WO2025130815A1 - Use of mitoxantrone in preparing lymphatic tracing agent - Google Patents

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

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application 2023117680923 filed on December 20, 2023, and the entire text of the above-mentioned Chinese patent application is incorporated herein by reference.

Technical Field

The present invention belongs to the field of pharmaceutical preparations, and in particular relates to the application of mitoxantrone in the manufacture of lymphatic tracers for diseases associated with gastrectomy.

Background Art

Gastric cancer is the fifth most common cancer in the world and the third most common cause of cancer death. Every year, there are about 1 million new gastric cancer patients and more than 700,000 deaths. In my country, there are more than 400,000 new gastric cancer patients every year. In the treatment of advanced gastric cancer, with the development of chemotherapy and small molecule targeted therapy, the survival of patients has been extended, but surgical resection is still the most effective way to cure cancer. The 2010 edition of Japan's "Gastric Cancer Treatment Protocol" defines D2 dissection as the standard surgical procedure for gastric cancer treatment, which has also been accepted by Eastern and Western scholars.

The 2010 edition of Japan's "Gastric Cancer Treatment 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 cure (D0 surgery), the second level is level 1 radical cure (D1 surgery), the third level is level 2 radical cure (D2 surgery), the fourth level is level 3 radical cure (D3 surgery), and the fifth level is level 4 radical cure (D4 surgery). D0 surgery is basically not used by surgeons except for very early gastric cancer due to its extremely low radicality. D1 surgery performs the first-stop lymph node dissection around the stomach, which has a certain radicality and high safety. D2 surgery clears the first and second lymph nodes around the stomach, which is highly radical, but the surgical risk is high, especially in non-large tumor centers, which are prone to postoperative complications. Due to the indications and surgical difficulty, D3 and D4 surgeries are usually only performed in large tumor centers for patients with special medical needs. The risks of this type of surgery are doubled compared to D2 surgery. There are two benefits to clearing as many lymph nodes as possible: 1. The surgery is thorough and minimizes tumor residues; 2. It provides more accurate postoperative pathological staging, which is conducive to the formulation of subsequent treatment (chemotherapy) plans. In other words, the most important and basic point about the quality of gastric cancer surgery is whether the lymph nodes are cleared.

The perigastric lymph nodes are numerous and widely distributed, but they are often hidden in the surrounding tissues and difficult to identify with the naked eye. It is often difficult to remove as many lymph nodes as possible. Using lymph tracers to dye and trace living lymph nodes during surgery to help doctors completely remove lymph nodes is the key to improving the treatment of malignant tumors.

Therefore, developing safe and effective lymphatic tracers and effectively clearing the lymph nodes in the tumor drainage area is an important means to improve the quality of life of patients and prolong their life. Lymphatic tracers are currently playing a vital role in the clinical treatment of malignant tumors at home and abroad. Lymphatic tracers are injected near the primary lesion to quickly stain the lymph nodes near the malignant tumor.

Currently known lymphatic tracers include nanocarbon, methylene blue, etc., but nanocarbon and the like have the following problems: nanocarbon and the like are often used for sentinel lymph node tracing during breast cancer and thyroid surgery, but nanocarbon is in an accumulated state and is not metabolized in the body. There is a risk of blocking capillaries when entering the blood and lymphatic circulation. In addition, the texture of cancerous tissue is hard and brittle, and direct intratumoral injection can cause tumor tissue necrosis and shedding, causing bleeding. Furthermore, nanocarbon is not easy to enter small lymph nodes, so the tracing effect on sentinel lymph nodes of gastric cancer is not good, resulting in the continued proliferation and metastasis of tumor cells remaining in small lymph nodes after gastric cancer surgery, ultimately leading to poor prognosis. In addition, gastric cancer resection and breast cancer resection have different requirements for the size of lymph nodes that can be cleared by tracers. Breast cancer tracing is more about tracing sentinel lymph nodes. If there is no metastasis in the sentinel lymph nodes, the possibility of metastasis in other lymph nodes in the area is very small. However, the metastatic pathways of gastric cancer and breast cancer are different. Gastric cancer resection requires tracing the smallest lymph nodes (≤1mm) and tracing as many lymph nodes as possible.

Therefore, seeking to develop a safe and effective lymph tracer for intraoperative lymph node tracing in gastric cancer 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

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 diseases associated with gastrectomy.

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present disclosure, the definitions and explanations of the relevant terms are provided below.

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.

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 a specific embodiment, the patient herein is a human. The patient may be suffering from, suspected of suffering from, or at risk of suffering from 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.

As used herein, "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.

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), phosphates 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 may 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.

As used herein, "pharmaceutically acceptable excipients" refer to ingredients in pharmaceutical preparations other than active ingredients that are non-toxic to subjects. Pharmaceutically acceptable excipients include, but are not limited to, buffers, carriers, stabilizers or preservatives.

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.

As used herein, "pharmaceutically acceptable acid addition salts" refers to those pharmaceutically acceptable salts formed with inorganic acids, the inorganic acid being selected from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and the organic acid being selected from aliphatic, alicyclic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic acid 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.

The term "pharmaceutical base addition salt" refers to those pharmaceutical 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 useful 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, methylglucosamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine and salts of polyamine resins.

As used herein, "treatment" refers to clinical intervention that attempts to change the natural course of the individual being treated, and can be used 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 relieving the disease state, and alleviating or improving prognosis.

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 infusions include, 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-term 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 infusions.

As used in this article, 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 evaluation.

As used in this article, 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 research subjects violating the inclusion or exclusion criteria). It is a collection of cases that met the inclusion criteria, did not meet the exclusion criteria, and completed the treatment plan.

As used in this article, "lymph node metastasis" refers to the metastasis of gastric cancer cells to lymph nodes somewhere along the lymphatic channels, and is an important way for gastric cancer to metastasize. Generally speaking, gastric cancer lymph node metastasis is near first and then far, and as the cancer tumor extends to the deeper layer, the chance of metastasis increases; it is divided into 3 stations according to the order of metastasis: the first station is the superficial lymph nodes closest to the cancer body and 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, the abdominal aorta, the hepatic hilum and the mesenteric root, and the 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").

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).

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 diseases associated with gastrectomy.

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

The inventors of the present disclosure have found through research that the mitoxantrone and/or its pharmaceutical salt described in the present disclosure can be used in the preparation of lymph tracers to trace lymph nodes during gastric cancer resection, and can stain lymph nodes less than or equal to 1 mm, and has a good lymph node staining effect during resection surgeries at various locations and directions of the stomach and perigastric area.

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%.

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

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

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

In some specific embodiments, the disease associated with gastrectomy is gastric polyp or gastric tumor.

In some embodiments, the gastric tumor includes benign gastric tumor and malignant gastric tumor.

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.

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

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

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

In some specific embodiments, the gastrectomy-related disease is gastric polyp or gastric tumor.

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

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

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

In some specific 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.

In some embodiments, the lymphatic tracer is an injectable.

In some specific embodiments, the dosage form of the injection is a solution, a lyophilized powder, an emulsion, a liposome, a nanoparticle, a nanocrystal, a microcrystal, a microsphere or a gel.

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

In some specific embodiments, the injection is formulated as a dosage form with 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.

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.

Preferably, the pharmaceutically acceptable salt is mitoxantrone hydrochloride.

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).

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%.

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

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

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

In some specific embodiments, 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 adsorbent is activated carbon.

In some embodiments, the lymphatic tracer comprises a pH adjuster.

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.

In some specific embodiments, the injection pH range is between 2.8-4.3.

In some specific embodiments, the tracer further comprises polyethylene glycol, which accounts for 0.01%-2% by mass concentration in the injection, preferably 1%-2%, which is beneficial to increase the water solubility of mitoxantrone hydrochloride, improve its stability (for example, help it not to decompose easily when in contact with water), and is used for the preparation of nanoparticle drug carriers and controlled release systems, control drug delivery, and promote lymphatic transport. Specifically, the addition of polyethylene glycol helps the drug to form a local reservoir after injection of the injection, thereby prolonging the duration of the drug's action.

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.

In some embodiments, the osmotic pressure is selected in the range of 285 to 2317 mmol/kg.

In some preferred embodiments, the osmotic pressure is selected in the range of 600-1200mmol/kg. When the osmotic pressure is too low, the lymphatic drainage power is insufficient. When the osmotic pressure is too high, the concentration of the osmotic pressure regulator in the injection is too high, resulting in irritation at the injection site. The osmotic pressure is maintained at 600-1200mmol/kg to facilitate better staining of the lymph nodes.

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.

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

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

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

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

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

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

In some embodiments, the lymphatic tracer comprises:

In some preferred embodiments, the lymphatic tracer comprises:

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.

In some preferred embodiments, the lymphatic tracer optionally further comprises polyethylene glycol, and preferably, the amount of the polyethylene glycol is 0-20 mg/mL.

In some preferred embodiments, the lymphatic tracer comprises:

In some specific embodiments, the solvent is selected from: water, sodium chloride solution or glucose solution.

Preferably, the water is water for injection.

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):

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 as shown in formula (III):

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):

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):

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;

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;

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;

Or, 23.28g 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;

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;

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;

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;

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 is added to 2000ml;

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;

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;

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;

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 added to 2000ml;

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;

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.

In some specific embodiments, 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 add the prescribed amount of mitoxantrone hydrochloride and/or its pharmaceutically acceptable salt after dissolution. In some specific embodiments, the solvent is water for injection.

It can be understood that the aforementioned osmotic pressure parameters can be adjusted by controlling the prescription and content of auxiliary materials, etc., and the specific details shall be subject to the test compliance standards.

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

(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.

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

In some specific embodiments, the method further comprises:

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

In some specific embodiments, the method further comprises:

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

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 them to the prescribed amount of water for injection, stirring to dissolve, and obtaining an excipient mixture.

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

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

In some preferred embodiments, the injection is administered at 4-6 injection points above, below, left and right of the greater curvature and lesser curvature of the stomach.

In another aspect of the present disclosure, there is provided a method for tracing lymph nodes in gastrectomy-related diseases, which comprises 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: Conduct statistical analysis on the obtained indicator data.

In the method, the usage, dosage, composition, preparation method, etc. of the tracer are partially consistent with the aforementioned uses.

In another aspect of the present disclosure, there is provided a lymphatic 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 consistent with the aforementioned use section.

Another aspect of the present disclosure provides a method for treating a patient suffering from a gastrectomy-related disease, 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 aforementioned use section;

Preferably, the tracing method is the aforementioned lymph node tracing method.

By adopting the above technical solution; Mitoxantrone itself has a tropism for the lymphatic system, and its own color (blue) can stain the lymph nodes near the tumor. It can be locally injected during gastric cancer surgery to stain the lymph nodes near the tumor, helping the clinic to locate and clear the lymph nodes. Through in-depth research on the preclinical pharmacodynamics of mitoxantrone hydrochloride, it was found that this product has a high affinity for lymph nodes when injected subcutaneously, can dye lymph nodes blue, and can be used as a lymph tracer. Using it for lymph node tracing in gastrectomy surgery can increase the number of lymph nodes detected, thereby reducing staging bias and improving the accuracy of gastric cancer staging.

By adopting the above technical scheme, the lymph nodes and the first station lymph nodes can be obviously blue-stained and clearly visible during surgery, thereby increasing the blue-stained rate of lymph nodes, and thus 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-stained rate. Compared with the traditional nano-carbon tracer method, it can further increase the number of detected lymph nodes, thereby reducing staging bias and improving the accuracy of gastric cancer staging.

According to the above technical scheme, the present invention can completely clean 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 of patients.

DETAILED DESCRIPTION

For purposes of clarity and concise description, 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.

Example 1. Preparation of Mitoxantrone Hydrochloride Injection Formula 1

Table 1: Prescription of Example 1

Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate and disodium edetate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride after dissolution, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, sterilize at 121℃ for 15 minutes, and the pH value is 3.5.

Example 2. Preparation of Mitoxantrone Hydrochloride Injection Formula 2

Table 2: Prescription of Example 2

Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, sodium pyrosulfite and sodium sulfate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride after dissolution, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, sterilize at 121℃ for 15 minutes, and the pH value is 3.4.

Example 3. Preparation of Mitoxantrone Hydrochloride Injection Formula 3

Table 3: Prescription of Example 3

Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate and disodium edetate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride after dissolution, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, sterilize at 121℃ for 15 minutes, and the pH value is 3.6.

Example 4. Preparation of Mitoxantrone Hydrochloride Injection Formula 4

Table 4: Prescription of Example 4

Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, sodium pyrosulfite and sodium sulfate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride after dissolution, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, sterilize at 121℃ for 15 minutes, and the pH value is 3.7.

Example 5. Preparation of Mitoxantrone Hydrochloride Injection Formula 5

Table 5: Prescription of Example 5

Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, sodium pyrosulfite and sodium sulfate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride after dissolution, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, sterilize at 121℃ for 15 minutes, and the pH value is 3.6.

Example 6. Preparation of Mitoxantrone Hydrochloride Injection Formula 6

Table 6: Prescription of Example 6

Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate and disodium edetate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride after dissolution, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, sterilize at 121℃ for 15 minutes, and the pH value is 3.7.

Example 7. Preparation of Mitoxantrone Hydrochloride Injection Formula 7

Table 7: Prescription of Example 7

Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate and sodium metabisulfite, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride after dissolution, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, sterilize at 121℃ for 15 minutes, and the pH value is 3.9.

Example 8. Preparation of Mitoxantrone Hydrochloride Injection Formula 8

Table 8: Prescription of Example 8

Weigh the prescribed amount of sodium chloride, acetic acid, sodium acetate, sodium pyrosulfite and sodium sulfate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride after dissolution, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, sterilize at 121℃ for 15 minutes, and the pH value is 3.5.

Example 9. Preparation of Mitoxantrone Hydrochloride Injection Formula 9

Table 9: Prescription of Example 9

Weigh the prescribed amount of sodium chloride, polyethylene glycol 400, sodium chloride, acetic acid, sodium acetate, and disodium edetate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, and sterilize at 121℃ for 15 minutes. The pH value is 3.5.

Example 10. Preparation of Mitoxantrone Hydrochloride Injection Formula 10

Table 10: Prescription of Example 10

Weigh the prescribed amount of sodium chloride, polyethylene glycol 1000, sodium chloride, acetic acid, sodium acetate, and disodium edetate, add them to the prescribed amount of water for injection, stir to dissolve, add the prescribed amount of mitoxantrone hydrochloride, stir for 30 minutes to dissolve, filter through 0.45μm and 0.22μm filter membranes, fill with nitrogen, roll cap, and sterilize at 121℃ for 15 minutes. The pH value is 3.5.

Example 11. Osmotic pressure study

11.1 Effect of Osmotic Pressure on Injection Site Irritation

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

11.2 Effect of Osmotic Pressure on Drug Targeting Ability

Using mitoxantrone hydrochloride as model drug, the osmotic pressure value of injection (embodiment 4) is regulated to be 196, 285, 307, 600, 900, 1200, 1503, 2317, 3011mmol/kg respectively, the administration mode of interstitial injection is selected, subcutaneous injection is carried out in the sole of Kunming mice, at the preset time point, mice are decapitated to death by cervical vertebra, one, two and three levels of lymph nodes are extracted, and their dyeing is observed. When the osmotic pressure value is less than 285mmol/kg, only one level of lymph nodes can be dyed. When the osmotic pressure value is between 285-2317mmol/kg, three levels of lymph nodes can all be dyed, but when the osmotic pressure value is 600, 900 and 1200mmol/kg, the third level of lymph nodes blue is dark blue, and when the osmotic pressure value is 285, 307 and 1503mmol/kg, the three levels of lymph nodes color is normal blue.

Example 12. Osmotic pressure study

12.1 Effect of Osmotic Pressure on Injection Site Irritation

Mitoxantrone hydrochloride was used as a model drug, and 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. The interstitial injection method was selected, and the Kunming mice in group A and group B were subcutaneously injected into the soles of their feet. At a 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, and other osmotic pressure values did not show obvious local irritation.

12.2 Effect of Osmotic Pressure on Drug Targeting Ability

Mitoxantrone hydrochloride was used as a model drug, and two groups of mice, group C and group D, were taken, and 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. The interstitial injection was selected as the administration method, and the Kunming mice of group C and group D were injected subcutaneously in the soles of their feet. At a preset time point, the mice were killed by cervical vertebrae dislocation, and the first, second, and third level lymph nodes were removed to observe their staining. For the lymph nodes of group C (Example 9) mice, when the osmotic pressure value was less than 285 mmol/kg, only the first level lymph nodes could be stained. When the osmotic pressure value is between 285 and 2317 mmol/kg, the three-level lymph nodes can all be stained, but when the osmotic pressure value is 600, 900 and 1200 mmol/kg, the blue color of the third-level lymph nodes is dark blue, and when the osmotic pressure value is 285, 307 and 1503 mmol/kg, the three-level lymph nodes can be stained but the color is dark blue. However, for the lymph nodes of the 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, the three-level lymph nodes can all be stained, but when the osmotic pressure value is 600, 900 and 1200 mmol/kg, the blue color of the third-level lymph nodes is normal blue, and when the osmotic pressure value is 285, 307 and 1503 mmol/kg, the three-level lymph nodes can be stained but the blue color is lighter. The above embodiment illustrates that after the injection, the mitoxantrone nanocrystals gradually precipitate out from the solution. The nanocrystals can pass through the endothelial stroma, be engulfed by the endothelial pinocytosis and enter the capillary lymphatic vessels, and then gather in the local lymph nodes through lymphatic drainage. Mitoxantrone enters and temporarily stays in the lymph nodes, making them blue. And under the effect of optimized formulation ingredients and osmotic pressure, its main active ingredient penetrates the lymphatic vessels faster, with a stronger lymph node staining effect. And the size of the nanocrystals is larger than the capillary container. Therefore, they rarely enter the blood circulation and cause systemic toxicity.

Test Example 1. Pharmacokinetics and pharmacodynamics study of mitoxantrone hydrochloride injection

In order to investigate the safety and effectiveness of mitoxantrone hydrochloride injection for lymphatic tracing in tracing the cancer-draining lymph nodes of gastric cancer patients, as well as the tolerance test and in vivo pharmacokinetic test of mitoxantrone hydrochloride injection for lymphatic tracing in gastric cancer subjects, and determine the safe dosage range, this example adopts a single-blind, single-center, parallel-controlled, pharmacokinetic, and tolerability clinical trial design, and according to the dose escalation principle, the human tolerance and pharmacokinetic study and safety study of mitoxantrone hydrochloride injection for lymphatic tracing in gastric cancer subjects are carried out group by group, and after obtaining the safe dosage range, the effectiveness study is carried out.

Specifically, 12 gastric cancer subjects were selected in this stage. Using the mitoxantrone hydrochloride injection (5 mg/ml) prepared in Example 4, 4 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 only participated in one dose group test. Observe the safety of the test drug and explore the optimal dosage range of the test drug. This stage of the test consists of a screening period, intraoperative observation, and safety evaluation after medication.

Experimental group: used during surgery, mitoxantrone hydrochloride injection for tracer was directly injected into the greater and lesser curvatures of the stomach around the gastric tumor. The posterior wall and the stomach in the blind area of the visual field were first freed, and the drug was injected after the tumor position was fully exposed. Specific method: After the tracer mitoxantrone hydrochloride injection was injected into the gastric serosa about 1.0 cm away from the edge of the tumor lesion, it was injected at multiple points, and the lymph nodes around the stomach were cleared after staining. The total doses for each subject were 1.0 ml, 1.5 ml, 2.0 ml, and 3.0 ml, respectively.

Cytological pathological analysis was performed during the operation; the number of lymph nodes detected in each subject, 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; the lymph node staining rate, the first station (N1) lymph node staining rate, and the second station (N2) lymph node staining rate of each subject were calculated. All lymph node specimens were routinely prepared and observed. 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;

Before the end of the study, the pathological test and analysis were completed, and the stained lymph node metastasis rate, the 1st station (N1) stained lymph node metastasis rate, the 2nd station (N2) stained lymph node metastasis rate and other indicators were calculated for each subject.
Metastatic lymph node staining rate = (total number of metastatic lymph nodes stained/total number of metastatic lymph nodes detected) × 100;
N1 metastatic lymph node staining rate = (total number of N1 metastatic lymph nodes stained/total number of N1 metastatic lymph nodes detected) × 100;
N2 metastatic lymph node staining rate = (total number of N2 metastatic lymph nodes stained/total number of N2 metastatic lymph nodes detected) × 100.

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

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 administration. If no DLT occurs, the next dose group will be observed. Safety evaluation will be observed until 28±3 days after administration. 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 CTCAE5.0 evaluation criteria".

Table 11: Pharmacokinetic data after injection of mitoxantrone hydrochloride injection

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

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

From the pharmacokinetic data in Table 11, it can be seen that after the tracer mitoxantrone hydrochloride injection is administered peritumorally to gastric cancer subjects, it is rapidly absorbed, the peak concentration and exposure are very low, and it is cleared quickly without causing toxic side effects.

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 240min, indicating that it can be metabolized in the body. Therefore, the safe dose range of the test drug was set at 2.0-3.0ml.

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

1. Clinical trial design

The experiment adopts a single-center, positive, self-controlled experimental design, and intraoperative injection (injection of Example 4) is administered in groups for human tolerance test and pharmacokinetic test, and the effectiveness of the experimental drug is observed at the same time. This experiment 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 experiment, and the subjects had good compliance, including 9 subjects in the experimental group and 8 subjects in the control group.

The effectiveness 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.

Test group: used during surgery, mitoxantrone hydrochloride injection for tracer was directly injected into the greater and lesser curvatures of the stomach around the gastric tumor. The posterior wall and the stomach in the blind area of the visual field were first freed, and the drug was injected after the tumor position was completely exposed. Specific method: The mitoxantrone hydrochloride injection for tracer was injected into the gastric serosa about 1.0 cm away from the edge of the tumor lesion, and then injected at multiple points. The lymph nodes around the stomach were cleared after staining. The total dose for each subject was 3.0 ml.

Control group: used during surgery. After the surgical field is exposed, 1 ml (50 mg) of nanocarbon suspension injection is taken and injected subserously at 4-6 points around the tumor using a skin test needle, 0.1-0.3 ml is injected at each point, and the injection is pushed slowly for about 3 minutes.

The safety evaluation of this clinical study was conducted until 28 days ± 3 days after the surgery. Safety was evaluated by comparing the results of the hospital's examinations within one week before the screening and the results of the postoperative laboratory tests, as well as by evaluating adverse events based on the "NCI CTCAE 5.0 evaluation criteria". The safety and efficacy data obtained in this trial were processed using statistical software and statistical methods approved by NMPA, and the conclusions were reliable.

Table 14: Lymph node staining rate after injection of mitoxantrone hydrochloride injection

Note: Statistical method of data: 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 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

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

According to the data in Table 14, it can be seen that the total number of lymph nodes detected in the experimental group was 445, the total number of lymph nodes stained was 265, and the staining rate was 59.6%; the total number of lymph nodes detected in the control group was 484, the total number of lymph nodes stained was 209, and the staining rate was 43.2%. Using the chi-square test, the P value is less than 0.0001, so according to the bilateral α=0.05 test level, it can be determined that the difference in lymph node staining rate between the experimental group and the control group is statistically significant.

In the experimental group, the total number of lymph nodes detected by N1 was 341, the total number of lymph nodes stained by N1 was 207, and the N1 lymph node staining rate was 60.7%; in the control group, the total number of lymph nodes detected by N1 was 366, the total number of lymph nodes stained by N1 was 175, and the N1 lymph node staining rate was 47.8%. Using the chi-square test, the P value was 0.0006, so according to the bilateral α=0.05 test level, it can be determined that the difference in the N1 lymph node staining rate between the experimental group and the control group is statistically significant.

In the experimental group, the total number of lymph nodes detected in N2 was 104, the total number of lymph nodes stained in N2 was 58, and the N2 lymph node staining rate was 55.8%; in the control group, the total number of lymph nodes detected in N2 was 118, the total number of lymph nodes stained in N2 was 34, and the N2 lymph node staining rate was 28.8%. Using the chi-square test, the P value was less than 0.0001, so according to the bilateral α=0.05 test level, it can be determined that the difference in the N2 lymph node staining rate between the experimental group and the control group is statistically significant.

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

The number of lymph nodes with short diameter ≤1mm detected in the experimental group was 7, with a detection rate of 77.8%; the number of lymph nodes with short diameter ≤1mm detected in the control group was 2, with a detection rate of 25.0%. Using the chi-square test, the P value was 0.0295, so at the bilateral α=0.05 test level, it can be determined that the difference in the detection rate of lymph nodes with short diameter ≤1mm between the experimental group and the control group is statistically significant.

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

The number of lymph nodes with short diameter ≤2mm detected in the experimental group was 8, with a detection rate of 88.9%; the number of lymph nodes with short diameter ≤2mm detected in the control group was 6, with a detection rate of 75.0%. Using the chi-square test, the P value was 0.4534, so according to the bilateral α=0.05 test level, it can be determined that the difference in the detection rate of lymph nodes with short diameter ≤2mm between the experimental group and the control group is not statistically significant.

2. Clinical trial effectiveness results

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.

Test Example 3. Tracing effect of mitoxantrone injection on tumors in different locations

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

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

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

In conclusion, the tracer mitoxantrone hydrochloride injection was rapidly absorbed after injection into the stomach and various parts of the stomach, and the peak value was basically reached 0.5h after injection, and the drug was eliminated quickly after entering the blood. The blood drug concentration after administration of different dose groups showed a dose-dependent trend. The highest concentration and maximum exposure detected were 37.40ng/ml in the 2ml dose group and 34.47h*ng/mL in the 3ml dose group, which were far lower than the peak concentration and exposure of mitoxantrone for intravenous chemotherapy reported in the literature. The highest concentration detected was only 7% of the Cmax (510±206ng/m) when mitoxantrone was administered as a chemotherapy drug by intravenous injection (10-12mg/ ^m2 /d) in the literature, indicating that the tracer mitoxantrone hydrochloride injection was injected into the peritumoral area of gastric cancer, and the exposure was very low and would not cause toxic side effects.

Among the subjects in the full analysis set of the clinical study, 4 subjects in different dose groups had 13 adverse event records, and no subject withdrew from the trial due to adverse events; 2 subjects had 2 serious adverse events; severity: 10 cases of grade 1, 1 case of grade 2, 2 cases of grade 3; relationship with the study drug: "may be unrelated" 3 cases, "definitely unrelated" 10 cases; adverse event outcomes: 3 cases of relief, 10 cases of disappearance. The occurrence of DLT in 12 subjects was observed until 14±2 days after administration. None of the 12 subjects experienced dose-limiting toxicity (DLT), that is, no toxic reactions of grade 3 or above in the blood or skin occurred. This indicates that the safety of the test drug is good.

The efficacy analysis of the subjects showed that the total number of lymph nodes detected by the test drug in the 2.0ml dose group was 60.3±14.57 (number), the total number of N1 lymph nodes detected was 57.3±13.58, and the total number of N2 lymph nodes detected was 3.0±1.00 (number). The total number of lymph nodes detected by the test drug in the 3.0ml dose group was 60.3±14.57 (number), the total number of N1 lymph nodes detected was 57.3±13.58, and the total number of N2 lymph nodes detected was 3.0±1.00 (number). The lymph node staining showed a dose-dependent trend overall, and the total number of lymph nodes detected in the 2.0ml and 3.0ml dose groups was higher; and the 3.0ml dose group was completely metabolized at 240 minutes, so the safe dose range of the test drug is 2.0-3.0ml.

In this clinical study, although the differences in the total number of lymph nodes detected, the total number of lymph nodes detected in the first station (N1) and the total number of lymph nodes detected in the second station (N2), and the detection rate of lymph nodes with a short diameter ≤ 2 mm between the experimental group and the control group were not statistically significant, the differences in the lymph node staining rate, N1 lymph node staining rate, N2 lymph node staining rate, metastatic lymph node staining rate, N1 metastatic lymph node staining rate, and the detection rate of lymph nodes with a short diameter ≤ 1 mm between the experimental group and the control group were statistically significant. This suggests that the total number of lymph nodes detected by the experimental drug is not inferior to that of the control drug, but the lymph node staining rate and the detection rate of lymph nodes with a short diameter ≤ 1 mm of the experimental drug are significantly better than those of the control drug. In addition, the control drug nanocarbon cannot be metabolized in the body and has the problem of permanent residue; while mitoxantrone has specific lymph affinity, fast staining, and long-lasting effect without accumulation. Among the subjects in the clinical study safety set, 1 subject had 1 adverse event record of "hypertension" with a severity of Grade 3. The relationship with the study drug was "definitely unrelated" and the adverse event was converted to "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.

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

The foregoing description of specific exemplary embodiments of the present disclosure is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present disclosure to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present disclosure and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present disclosure and various different selections and changes. The scope of the present disclosure is intended to be defined by the claims and their equivalents.

Claims (14)

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 diseases associated with gastrectomy. 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%. 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. The use according to any one of claims 1 to 3, wherein the gastrectomy is selected from total gastrectomy, subtotal gastrectomy, hemigastrectomy or antrectomy. According to the use according to any one of claims 1 to 4, 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. 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. 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%. 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. 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. 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 by injection at each point of the greater curvature and lesser curvature of the stomach; preferably, the injection is administered by injection at each point 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. 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. 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. 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. A method for treating a patient suffering from a gastrectomy-related disease, 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.