Use of mitoxantrone in preparing lymphatic tracing agent

Abstract

The present disclosure provides use of mitoxantrone and / or a pharmaceutically acceptable salt thereof in preparing a lymphatic tracing agent. The lymphatic tracing agent is used for lymph node tracing in gastrectomy-associated diseases, demonstrating a good staining effect on gastric cancer lymph nodes.

Description

Application of Mitoxantrone in the Preparation of Lymphatic Tracers

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 2023117680923 filed on December 20, 2023, and the entire text of the above-mentioned Chinese patent application is incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the field of pharmaceutical preparations, and particularly relates to the use of mitoxantrone in the manufacture of lymphatic tracers for diseases associated with gastrectomy. Background Art

[0004] Gastric cancer is the fifth most common cancer worldwide and the third most common cause of cancer-related deaths. Approximately one million new cases of gastric cancer are diagnosed each year, with over 700,000 deaths. In my country, over 400,000 new cases are diagnosed annually. While the development of chemotherapy and small molecule targeted therapies has prolonged survival in the treatment of advanced gastric cancer, surgical resection remains the most effective cure. The 2010 edition of Japan's "Gastric Cancer Management Guidelines" designated D2 dissection as the standard surgical procedure for gastric cancer, a procedure widely accepted by both Eastern and Western researchers.

[0005] The 2010 edition of Japan's "Gastric Cancer Management Protocol" divides the lymph nodes involved in gastric cancer lymph node dissection into multiple groups, such as NO.1-right cardia lymph nodes, NO.2-left cardia lymph nodes, NO.3-lesser curvature lymph nodes, NO.4-greater curvature lymph nodes, and other groups of lymph nodes. Currently, there are five standards for lymph node dissection in gastric cancer surgery: the first level is level 0 radical resection (D0 surgery), the second level is level 1 radical resection (D1 surgery), the third level is level 2 radical resection (D2 surgery), the fourth level is level 3 radical resection (D3 surgery), and the fifth level is level 4 radical resection (D4 surgery). Due to its extremely low radicality, D0 surgery is basically not used by surgeons except for very early gastric cancer. D1 surgery performs the first-stage lymph node dissection around the stomach, which has a certain degree of radicality and is very safe. D2 surgery involves clearing the first and second lymph nodes around the stomach, and is highly curative, but carries a high risk of surgery, especially in non-major cancer centers, which are prone to postoperative complications. Due to their indications and difficulty, D3 and D4 surgeries are typically only performed in large cancer centers for patients with special medical conditions, and the risks of such surgeries are exponentially greater than those of D2 surgery. Clearing as many lymph nodes as possible has two benefits: 1. The surgery is thorough, minimizing residual tumor; 2. It provides more accurate postoperative pathological staging, facilitating the development of subsequent treatment (chemotherapy) plans. In other words, the most important and fundamental factor in determining the success of gastric cancer surgery is whether the lymph nodes are completely cleared.

[0006] Perigastric lymph nodes are numerous and widely distributed. However, they are often hidden in surrounding tissues, making them difficult to discern with the naked eye. Removing as many lymph nodes as possible is often challenging. Using lymphatic tracers to stain and trace living lymph nodes during surgery, allowing doctors to completely remove them, is key to improving the effectiveness of malignant tumor treatment.

[0007] Therefore, developing safe and effective lymphatic tracers to effectively clear the lymph nodes in the tumor-draining area is an important means to improve patients' quality of life and prolong their lifespan. Lymphatic tracers currently play a vital role in the clinical treatment of malignant tumors both domestically and internationally. Injecting lymphatic tracers near the primary lesion rapidly stains the lymph nodes near the tumor.

[0008] Currently known lymphatic tracers include nanocarbon and methylene blue, but nanocarbon and other agents present several challenges. While nanocarbon is often used for intraoperative sentinel lymph node tracing in breast cancer and thyroid cancer surgery, it accumulates and is not metabolized in the body. Entry into the blood and lymphatic circulation poses a risk of capillary blockage. Furthermore, given the hard and brittle texture of cancerous tissue, direct intratumoral injection can cause tumor necrosis and detachment, leading to bleeding. Furthermore, nanocarbon does not readily enter small lymph nodes, making it ineffective for tracing sentinel lymph nodes in gastric cancer. This can lead to continued proliferation and metastasis of tumor cells remaining in small lymph nodes after gastric cancer surgery, ultimately leading to a poor prognosis. Furthermore, gastric cancer resection and breast cancer resection have significantly different requirements for the size of lymph nodes that can be cleared with the tracer. Breast cancer tracing primarily targets sentinel lymph nodes, and if there is no metastasis in a sentinel lymph node, the likelihood of metastasis to other lymph nodes in the area is low. However, gastric cancer and breast cancer have different metastatic pathways. Gastric cancer resection requires tracing the smallest lymph nodes (≤1mm) and tracing as many lymph nodes as possible.

[0009] Therefore, seeking to develop a safe and effective lymph node tracer for intraoperative lymph node tracing in gastric cancer surgery and predicting whether the tumor has metastasized is an important means to improve the quality of life of gastric cancer patients and prolong their lifespan. Summary of the Invention

[0010] The present disclosure aims to provide a use of mitoxantrone and / or a pharmaceutically acceptable salt thereof in preparing a lymphatic tracer, wherein the lymphatic tracer is used for lymph node tracing in gastrectomy-related diseases.

[0011] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0012] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0013] As used herein, the terms "patient," "individual," and "subject" are used interchangeably and refer to any single animal, more preferably a mammal (including, for example, non-human animals such as cats, dogs, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates) for which treatment is desired. In certain embodiments, the patient herein is a human. The patient may be someone who has, is suspected of having, or is at risk of having a gastric tumor. As used herein, a "disorder" is any condition that would benefit from treatment, including, but not limited to, chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the condition in question.

[0014] As used herein, a "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and that contains no other components that are unacceptably toxic to a subject to which the formulation is administered.

[0015] As used herein, "pH adjusting agent" refers to a compound or a mixture of compounds that can be used to ensure that the pH of the reconstituted kit is within an acceptable range for human or mammalian administration (pH of about 4.0-10.5). Suitable pH adjusting agents include pharmaceutical buffers such as tris(hydroxymethyl)methylglycine (tricine), phosphate or TRIS (i.e., tris(hydroxymethyl)aminomethane); pharmaceutical acids such as pharmaceutical organic acids (such as formic acid, acetic acid) or mixtures thereof or inorganic acids (such as hydrochloric acid, phosphoric acid) or mixtures thereof, and pharmaceutical bases such as sodium carbonate, sodium bicarbonate or mixtures thereof. When the conjugate used is in the form of an acid salt, the pH adjusting agent can optionally be provided in a separate vial or container so that the user of the kit can adjust the pH as part of a multi-step operation.

[0016] As used herein, "pharmaceutically acceptable excipients" refer to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, carriers, stabilizers, or preservatives.

[0017] As used herein, "pharmaceutically acceptable salts" means salts that are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include acid and base addition salts. The phrase "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients forming the formulation and / or the mammal to be treated therewith.

[0018] As used herein, "pharmaceutically acceptable acid addition salts" refers to those pharmaceutically acceptable salts formed with inorganic acids selected from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid.

[0019] The term "pharmaceutically acceptable base addition salt" refers to those pharmaceutically acceptable salts formed with organic or inorganic bases. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and salts of basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrazine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine and polyamine resins.

[0020] As used herein, "treatment" refers to clinical intervention that attempts to alter the natural course of a disease in the individual being treated, and can be performed for prevention or during clinical pathology. Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, alleviating or palliating the disease state, and alleviating or improving prognosis.

[0021] As used herein, "administration" refers to a method of administering a dose of a compound (e.g., mitoxantrone hydrochloride injection) or a pharmaceutical composition (e.g., a pharmaceutical composition comprising an inhibitor or antagonist) to a subject (e.g., a patient). Administration can be performed in any suitable manner, including parenteral, intrapulmonary, and intranasal administration, and if necessary for local treatment, intralesional administration can be performed. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be performed by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at different time points, push administration, and pulse infusion.

[0022] As used herein, the Full Analysis Set (FAS) refers to the subject set based on the Intention To Treat (ITT) principle. It refers to the data set consisting of all subjects who participated in the trial, received treatment, and had baseline efficacy evaluations.

[0023] As used in this article, the Per Protocol Set (PPS) refers to all subgroups of the treatment population that completed the trial and excluded serious violations of the protocol (referring to the research subjects violating the inclusion or exclusion criteria), and is a collection of cases that met the inclusion criteria, did not meet the exclusion criteria, and completed the treatment plan.

[0024] As used in this article, "lymph node metastasis" refers to the metastasis of gastric cancer cells to lymph nodes somewhere along the lymphatic system, which is an important way for gastric cancer to metastasize. Generally speaking, gastric cancer lymph node metastasis starts from near and then to far. As the cancer tumor extends to the deeper layer, the chance of metastasis increases. It is divided into three stations according to the order of metastasis: the first station is the superficial lymph nodes closest to the cancer body, attached to the stomach wall, such as the greater curvature of the stomach, the lesser curvature of the stomach, the upper and lower pyloric nodes, and the lymph nodes beside the cardia (hereinafter, the first station lymph nodes are sometimes referred to as "N1 lymph nodes", and the first station stained lymph nodes are referred to as "N1 stained lymph nodes"); the second station is the deep lymph nodes that drain the superficial lymph nodes, such as The splenic hilum, common hepatic, left gastric artery trunk, and pancreaticoduodenal artery lymph nodes (hereinafter, the second-station lymph nodes are sometimes referred to as "N2 lymph nodes," and the second-station stained lymph nodes are referred to as "N2-stained lymph nodes"); the third station includes the lymph nodes around the celiac artery, para-aorta, hepatic hilum and mesenteric root, and middle colic artery, and sometimes metastasizes to the left supraclavicular lymph nodes (hereinafter, the third-station lymph nodes are sometimes referred to as "N3 lymph nodes," and the third-station stained lymph nodes are referred to as "N3-stained lymph nodes").

[0025] As used in this article, "TNM tumor staging" refers to the AJCC / UICC TNM staging for gastric cancer. For specific staging standards, please refer to the Guidelines for the Diagnosis and Treatment of Gastric Cancer (2022).

[0026] The present disclosure provides a use of mitoxantrone and / or a pharmaceutically acceptable salt thereof in preparing a lymphatic tracer, wherein the lymphatic tracer is used for lymph node tracing in gastrectomy-related diseases.

[0027] In some specific embodiments, the lymph node is a lymph node with a short diameter less than or equal to 2 mm, and may further be a lymph node with a short diameter less than or equal to 1.5 mm, and preferably a lymph node with a short diameter less than or equal to 1 mm.

[0028] The inventors of the present disclosure have discovered that the mitoxantrone and / or its pharmaceutically acceptable salts described in the present disclosure can be used in the preparation of lymph tracers for tracing lymph nodes during gastric cancer resection, and can stain lymph nodes smaller than or equal to 1 mm in size. Furthermore, the lymph node staining effect is excellent during resection surgeries at various locations and orientations of the stomach and perigastric region.

[0029] In some specific embodiments, the detection rate of the tracer for lymph nodes with a short diameter of 1 mm or less is greater than 70%, and further greater than 75%.

[0030] In the present disclosure, the color development time of the tracer is less than 25 minutes, and further less than 20 minutes.

[0031] In some embodiments, the tracer has a fade time of greater than 180 minutes.

[0032] In some embodiments, the tracer has a lymph node staining rate greater than 50%, further greater than 55%, in gastric cancer resection.

[0033] In some specific embodiments, the disease associated with gastrectomy is gastric polyps or gastric tumors.

[0034] In some embodiments, the gastric tumor includes benign gastric tumors and malignant gastric tumors.

[0035] In some specific embodiments, the benign gastric tumor and the malignant gastric tumor are selected from gastric fundus and cardia cancer, gastric body cancer, and gastric antrum cancer.

[0036] In some specific embodiments, the disease associated with gastrectomy is selected from one or more of tumors located in the following locations: the cardia, the cardia curvature side, the posterior wall of the cardia, the lesser curvature of the cardia, the lesser curvature side of the cardia and the gastric body, the anterior wall of the lesser curvature of the gastric fundus, the gastric fundus, the lesser curvature of the gastric fundus, the anterior wall of the gastric antrum, the greater curvature of the gastric fundus, the gastric antrum junction, the lesser curvature of the gastric antrum, the upper posterior wall of the gastric body, the posterior wall of the lower gastric body, the greater curvature side of the gastric fundus and the gastric body, the posterior wall of the lesser curvature of the gastric fundus, the anterior and posterior walls of the lesser curvature, the gastric fundus, and the posterior wall of the lesser curvature of the gastric body.

[0037] In some embodiments, the tumor is a TNM tumor stage I, II, III, or IV tumor.

[0038] In some embodiments, the gastrectomy is selected from total gastrectomy, subtotal gastrectomy, hemigastrectomy, or antrectomy.

[0039] In some embodiments, the gastrectomy-related disease is gastric polyps or gastric tumors.

[0040] In some embodiments, the lymphatic tracer is used for lymphatic tracing in the gastric polyp or the gastric tumor.

[0041] In some embodiments, the lymphatic tracer is used for lymphatic tracing in gastric tumors.

[0042] In some embodiments, the lymphatic tracer comprises mitoxantrone and / or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0043] In some embodiments, the pharmaceutical excipients include, but are not limited to, one or more of an osmotic pressure regulator, an antioxidant, an adsorbent, a filler, a buffer, a carrier, a stabilizer, or a preservative.

[0044] In some embodiments, the lymphatic tracer is an injectable agent.

[0045] In some embodiments, the injection is in the form of a solution, a lyophilized powder, an emulsion, a liposome, a nanoparticle, a nanocrystal, a microcrystal, a microsphere, or a gel.

[0046] In some preferred embodiments, the solution is sodium chloride injection or glucose injection.

[0047] In some specific embodiments, the injection is formulated as a dosage form at a concentration of 2-10 mg / mL, and a single dose of the drug is 0.5-3 mL, for example, 0.5 mL, 1 mL, 1.5 mL, 2.5 mL or 3 mL, preferably 2-3 mL.

[0048] In some preferred embodiments, the pharmaceutically acceptable salt is selected from one or more of mitoxantrone hydrochloride, mitoxantrone oxalate, mitoxantrone sulfate, mitoxantrone phosphate, mitoxantrone acetate, and mitoxantrone citrate.

[0049] Preferably, the pharmaceutically acceptable salt is mitoxantrone hydrochloride.

[0050] In some specific embodiments, the tracer is an injection, and its components include mitoxantrone or mitoxantrone salts: 0.05%-5%, and osmotic pressure regulator: 0.1%-10% according to the mass volume ratio (g / mL).

[0051] In some specific embodiments, the tracer further contains a buffer: 0.01%-0.1%, an antioxidant: 0.01%-0.1%, an adsorbent: 0.05%-1%, and a filler: 0%-20%.

[0052] In some specific embodiments, the osmotic pressure regulator is one or a mixture of sodium chloride, glucose, sorbitol, mannitol, glycerol, phosphate, and citrate.

[0053] In some specific embodiments, the buffer is one or more of acetic acid, sodium acetate, citric acid, and sodium citrate.

[0054] In some specific embodiments, the antioxidant is one or more of sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, and disodium edetate. Polyethylene glycol is unstable at high temperatures, and antioxidants (such as sodium metabisulfite) help inhibit its oxidation to produce impurities.

[0055] In some specific embodiments, the filler is one or more of the monosaccharides glucose, fructose, galactose, ribose or deoxyribose, or the disaccharides sucrose, trehalose, maltose, lactose, or the polymeric sugars mannitol, sorbitol, lactitol, xylitol, maltitol, and erythritol.

[0056] Preferably, the adsorbent is activated carbon.

[0057] In some embodiments, the lymphatic tracer comprises a pH modifier.

[0058] In some preferred embodiments, the pH adjuster is selected from one or more of hydrochloric acid, phosphoric acid, sulfuric acid, oxalic acid, acetic acid and citric acid.

[0059] In some specific embodiments, the pH value of the injection is in the range of 2.8-4.3.

[0060] In some embodiments, the tracer also includes polyethylene glycol, with a concentration of 0.01%-2% by mass in the injection, preferably 1%-2%. This helps increase the water solubility and stability of mitoxantrone hydrochloride (for example, by preventing it from decomposing in water). It is also used in the formulation of nanoparticle drug carriers and controlled-release systems, controlling drug delivery, and promoting lymphatic transport. Specifically, the addition of polyethylene glycol helps form a local reservoir of the drug after injection, thereby prolonging the duration of the drug's effect.

[0061] In one embodiment, the molecular weight of the polyethylene glycol is less than 2000, preferably less than 1000, further preferably 200, 400 or 600, more preferably 400 or 600, which is more conducive to the staining of mitoxantrone hydrochloride.

[0062] In some embodiments, the osmolality is selected in the range of 285 to 2317 mmol / kg.

[0063] In some preferred embodiments, the osmotic pressure range is 600-1200 mmol / kg. When the osmotic pressure is too low, lymphatic drainage is insufficient, while when the osmotic pressure is too high, the concentration of the osmotic pressure regulator in the injection is too high, causing irritation at the injection site. Maintaining the osmotic pressure between 600-1200 mmol / kg facilitates better staining of the lymph nodes.

[0064] In some specific embodiments, the nanoparticle size of the tracer under physiological conditions is 20-100 nm, which can pass through the gaps between lymphatic endothelial cells and produce a good lymphatic targeting effect, preferably 30-60 nm, and further can be 40-60 nm.

[0065] In some preferred embodiments, the content of mitoxantrone in the mitoxantrone or its salt is 1-15 mg / ml by weight and volume; preferably 2-10 mg / ml; more preferably 2 mg / ml, 5 mg / ml or 10 mg / ml.

[0066] In some preferred embodiments, the content of sodium chloride is 3-18 mg / ml by weight and volume; preferably 4-16 mg / ml; more preferably 4 mg / ml, 8 mg / ml or 16 mg / ml.

[0067] In some preferred embodiments, the content of acetic acid is 0.15-1 mg / ml by weight and volume; preferably 0.23-0.92 mg / ml; more preferably 0.23 mg / ml, 0.46 mg / ml or 0.92 mg / ml.

[0068] In some preferred embodiments, the content of sodium acetate is 0.03-0.15 mg / ml by weight and volume; preferably 0.05-0.1 mg / ml; more preferably 0.05 mg / ml or 0.1 mg / ml.

[0069] In some preferred embodiments, the content of the antioxidant is 0.05-0.3 mg / ml by weight and volume; preferably 0.8-0.12 mg / ml; more preferably 0.1 mg / ml, 0.2 mg / ml or 0.3 mg / ml.

[0070] In some preferred embodiments, the content of sodium sulfate is 0.05-0.6 mg / ml by weight and volume; preferably 0.15-0.45 mg / ml; more preferably 0.15 mg / ml, 0.3 mg / ml or 0.45 mg / ml.

[0071] In some embodiments, the lymphatic tracer comprises:

[0072] In some preferred embodiments, the lymphatic tracer comprises:

[0073] In some preferred embodiments, the lymphatic tracer optionally further comprises edetate disodium, and preferably, the amount of edetate disodium is 0-0.3 mg / mL.

[0074] In some preferred embodiments, the lymphatic tracer optionally further comprises polyethylene glycol. Preferably, the amount of the polyethylene glycol is 0-20 mg / mL.

[0075] In some preferred embodiments, the lymphatic tracer comprises:

[0076] In some embodiments, the solvent is selected from water, sodium chloride solution or glucose solution.

[0077] Preferably, the water is water for injection.

[0078] In a preferred embodiment, the content of impurity I in the mitoxantrone preparation is between 0% and 1.5%, and the structural formula of impurity I is shown in formula (II):

[0079] In a preferred embodiment, the content of impurity II in the mitoxantrone preparation is between 0% and 1.5%, and the structural formula of impurity II is shown in formula (III):

[0080] In a preferred embodiment, the content of impurity III in the mitoxantrone preparation is between 0% and 1.5%, and the structural formula of impurity III is shown in formula (IV):

[0081] In a preferred embodiment, the content of impurity IV in the mitoxantrone preparation is between 0% and 1.5%, and the structural formula of impurity IV is shown in formula (V):

[0082] In some specific embodiments, the lymphatic tracer comprises: 2.91 g mitoxantrone, 16 g sodium chloride, 0.92 g acetic acid, 0.1 g sodium acetate, 0.4 g sodium metabisulfite, 0.9 g sodium sulfate, and water for injection is added to 2000 ml;

[0083] Or, 5.82g mitoxantrone, 16g sodium chloride, 0.92g acetic acid, 0.1g sodium acetate, 0.4g sodium metabisulfite, 0.9g sodium sulfate, water for injection added to 2000ml;

[0084] Or, 11.64g mitoxantrone, 16g sodium chloride, 0.92g acetic acid, 0.1g sodium acetate, 0.4g sodium metabisulfite, 0.9g sodium sulfate, water for injection added to 2000ml;

[0085] Alternatively, 23.28g mitoxantrone, 16g sodium chloride, 0.92g acetic acid, 0.1g sodium acetate, 0.4g sodium metabisulfite, 0.9g sodium sulfate, and water for injection are added to 2000ml;

[0086] Or, 11.64g mitoxantrone, 32g sodium chloride, 0.92g acetic acid, 0.1g sodium acetate, 0.4g sodium metabisulfite, 0.9g sodium sulfate, water for injection added to 2000ml;

[0087] Or, 11.64g mitoxantrone, 16g sodium chloride, 0.92g acetic acid, 0.1g sodium acetate, 0.2g sodium metabisulfite, 0.3g sodium sulfate, water for injection added to 2000ml;

[0088] Or, 11.64g mitoxantrone, 16g sodium chloride, 0.92g acetic acid, 0.1g sodium acetate, 0.3g sodium metabisulfite, 0.3g sodium sulfate, water for injection added to 2000ml;

[0089] Or, 11.64g mitoxantrone, 16g sodium chloride, 1.84g acetic acid, 0.2g sodium acetate, 0.2g sodium metabisulfite, 0.3g sodium sulfate, water for injection added to 2000ml;

[0090] Or, 11.64g mitoxantrone, 16g sodium chloride, 1.84g acetic acid, 0.2g sodium acetate, 0.4g sodium metabisulfite, 0.3g sodium sulfate, water for injection added to 2000ml;

[0091] Or, 11.64g mitoxantrone, 16g sodium chloride, 1.84g acetic acid, 0.2g sodium acetate, 0.6g sodium metabisulfite, 0.3g sodium sulfate, water for injection added to 2000ml;

[0092] Or, 11.64g mitoxantrone, 32g sodium chloride, 1.84g acetic acid, 0.2g sodium acetate, 0.3g sodium metabisulfite, 0.3g sodium sulfate, water for injection added to 2000ml;

[0093] Or, 11.64g mitoxantrone, 32g sodium chloride, 1.84g acetic acid, 0.2g sodium acetate, 0.3g sodium metabisulfite, 0.6g sodium sulfate, water for injection to 2000ml;

[0094] Or, 11.64g mitoxantrone, 16g sodium chloride, 0.92g acetic acid, 0.1g sodium acetate, 40g polyethylene glycol 400, 0.2g sodium metabisulfite, 0.3g sodium sulfate, water for injection added to 2000ml;

[0095] Or, 11.64g mitoxantrone, 16g sodium chloride, 0.92g acetic acid, 0.1g sodium acetate, 20g polyethylene glycol 1000, 0.2g sodium metabisulfite, 0.3g sodium sulfate, add water for injection to 2000ml.

[0096] In some specific embodiments, the lymphatic tracer is an injectable preparation, which is prepared by a method comprising the following steps:

[0097] Weigh the prescribed amount of excipients, add them to the prescribed amount of solvent, stir to dissolve, and then add the prescribed amount of mitoxantrone hydrochloride and / or a pharmaceutically acceptable salt thereof. In some specific embodiments, the solvent is water for injection.

[0098] It is understandable that the aforementioned osmotic pressure parameters can be adjusted by controlling the formulation and content of excipients, etc., and the specific details shall be subject to the test compliance standards.

[0099] In some specific embodiments, the injection is prepared by the following method:

[0100] (1) Weigh the prescribed amount of acetic acid, sodium acetate, sodium chloride, and disodium edetate; or weigh the prescribed amount of acetic acid, sodium acetate, sodium chloride, and sodium metabisulfite; or weigh the prescribed amount of acetic acid, sodium acetate, sodium chloride, sodium metabisulfite, and sodium sulfate, add them to the prescribed amount of water for injection, stir to dissolve, and obtain an excipient mixture.

[0101] (2) Adding the prescribed amount of mitoxantrone hydrochloride to the excipient mixture obtained in step (1), stirring to dissolve, and obtaining mitoxantrone hydrochloride injection; preferably, stirring for 10-30 minutes to dissolve.

[0102] In some embodiments, the method further comprises:

[0103] (3) Filtration; preferably, fine filtration through 0.45 μm and 0.22 μm filter membranes.

[0104] In some embodiments, the method further comprises:

[0105] (4) Fill with nitrogen, 2 ml per tube, cap, and sterilize at 121℃ for 15 min; the pH value range is between 2.8-4.3.

[0106] In some specific embodiments, step (1) further comprises: weighing the prescribed amount of polyethylene glycol, acetic acid, sodium acetate, sodium chloride and disodium edetate; or weighing the prescribed amount of polyethylene glycol, acetic acid, sodium acetate, sodium chloride and sodium metabisulfite; or weighing the prescribed amount of polyethylene glycol, acetic acid, sodium acetate, sodium chloride, sodium metabisulfite and sodium sulfate, adding the mixture to the prescribed amount of water for injection, stirring to dissolve, and obtaining an excipient mixture.

[0107] In some preferred embodiments, the injection is prepared in a specification of 2 ml:10 mg, 1 ml:5 mg, or 0.5 ml:2.5 mg.

[0108] In some specific embodiments, the injection is administered as peritumoral injection, or at points in the greater curvature and lesser curvature of the stomach.

[0109] In some preferred embodiments, the injection is administered subcutaneously at 4-6 injection points above, below, on the left, and on the right of the greater and lesser curvatures of the stomach.

[0110] In another aspect of the present disclosure, a method for tracing lymph nodes in gastrectomy-related diseases is provided, which comprises the following main steps:

[0111] S1: Prepare pathological information of experimental patients;

[0112] S2: Use mitoxantrone and / or its pharmaceutically acceptable salts to prepare lymphatic tracers and conduct tracing experiments on patients who meet the requirements;

[0113] S3: Observing the patient's indicators after using mitoxantrone and / or its pharmaceutically acceptable salts to prepare lymphatic tracers;

[0114] S4: Perform statistical analysis on the obtained indicator data.

[0115] In the method, the usage, dosage, composition, preparation method, etc. of the tracer are consistent with the aforementioned usage.

[0116] In another aspect of the present disclosure, a lymph node tracer comprising mitoxantrone and / or a pharmaceutically acceptable salt thereof is provided, which is used for lymph node tracing in gastrectomy-related diseases;

[0117] Preferably, the composition, preparation method, usage and / or dosage of the tracer are consistent with the aforementioned use section.

[0118] Another aspect of the present disclosure provides a method for treating a patient suffering from a gastrectomy-related disease, comprising:

[0119] administering a lymphatic tracer comprising mitoxantrone and / or a pharmaceutically acceptable salt thereof to a patient diagnosed with a gastrectomy-related disease to trace the lymph nodes in the stomach and surrounding areas;

[0120] administering to the patient a treatment comprising gastrectomy;

[0121] Preferably, the composition, preparation method, usage and / or dosage of the tracer are consistent with the aforementioned uses;

[0122] Preferably, the tracing method is the aforementioned lymph node tracing method.

[0123] By adopting the above technical solution, mitoxantrone itself has a tropism for the lymphatic system, and its own color (blue) stains the lymph nodes near the tumor. Local injection during gastric cancer surgery can stain the lymph nodes near the tumor, assisting in the clinical location and clearance of lymph nodes. In-depth preclinical pharmacodynamics studies of mitoxantrone hydrochloride have shown that this product has a high affinity for lymph nodes when injected subcutaneously, staining them blue and facilitating its use as a lymphatic tracer. Its use in lymph node tracing during gastrectomy surgery can increase the number of lymph nodes detected, thereby reducing staging bias and improving the accuracy of gastric cancer staging.

[0124] By adopting the above technical solution, the lymph nodes and the first-station lymph nodes can be clearly blue-stained and clearly visible during surgery, thereby improving the blue-staining rate of lymph nodes and significantly increasing the total number of detected lymph nodes. It has the characteristics of convenience, fast staining, long staining time and high lymph node blue-staining rate. Compared with the traditional nanocarbon tracer method, it can further increase the number of detected lymph nodes, thereby reducing staging bias and improving the accuracy of gastric cancer staging.

[0125] According to the above technical solution, the present invention can completely clear the lymph nodes (especially the smallest lymph nodes) during the metastasis of gastric cancer, prevent the recurrence and secondary metastasis of gastric cancer, improve the quality of life of gastric cancer patients, and delay the life span of patients. DETAILED DESCRIPTION

[0126] For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments; however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0127] Example 1. Preparation of Mitoxantrone Hydrochloride Injection Formula 1

[0128] Table 1: Prescription of Example 1

[0129] Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, and edetate disodium, add to the prescribed amount of water for injection, and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value should be 3.5.

[0130] Example 2. Preparation of Mitoxantrone Hydrochloride Injection Formula 2

[0131] Table 2: Prescription of Example 2

[0132] Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, sodium metabisulfite, and sodium sulfate, add to the prescribed amount of water for injection, and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value should be 3.4.

[0133] Example 3. Preparation of Mitoxantrone Hydrochloride Injection Formula 3

[0134] Table 3: Prescription of Example 3

[0135] Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, and edetate disodium, add to the prescribed amount of water for injection, and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value is 3.6.

[0136] Example 4. Preparation of Mitoxantrone Hydrochloride Injection Formula 4

[0137] Table 4: Prescription of Example 4

[0138] Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, sodium metabisulfite, and sodium sulfate, add to the prescribed amount of water for injection, and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value should be 3.7.

[0139] Example 5. Preparation of Mitoxantrone Hydrochloride Injection Formula 5

[0140] Table 5: Prescription of Example 5

[0141] Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, sodium metabisulfite, and sodium sulfate, add to the prescribed amount of water for injection, and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value should be 3.6.

[0142] Example 6. Preparation of Mitoxantrone Hydrochloride Injection Formula 6

[0143] Table 6: Prescription of Example 6

[0144] Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, and edetate disodium, add to the prescribed amount of water for injection, and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value should be 3.7.

[0145] Example 7. Preparation of Mitoxantrone Hydrochloride Injection Formula 7

[0146] Table 7: Prescription of Example 7

[0147] Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, and sodium metabisulfite and add them to the prescribed amount of water for injection. Stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value is 3.9.

[0148] Example 8. Preparation of Mitoxantrone Hydrochloride Injection Formula 8

[0149] Table 8: Prescription of Example 8

[0150] Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, sodium metabisulfite, and sodium sulfate, add to the prescribed amount of water for injection, and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. Measure the pH value to be 3.5.

[0151] Example 9. Preparation of Mitoxantrone Hydrochloride Injection Formula 9

[0152] Table 9: Prescription of Example 9

[0153] Weigh the prescribed amount of sodium chloride, polyethylene glycol 400, sodium chloride, acetic acid, sodium acetate, and edetate disodium into the prescribed amount of water for injection and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45 μm and 0.22 μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value should be 3.5.

[0154] Example 10. Preparation of Mitoxantrone Hydrochloride Injection Formula 10

[0155] Table 10: Prescription of Example 10

[0156] Weigh the prescribed amount of sodium chloride, polyethylene glycol 1000, sodium chloride, acetic acid, sodium acetate, and edetate disodium into the prescribed amount of water for injection and stir to dissolve. Once dissolved, add the prescribed amount of mitoxantrone hydrochloride and stir for 30 minutes to dissolve. Fine filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, cap, and sterilize at 121°C for 15 minutes. The pH value should be 3.5.

[0157] Example 11. Osmotic pressure investigation

[0158] 11.1 Effect of Osmotic Pressure on Injection Site Irritation

[0159] Mitoxantrone hydrochloride was used as a model drug. The osmotic pressure values ​​of the injection solution (Example 4) were adjusted to 196, 285, 307, 600, 900, 1200, 1503, 2317, and 3011 mmol / kg, respectively. Interstitial injection was selected as the administration method and subcutaneous injection was performed on the paws of Kunming mice. At the preset time point, the mice were killed by cervical dislocation. The injection site of the mice was removed and H&E staining was performed to evaluate local irritation. When the osmotic pressure of the injection solution was 3011 mmol / kg, redness and swelling occurred. The H&E staining results showed local irritation. Other osmotic pressure values ​​did not show obvious local irritation.

[0160] 11.2 Effect of Osmotic Pressure on Drug Targeting Ability

[0161] Using mitoxantrone hydrochloride as model drug, the osmotic pressure value of regulating injection (embodiment 4) is respectively 196,285,307,600,900,1200,1503,2317,3011mmol / kg respectively, select the administration mode of interstitial injection, in Kunming plant mouse paw subcutaneous injection, at preset time point, mice are taken off cervical vertebra and killed, extract one, two, three-level lymph node, observe its dyeing situation.When osmotic pressure value is less than 285mmol / kg, only one-level lymph node can dye.When osmotic pressure value is between 285-2317mmol / kg, three-level lymph node all can dye, but when osmotic pressure value is 600,900 and 1200mmol / kg, the third-level lymph node blue is dark blue, and when osmotic pressure value is 285,307 and 1503mmol / kg, the third-level lymph node color is normal blue.

[0162] Example 12. Osmotic pressure investigation

[0163] 12.1 Effect of Osmotic Pressure on Injection Site Irritation

[0164] Mitoxantrone hydrochloride was used as a model drug. Two groups of mice, group A and group B, were taken. The osmotic pressure values ​​of the injection solutions of Example 9 and Example 1 were adjusted to 196, 285, 307, 600, 900, 1200, 1503, 2317, and 3011 mmol / kg, respectively. Interstitial injection was selected as the administration method, and subcutaneous injection was performed on the soles of the feet of Kunming mice in group A and group B, respectively. At the preset time point, the mice were killed by cervical dislocation, and the administration sites of the mice were removed for H&E staining to evaluate local irritation. When the osmotic pressure of the injection solution was 3011 mmol / kg, redness and swelling occurred. The H&E staining results showed local irritation. Other osmotic pressure values ​​did not show obvious local irritation.

[0165] 12.2 Effect of Osmotic Pressure on Drug Targeting Ability

[0166] Mitoxantrone hydrochloride was used as a model drug. Two groups of mice, Group C and Group D, were taken. The osmotic pressure values ​​of the injections of Example 9 and Example 10 were adjusted to 196, 285, 307, 600, 900, 1200, 1503, 2317, and 3011 mmol / kg, respectively. Interstitial injection was selected as the administration method. Subcutaneous injection was performed on the soles of the feet of Kunming mice in Groups C and D, respectively. At a preset time point, the mice were killed by cervical dislocation, and the primary, secondary, and tertiary lymph nodes were removed and their staining was observed. For the lymph nodes of mice in Group C (Example 9), when the osmotic pressure value was less than 285 mmol / kg, only the primary lymph nodes could be stained. When the osmotic pressure value is between 285 and 2317 mmol / kg, all three levels of lymph nodes can be stained. However, when the osmotic pressure value is 600, 900, and 1200 mmol / kg, the blue color of the third level lymph nodes is dark blue. When the osmotic pressure value is 285, 307, and 1503 mmol / kg, the third level lymph nodes can be stained, but the color is dark blue. However, for the lymph nodes of mice in Group D (Example 10), when the osmotic pressure value is less than 285 mmol / kg, only the first level lymph nodes can be stained. When the osmotic pressure value is between 285 and 2317 mmol / kg, all three levels of lymph nodes can be stained. However, when the osmotic pressure value is 600, 900, and 1200 mmol / kg, the blue color of the third level lymph nodes is normal blue. When the osmotic pressure value is 285, 307, and 1503 mmol / kg, the third level lymph nodes can be stained, but the blue color is lighter. The above examples illustrate that after injection of the above-mentioned injection solution, mitoxantrone nanocrystals gradually precipitate from the solution. The nanocrystals can pass through the endothelial interstitium, be engulfed by endothelial pinocytosis, and enter the capillary lymphatic vessels. They then accumulate in local lymph nodes through lymphatic drainage. Mitoxantrone enters and temporarily resides in the lymph nodes, causing them to appear blue. The optimized formulation composition and osmotic pressure allow the main active ingredient to more rapidly penetrate the lymphatic vessels, resulting in a strong lymph node staining effect. Furthermore, the nanocrystals are larger than the capillary vessels. Therefore, they are less likely to enter the blood circulation and cause systemic toxicity.

[0167] Test Example 1. Pharmacokinetic and Pharmacodynamic Study of Mitoxantrone Hydrochloride Injection

[0168] To investigate the safety and efficacy of mitoxantrone hydrochloride injection for lymph node tracing in tracing cancer-draining lymph nodes in gastric cancer patients, as well as to conduct a tolerability test and in vivo pharmacokinetic study of mitoxantrone hydrochloride injection for lymph node tracing in gastric cancer subjects, and to determine a safe dosage range, this example employed a single-blind, single-center, parallel-controlled, pharmacokinetic, and tolerability clinical trial design. Following the dose-escalation principle, human tolerability, pharmacokinetic, and safety studies of mitoxantrone hydrochloride injection for lymph node tracing in gastric cancer subjects were conducted group by group. After determining the safe dosage range, an efficacy study was conducted.

[0169] Specifically, 12 gastric cancer subjects were selected in this stage. Using the mitoxantrone hydrochloride injection (5 mg / ml) prepared in Example 4, four dose groups of 1.0 ml, 1.5 ml, 2.0 ml, and 3.0 ml were tested from low to high, with 3 cases in each group. Each subject participated in only one dose group test. The safety of the test drug was observed and the optimal dosage range of the test drug was explored. This phase of the trial consists of a screening period, intraoperative observation, and safety evaluation after medication.

[0170] The experimental group received a tracer injection of mitoxantrone hydrochloride injection directly into the greater and lesser curvatures of the stomach surrounding the gastric tumor during surgery. The posterior wall and the blind area of ​​the stomach were first freed, and the drug was injected after the tumor was fully exposed. Specific methods: The tracer mitoxantrone hydrochloride injection was injected subserously approximately 1.0 cm from the edge of the tumor lesion. Multiple injections were then made. Lymph nodes in the perigastric region were cleared after staining. The total dose per subject was 1.0 ml, 1.5 ml, 2.0 ml, and 3.0 ml, respectively.

[0171] Cytological pathological analysis was performed intraoperatively. The number of lymph nodes detected, the number of lymph nodes detected in the first station (N1), and the number of lymph nodes detected in the second station (N2) were recorded for each subject. The lymph node staining rate, the lymph node staining rate in the first station (N1), and the lymph node staining rate in the second station (N2) were calculated for each subject. All lymph node specimens were prepared and examined routinely. Among them, the total number of lymph nodes detected = the number of stained lymph nodes + the number of unstained lymph nodes; the total number of N1 lymph nodes detected = the number of N1 stained lymph nodes + the number of N1 unstained lymph nodes; the total number of N2 lymph nodes detected = the number of N2 stained lymph nodes + the number of N2 unstained lymph nodes; lymph node staining rate = (total number of stained lymph nodes / total number of detected lymph nodes) × 100; N1 lymph node staining rate = (total number of N1 stained lymph nodes / total number of N1 detected lymph nodes) × 100; N2 lymph node staining rate = (total number of N2 stained lymph nodes / total number of N2 detected lymph nodes) × 100;

[0172] Pathological examination and analysis were completed before the end of the study, and indicators such as the stained lymph node metastasis rate, the stained lymph node metastasis rate of the first station (N1), and the stained lymph node metastasis rate of the second station (N2) were calculated for each subject. Metastatic lymph node staining rate = (total number of stained metastatic lymph nodes / total number of detected metastatic lymph nodes) × 100; N1 metastatic lymph node staining rate = (total number of stained metastatic lymph nodes in N1 / total number of detected metastatic lymph nodes in N1) × 100; N2 metastatic lymph node staining rate = (total number of stained metastatic lymph nodes in N2 / total number of detected metastatic lymph nodes in N2) × 100.

[0173] Blood sample collection: 4 ml of venous blood samples were collected before administration (within 60 minutes) and at 15±1 minutes, 30±1 minutes, 60±2 minutes, 120±2 minutes, 240±2 minutes, and 360±2 minutes after administration.

[0174] All subjects in this phase will be included in the safety and efficacy evaluation. Dose-limiting toxicity (DLT) will be observed until 14±2 days after dosing. If no DLT occurs, the patient will be transferred to the next dose group for observation. Safety evaluation will be observed until 28±3 days after dosing. Safety will be evaluated by comparing the results of our hospital examinations within one week before screening and postoperative laboratory test results, as well as by evaluating adverse events based on the "NCI CTCAE 5.0 evaluation criteria."

[0175] Table 11: Pharmacokinetic data after injection of mitoxantrone hydrochloride injection

[0176] Table 12: Total number of lymph nodes detected after injection of mitoxantrone hydrochloride injection

[0177] Table 13: Metastatic lymph node staining rate after injection of mitoxantrone hydrochloride injection

[0178] From the pharmacokinetic data in Table 11, it can be seen that after peritumoral injection of tracer mitoxantrone hydrochloride injection to gastric cancer subjects, it is rapidly absorbed, with very low peak concentrations and exposure, and is quickly cleared without causing toxic side effects.

[0179] After the subjects were injected with the test drug, the lymph node staining in the 1.0mL-2mL group generally showed a dose-dependent trend, and the total number of lymph nodes detected in the 2.0ml and 3.0ml dose groups was higher; and its components in the 3.0ml dose group could no longer be detected at 240 minutes, suggesting that it can be metabolized in the body. Therefore, the safe dose range of the test drug was set at 2.0-3.0ml.

[0180] Test Example 2. Application of Mitoxantrone Hydrochloride Injection in Lymphatic Tracing in Patients Undergoing Gastric Cancer Surgery

[0181] 1. Clinical trial design

[0182] The trial adopted a single-center, positive, self-controlled trial design, and intraoperative injection of the drug (injection of Example 4) was performed in groups to conduct human tolerance test and pharmacokinetic test, and the effectiveness of the test drug was observed at the same time. This trial intends to enroll 20 gastric cancer subjects, respectively, in the experimental group and the control group, and randomly assign them to each group, with 10 subjects in each group. Finally, 17 subjects completed the trial with good subject compliance, including 9 subjects in the experimental group and 8 subjects in the control group.

[0183] The efficacy and safety of the test drug were observed based on the dose range of 2.0-3 ml determined in Test Example 1. This phase of the trial consisted of a screening period, intraoperative observation, and postoperative safety evaluation.

[0184] The experimental group received a tracer injection of mitoxantrone hydrochloride injection directly into the greater and lesser curvatures of the stomach surrounding the gastric tumor during surgery. The posterior wall and any blind spots in the stomach were first freed, and the drug was then injected after the tumor was fully exposed. Specific procedures: The tracer mitoxantrone hydrochloride injection was injected subserously approximately 1.0 cm from the tumor edge. Multiple injections were then made. Lymph nodes in the surrounding area were cleared after staining. Each subject received a total dose of 3.0 ml.

[0185] Control group: Used during surgery. After exposing the surgical field, 1 ml (50 mg) of nanocarbon suspension injection was injected subserously at 4-6 points around the tumor using a skin test needle. 0.1-0.3 ml was injected at each point, slowly pushing the solution in about 3 minutes.

[0186] Safety in this clinical study was evaluated up to 28 days ± 3 days after surgery. Safety was assessed by comparing in-hospital examination results within one week before enrollment and postoperative laboratory test results, as well as adverse event evaluation according to the NCI CTCAE 5.0 criteria. The safety and efficacy data from this trial were processed using NMPA-approved statistical software and methods, ensuring reliable conclusions.

[0187] Table 14: Lymph node staining rate after injection of mitoxantrone hydrochloride injection Note: Statistical method: Chi-square test *: P < 0.05, statistically significant difference Lymph node staining rate = (total number of stained lymph nodes / total number of detected lymph nodes) × 100 N1 lymph node staining rate = (total number of stained lymph nodes in N1 / total number of detected lymph nodes in N1) × 100 N2 lymph node staining rate = (total number of stained lymph nodes in N2 / total number of detected lymph nodes in N2) × 100

[0188] (1) Comparison of lymph node staining rates between the experimental group and the control group

[0189] The data in Table 14 show that in the experimental group, a total of 445 lymph nodes were detected, 265 of which were stained, for a staining rate of 59.6%. In the control group, a total of 484 lymph nodes were detected, 209 of which were stained, for a staining rate of 43.2%. Using the chi-square test, the P value was less than 0.0001. Therefore, at a two-sided α-level of 0.05, the difference in lymph node staining rates between the experimental and control groups was considered statistically significant.

[0190] In the experimental group, a total of 341 N1 lymph nodes were detected, 207 of which were stained, for a N1 lymph node staining rate of 60.7%. In the control group, a total of 366 N1 lymph nodes were detected, 175 of which were stained, for a N1 lymph node staining rate of 47.8%. Using a chi-square test, the P value was 0.0006, thus confirming a statistically significant difference in the N1 lymph node staining rate between the experimental and control groups at a two-sided α-level of 0.05.

[0191] In the experimental group, a total of 104 N2 lymph nodes were detected, 58 of which were stained, for an N2 lymph node staining rate of 55.8%. In the control group, a total of 118 N2 lymph nodes were detected, 34 of which were stained, for an N2 lymph node staining rate of 28.8%. Using a chi-square test, the P value was less than 0.0001, thus confirming statistical significance at a two-sided α-level of 0.05 in the difference in N2 lymph node staining rates between the experimental and control groups.

[0192] (2) Comparison of the detection rate of lymph nodes with a short diameter ≤ 1 mm between the experimental group and the control group

[0193] In the experimental group, 7 lymph nodes with a short diameter ≤1 mm were detected, for a detection rate of 77.8%. In the control group, 2 lymph nodes with a short diameter ≤1 mm were detected, for a detection rate of 25.0%. Using a chi-square test, the P value was 0.0295. Therefore, at a two-sided α = 0.05 test level, the difference in the detection rate of lymph nodes with a short diameter ≤1 mm between the experimental and control groups was considered statistically significant.

[0194] (3) Comparison of the detection rate of lymph nodes with a short diameter ≤ 2 mm between the experimental group and the control group

[0195] In the experimental group, 8 lymph nodes with a short diameter ≤2 mm were detected, for a detection rate of 88.9%. In the control group, 6 lymph nodes with a short diameter ≤2 mm were detected, for a detection rate of 75.0%. Using a chi-square test, the P value was 0.4534. Therefore, at a two-sided α = 0.05 test level, it was determined that the difference in the detection rate of lymph nodes with a short diameter ≤2 mm between the experimental and control groups was not statistically significant.

[0196] 2. Clinical trial effectiveness results

[0197] According to the above experimental data, it can be seen that the differences in lymph node staining rate, N1 lymph node staining rate, N2 lymph node staining rate, and lymph node detection rate with short diameter ≤ 1 mm between the experimental group and the control group are statistically significant, and the experimental group is better than the control group.

[0198] Test Example 3. Tracing Effect of Mitoxantrone Injection on Tumors in Different Locations

[0199] For the patients enrolled in Test Case 1 and Test Case 2, lymph node staining of tumors in different locations was statistically analyzed, and the results are shown in Tables 15 and 16.

[0200] Table 15: Effects of different doses of mitoxantrone hydrochloride injection on tumor tracing in different locations

[0201] Table 16: Tracing effect of mitoxantrone and nanocarbon suspension on tumors in different locations

[0202] In summary, the tracer mitoxantrone hydrochloride injection was rapidly absorbed after injection into the stomach and surrounding areas, with peak absorption generally reaching 0.5 hours after injection. The drug was also rapidly eliminated from the bloodstream. Plasma concentrations in the different dose groups generally showed a dose-dependent trend, with the highest concentration and maximum exposure detected being 37.40 ng / ml in the 2 ml dose group and 34.47 h*ng / mL in the 3 ml dose group, respectively. These were significantly lower than the peak concentration and exposure reported in the literature for intravenous chemotherapy with mitoxantrone. The highest concentration detected was only 10-12 mg / mL in the literature for intravenous chemotherapy with mitoxantrone. 2 / d) was 7% of Cmax (510±206ng / m), indicating that the exposure of tracer mitoxantrone hydrochloride injection after peritumoral injection in gastric cancer was very low and would not cause toxic side effects.

[0203] Among the subjects in the full analysis set of the clinical study, a total of 13 adverse event records were reported in four subjects across different dose groups, with no subjects withdrawing from the trial due to adverse events. Two serious adverse events occurred in two subjects, with severity levels of: Grade 1 in 10 cases, Grade 2 in 1 case, and Grade 3 in 2 cases. Relationship to study drug: "Possibly unrelated" in 3 cases, "Definitely unrelated" in 10 cases. Adverse event outcomes: Remission in 3 cases, and resolution in 10 cases. Twelve subjects were observed for the occurrence of dose-limiting toxicities (DLTs) up to 14 ± 2 days after dosing. None of the 12 subjects experienced dose-limiting toxicities (DLTs), defined as Grade 3 or higher hematologic or skin toxicities. This suggests that the study drug is safe.

[0204] Efficacy analysis of the subjects showed that in the 2.0ml dose group, the total number of lymph nodes detected by the test drug was 60.3±14.57 (nodes), the total number of N1 lymph nodes detected was 57.3±13.58 (nodes), and the total number of N2 lymph nodes detected was 3.0±1.00 (nodes). In the 3.0ml dose group, the total number of lymph nodes detected by the test drug was 60.3±14.57 (nodes), the total number of N1 lymph nodes detected was 57.3±13.58 (nodes), and the total number of N2 lymph nodes detected was 3.0±1.00 (nodes). Lymph node staining generally showed a dose-dependent trend, with a higher total number of lymph nodes detected in the 2.0ml and 3.0ml dose groups. Furthermore, the 3.0ml dose group was completely metabolized by 240 minutes, so the safe dose range of the test drug is 2.0-3.0ml.

[0205] In this clinical study, although the differences between the experimental and control groups in the total number of lymph nodes detected, the total number of lymph nodes detected in the first (N1) and second (N2) stations, and the detection rate of lymph nodes with a short diameter ≤2 mm were not statistically significant, the differences in the lymph node staining rate, the N1 lymph node staining rate, the N2 lymph node staining rate, the metastatic lymph node staining rate, the N1 metastatic lymph node staining rate, and the detection rate of lymph nodes with a short diameter ≤1 mm were statistically significant. This suggests that the experimental drug was non-inferior to the control drug in terms of the total number of lymph nodes detected, but the lymph node staining rate and the detection rate of lymph nodes with a short diameter ≤1 mm were significantly superior to the control drug. Furthermore, the control drug, nanocarbon, cannot be metabolized in the body, resulting in permanent residual disease. In contrast, mitoxantrone has specific lymph node affinity, rapid staining, and a long-lasting effect without accumulation. Among the subjects in the clinical study safety set, a total of 1 subject had 1 adverse event record of "hypertension", with a severity of Grade 3, and the relationship with the study drug was "definitely unrelated", and the adverse event outcome was "disappeared", indicating that the clinical trial of the 2ml dose group was safe and effective, and there were no adverse reactions related to the study drug.

[0206] The above studies show that mitoxantrone hydrochloride injection for lymph node tracing in gastric cancer patients is stable, efficient and safe.

[0207] The foregoing descriptions of specific exemplary embodiments of the present disclosure are for purposes of illustration and description. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the present disclosure and various options and modifications. The scope of the present disclosure is intended to be defined by the claims and their equivalents.

Claims

1. Use of mitoxantrone and / or a pharmaceutically acceptable salt thereof in the preparation of a lymphatic tracer, wherein the lymphatic tracer is used for lymph node tracing in diseases associated with gastrectomy.

2. The use according to claim 1, wherein The lymph node is a lymph node with a short diameter less than or equal to 2 mm, preferably a lymph node with a short diameter less than or equal to 1 mm; Preferably, the detection rate of the tracer for lymph nodes with a short diameter of less than or equal to 1 mm is greater than 70%, and more preferably greater than 75%; Preferably, the color development time of the tracer is less than 25 min, more preferably less than 20 min; Preferably, the tracer fades for more than 180 minutes; Preferably, the lymph node staining rate of the tracer in gastric cancer resection is greater than 50%, and more preferably greater than 55%.

3. The use according to claim 1 or 2, wherein The gastrectomy-related disease is gastric polyp or gastric tumor; Preferably, the gastric tumor includes benign gastric tumor and malignant gastric tumor; Further preferably, the benign gastric tumor and the malignant gastric tumor are selected from gastric fundus and cardia cancer, gastric body cancer, and gastric antrum cancer; Preferably, the disease associated with gastrectomy is selected from one or more of tumors located in the following locations: the cardia, the cardia mucocurvature side, the posterior wall of the cardia, the lesser curvature of the cardia, the lesser curvature side of the cardia and the gastric body, the anterior wall of the lesser curvature of the gastric fundus, the gastric fundus, the lesser curvature of the gastric fundus, the anterior wall of the gastric antrum, the greater curvature of the gastric fundus, the gastric antrum junction, the lesser curvature of the gastric antrum, the upper posterior wall of the gastric body, the posterior wall of the lower segment of the gastric body, the greater curvature side of the gastric fundus and the gastric body, the posterior wall of the lesser curvature of the gastric body, the anterior and posterior walls of the lesser curvature, the gastric fundus, and the posterior wall of the lesser curvature of the gastric body.

4. The use according to any one of claims 1 to 3, wherein The gastrectomy is selected from total gastrectomy, subtotal gastrectomy, hemigastrectomy or antrectomy.

5. The use according to any one of claims 1 to 4, wherein the gastrectomy-related disease is gastric polyp or gastric tumor, and the lymphatic tracer is used for lymphatic tracing in the gastric polyp or the gastric tumor.

6. The use according to any one of claims 1 to 5, wherein The lymphatic tracer comprises mitoxantrone and / or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient; Preferably, the pharmaceutical excipients include, but are not limited to, one or more of an osmotic pressure regulator, an antioxidant, an adsorbent, a filler, a buffer, a carrier, a stabilizer or a preservative; Preferably, the lymphatic tracer is an injection; further preferably, the injection is in the form of a solution, a lyophilized powder, an emulsion, a liposome, a nanoparticle, a nanocrystal, a microcrystal, a microsphere or a gel.

7. The use according to claim 6, wherein The tracer contains, by mass percentage, mitoxantrone or a pharmaceutically acceptable salt thereof: 0.05%-5% and an osmotic pressure regulator: 0.1%-10%; Preferably, the tracer further contains a buffer: 0.01%-0.1%, an antioxidant: 0.01%-0.1%, an adsorbent: 0.05%-1%, and a filler: 0%-20%; Preferably, the osmotic pressure regulator is one or a mixture of several substances selected from the group consisting of sodium chloride, glucose, sorbitol, mannitol, glycerol, phosphate, and citrate; Preferably, the buffer is one or more of acetic acid, sodium acetate, citric acid, and sodium citrate; Preferably, the antioxidant is one or more of sodium sulfite, sodium bisulfite, sodium pyrosulfite, sodium sulfate, sodium thiosulfate, and disodium edetate; Preferably, the filler is one or more of monosaccharides such as glucose, fructose, galactose, ribose or deoxyribose, or disaccharides such as sucrose, trehalose, maltose, lactose, or polymeric sugars such as mannitol, sorbitol, lactitol, xylitol, maltitol, and erythritol; Preferably, the tracer further comprises a pH adjuster, and the pH adjuster is selected from one or more of hydrochloric acid, phosphoric acid, sulfuric acid, oxalic acid, acetic acid and citric acid; Preferably, the tracer further comprises polyethylene glycol, and the molecular weight of the polyethylene glycol is less than 2000, more preferably less than 1000, further preferably 200, 400 or 600; Preferably, the mass concentration of the polyethylene glycol in the injection is 0.01%-2%; More preferably, the mass concentration of the polyethylene glycol in the injection solution is 1%-2%; Preferably, the impurity content in the tracer is 0% to 1.5%.

8. The use according to claim 6 or 7, wherein The injection osmotic pressure is 285-2317 mmol / kg, preferably 600-1200 mmol / kg; and / or, The injection pH value range is between 2.8 and 4.3; and / or, The nanoparticle size of the tracer under physiological conditions is 20-100 nm, preferably 30-60 nm, and more preferably 40-60 nm; and / or, The content of mitoxantrone in the mitoxantrone or its salt in the injection is 1-15 mg / ml by weight to volume ratio; preferably 2-10 mg / ml; more preferably 2 mg / ml, 5 mg / ml or 10 mg / ml; and / or, The single dose of the injection is 0.5-3 mL, preferably 2-3 mL; Preferably, the content of mitoxantrone in the mitoxantrone or its salt in the injection is 5 mg / ml in terms of weight to volume ratio, and the single dose of the injection is 2 ml.

9. The use according to any one of claims 1 to 8, wherein The lymphatic tracer comprises: Preferably, the lymphatic tracer comprises: Preferably, the lymphatic tracer optionally further comprises edetate disodium; preferably, the amount of edetate disodium is 0-0.3 mg / mL; Preferably, the lymphatic tracer optionally further comprises polyethylene glycol; preferably, the amount of the polyethylene glycol is 0-20 mg / mL; Preferably, the lymphatic tracer comprises: Preferably, the solvent is selected from water, sodium chloride solution or glucose solution.

10. The use according to any one of claims 1 to 9, wherein The lymphatic tracer is an injection. Preferably, the injection is administered by peritumoral injection or at various points of the greater curvature and lesser curvature of the stomach. Preferably, the injection is administered by injection at various points of the greater curvature and lesser curvature of the stomach. and / or, The pharmaceutically acceptable salt of mitoxantrone is one or more selected from the group consisting of mitoxantrone hydrochloride, mitoxantrone oxalate, mitoxantrone sulfate, mitoxantrone phosphate, mitoxantrone acetate and mitoxantrone citrate.

11. The use according to any one of claims 1 to 10, wherein The lymphatic tracer is an injection, which is prepared by a method comprising the following steps: Weigh the prescribed amount of excipients, add them to the prescribed amount of solvent, stir to dissolve, and after dissolution, add the prescribed amount of mitoxantrone hydrochloride and / or its pharmaceutically acceptable salt; Preferably, the excipients are polyethylene glycol, acetic acid, sodium acetate, sodium chloride and disodium edetate, or polyethylene glycol, acetic acid, sodium acetate, sodium chloride and sodium metabisulfite, or polyethylene glycol, acetic acid, sodium acetate, sodium chloride, sodium metabisulfite and sodium sulfate; Preferably, the solvent is water for injection; Preferably, the method further comprises the steps of filtering and filling with nitrogen; Preferably, the filtration is fine filtration through 0.45 μm and 0.22 μm filter membranes.

12. A method for tracing lymph nodes in gastrectomy-related diseases, comprising the following main steps; S1: Prepare pathological information of experimental patients; S2: Use mitoxantrone and / or its pharmaceutical salts to prepare lymphatic tracers and conduct tracing experiments on patients who meet the requirements; S3: Observe the indicators of patients after using mitoxantrone and / or its pharmaceutically acceptable salts to prepare lymphatic tracers; S4: Statistical analysis of the obtained indicator data; Preferably, the usage, dosage, composition and / or preparation method of the tracer is compatible with the use described in any one of claims 1-11.

13. A lymph node tracer comprising mitoxantrone and / or a pharmaceutically acceptable salt thereof, which is used for lymph node tracing in gastrectomy-related diseases; Preferably, the composition, preparation method, usage and / or dosage of the tracer are compatible with the use described in any one of claims 1-11.

14. A method for treating a disease associated with gastrectomy, comprising: administering a lymphatic tracer comprising mitoxantrone and / or a pharmaceutically acceptable salt thereof to a patient diagnosed with a gastrectomy-related disease to trace lymph nodes in the stomach and surrounding areas; administering to the patient a treatment comprising gastrectomy; Preferably, the composition, preparation method, usage and / or dosage of the tracer are compatible with the use described in any one of claims 1 to 11; Preferably, the tracing method is the method according to claim 12.

Images