[0138] 10. A solution of citric acid and sodium hydroxide Sample 10-1 (pH=7.00): Under nitrogen atmosphere, 0.3006 mol (12.0 g) NaOH solid was added to 50 ml of sterile distilled water and cooled to 10 °C; it was then added dropwise to a solution of 0.1 mol (19.2 g) citric acid in 20 ml of sterile distilled water, and the mixture was heated to 40 °C over 30 minutes; sterile distilled water was added to a final volume of 100 ml; the pH was measured to be 6.53; 1.8 ml of 1 mmol / ml NaOH was added dropwise, and the pH was measured to be 7.00 (25.0 °C); 20 ml of the solution was removed, sealed in argon gas into 2 ml ampoules, and stored in the dark and at low temperature.

[0139] Sample 10-2 (pH=10.00): Take 20 ml of the above pH=7.00 solution, add 0.3 ml of 1 mmol / ml NaOH solution under nitrogen atmosphere, and measure pH=10.00 (25.0℃); seal it in a 2 ml ampoule under argon atmosphere and store it in the dark and at low temperature.

[0140] Sample 10-3 (pH=11.81): Take 20 ml of the above pH=7.00 solution, add 0.35 ml of 1 mmol / ml NaOH solution under nitrogen atmosphere, and measure pH=11.81 (25.0℃); seal it in a 2 ml ampoule under argon atmosphere and store it in the dark and at low temperature.

[0141] Sample 10⁻⁴ (pH=13.50): Take 20 ml of the above pH=7.00 solution, add 0.98 g of solid NaOH under nitrogen atmosphere, and measure the pH=13.50 (25.0℃); seal it in a 2 ml ampoule under argon atmosphere, and store it in the dark and at low temperature; Sample 10-5 (pH=13.62): Under argon atmosphere and room temperature, 1.5 mol (60 g) of solid sodium hydroxide was dissolved in a small amount of water, stirred slowly, and then 100 ml of water was added to prepare a saturated sodium hydroxide solution. Under argon atmosphere and room temperature, 0.3125 mol (60 g) of anhydrous citric acid solid was accurately weighed using a balance, dissolved in a small amount of water, stirred slowly, and then 100 ml of water was added to prepare a saturated citric acid solution. Under room temperature and nitrogen atmosphere, 14.265 g of the above saturated solution containing 0.028 mol of citric acid was taken, and 15 g of the above saturated solution containing 0.140 mol of sodium hydroxide was added dropwise using a syringe. The mixture was stirred for 30 minutes to obtain the sample, and the pH was measured to be 13.62 (25℃). The sample was then sealed in a 2 ml ampoule under nitrogen atmosphere and stored in the dark and at low temperature.

[0142] 11. Solutions of L-ascorbic acid with other bases Sample 11-1 (pH=13.30): Under argon atmosphere and at room temperature, take 15.046 g of a saturated solution containing 17.09 mmol of L-ascorbic acid, add 4.629 g of a saturated solution containing potassium hydroxide dropwise with a syringe needle, mix and stir to obtain a clear solution sample, and measure pH=13.30 (25℃); seal in a 2 ml ampoule under nitrogen atmosphere, protect from light, and store at low temperature.

[0143] Sample 11-2 (pH=13.30): Under argon atmosphere and at room temperature, take 15.046 g of a saturated solution containing 17.09 mmol of L-ascorbic acid, add 6.280 g of a saturated solution containing sodium ethoxide dropwise with a syringe needle, mix and stir to obtain a clear solution sample, and measure pH=13.30 (25℃); seal in a 2 ml ampoule under nitrogen atmosphere, protect from light, and store at low temperature.

[0144] 12. A solution of L-ascorbic acid, citric acid, and sodium hydroxide. Sample 12-1 (pH=13.30, 50 / 50 saturated solution mass ratio): Under argon atmosphere and at room temperature, take 290g of a saturated solution containing 0.329mol L-ascorbic acid and mix thoroughly with 290g of a saturated solution containing 0.566mol citric acid. Then, use 280.08g of a saturated solution containing 2.625mol sodium hydroxide to adjust the pH of the mixture to 13.30 (25.0℃). Seal the mixture with argon gas into 2ml ampoules and store in a dark, low-temperature environment.

[0145] Sample 12-2 (pH=13.30, 30 / 70 saturated solution mass ratio): Under argon atmosphere and at room temperature, take 177g of a saturated solution containing 0.201mol L-ascorbic acid and mix thoroughly with 413g of a saturated solution containing 0.806mol citric acid. Then, use 311.8g of a saturated solution containing 2.923mol sodium hydroxide to adjust the pH of the mixture to 13.30 (25.0℃). Seal the mixture with argon gas into 2ml ampoules and store in a dark, low-temperature environment.

[0146] Sample 12-3 (pH=13.30, 70 / 30 saturated solution mass ratio): Under argon atmosphere and at room temperature, take 413g of a saturated solution containing 0.469mol L-ascorbic acid and mix thoroughly with 177g of a saturated solution containing 0.345mol citric acid. Then, use 291.09g of a saturated solution containing 2.728mol sodium hydroxide to adjust the pH of the mixture to 13.30 (25.0℃). Seal the mixture with argon gas into 2ml ampoules and store in a dark, low-temperature environment.

[0147] Active Examples Methods for constructing tumor animal models 1) Construction of human tumor xenotransplantation (PDX) model Patient-Derived Xenograft (PDX) models are tumor models established by directly inoculating fresh tumor tissue from clinical patients into immunodeficient mice and then preserving it through in vivo passage. This model preserves the tissue morphology, gene expression, heterogeneity, and drug sensitivity of the patient's tumor to the greatest extent possible, making it the gold standard in vivo model for personalized cancer treatment, screening of new anti-tumor drugs, development of biomarkers, and mechanistic studies.

[0148] The experimental animals were Nude mice, 5-6 weeks old, half male and half female (the sexes were fixed according to the cell line characteristics and could not be mixed), weighing 16-20g, and were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in the animal laboratory of Xi'an International Medical Safety Evaluation Co., Ltd. in a specific pathogen-free SPF-grade environment with constant temperature and humidity, 12h / 12h light and dark cycle, independent ventilated cages, and free access to food and water.

[0149] Fresh tissue specimens from clinical oncology patients, after being pathologically confirmed as the target tumor, are collected under aseptic conditions and immediately transferred to an SPF-grade animal facility for inoculation and passage. Strict aseptic procedures are followed throughout the process to avoid tissue contamination.

[0150] The main reagents are as follows:

[0151] The main instruments are as follows:

[0152] The specific experimental method is as follows: Tumor tissue preparation: Fresh tumor specimens were placed in pre-cooled Hank's balanced salt solution (HBSS) containing triple antibodies within 2 hours of ex vivo transport on ice. The specimens were repeatedly rinsed 3–5 times in HBSS in a biosafety cabinet to remove blood, fat, necrotic tissue, and normal tissue. Suitable tumor tissue was then trimmed to 2–3 mm. 3 Prepare uniform small pieces and place them in pre-cooled sterile petri dishes for later use.

[0153] Subcutaneous inoculation in mice: Nude mice were weighed and labeled using an electronic balance. The inoculation area (bilateral dorsal subcutaneous tissue) was disinfected with povidone-iodine. Sterile forceps were used to cut open the skin on the mouse's back, making a small subcutaneous incision. The subcutaneous space was bluntly dissected, and the prepared tumor tissue block was grasped. With the aid of a small amount of matrix adhesive, it was inserted into the puncture needle and implanted into the subcutaneous cavity. The skin wound was closed, and the wound was disinfected again with povidone-iodine. The mice were then housed together.

[0154] Tumor observation and feeding: (1) After tumor inoculation, the mice were fed normally, and their mental state, food and water intake, activity and fur luster were observed daily. (2) Monitor tumor growth daily, observe whether there is ulceration, bleeding or infection at the tumor site, and observe whether the mice show abnormalities such as emaciation, hair loss, ascites, or limb swelling. (3) Mice with serious abnormalities (such as large-area tumor ulceration, sudden weight loss, or near death) should be removed in time and recorded to avoid affecting the experimental data.

[0155] (4) After tumor inoculation, on days 7, 10, 13, 16, etc., the tumor volume is measured with calipers. The tumor volume is maintained until the average tumor volume reaches the preset inclusion standard (generally 100-300 mm). 3 When the drug administration experiment begins, the formal drug administration experiment will commence.

[0156] Tumor volume calculation formula: V = L × W 2 / 2 (where V is the tumor volume, L is the longest diameter of the tumor, and W is the shortest diameter of the tumor. Mouse body weight was recorded simultaneously to monitor animal health status.)

[0157] 2) Construction of human tumor cell line xenotransplantation (CDX) model The cell line derived xenograft (CDX) model is a standardized in vivo model in which human tumor cells, stably passaged in vitro, are inoculated into immunodeficient mice to form solid tumor tissue by relying on the host's nutritional supply. It is used for in vivo tumor growth characteristics research, anti-tumor drug screening and pharmacodynamic evaluation.

[0158] The experimental animals were BALB / c Nude mice, 5-6 weeks old, half male and half female (fixed according to cell line characteristics, not mixed), weighing 16-20g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; they were housed in a specific pathogen-free SPF-grade environment in the animal laboratory of Xi'an International Medical Safety Evaluation Co., Ltd., with constant temperature and humidity, 12h / 12h light-dark cycle, independent ventilated cages, and free access to food and water.

[0159] All cells from human cancer cell lines (such as the liver cancer cell line Huh-7 and the human colon cancer cell line HT-29) were correctly identified by STR and were free of mycoplasma contamination. Log-phase cells were used in the experiments. The cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0160] The main reagents are as follows:

[0161] The main instruments are as follows:

[0162] The experimental method is as follows: Preparation of tumor cell suspension: Human tumor cells were resuscitated, cultured, expanded, and passaged three times. Tumor cells from passages three or more were used as the seed cell line. The cell culture supernatant was discarded, and the cell monolayer was washed once with PBS. 0.25% trypsin-EDTA digestion solution was added, and the cells were incubated at 37°C until they shrank and became rounded. Serum-containing complete culture medium was added to terminate digestion, and the cells were gently pipetted to obtain a single-cell suspension. The suspension was centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. The cells were resuspended in sterile PBS, stained with trypan blue, and counted. Cells with a viability ≥95% were used for seeding. The cell density was adjusted, and the cell suspension and Matrigel were mixed at a 1:1 volume ratio on ice (50 μL cell suspension, 50 μL Matrigel), to prepare a final concentration of 1 × 10⁻⁶. 7 Use 100 μL of inoculum suspension and keep it in an ice bath throughout the process to prevent solidification.

[0163] Subcutaneous inoculation in mice: Nude mice were weighed and labeled using an electronic balance. The inoculation sites (bilateral dorsal subcutaneous tissue) were disinfected with povidone-iodine. Using a 1 mL syringe, 100 μL of cell suspension was drawn up, and air bubbles were removed. The needle was inserted subcutaneously at a 15°-30° angle, and after probing 0.8-1.0 cm, the injection was slowly pushed in to form a stable wheal. After injection, the needle was left in place for 10 seconds before being slowly withdrawn. A sterile cotton swab was used to gently press the puncture site for 5-10 seconds to prevent leakage. Each mouse was inoculated bilaterally, and the inoculation site and procedure were standardized to ensure consistency between groups.

[0164] Tumor observation and volume measurement: After inoculation, mice were observed daily for their mental state, activity, diet, and for any redness, swelling, leakage, infection, or ulceration at the inoculation site. Once the tumor was palpable, the long diameter (L) and short diameter (W) of the tumor were measured every 2 days using electronic calipers.

[0165] Tumor volume calculation formula: V = L × W 2 / 2 (where V is the tumor volume, L is the longest diameter of the tumor, and W is the shortest diameter of the tumor. Mouse body weight was recorded simultaneously to monitor animal health status.)

[0166] The following embodiments of the present invention employ the two model construction methods described above to construct the following human tumor animal models: gastric cardia squamous cell carcinoma, tracheoesophageal groove squamous cell carcinoma, mandibular squamous cell carcinoma, lung squamous cell carcinoma, lung adenocarcinoma, colonic adenocarcinoma, esophageal adenocarcinoma, ovarian adenocarcinoma, small intestinal adenocarcinoma, greater omentum adenocarcinoma, metastatic intestinal adenocarcinoma of moderately differentiated pelvic adenocarcinoma, colonic adenocarcinoma, small cell lung cancer brain metastasis, pancreatic cancer, glioma, lung adenosquamous carcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal squamous cell carcinoma, small cell neuroendocrine gastric cancer, gastric squamous cell carcinoma, liver cancer, and pelvic adenocarcinoma. The antitumor activity of the samples and / or control samples of the present invention is investigated and verified in one or more of the above animal models.

[0167] 3) Animal pharmacodynamic studies: administration methods and activity evaluation methods Animal grouping 1. When the average tumor volume in mice reaches 100–300 mm. 3 The subjects were grouped using a stratified randomization method, and one or more of the following groups were set up in different experiments: blank control group, solvent control group, positive control group (selected), and low / medium / high dose of test substance group, with no less than 6 subjects in each group.

[0168] 2. The day of administration is recorded as day 0 (D0), and the initial tumor volume and body weight are recorded.

[0169] Animal administration methods Administration method: Intratumoral injection; fixed administration frequency: single dose.

[0170] Dosage volume: The dosage was calculated based on the tumor volume in mice, and the dosage volume was strictly controlled. The dosing time, dosage volume, and any abnormal animal reactions were also fully recorded.

[0171] Post-drug administration observation and index detection General condition observation: Observe the animal's mental state, activity level, fur, diet and water intake, and excretion daily.

[0172] Weight monitoring: Mouse weight was recorded twice a week. Any weight loss or emaciation was promptly assessed.

[0173] Tumor measurement: The long and short diameters of the tumor are measured twice a week, the tumor volume is calculated, and the presence of ulceration, bleeding, necrosis, or infection is observed.

[0174] Euthanasia shall be performed immediately if any of the following conditions are met: 1. The volume of a single tumor is >2000 mm. 3 Or the tumor diameter is >20mm; 2. The mouse's weight decreases by ≥20% from the baseline; 3. The tumor is severely ulcerated, infected, or has large-area necrosis; 4. The animal exhibits severe pathological conditions such as cachexia, inability to eat or move independently, and difficulty breathing.

[0175] Trial termination and specimen collection In the control group, all animals were euthanized when tumors reached the set upper limit or the dosing cycle ended. Subcutaneous tumor tissue was completely dissected, excess connective tissue was removed, and the tissues were weighed and photographed for record-keeping. Specimens were aliquoted and processed: some tissues were fixed in 4% paraformaldehyde for pathological studies. Major organs such as the heart, liver, spleen, lungs, and kidneys were harvested as needed for organ toxicity assessment.

[0176] Data computation and statistical analysis 1. Core parameters include (mean) tumor volume and / or (mean) tumor weight, etc. Core calculation indicator: %T / C = (Tumor volume in DN treatment group - Tumor volume in D0 treatment group) / (Tumor volume in DN model control group - Tumor volume in D0 model group) 100; (Note: DN refers to day N; D0 refers to day 0) %TGI=100-%T / C.

[0177] 2. Statistical methods: One-way ANOVA was used for measurement data that conformed to a normal distribution; non-normal data were tested using non-parametric tests; P < 0.05 was considered statistically significant between groups.

[0178] 3. Compile tumor growth curves and weight change curves.

[0179] Example 1: Tumor volume data study of the representative sample of the present invention 3 days after administration This embodiment aims to investigate the effect of the sample of the present invention on tumor volume 3 days after administration in an animal model. A mouse pancreatic cancer tumor model was constructed according to the tumor model construction method listed above, and L-ascorbic acid and sodium hydroxide solution samples (samples 1-1, 1-2, 1-3, and 1-4) were used for testing. The indicators examined were tumor volume and tumor growth inhibition rate 3 days after administration. Specific experimental results are shown in Table 1 below.

[0180] Table 1. Tumor volume and tumor growth inhibition rate in mice 3 days after administration of the sample of the present invention.

[0181] Remark: 1. Dosage volume: 100µL / 100mm 3 Inject the undiluted solution at 100% of the tumor volume. Dosage volume: 50 µL / 100 mm. 3 1. Inject the undiluted solution at 50% of the tumor volume. 2. TGI represents the tumor growth inhibition rate. 3. P Value: Difference in tumor volume between the treatment group and the control group. 4. TGI < 40%, no tumor-inhibiting effect. TGI 40%–60%: Moderate tumor-inhibiting activity; TGI 60%–80%: Significant inhibitory effect, tumor growth is significantly limited; TGI > 80%: Strong tumor inhibition, tumor growth is basically stopped; TGI > 100%: Tumor no longer grows and its volume even decreases; TGI > 200%: Tumor volume is significantly reduced, proving a very strong tumor-eliminating effect.

[0182] In addition, the tumor growth curves of representative samples 1-2 and 1-4, as well as the changes in mouse body weight after intratumoral injection, are shown in [reference 1]. Figure 1 and Figure 2 .

[0183] The above experimental results demonstrate that the sample of this invention has a significant effect on tumor volume in animal models after administration, significantly reducing tumor volume within 3 days after administration. This proves that it has a highly potent and rapidly acting tumor-suppressive effect, enabling effective tumor elimination. Furthermore, the body weight of mice in each experimental group remained stable without significant changes, indicating that the sample of this invention has good safety.

[0184] Example 2: Tumor volume data study of the representative sample of the present invention 21 days after administration This embodiment aims to investigate the effect of the sample of the present invention on tumor volume after 21 days of administration in an animal model. Various mouse tumor models were constructed using the tumor model construction methods listed above, and tests were conducted using L-ascorbic acid and sodium hydroxide solution samples (samples 1-5 (pH=13.30)). The tumor volume after 21 days of administration was the key performance indicator. Specific experimental results are shown in Table 2 below.

[0185] Table 2. Tumor volume and tumor growth inhibition rate in mice 21 days after administration of the samples of this invention.

[0186] Remark: 1. 100% Dosage Volume: Inject the undiluted solution at 100% of the tumor volume. 75% Dosage Volume: Inject the undiluted solution at 75% of the tumor volume. The same applies below. 2. P Value: Difference in tumor volume between the treatment group and the control group.

[0187] Furthermore, as an example, comparative photographs of tumor sites in mice with lung squamous cell carcinoma (samples 1-5 of the present invention) before administration, on day 3, day 7, and day 21 after administration are provided. Figure 3 .

[0188] The above experimental results show that the sample of this invention has a significant effect on the volume of various tumors after administration to animal models, with an efficacy rate of 100%. Furthermore, after 21 days of administration, the tumor volume in mice decreased to 0 (i.e., invisible / undetectable level), indicating that the sample of this invention can significantly reduce tumor volume and even achieve the technical effect of tumor elimination. In addition, the body weight of mice in each experimental group remained stable without significant changes, indicating that the sample of this invention has good safety. In summary, the sample of this invention has a highly potent and rapid tumor-suppressive effect, which can effectively eliminate tumors and has broad prospects for achieving "effective cure" of tumors.

[0189] Example 3: Tumor volume inhibition data of other samples after 21 days of administration according to the present invention This embodiment aims to investigate the effect of other samples of the present invention on tumor volume in animal models after administration for 21 days. Various mouse tumor models were constructed using the tumor model construction methods listed above, and different samples were used for testing. The evaluation index was the tumor growth inhibition rate (TGI%) after 21 days of administration. Specific experimental results are shown in Table 3 below.

[0190] Table 3. Tumor growth inhibition rate in mice 21 days after administration of other samples of the present invention.

[0191] The above experimental results show that the various samples of the present invention have a significant effect on the volume of various tumors after administration in animal models, indicating that the samples of the present invention can significantly reduce tumor volume and even achieve the technical effect of eliminating tumors. Furthermore, the body weight of mice in each experimental group remained stable without significant changes, indicating that the samples of the present invention have good safety. In addition, the above experimental results further show that when the sample components are the same, a pH greater than 12 (preferably greater than 13) can achieve a further improved tumor inhibition effect. For details, please refer to the TGI comparison data between samples 2-3 (pH=11.80) and 2-4 (pH=13.50), and the TGI comparison data between samples 8-2 (pH=11.80) and 8-3 (pH=13.50).

[0192] Example 4: Study on the effect of NaOH concentration in the sample of the present invention on antitumor activity This embodiment aims to evaluate the effect of NaOH concentration in the samples of the present invention on antitumor activity. The added concentrations of samples 1-10, 1-11, and 1-12 were 10%, 30%, and 80% of the NaOH concentration of samples 1-3, respectively. Before a single injection of the sample solution into the tumor of a tumor model mouse, and 2 and 4 hours after injection, the maximum surface area of ​​the tumor and the necrotic surface area of ​​the maximum surface area of ​​the tumor (by contrast imaging) were measured. The ratio of the necrotic surface area of ​​the maximum surface area of ​​the tumor to the maximum surface area of ​​the tumor characterized the antitumor activity of the sample; a higher ratio indicated higher antitumor activity. Specific experimental results are shown in Table 4 below.

[0193] Table 4. Effect of different NaOH concentrations on antitumor activity in the samples of this invention.

[0194] As shown in Table 4 above, samples 1-10, 1-11, and 1-12 all achieved significant tumor inhibition 2 hours and 4 hours after intratumoral injection. The ratio of the largest necrotic area to the largest tumor area was greater than 70% in all samples, especially for sample 1-12, where the ratio was close to 100%. These data indicate that, within the scope of this invention, even with a reduction in the concentration of NaOH added to the samples, excellent tumor inhibition effects can still be achieved.

[0195] Example 5: Study on the antitumor activity of the invented sample compared to L-ascorbic acid alone and NaOH alone This embodiment aims to evaluate the antitumor activity of the invention sample containing both L-ascorbic acid and NaOH compared to L-ascorbic acid solution alone or NaOH solution alone. This embodiment uses samples 1-5 (pH=13.30) (A / B composition), control sample 1-1 (1% NaOH solution alone (pH=13.33)) (component B) and control sample 2-1 (ascorbic acid solution alone (pH=2.013)) (component A).

[0196] Eighteen SPF-grade Nu / Nu nude mice (8 females and 10 males), aged 5–6 weeks and weighing 18–22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK(Sichuan)2023-0040; laboratory unit usage license number: SYXK(Shaanxi)2025-014. They were housed in a specific pathogen-free SPF-grade environment at the Xi'an International Medical Animal Laboratory, with constant temperature and humidity, 12h / 12h light / dark cycle, individually ventilated cages, and free access to food and water. Animal handling during the experiment complied with the "Guiding Opinions on the Humane Treatment of Laboratory Animals" issued by the Ministry of Science and Technology in 2006.

[0197] In this experiment, all test samples, including component A, component B, and the A / B combination, were sterile, filtered for sterility, and stored at room temperature in the dark. Fresh surgical tissue specimens from clinical oncology patients, after pathological confirmation of tumors, were collected under aseptic conditions and immediately transferred to an SPF-grade animal facility for inoculation and passage. Strict aseptic techniques were followed throughout the process to avoid tissue contamination.

[0198] Fresh glioma tumor specimens were placed in pre-cooled Hank's balanced salt solution (HBSS) containing triple antibodies within 2 hours of extubation and transported on ice. The specimens were repeatedly rinsed 3–5 times in the biosafety cabinet with HBSS to remove blood, fat, necrotic tissue, and normal tissue. Suitable tumor tissue was then trimmed to 2–3 mm. 3 Uniform small pieces were placed in pre-cooled sterile culture dishes for later use. 15 μL of Matrigel was added to each tumor tissue piece. Nude mice were weighed and labeled using an electronic balance. The inoculation area (bilateral dorsal subcutaneous tissue) was disinfected with povidone-iodine. The skin on the back of the NCG mice was cut open with sterile forceps, a small subcutaneous incision was made, and the subcutaneous space was bluntly dissected. The prepared tumor tissue piece was picked up, and with the aid of a small amount of Matrigel, it was inserted into the subcutaneous cavity through a puncture needle. The skin wound was closed, and the wound was disinfected again with povidone-iodine. The mice were then housed together. The survival and tumor formation of the NCG mice were observed. The inoculation continued until the subcutaneous tumor in the nude mice reached 1000-2000 mm in size. 3 Afterwards, the nude mice were euthanized, and the tumor tissue was removed by aseptic surgery. The tissue was then washed in Hanks' balanced salt solution (HBSS) pre-cooled with triple antibodies, and the tumor was then cut into pieces of approximately 2–3 mm. 3Prepare tissue fragments of varying sizes and transplant them into Nu / Nu mice using the method described above. After transplantation, monitor tumor growth regularly and measure and calculate tumor volume using calipers.

[0199] The experiment consisted of three groups: Group A (component 1), Group B (component 2), and Group A / B combination group (component 3). Six tumor-bearing mice were used in each group. Different doses were administered based on the tumor volume, with a single single dose. Changes in the mice's physical signs, tumor shape, and morphology were observed before and at different time points after administration. The antitumor activity of the three different administration groups was evaluated. The experimental design is shown in Tables 5-1, 5-2, and 5-3. TV refers to tumor volume.

[0200] Table 5-1 Dosage Design Table for Experiment 1

[0201] Table 5-2 Dosage Design Table for Experimental Group 2

[0202] Table 5-3 Dosage Design Table for Experimental Groups 3

[0203] The observation indicators include the following: General condition observation: Animals in each group were observed in their general condition from the acclimatization period to the end of the experiment, at least once a day. This included, but was not limited to, the animals' appearance, activity, and condition.

[0204] Body weight: Animals to be measured: Surviving animals in each group. Measurement time: At least once during the adaptation period; at least once during the modeling period; at least once a week during the drug administration period.

[0205] Tumor volume: Animals used for measurement: surviving animals from each group. Measurement time: Tumor volume was measured before and at different time points after drug administration. Measurement method: The major axis (a) and minor axis (b) of the tumor were measured using vernier calipers. Tumor volume was calculated as V = 1 / 2 × a × b. 2 The tumor inhibition rate (%) is calculated based on the measured tumor volume. The formula is: Tumor inhibition rate = (1 - Tumor volume after drug administration / Tumor volume of control group) × 100%. The tumor inhibition rate measures the inhibitory effect of the drug on tumor growth.

[0206] Data collection and analysis: Excel software was used for data analysis, and GraphPad Prism 9.5 was used for drawing statistical charts.

[0207] A comparison of the effects of component A, component B, and the A / B combination on tumor growth in tumor-bearing mice revealed significant differences in tumor growth among the different treatment groups. In the A / B combination group, tumor volume decreased rapidly after administration, approaching baseline levels from day 3 onwards, and remained at extremely low levels thereafter, with complete tumor regression by day 7, achieving a tumor inhibition rate of (99.00±2.44)%, demonstrating a stable and sustained inhibitory effect. In the component B group, tumor volume showed a continuous decreasing trend after administration, but the decrease was less pronounced than in the A / B combination group, with a tumor inhibition rate of (28.51±44.53)%. In the component A group, tumor volume decreased significantly on day 3 after administration, gradually increasing in a fluctuating manner, with a significantly increased standard deviation over time. Individual variability was significant in this experimental group, with a tumor inhibition rate of only (45.29±88.69)%, as detailed in Tables 5-4 and 5-5. Figure 4 (Trend chart of tumor volume change).

[0208] Table 5-4 Tumor inhibition rate (%) in tumor-bearing mice after administration of different drugs ( - (x±s, n=6)

[0209] Overall, component A showed tumor growth inhibition and tumor regression in some tumor-bearing mice, but a certain proportion of mice also experienced tumor growth after administration, indicating a tendency for tumor recurrence, with significant inter-individual differences. Component B showed tumor inhibition in the short term, but some mice also showed an increase in tumor volume on the 2nd to 3rd day after administration, indicating a tendency for tumor recurrence. The tumor inhibition rate of tumor-bearing mice reached 100% after 7 days of administration of the A / B combination, and no tumor recurrence was observed within 21 days. Therefore, the A / B combination has a very strong tumor growth inhibition and clearance ability in the tumor-bearing mice in this experiment, achieving a significant synergistic effect compared to component A and component B alone.

[0210] Furthermore, morphological observations showed that before administration of the A / B combination, the morphology and color of the tumors in all tumor-bearing mice were normal. Immediately after administration, all six mice exhibited redness of the tumor and surrounding tissue, with some mice also showing tumor swelling. Two hours after administration, the redness of the tumor and surrounding tissue expanded. Twenty-one days after administration, complete tumor regression was observed in all mice, leaving only scars, small areas of unhealed ulceration, or peripheral erythema. No mouse deaths or other acute or chronic safety events occurred throughout the experimental period. For detailed morphological observations, please refer to [link to relevant documentation]. Figures 5A-5C The above experimental results indicate that the A / B combination exhibits excellent safety and efficacy when administered.

[0211] Example 6: Antitumor safety study of the inventive sample compared to NaOH alone This embodiment aims to evaluate the antitumor safety of the invention sample containing both L-ascorbic acid and NaOH compared to NaOH solution alone. This embodiment uses samples 1-5 (pH=13.30) (A / B composition) and control samples 1-2 (stock solution B (without A) used to prepare the A / B composition).

[0212] Twelve SPF-grade Nu / Nu nude mice (5 females and 7 males), aged 5–6 weeks and weighing 18–22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK(Sichuan)2023-0040; experimental unit usage license number: SYXK(Shaanxi)2025-014. They were housed in a specific pathogen-free SPF-grade environment at Xi'an International Medical Safety Evaluation Co., Ltd., with constant temperature and humidity, 12h / 12h light / dark cycle, individually ventilated cages, and free access to food and water. Animal handling during the experiment complied with the "Guiding Opinions on the Humane Treatment of Laboratory Animals" issued by the Ministry of Science and Technology in 2006.

[0213] Fresh tumor specimens were placed in pre-cooled Hank's balanced salt solution (HBSS) containing triple antibodies within 2 hours of extubation and transported on ice. The specimens were repeatedly rinsed 3–5 times in the biosafety cabinet with HBSS to remove blood, fat, necrotic tissue, and normal tissue. Suitable tumor tissue was then trimmed to 2–3 mm. 3 Uniform small pieces were placed in pre-cooled sterile culture dishes for later use. 15 μL of Matrigel was added to each tumor tissue piece. Nude mice were weighed and labeled using an electronic balance. The inoculation area (bilateral dorsal subcutaneous tissue) was disinfected with povidone-iodine. The skin on the back of the NCG mice was cut open with sterile forceps, a small subcutaneous incision was made, and the subcutaneous space was bluntly dissected. The prepared tumor tissue piece was picked up, and with the aid of a small amount of Matrigel, it was inserted into the subcutaneous cavity through a puncture needle. The skin wound was closed, and the wound was disinfected again with povidone-iodine. The mice were then housed together. The survival and tumor formation of the NCG mice were observed. The inoculation continued until the subcutaneous tumor in the nude mice reached 1000-2000 mm in size. 3 Afterwards, the nude mice were euthanized, and the tumor tissue was removed by aseptic surgery. The tissue was then washed in Hanks' balanced salt solution (HBSS) pre-cooled with triple antibodies, and the tumor was then cut into pieces of approximately 2–3 mm. 3 Prepare tissue fragments of varying sizes and transplant them into Nu / Nu mice using the method described above. After transplantation, monitor tumor growth regularly and measure and calculate tumor volume using calipers.

[0214] The experiment consisted of two groups: Group 1 was the component B stock solution group (control samples 1-2, calculated to correspond to 22% of component B per unit volume in the A / B composition), and Group 2 was the A / B composition group. Six tumor-bearing mice were used in each group. Different doses were administered based on the tumor volume, with a single administration. Changes in the mice's physical signs, tumor shape, and morphology were observed before and at different time points after administration. The experimental design is shown in Tables 6 and 7.

[0215] Table 6 Dosage Design Table for Experiment 1

[0216] Table 7 Dosage Design Table for Experiment 2

[0217] Morphological observation showed that all test animals in both experimental groups experienced varying degrees of drug extravasation immediately after administration, with some cases involving extensive extravasation. The tumor and surrounding normal skin tissue exhibited reactions such as tumor hemorrhage and localized skin redness. Two hours after administration, cyanotic or black necrosis appeared in the tumor and surrounding tissues, and some tumor-bearing mice experienced tumor rupture. Four hours after administration, the necrosis area further expanded, accompanied by tissue necrosis and hemorrhage, indicating a worsening trend in tumor and surrounding tissue damage. These results suggest that drug extravasation during administration led to severe damage to the tumor and surrounding normal tissues. For detailed morphological observations, please refer to [link to relevant documentation]. Figures 6A-6C Compared to the morphological observations of the test animals in Experiment 1 (see...), Figures 5A-5C In experiment 2, the tissue damage in all test animals was significantly more severe.

[0218] The above experimental results show that the sample of the present invention has significantly improved antitumor safety compared with NaOH solution alone, and the introduction of component A (L-ascorbic acid) significantly reduces tissue damage caused by component B.

[0219] Example 7: The effect of the selection of other component A in the composition on antitumor activity This embodiment aims to evaluate the effect of the selection of other component A in the composition on the antitumor activity. The selected sample is control sample 3-1 (a solution of hydrochloric acid and sodium hydroxide (pH=7.00)), that is, hydrochloric acid is used instead.

[0220] Nude mice were inoculated with human pancreatic cancer tumors (tumor volume 100 mm²). 3 -300mm 3 The efficacy of the drug on tumors in mice was observed by monitoring changes in body weight, tumor growth curves, and tumor growth inhibition rate (TGI%) through intratumoral injection. Six mice (half male and half female) were used in the experimental group, and the drug was administered at a volume of 100 µL / 100 mm².3 / time, the number of times to administer the medication is 1.

[0221] For control sample 3-1, its TGI on day 21 was less than 10% (P > 0.05), and it did not exhibit statistically significant antitumor activity compared to the solvent control. The experiment demonstrates that the selection of component A has a significant impact on the antitumor activity of the composition, and the selection of specific component A in this invention is crucial for achieving the potent antitumor effect of the composition.

[0222] Example 8: The effect of the selection of other component B in the composition on antitumor activity This embodiment aims to evaluate the effect of the selection of other component B (base) in the composition on the antitumor activity. The selected samples are samples 11-1 (L-ascorbic acid + potassium hydroxide (pH=13.30)) and 11-2 (L-ascorbic acid + sodium ethoxide (pH=13.30)); and control samples 4-1 (L-ascorbic acid + berberine (pH=10.05)), 4-2 (L-ascorbic acid + berberine (pH=13.50)), 5-1 (L-ascorbic acid + matrine (pH=6.27)), 6-1 (citric acid + matrine (pH=3.67)), 6-2 (citric acid + matrine (pH=7.216)), and 7-1 (citric acid + berberine (pH=6.105)).

[0223] This experiment was conducted in two batches, with the first batch targeting samples 11-1 and 11-2. Nude mice were inoculated with human-derived tumors (tumor volume 100 mm²). 3 -300mm 3 The tumor volume (TV / mm²) in mice was monitored before drug administration and on days 1 and 2 after drug administration via intratumoral injection. 3 The administration volume is 100µL / 100mm. 3 / time, administration frequency is 1 time. See Table 8 below for specific experimental results.

[0224] Table 8 Tumor volume on day 1 and day 2 after drug administration

[0225] Note: " / " indicates that it was not measured.

[0226] As shown in Table 8, when potassium hydroxide or sodium ethoxide is selected as component B, the resulting composition also has excellent antitumor activity, and a significant reduction in tumor volume was observed on the first or second day after administration (P<0.05).

[0227] The second batch of experiments targeted control samples 4-1, 4-2, 5-1, 6-1, 6-2, and 7-1. Nude mice were inoculated with human-derived tumors (tumor volume 100 mm²).3 -300mm 3 The tumor growth inhibition rate (TGI%) in mice was monitored on day 21 after administration via intratumoral injection. Specific experimental results are shown in Table 9 below.

[0228] Table 9 Tumor growth inhibition rate after drug administration

[0229] As shown in Table 9, when matrine or berberine was selected as component B, the resulting composition did not have substantial tumor-suppressive effects, and there were significant safety issues (mouse deaths) in multiple experimental groups.

[0230] The experiments described above in this embodiment demonstrate that the selection of component B also has a significant impact on the antitumor activity of the composition. The selection of specific component B in this invention is crucial for achieving the potent antitumor effect of the composition.

[0231] Example 9: Study on the long-term antitumor efficacy and safety of the composition of the present invention This embodiment aims to study the long-term antitumor efficacy and safety of the compositions of the present invention (samples 1-5) after intratumoral injection (Note: mice older than 9 months are considered elderly).

[0232] SPF grade Nu / Nu nude mice, 5–8 weeks old, 18–22g; purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Sichuan) 2023-0040; laboratory unit use license number: SYXK (Shaanxi) 2025-014; housed in the specific pathogen-free SPF grade environment of Xi'an International Medical Safety Evaluation Co., Ltd., with constant temperature and humidity, 12h / 12h light and dark cycle, independent ventilated cages, and free access to food and water.

[0233] Fresh tumor specimens were placed in pre-cooled Hank's balanced salt solution (HBSS) containing triple antibodies within 2 hours of extubation and transported on ice. The specimens were repeatedly rinsed 3–5 times in the biosafety cabinet with HBSS to remove blood, fat, necrotic tissue, and normal tissue. Suitable tumor tissue was then trimmed to 2–3 mm. 3 Prepare uniform small pieces and place them in pre-cooled sterile culture dishes for later use. Add 15 μL of Matrigel to each piece of tumor tissue.

[0234] Nude mice were weighed and labeled using an electronic balance. The inoculation sites (bilateral dorsal subcutaneous tissue) were disinfected with povidone-iodine. Sterile forceps were used to cut open the skin on the back of the NCG mice, making a small subcutaneous incision. The subcutaneous space was bluntly dissected, and the prepared tumor tissue block was grasped. With the aid of a small amount of matrix gel, it was inserted into the subcutaneous cavity through a puncture needle. The skin wound was closed, and the wound was disinfected again with povidone-iodine. The mice were then housed together. The survival and tumor formation of the NCG mice were observed. The inoculation continued until the subcutaneous tumors in the nude mice reached 1000-2000 mm in size. 3 Afterwards, the nude mice were euthanized, and the tumor tissue was removed using aseptic surgery. The tissue was then washed in physiological saline, and the tumor was cut into pieces of approximately 2–3 mm. 3 Prepare tissue fragments of different sizes and transplant them into Nu / Nu mice as described above. After transplantation, monitor tumor growth regularly, measure and calculate tumor volume using calipers, and aseptically dissect the tumor after it has grown to a suitable volume. Remove necrotic tissue and perform continuous passage in mice. Generally, passage is performed for 3 to 5 generations to establish 14 tumor models, as shown in Table 10 below.

[0235] Table 10 Established tumor models

[0236] The administration method can be either of the following two: 1. Constructing a PDX model by subcutaneous inoculation in the right dorsal scapular region: intratumoral injection based on tumor volume. 2. Constructing a PDX model by subcutaneous inoculation in the left and right dorsal scapular regions, administering 100% of the full tumor volume to one tumor; considering the systemic exposure of the contralateral ipsilateral mice and the synchronous growth of the contralateral tumor, after 5 days of observation following the initial administration, administer 75% (or 67%) of the tumor volume to the contralateral tumor. See the table below for specific dosage design.

[0237] Table 11-1 Dosage Design Table for Experiment 1

[0238] Table 11-2 Dosage Design Table for Experimental Group 2

[0239] Table 11-3 Dosage Design Table for Experimental Groups 3

[0240] Table 11-4 Dosage Design Table for Experimental Groups 4

[0241] Table 11-5 Dosage Design Table for Experimental Groups 5

[0242] Table 11-6 Dosage Design Table for Experimental Groups 6

[0243] Table 11-7 Dosage Design Table for Experiment 7 Groups

[0244] Table 11-8 Dosage Design Table for Experimental Groups 8

[0245] Table 11-9 Dosage Design Table for Experimental Groups 9

[0246] Table 11-10 Dosage Design Table for Experimental Groups 10

[0247] Table 11-11 Dosage Design Table for Experiment 11

[0248] Table 11-12 Dosage Design Table for Experiment 12

[0249] Table 11-13 Dosage Design Table for Experiment 13

[0250] Table 11-14 Dosage Design Table for Experiment 14

[0251] The observation indicators include the following: General condition observation: Animals in each group were observed in their general condition from the acclimatization period to the end of the experiment, at least once a day. This included, but was not limited to, the animals' appearance, activity, and condition.

[0252] Body weight: Animals to be measured: Surviving animals in each group. Measurement time: At least once during the adaptation period; at least once during the modeling period; at least once a week during the drug administration period.

[0253] Tumor volume: Animals used for measurement: surviving animals from each group. Measurement time: Tumor volume was measured before and at different time points after drug administration. Measurement method: The major axis (a) and minor axis (b) of the tumor were measured using vernier calipers. Tumor volume was calculated as V = 1 / 2 × a × b. 2 The tumor inhibition rate (%) is calculated based on the measured tumor volume. The formula is: Tumor inhibition rate = (1 - Tumor volume after drug administration / Tumor volume before drug administration) × 100%. The tumor inhibition rate measures the inhibitory effect of the drug on tumor growth.

[0254] Data collection and analysis: Excel software was used to analyze the data.

[0255] 9.1 Effects of drugs on tumor growth in tumor-bearing mice (lung squamous cell carcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group ranged from 80 to 228 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>11 months and 15 days), the calculated tumor inhibition rate was 100%. Therefore, the drug has antitumor effects in vivo, and no recurrence was observed during long-term observation, demonstrating excellent long-term safety. Specifically... Table 12-1 Effects of drug administration on tumor growth in tumor-bearing mice

[0256] 9.2 Effects of drugs on tumor growth in tumor-bearing mice (lung adenocarcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group was 80–118 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>10 months), the calculated tumor inhibition rate was 100%. Therefore, the drug has an antitumor effect in vivo, and no recurrence was observed during long-term observation, demonstrating excellent long-term safety.

[0257] Table 12-2 Effects of drug administration on tumor growth in tumor-bearing mice

[0258] 9.3 Effects of drugs on tumor growth in tumor-bearing mice (lung adenosquamous carcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group was 123–177 mm. 3 After local injection into the tumor, the tumor inhibition rate was 100% after 21 days of treatment and long-term observation (>14 months). Therefore, the drug has an antitumor effect in vivo and no recurrence was observed during long-term observation, and it has excellent long-term safety.

[0259] Table 12-3 Effects of drug administration on tumor growth in tumor-bearing mice

[0260] 9.4 Effects of drugs on tumor growth in tumor-bearing mice (gastric adenocarcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group ranged from 170 to 490 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>11 months and 13 days), the calculated tumor inhibition rate was 100%. Therefore, the drug has an antitumor effect in vivo, and no recurrence was observed during long-term observation, demonstrating excellent long-term safety.

[0261] Table 12-4 Effects of drug administration on tumor growth in tumor-bearing mice

[0262] 9.5 Effects of drugs on tumor growth in tumor-bearing mice (rectal adenocarcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group ranged from 84 to 331 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>11 months), the calculated tumor inhibition rate was 100%. Therefore, the drug has a tumor-suppressing effect in vivo, and no recurrence was observed during long-term observation. It also has excellent long-term safety.

[0263] Table 12-5 Effects of drug administration on tumor growth in tumor-bearing mice

[0264] 9.6 Effects of drugs on tumor growth in tumor-bearing mice (colon adenocarcinoma) Before drug administration, the tumor volume of tumor-bearing mice in this experimental group ranged from 98 to 316 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>11 months), the calculated tumor inhibition rate was 100%. Therefore, the drug has a tumor-suppressing effect in vivo, and no recurrence was observed during long-term observation. It also has excellent long-term safety.

[0265] Table 12-6 Effects of drug administration on tumor growth in tumor-bearing mice

[0266] 9.7 Effects of drugs on tumor growth in tumor-bearing mice (esophageal squamous cell carcinoma) Before drug administration, the tumor volume of tumor-bearing mice in this group ranged from 95 to 398 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>10 months), the calculated tumor inhibition rate was 100%. Therefore, the drug has an antitumor effect in vivo, and no recurrence was observed during long-term observation, demonstrating excellent long-term safety.

[0267] Table 12-7 Effects of drug administration on tumor growth in tumor-bearing mice

[0268] 9.8 Effects of drugs on tumor growth in tumor-bearing mice (greater omental adenocarcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group was 75–223 mm. 3After local injection into the tumor, and following 21 days of treatment and long-term observation (>14 months and 15 days), the calculated tumor inhibition rate was 100%. Therefore, the drug has an antitumor effect in vivo, and no recurrence was observed during long-term observation, demonstrating excellent long-term safety.

[0269] Table 12-8 Effects of drug administration on tumor growth in tumor-bearing mice

[0270] 9.9 Effects of drugs on tumor growth in tumor-bearing mice (ovarian adenocarcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this group ranged from 70 to 365 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>11 months), the calculated tumor inhibition rate was 100%. Therefore, the drug has a tumor-suppressing effect in vivo, and no recurrence was observed during long-term observation. It also has excellent long-term safety.

[0271] Table 12-9 Effects of drug administration on tumor growth in tumor-bearing mice

[0272] 9.10 Effects of drugs on tumor growth in tumor-bearing mice (esophageal adenocarcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group ranged from 106 to 432 mm. 3 After local injection into the tumor, and after 21 days of treatment and long-term observation (>10 months and 17 days), the tumors in the two treated mice disappeared on day 21 after administration. However, because the tumors in the untreated mice were too large, the mice were collected. The tumor inhibition rate was calculated to be 100%. Therefore, the drug has a tumor-suppressing effect in vivo and no recurrence was observed during long-term observation. It also has excellent long-term safety.

[0273] Table 12-10 Effects of drug administration on tumor growth in tumor-bearing mice

[0274] 9.11 Effects of drugs on tumor growth in tumor-bearing mice (small intestinal adenocarcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group was 135–137 mm. 3After local injection into the tumor, and during long-term observation (>10 months and 14 days) 21 days after administration, the tumor disappeared in one mouse on day 21 after administration. However, because the tumor on the unadministered side was too large, the mice were collected. The tumor inhibition rate was calculated to be 100%. Therefore, the drug has a tumor-suppressing effect in vivo and no recurrence was observed during long-term observation. It also has excellent long-term safety.

[0275] Table 12-11 Effects of drug administration on tumor growth in tumor-bearing mice

[0276] 9.12 Effects of drugs on tumor growth in tumor-bearing mice (small cell neuroendocrine gastric carcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group was 99–146 mm. 3 After local injection into the tumor, the tumor inhibition rate was 100% after 21 days of treatment and long-term observation (>12 months and 11 days). Therefore, the drug has a tumor-suppressing effect in vivo and no recurrence was observed during long-term observation, and it has excellent long-term safety.

[0277] Table 12-12 Effects of drug administration on tumor growth in tumor-bearing mice

[0278] 9.13 Effects of drugs on tumor growth in tumor-bearing mice (gastric squamous cell carcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group was 104–186 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>10 months), the calculated tumor inhibition rate was 100%. Therefore, the drug has an antitumor effect in vivo, and no recurrence was observed during long-term observation, demonstrating excellent long-term safety.

[0279] Table 12-13 Effects of drug administration on tumor growth in tumor-bearing mice 9.14 Effects of drugs on tumor growth in tumor-bearing mice (pelvic adenocarcinoma) Before drug administration, the tumor volume of the tumor-bearing mice in this experimental group was 112–174 mm. 3 After local injection into the tumor, and following 21 days of treatment and long-term observation (>11 months), the calculated tumor inhibition rate was 100%. Therefore, the drug has a tumor-suppressing effect in vivo, and no recurrence was observed during long-term observation. It also has excellent long-term safety.

[0281] Table 12-14 Effects of drug administration on tumor growth in tumor-bearing mice

[0282] 9.15 Effects of intratumoral drug administration on tumor morphology in 14 human tumor models Tumor morphology observation results: Before drug administration, no abnormalities were observed in the morphology and color of the tumors in all tumor-bearing mice. On day 3 after drug administration, the tumor wound had completely scabbed over, the scab was dry with no bleeding or exudation, and the surrounding skin showed no redness or swelling. On day 7, the scab on the tumor wound was firm and stable, with a slightly dry and hard texture, no ulceration or exudation, and the surrounding skin had normal color without redness or inflammation; the wound gradually and steadily healed. On day 21 and during long-term observation, the mouse wounds were completely healed, the scar tissue tended to flatten and soften, the skin color was basically close to that of the surrounding normal skin, there was no redness, swelling, ulceration, or recurrence, and the local skin and the mouse's condition remained stable. See details. Figures 7A-7E .

[0283] Furthermore, after the primary tumor lesions in mice completely regressed, there were no signs of local recurrence or distant metastasis, and the mice were in good condition. These results indicate that the drug, when administered intratumorally, can effectively clear lesions in human PDX tumor models and has the potential to radically cure tumors. This provides important evidence of long-term efficacy and safety in vivo for subsequent clinical local administration of the drug to treat cancer.

[0284] Example 10: In vivo pharmacokinetic study of the composition of the present invention This embodiment aims to study the pharmacokinetic characteristics of the composition of the present invention (samples 1-5) after a single local injection into the tumor in tumor-bearing mice, and to investigate the absorption and metabolism of the composition of the present invention in tumor-bearing mice, so as to provide a basis for subsequent in vivo pharmacokinetic studies.

[0285] Thirty-three SPF-grade male Nu / Nu nude mice, 5–8 weeks old, 18–22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Sichuan) 2023-0040; experimental unit usage license number: SYXK (Shaanxi) 2025-014. They were housed in a specific pathogen-free SPF-grade environment at Xi'an International Medical Safety Evaluation Co., Ltd., with constant temperature and humidity, 12h / 12h light-dark cycle, independent ventilated cages, and free access to food and water.

[0286] Fresh lung squamous cell carcinoma tumor specimens were placed in pre-cooled Hank's balanced salt solution (HBSS) containing triple antibodies within 2 hours of ex vivo transport on ice. The specimens were repeatedly rinsed 3–5 times in the biosafety cabinet with HBSS to remove blood, fat, necrotic tissue, and normal tissue. Suitable tumor tissue was then trimmed to 2–3 mm. 3 Prepare uniform small pieces and place them in pre-cooled sterile culture dishes for later use. Add 15 μL of Matrigel to each piece of tumor tissue.

[0287] Nude mice were weighed and labeled using an electronic balance. The inoculation sites (bilateral dorsal subcutaneous tissue) were disinfected with povidone-iodine. Sterile forceps were used to cut open the skin on the back of the NCG mice, making a small subcutaneous incision. The subcutaneous space was bluntly dissected, and the prepared tumor tissue block was grasped. With the aid of a small amount of matrix gel, it was inserted into the subcutaneous cavity through a puncture needle. The skin wound was closed, and the wound was disinfected again with povidone-iodine. The mice were then housed together. The survival and tumor formation of the NCG mice were observed. The inoculation continued until the subcutaneous tumors in the nude mice reached 1000-2000 mm in size. 3 Afterwards, the nude mice were euthanized, and the tumor tissue was removed using aseptic surgery. The tissue was then washed in physiological saline, and the tumor was cut into pieces of approximately 2–3 mm. 3 Prepare tissue fragments of varying sizes and transplant them into Nu / Nu mice using the method described above. After transplantation, monitor tumor growth regularly, measure and calculate tumor volume using calipers, and aseptically dissect the tumor after it has grown to a suitable size. Remove necrotic tissue and then pass the tumor continuously in mice, generally for 3 to 5 generations of colon adenocarcinoma.

[0288] The experiment consisted of 11 groups, with 3 tumor-bearing mice in each group. Different doses of the drug were administered based on the tumor volume (100 μL / 100 mmHg). 3 ), administer once per dose.

[0289] Set the following sampling time points: Before administration: (0 h); After administration: 0.08h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h.

[0290] Note: 1) The acceptable error is ±1 min when sampling for ≤30 min, ±2 min when sampling for 31~60 min, ±5 min when sampling for 61~359 min, ±10 min when sampling for 360~1440 min, and ±15 min when sampling for ≥1440 min.

[0291] Plasma Sample Collection and Preservation: Sampling Method: Whole blood was collected from mice by enucleation. After collection, the whole blood sample was placed in an EP tube containing EDTA-K2 anticoagulant, mixed thoroughly, and centrifuged at 3500 rpm for 10 min at 4°C using a high-speed refrigerated centrifuge. The supernatant plasma was then transferred to an EP tube. Samples should not be left at room temperature for more than 4 hours. At the sampling site, blood samples were stored in a foam box containing ice. Plasma samples were then transferred to the analytical laboratory for preservation in a freezer at -60°C to -80°C.

[0292] Tumor tissue sample collection and preservation: Sampling method: Whole tumor tissue was obtained by dissection. Mice were anesthetized and euthanized immediately after whole blood collection, and the entire tumor tissue was dissected and removed. If the tumor was contaminated with blood from other sites, the surface was rinsed with physiological saline, dried, weighed, and placed in EP tubes. After collection, the samples should not be left at room temperature for more than 4 hours. At the sampling site, samples were stored in foam boxes containing ice. Samples were then transferred to the analytical laboratory and stored at -60℃ to -80℃.

[0293] Sample pretreatment analysis is as follows: Plasma: After collecting plasma samples, the acetonitrile protein precipitation method was used. Plasma was mixed with acetonitrile at a volume ratio of 1:6 (v / v), ultrasonically homogenized, and centrifuged at 12000 rpm / min for 5 min at 4°C. The supernatant was collected, concentrated by nitrogen blowing, and the samples were reconstituted before LC-MS / MS analysis.

[0294] Tumor tissue: After obtaining tissue specimens, tissue homogenization was performed. 1 mL of sterile water for injection was added to every 0.1 g of tissue and homogenized. The homogenate was then subjected to acetonitrile protein precipitation at a 1:6 (v / v) volume ratio of tissue homogenate to acetonitrile, ultrasonically mixed, and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was then analyzed by LC-MS / MS.

[0295] Based on the measured blood drug concentration-time data for each animal, Phoenix WinNonlin was used to plot the blood drug concentration-time curve and the mean blood drug concentration-time curve for each animal. Pharmacokinetic parameters, including peak concentration (Cmax), peak time (Tmax), terminal elimination rate (Ke), area under the curve (AUC), clearance rate (CL), apparent volume of distribution (Vd), and mean residence time (MRT), were calculated using the Phoenix WinNonlin non-compartmental model.

[0296] The experimental results are shown in Table 13 below.

[0297] Table 13 Drug concentrations in mouse plasma and tumor tissue at different time points

[0298] Based on the concentrations of drug components in mouse plasma and tumor tissue measured at different time points, pharmacokinetic parameters were calculated using the Phoenix WinNonlin non-compartmental model, and the results are shown in Table 14 below.

[0299] Table 14 Pharmacokinetic parameters in mouse plasma and tumor tissue

[0300] After local injection of the drug of this invention into tumor tissue, a peak concentration (1.32 mg / ml) is achieved within a short time (0.5 h). This demonstrates that the drug of this invention can rapidly exert its effect in tumor tissue within a short period of time. The peak concentration of the drug at the tumor site is more than 500 times higher than the peak concentration detected in peripheral plasma (2.45 ug / mL). This demonstrates that the amount of the drug of this invention exposed in peripheral plasma is extremely small. After local injection of the drug of this invention into tumor tissue, its maximum residence time is 12 h, and the average residence time is 4.5 h. Compared with the average drug residence time detected in peripheral plasma (0.14 h), the exposure time within the tumor is more than 30 times longer. This demonstrates that the residence time of the composition of this invention within tumor tissue is sufficiently long, and even when exposed outside the tumor, the residence time is extremely short. After local injection of the drug composition of this invention into tumor tissue, the drug clearance rate in peripheral plasma is more than 12,000 times that in the local tumor tissue, demonstrating that the composition of this invention is readily absorbed and metabolized in vivo. Furthermore, after local injection of the drug composition of the present invention into tumor tissue, the drug half-life in the tumor tissue (3.3h) is 17 times that of the drug half-life exposed in peripheral plasma (0.19h), proving that the composition of the present invention exerts its efficacy in tumor tissue for a longer period of time and is less likely to be exposed in peripheral plasma.

[0301] Example 11: Study on pH changes in tumor and adjacent tissues after administration of the composition of the present invention This embodiment aims to study the changes in pH value of the tumor and adjacent tissues after a single local injection of the composition of the present invention (samples 1-5).

[0302] Thirty-nine SPF-grade Nu / Nu nude mice, 5–8 weeks old, 18–22g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Sichuan) 2023-0040; experimental unit usage license number: SYXK (Shaanxi) 2025-014. They were housed in a specific pathogen-free SPF-grade environment at Xi'an International Medical Safety Evaluation Co., Ltd., with constant temperature and humidity, 12h / 12h light-dark cycle, independent ventilated cages, and free access to food and water.

[0303] Fresh human tumor specimens were placed in pre-cooled Hank's balanced salt solution (HBSS) containing triple antibodies within 2 hours of extubation and transported on ice. The specimens were repeatedly rinsed 3–5 times in the biosafety cabinet with HBSS to remove blood, fat, necrotic tissue, and normal tissue. Suitable tumor tissue was then trimmed to 2–3 mm. 3 Prepare uniform small pieces and place them in pre-cooled sterile culture dishes for later use. Add 15 μL of Matrigel to each piece of tumor tissue.

[0304] Nude mice were weighed and labeled using an electronic balance. The inoculation sites (bilateral dorsal subcutaneous tissue) were disinfected with povidone-iodine. Sterile forceps were used to cut open the skin on the back of the NCG mice, making a small subcutaneous incision. The subcutaneous space was bluntly dissected, and the prepared tumor tissue block was grasped. With the aid of a small amount of matrix gel, it was inserted into the subcutaneous cavity through a puncture needle. The skin wound was closed, and the wound was disinfected again with povidone-iodine. The mice were then housed together. The survival and tumor formation of the NCG mice were observed. The inoculation continued until the subcutaneous tumors in the nude mice reached 1000-2000 mm in size. 3 Afterwards, the nude mice were euthanized, and the tumor tissue was removed using aseptic surgery. The tissue was then washed in physiological saline, and the tumor was cut into pieces of approximately 2–3 mm. 3 Prepare tissue fragments of varying sizes and transplant them into Nu / Nu mice using the method described above. After transplantation, monitor tumor growth regularly, measure and calculate tumor volume using calipers, and aseptically dissect the tumor after it has grown to a suitable size. Remove necrotic tissue and then pass the tumor continuously in mice, generally for 3 to 5 generations of colon adenocarcinoma.

[0305] This experiment used 39 immune tumor-bearing mice and divided them into 13 time periods (3 mice per time period). Different doses were administered based on the tumor volume of the mice, with each dose administered once.

[0306] Animals tested: Animals at various administration time points. Test times: before administration, 0.08h, 0.5h, 1h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, D5, and D7 after administration.

[0307] Measurement Method: This experiment used a pH meter to measure the pH values ​​of tumor tissue (left, middle, and right sites) and adjacent tissues (2mm, 5mm, 10mm, and 20mm). Due to the influence of tissue sampling method, probe contact angle, local tissue heterogeneity, and inherent equipment error (±0.5 pH units), single-sample data exhibited some fluctuations. Subsequent analyses interpreted trends within the error range. Excel software was used for data analysis and related methods.

[0308] The overall trend of pH changes within the tumor is shown in Table 15.

[0309] Table 15 pH values ​​of the left, right, and central parts of the tumor at different time points.

[0310] Using analysis software such as Excel, the data in Table 15 was plotted into a graph, and the result is as follows. Figure 8AAs shown in the figure, the horizontal axis represents 13 different time points after drug administration to the tumor, and the vertical axis represents the corresponding pH value at each time point. The blue, purple, and yellow lines represent the pH value fluctuation curves of three parts of the tumor (left, right, and center) over time after drug administration. The red box indicates the normal physiologically acceptable pH range.

[0311] from Figure 8A It is evident that within a short period after drug administration (0.08 h), the pH values ​​of the left, middle, and right sites of the tumor all reached their peak values. The middle site had the highest pH value at 12.67, followed by the left site at 12.24, and the right site had the lowest at 11.99. After the peak values, the pH values ​​of all three sites gradually decreased (from 1 h to 48 h), with a faster rate of decrease in the first 12 h, followed by a slower rate of decrease after 24 h. Finally, the pH values ​​of all three sites gradually stabilized after 48 h. Furthermore, from 72 h to D7, the pH value of the entire tumor remained between 7.0 and 7.5, close to normal levels and consistent with the physiological variation patterns.

[0312] Furthermore, the pH changes of tissues at different distances from the tumor (2 mm, 5 mm, 10 mm, 20 mm) before and after drug administration were dynamically monitored to investigate the pH change trend of the adjacent tissues after intratumoral injection of the drug composition of the present invention. The results are shown in Table 16.

[0313] Table 16 pH values ​​at different time points and different tumor-peripheral distances

[0314] Using analysis software such as Excel, the data in Table 16 was plotted into a graph, and the result is as follows. Figure 8B As shown in the figure, the horizontal axis represents different time points after drug administration to the tumor, and the vertical axis represents the corresponding pH value at each time point. The four lines (blue, purple, yellow, and red) represent the fluctuation curves of pH value in the tissue adjacent to the tumor (2mm, 5mm, 10mm, and 20mm) over time, respectively. The red box indicates the normal physiologically acceptable pH range.

[0315] from Figure 8B It is evident that at 2 mm from the tumor: the pH value reached its peak (8.751) shortly after administration (0.08 h), then gradually decreased, returning to near pre-administration levels after 12 h. At 5 mm from the tumor: it also reached its peak (8.475) shortly after administration (0.08 h), but the rate of decrease was faster, returning to the normal physiological pH range after 1 h. At 10 mm from the tumor: the overall pH change was relatively gradual, with less influence from fluctuations in tumor-related drug administration. At 20 mm from the tumor: the overall change was the most stable, remaining within the normal physiological pH range throughout the monitoring process.

[0316] In summary, after intratumoral injection of the drug composition of the present invention, the intratumoral pH rises to 12.67 in a short period of time, while the pH of normal tissue 2 mm and 5 mm away from the tumor only reaches a maximum of 8.7, and the pH of tissue 10 mm and 20 mm away from the tumor is within the normal physiological range of 7.0 to 7.75. Therefore, the high pH value inside the tumor has little impact on the surrounding tissues, demonstrating excellent safety.

[0317] Example 12: Drug toxicity test of single intramuscular quantitative injection of the composition of the present invention in SD rats and Nu / Nu nude mice This embodiment aims to study the nature, extent, time-dependent relationship, and reversibility of toxic reactions that may be caused by a single intramuscular quantitative injection of the composition of the present invention (samples 1-5) into SD rats and Nu / Nu nude mice, providing a reference for the safety evaluation of the A / B composition drug.

[0318] Group design: SD rat group, Nu / Nu blank rat group, Nu / Nu tumor-bearing rat group; Animal numbers: SD rats: 6 per group, half male and half female; Nu / Nu blank rats: 6 per group, half male and half female; Nu / Nu tumor-bearing rats: 6 per group, half male and half female.

[0319] Grouping method: SD rats, Nu / Nu blank rats, and Nu / Nu tumor-bearing rats were randomly grouped by sex according to their body weight; See Table 17-1 for specific group information.

[0320] Table 17-1 Grouping Information

[0321] Excel software was used for data analysis, and GraphPad Prism 10.1.2 was used for creating statistical charts.

[0322] The composition of the present invention is administered by intramuscular injection of 100 μL into the hind limb of each SD rat according to its body weight; and by intramuscular injection of 5 μL into the hind limb of each Nu / Nu nude mouse according to its body weight. The sterile water for injection used in the control group of this experiment is administered at the same dose as the composition of the present invention.

[0323] To ensure more accurate experimental results, this experiment employed a self-comparison method between the left and right sides of the same body. All experimental groups received an intramuscular injection of sterile water for injection into the left hind limb and an intramuscular injection of the drug composition of this invention into the right hind limb.

[0324] The specific dosage design is shown in Table 17-2.

[0325] Table 17-2 Dosage Design Table

[0326] Route of administration: Intramuscular injection. Dosage frequency: Once a day, observation period of 28 days. Dosage volume: 12 mL / kg. Injection site: Bilateral hind limb muscles of rats or nude mice. Before administration to rats, prepare the skin of the left and right hind limbs to be injected, disinfect with 75% alcohol, and administer the drug. The day of administration is defined as day 0 of the experiment.

[0327] General observation: Observe once immediately after administration and once 2 hours later; observe once on days 0 (before and immediately after administration), 1, 2, 3, 7, 11, and 15 of the experiment. Observation indicators or contents: including but not limited to the physical signs of rats or nude mice, injection site, general behavior, mental state, glandular secretion, respiratory status, fecal characteristics, genitals, death, and other toxic manifestations.

[0328] Body weight was measured once each on days 0 (before and immediately after drug administration), 1, 2, 3, 7, 11, and 15 of the experiment. Animals used in the study: all surviving rats or Nu / Nu nude mice.

[0329] Hematological testing time: before and immediately after administration of experimental drugs; tested animals: surviving rats; blood collection method: rats in each group were anesthetized with isoflurane, and then approximately 0.5 mL of blood was collected from the abdominal aorta.

[0330] In general observation, no abnormal reactions were observed in the left hind limbs of all test animals in the SD rat group, Nu / Nu blank rat group, and Nu / Nu tumor-bearing rat group after intramuscular injection of sterile water for injection.

[0331] All SD rats showed no abnormalities in right hindlimb movement before administration. 30 minutes after injection of 100 μL of the composition of the present invention into the right hindlimb, only female rat 3 showed slight lameness, which returned to normal on the 3rd day after administration. No serious or irreversible motor impairment was observed. The other rats showed normal right hindlimb movement during the observation period, with no lameness, weakness or other motor abnormalities.

[0332] Before administration, both Nu / Nu blank mice and Nu / Nu tumor-bearing mice showed no abnormalities in right hind limb movement. Immediately after administration, a few mice exhibited mild local reactions such as hind limb lameness and weakness. On day 1 post-administration, most mice were in normal condition, with a few individuals showing hind limb weakness or mild lameness. On day 2 post-administration, some mice showed local reactions such as hind limb weakness, swelling, crusting, or mild lameness. On day 3 post-administration, only two mice in both experimental groups showed mild lameness, but these were all mild manifestations. No serious systemic adverse reactions or deaths occurred during the observation period, and there was no significant difference between the two groups. See Tables 17-3, 17-4, and 17-5 below for specific results.

[0333] Table 17-3 Summary of observations on the effects of intramuscular injection of the drug composition of the present invention on SD rats

[0334] Table 17-4 Summary of observations on the effects of intramuscular injection of the drug composition of the present invention on Nu / Nu blank mice

[0335] Table 17-5 Summary of observations on the effects of intramuscular injection of the drug composition of the present invention on Nu / Nu tumor-bearing mice

[0336] After intramuscular administration to the hind limbs of SD rats, the body weight changes over three consecutive days showed that neither male nor female rats experienced a statistically or toxicologically significant sustained decrease in body weight, indicating that the rats under this administration method had good local and systemic tolerance during the observation period.

[0337] For male rats, the weight of the rats after administration showed reversible fluctuations: some rats experienced a slight decrease in weight on the second day after administration, and by the third day all rats showed a recovery trend in weight. The weight change of female rats was generally a positive fluctuation based on normal growth. Therefore, the weight changes of male and female rats within 3 days after administration were within the normal physiological fluctuation range, with no obvious sex differences, and no serious weight suppression or toxic reactions related to administration were observed.

[0338] Following intramuscular administration to the hind limbs of Nu / Nu control mice and Nu / Nu tumor-bearing mice, the body weight showed a sustained and stable upward trend. From before administration to day 1, day 2, and day 3, the mice's body weight increased compared to baseline. Throughout the observation period, no mice experienced weight loss or significant fluctuations, demonstrating that the composition of this invention had no adverse effects on the overall health of the mice, and the animals were in good condition. Specific results are shown in Tables 17-6, 17-7, 17-8, and 17-9 below.

[0339] Table 17-6 Effects of intramuscular injection of the composition of the present invention on body weight of male SD rats

[0340] Table 17-7 Effect of intramuscular injection of the composition of the present invention on the body weight (g) of female SD rats

[0341] Table 17-8 Effect of intramuscular injection of the composition of the present invention on body weight of Nu / Nu blank mice

[0342] Table 17-9 Effects of intramuscular injection of the composition of the present invention on body weight in Nu / Nu tumor-bearing mice

[0343] Immediately after administration, compared with pre-administration levels, the main hematological parameters of the six SD rats, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT), did not show significant changes exceeding the normal physiological fluctuation range. Red blood cell and platelet-related parameters (MCV, MCH, MCHC, RDW, PDW, MPV, etc.) also remained stable. The experimental results demonstrate that the drug composition of this invention did not cause significant hematological toxicity under this administration method. Specific results are shown in Tables 17-10 and 17-11 below.

[0344] Table 17-10 Effects of intramuscular injection of the composition of the present invention on hematology in male SD rats

[0345] Table 17-11 Effects of intramuscular injection of the composition of the present invention on hematology in female SD rats

[0346] Example 13: Pathological study of tumor and organs after intratumoral injection of the composition of the present invention This embodiment aims to conduct a pathological study of the tumor and organs after intratumoral injection of the composition of the present invention.

[0347] Histological observation of the subcutaneous tumor tissue and surrounding tissue damage at the tumor implantation site from day 1 to day 21 after intratumoral injection of the composition of the present invention (samples 1-5) was conducted. The results showed that: (1) Tumor: After injection of the experimental drug, the active tumor showed pathological processes such as apoptosis and necrosis, and the tumor disappeared in the final section; (2) Fibrous connective tissue around the tumor: The tissue went through the process of acute injury - inflammation outbreak - inflammation subsidence - granulation tissue formation - tissue remodeling, and the injected drug did not have a significant effect on it; (3) There were no obvious damage changes in the rib and intercostal striated muscle tissue; (4) The skin at the injection site went through the process of acute injury - scab formation - epidermal healing and hyperplasia, with a process time of 21 days, which is basically consistent with the physiological repair time of skin lesions in nude mice. This suggests that the experimental drug does not affect the skin healing and repair process; (5) The drug did not produce pathological drug and toxin damage to the five important organs (liver, heart, lung, kidney and spleen) of nude mice.

[0348] Specifically, at 24 hours after administration, no obvious degeneration or necrosis was observed in the tumors of the experimental group. The epidermis around the tumor was thinned, with focal acute skin lesions; some skin appendages atrophied; mild acute inflammation was observed in the dermal fibers, adipose tissue, and deep striated muscle tissue, but no significant changes were seen in the fiber, fat, and striated muscle structures. In the control group (tumors without medication): mild inflammatory reaction was observed in the tissue adjacent to the tumor. See [link to detailed results] for further information. Figure 9A and 9B .

[0349] At 48 hours after administration, the drug showed a killing effect on the tumor (necrosis rate of approximately 15%, within the black circle). The epidermis around the tumor thinned, and skin appendages atrophied; mild hyalinization of dermal fibers and adipose tissue was observed, with no clear acute or chronic inflammatory response; no pathological lesions were observed in the deep fibrous striated muscle tissue structure. The black arrows indicate folds during slide preparation and are not related to the interpretation of experimental results. Control group (tumors without drug administration): No inflammatory response or hyalinization was observed in the tissue adjacent to the tumor. See details for further information. Figure 9C and 9D .

[0350] At 72 hours after administration, the drug showed a killing effect on the tumor (necrosis rate approximately 45%, indicated by the black circle). The epidermis surrounding the tumor thinned, and the skin appendages atrophied; mild hyalinization of the dermal fibers and adipose tissue was observed, with no clear acute or chronic inflammatory response; focal acute suppurative inflammation was observed in the interstitial spaces between the deep fibrous tissue and striated muscle tissue (black boxed area). The black arrows indicate folds during slide preparation and are not related to the interpretation of experimental results. Control group (tumors without drug administration): No inflammatory response or hyalinization was observed in the tissue adjacent to the tumor. See details for further information. Figure 9E and 9F .

[0351] On day 7 of drug administration, epidermal crusts formed in the experimental group, with no tumor tissue observed in the dermis. Granulation tissue (black line area) composed of lymphocytes, neutrophils, plasma cells, proliferating fibroblasts, and immature capillaries was observed in the deep dermis. No pathological lesions were observed in the deep chest wall, intermuscular striated muscle tissue, or pericostal cartilage tissue. In the control group (tumors without drug administration), no inflammatory reaction was observed in the tissue adjacent to the tumor (black line area). See details for further information. Figure 9G and 9H .

[0352] On day 21 of drug administration, the experimental group showed proliferation of the acanthosis and granular layer of the epidermis, with proliferating fibrous tissue and capillaries observed in the dermis, presenting as chronic granulomatous disease; no skin appendages or pathological damage were observed. For detailed results, please refer to [link to relevant documentation]. Figure 9I .

[0353] Furthermore, the pathological evaluation results of tissues and organs from day 1 to day 21 after intratumoral injection of the composition of the present invention are as follows: No significant drug or toxin damage was observed in liver tissue from 24 hours to 21 days after administration. See details below. Figure 10A .

[0354] No significant drug or toxin damage was observed in myocardial tissue from 24 hours to 21 days after administration. See details below. Figure 10B .

[0355] No significant drug or toxin damage was observed in lung tissue from 24 hours to 21 days after administration. See details for further information. Figure 10C .

[0356] No significant drug or toxin damage was observed in the renal tissue from 24 hours to 21 days after administration. See details below. Figure 10D .

[0357] No significant drug or toxin damage was observed in the spleen tissue from 24 hours to 21 days after administration. See details for further information. Figure 10E .

[0358] Example 14: Study on the antitumor activity of the combination of L-ascorbic acid and citric acid This embodiment aims to evaluate the antitumor activity of the samples of the present invention when L-ascorbic acid and citric acid are used in combination. Samples 12-1, 12-2, and 12-3 contain different ratios of L-ascorbic acid and citric acid, respectively. Mice were inoculated with human-derived tumors (tumor volume 100 mm²). 3 -300mm 3 The tumor volume (TV / mm²) in mice was monitored before drug administration and on days 3, 11, and 21 after drug administration. 3 The administration volume is 100µL / 100mm. 3 / time, administration frequency is 1 time. See Table 18 below for specific experimental results.

[0359] Table 18 Antitumor activity of the combination of L-ascorbic acid and citric acid

[0360] As shown in Table 18 above, samples 12-1, 12-2, and 12-3 all resulted in a significant reduction in tumor volume 3 days after intratumoral injection, and no tumors were observed in the experimental mice on days 11 and 21 after administration. These data indicate that, within the scope of this invention, combining L-ascorbic acid and citric acid in different ratios can also achieve excellent antitumor effects.

[0361] Having fully described the invention, those skilled in the art will understand that the invention can be carried out in a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of any embodiments provided herein.

[0362] All patents, patent applications and publications cited in this invention are incorporated herein by reference in their entirety.

Claims

1. A pharmaceutical composition for treating tumors, comprising, as an active ingredient: 1) Component A, which is selected from Compounds of formula (I) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (I) in, R1, R2, and R3 are each independently selected from H, C1-C 10 Alkyl, C1-C 18 Alkyl group, -P(=O)(OH)2, -S(=O)2(OH) and glucoside; R4 and R5 are each independently selected from H, OH, and Cl-C. 10 Alkoxy and C1-C 18 Alkyloxy group; or R4 and R5 together with the C atom attached to them to form an oxo group (-C(=O)); and / or Compounds of formula (II) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (II) Among them, R6, R7, R8, and R9 are each independently selected from H, C1-C 10 Alkyl, C1-C 18 Alkyl groups, -P(=O)(OH)2, -S(=O)2(OH) and glucosides; R 10 Selected from H and OH; and 2) Component B is a compound selected from the following: sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, sodium ethoxide, and potassium ethoxide; Based on the total weight of the composition, the mass percentage of component A is 0.5-50%; the mass percentage of component B is 0.5-50%. And optional pharmaceutically acceptable carriers or excipients.

2. The composition according to claim 1, wherein the compound of formula (I) is selected from L-ascorbic acid, L-ascorbyl glucoside, L-ascorbyl palmitate, L-ascorbyl-2-phosphate, L-ascorbyl-2-sulfate, D-ascorbic acid, 5-O-acetyl-L-ascorbic acid, 5-keto-L-ascorbic acid, and 5-diether-L-ascorbic acid.

3. The composition according to claim 1, wherein the compound of formula (II) is selected from citric acid.

4. The composition according to any one of claims 1-3, wherein component B is selected from sodium hydroxide.

5. The composition according to any one of claims 1-3, wherein the molar ratio between component A and component B is 20:1 to 1:

20.

6. The composition according to any one of claims 1-3, wherein the molar ratio between component A and component B is 5:1 to 1:

5.

7. The composition according to any one of claims 1-3, wherein, based on the total weight of the composition, the mass percentage of component A is 5-50%; and / or the mass percentage of component B is 5-50%.

8. The composition according to any one of claims 1-3, wherein the composition comprises component A, component B, and optionally a pharmaceutically acceptable carrier or excipient.

9. The composition according to any one of claims 1-3, wherein the composition is an injectable preparation.

10. The composition according to claim 9, wherein the composition is an intratumoral injection agent.

11. The composition according to claim 9, wherein the composition is an injection solution or a lyophilized powder for injection.

12. The composition according to claim 11, wherein the mass-volume concentration of component A in the injection solution is 0.5-50%; and / or the mass-volume concentration of component B is 0.5-50%.

13. The composition according to claim 11, wherein the pH of the injection solution is 9-14.

14. The composition according to claim 13, wherein the pH of the injection solution is 12-14.

15. A pharmaceutical composition for treating tumors, prepared by mixing the following components: 1) Component A, which is selected from Compounds of formula (I) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (I) in, R1, R2, and R3 are each independently selected from H, C1-C 10 Alkyl, C1-C 18 Alkyl group, -P(=O)(OH)2, -S(=O)2(OH) and glucoside; R4 and R5 are each independently selected from H, OH, C1-C6 alkoxy groups and C1-C6 alkoxy groups. 18 Alkyloxy group; or R4 and R5 together with the C atom attached to them to form an oxo group (-C(=O)); and / or Compounds of formula (II) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (II) Among them, R6, R7, R8, and R9 are each independently selected from H, C1-C 10 Alkyl, C1-C 18 Alkyl groups, -P(=O)(OH)2, -S(=O)2(OH) and glucosides; R 10 Selected from H and OH; and 2) Component B, which is a compound selected from the following: sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, sodium ethoxide, potassium ethoxide; wherein, based on the total weight of the composition, the mass percentage of component A is 0.5-50%; and the mass percentage of component B is 0.5-50%. And 3) optional pharmaceutically acceptable carriers or excipients.

16. An intratumoral injection composition for treating tumors, comprising, as an active ingredient: 1) Component A, which is selected from Compounds of formula (I) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (I) in, R1, R2, and R3 are each independently selected from H, C1-C 10 Alkyl, C1-C 18 Alkyl group, -P(=O)(OH)2, -S(=O)2(OH) and glucoside; R4 and R5 are each independently selected from H, OH, C1-C6 alkoxy groups and C1-C6 alkoxy groups. 18 Alkyloxy group; or R4 and R5 together with the C atom attached to them to form an oxo group (-C(=O)); and / or Compounds of formula (II) below, or pharmaceutically acceptable salts, solvates or stereoisomers thereof: (II) Among them, R6, R7, R8, and R9 are each independently selected from H, C1-C 10 Alkyl, C1-C 18 Alkyl groups, -P(=O)(OH)2, -S(=O)2(OH) and glucosides; R 10 Selected from H and OH; and 2) Component B is a compound selected from the following: sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, sodium ethoxide, and potassium ethoxide; And optionally a pharmaceutically acceptable carrier or excipient, wherein the pH of the injection solution is 9-14.

17. The composition according to claim 16, wherein the pH of the injection solution is 12-14.

18. The composition according to claim 16 or 17, wherein the mass-volume concentration of component A in the injection solution is 0.5-50%; and / or the mass-volume concentration of component B is 0.5-50%.

19. The composition according to claim 16 or 17, wherein the molar ratio between component A and component B is 20:1 to 1:

20.

20. A method for preparing the composition according to any one of claims 1-19, comprising the step of mixing component A, component B, and optionally a pharmaceutically acceptable carrier or excipient.

21. Use of the composition of any one of claims 1-19 in the preparation of a medicament for treating tumors.

22. The use according to claim 21, wherein the tumor is a solid tumor.

23. The use according to claim 21 or 22, wherein the tumor is selected from: squamous cell carcinoma of the lung, adenocarcinoma of the lung, adenosquamous carcinoma of the lung, gastric adenocarcinoma, rectal adenocarcinoma, colonic adenocarcinoma, esophageal squamous cell carcinoma, greater omentum adenocarcinoma, ovarian adenocarcinoma, esophageal adenocarcinoma, small intestinal adenocarcinoma, small cell neuroendocrine gastric carcinoma, gastric squamous cell carcinoma, pelvic adenocarcinoma, pancreatic cancer, glioma, pelvic adenocarcinoma, and liver cancer.

24. The use as described in claim 21 or 22, wherein the medicament further comprises one or more additional therapeutic agents, or is administered therewith.

25. The composition of any one of claims 1-19, for treating tumors.

Images