Mitoxantrone albumin preparation, its preparation method and application in anti-tumor

Abstract

The present application relates to a kind of mitoxantrone albumin preparation and its preparation method and application in antitumor, the mitoxantrone albumin preparation is prepared by the following method: (1) mitoxantrone or its pharmaceutically acceptable salt is dissolved in solvent 1, obtain solution 1;(2) albumin is dissolved in solvent 2, obtain solution 2;(3) the solution 1 and solution 2 are mixed, namely obtained;The solvent 2 is buffer solution with pH value of 7~9.5.The present application mitoxantrone albumin preparation release effect is good, and the size of particle size can be adjusted, high efficiency low toxicity, good stability.In addition, its preparation process is simple, control is convenient, easy to mass production.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a mitoxantrone albumin preparation, its preparation method, and its application in anti-tumor therapy. Background Technology

[0002] Cancer is the number one killer threatening human health.

[0003] Mitoxantrone hydrochloride (MTO) is a broad-spectrum antitumor drug, appearing as a blue-black crystalline powder, readily soluble in water. After entering the human body, it accumulates in the cell nucleus, inhibiting DNA topoisomerase II and blocking DNA-RNA synthesis by intercalating into the DNA double-stranded structure. Numerous clinical studies have demonstrated that mitoxantrone hydrochloride is significantly effective in treating leukemia, breast cancer, and malignant lymphoma, and also shows some efficacy against bladder cancer, ovarian cancer, and gastrointestinal tumors. As an anthracycline antitumor drug, mitoxantrone's adverse reactions mainly manifest as cardiotoxicity, bone marrow suppression, and gastrointestinal reactions. While bone marrow suppression and gastrointestinal reactions can be resolved with appropriate medication, cardiotoxicity often leads to serious consequences. Therefore, pharmaceutical researchers have conducted a series of studies focusing on improving its selectivity and stability, and reducing its toxic side effects.

[0004] CN114601791B discloses a mitoxantrone hydrochloride liquid formulation and its preparation method. By using low-oxygen water with low oxygen content, the use of sulfite antioxidants and chelating agents is avoided, and the product still maintains excellent stability. This method prevents antioxidants from reacting with the main components to generate impurities. The simple composition reduces the risk of side effects and improves product quality and safety. CN114569545B discloses a stable mitoxantrone liquid formulation. Using this formulation solves the technical problem of significant impurity growth during the preparation of mitoxantrone formulations. Its stable properties meet the requirements of clinical treatment.

[0005] However, mitoxantrone injections suffer from poor tumor selectivity and, due to their systemic distribution after intravenous injection, are prone to causing severe cardiotoxicity, bone marrow suppression, and other adverse reactions. From both efficacy and safety perspectives, they still fail to meet clinical needs, posing a significant challenge to the development and application of mitoxantrone formulations. Therefore, it is necessary to redesign novel mitoxantrone carrier delivery formulations with strong tumor selectivity, significant efficacy, and minimal toxic side effects.

[0006] CN101209243B discloses a liposomal drug and its preparation method, particularly a liposomal formulation of mitoxantrone and its preparation method. Significant improvements have been made in reducing mitoxantrone toxicity and prolonging the drug's half-life, and related liposomal products are already on the market. CN116650442B discloses a biomimetic nanoparticle loaded with mitoxantrone, its preparation method, and its application. This biomimetic nanoparticle loaded with mitoxantrone can effectively accumulate at the tumor site through long-term circulation under the action of erythrocyte membranes. The mitoxantrone within it can generate high heat and reactive oxygen species under 660 nm light irradiation, and the gas carried by perfluoroalkane can further promote the generation of more reactive oxygen species by mitoxantrone, thereby achieving good photothermal and photodynamic therapy effects. Furthermore, reactive oxygen species further trigger the self-accelerated release of mitoxantrone from the hydrophobic core of the reactive oxygen species-sensitive polyphosphate, thereby simultaneously achieving chemotherapy and greatly improving the antitumor effect of the chemotherapeutic drug mitoxantrone.

[0007] The construction of novel drug delivery systems relies on various carrier materials. During formulation design, issues such as the stability of the delivery carrier in the biological environment, drug encapsulation efficiency, and safety must be considered. However, the aforementioned formulations use numerous excipients, and the preparation process involves the use of organic reagents, raising concerns about potential safety hazards. Furthermore, the complex preparation process and the uncertainty surrounding whether excipients interact with the drug to generate impurities significantly increase the difficulty of converting anthraquinone formulations.

[0008] Therefore, there is a need in the field for a mitoxantrone formulation that is highly effective, low in toxicity, stable, and has a good sustained-release effect for the treatment of tumors. Summary of the Invention

[0009] Based on this, the purpose of the present invention is to provide a mitoxantrone albumin preparation, which is formed by self-assembly of albumin and mitoxantrone or a pharmaceutically acceptable salt thereof. The mitoxantrone albumin preparation is highly efficient, low in toxicity, stable, and has a good sustained-release effect, and the raw materials and methods for its preparation are simple.

[0010] In a first aspect, the present invention provides a method for preparing a mitoxantrone albumin formulation, comprising the following steps:

[0011] (1) Dissolve mitoxantrone or a pharmaceutically acceptable salt thereof in solvent 1 to obtain solution 1;

[0012] (2) Dissolve albumin in solvent 2 to obtain solution 2;

[0013] (3) Mix solution 1 and solution 2 to obtain the final product;

[0014] Solvent 2 is a buffer solution with a pH value of 7 to 9.5.

[0015] In some embodiments, the solvent 2 is a buffer solution with a pH value of 8 to 9.5.

[0016] In some embodiments, the mass ratio of albumin to mitoxantrone or a pharmaceutically acceptable salt thereof is (1-15):(1-5), preferably (1-10):(1-3).

[0017] In some embodiments, the mass ratio of albumin to mitoxantrone or a pharmaceutically acceptable salt thereof is (1-10):1, preferably (4-10):1.

[0018] In some embodiments, the pharmaceutically acceptable salt of mitoxantrone includes at least one of mitoxantrone hydrochloride, mitoxantrone citrate, mitoxantrone oxalate, mitoxantrone sulfate, mitoxantrone acetate, and mitoxantrone phosphate.

[0019] In some embodiments, the pharmaceutically acceptable salt of mitoxantrone is mitoxantrone hydrochloride.

[0020] In some embodiments, the albumin is at least one of human albumin, bovine albumin, mouse albumin, and albumin fusion protein; preferably at least one of human serum albumin, bovine serum albumin, mouse serum albumin, and albumin fusion protein; more preferably at least one of human serum albumin, bovine serum albumin, and albumin fusion protein.

[0021] In some embodiments, the albumin fusion protein is a fusion protein of albumin and an anti-tumor targeted therapy antibody.

[0022] In some embodiments, the anti-tumor targeted therapeutic antibody includes therapeutic antibodies targeting HER2, therapeutic antibodies targeting EGFR, therapeutic antibodies targeting immune checkpoints, etc.

[0023] In some embodiments, the therapeutic antibodies targeting immune checkpoints include anti-PD-1 antibodies, anti-PD-L1 antibodies, etc.

[0024] In some embodiments, solvent 1 is water.

[0025] In some embodiments, the buffer solution is selected from at least one of the following: disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (also known as phosphate buffer, PBS), sodium carbonate-sodium bicarbonate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, Tris-hydrochloric acid buffer, borate buffer, borax-hydrochloric acid buffer, boric acid-borax buffer, glycine-sodium hydroxide buffer, disodium hydrogen phosphate-citric acid buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, sodium barbital-hydrochloric acid buffer, and phosphate-buffered saline.

[0026] In some embodiments, the buffer solution is selected from at least one of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, phosphate buffer, sodium carbonate-sodium bicarbonate buffer, and phosphate buffer saline.

[0027] In some embodiments, the solvent 1 is water for injection.

[0028] In some embodiments, the concentration of mitoxantrone or a pharmaceutically acceptable salt thereof in solution 1 is 0.5 mg / mL to 30 mg / mL, preferably 1 mg / mL to 20 mg / mL, and more preferably 1 mg / mL to 5 mg / mL.

[0029] In some embodiments, the concentration of albumin in solution 2 is 0.5 mg / mL to 30 mg / mL, preferably 1 mg / mL to 20 mg / mL, and more preferably 1 mg / mL to 5 mg / mL.

[0030] In some embodiments, step (3) is: ultrasonically mixing the solution 1 and the solution 2 to obtain the solution.

[0031] In some embodiments, step (3) is: mixing solution 1 and solution 2 using microfluidic technology to obtain the solution.

[0032] In some embodiments, step (3) is: mixing solution 1 and solution 2, and then homogenizing under high pressure to obtain the solution.

[0033] In some embodiments, step (3) is: mixing solution 1 and solution 2 and stirring magnetically to obtain the solution.

[0034] In some embodiments, step (3) is: mixing solution 1 and solution 2 and letting it stand to obtain the solution.

[0035] In some embodiments, the settling time is 1 h to 48 h, preferably 12 h to 48 h, and more preferably 24 h to 48 h.

[0036] In a second aspect, the present invention provides a mitoxantrone albumin formulation prepared by the preparation method described above.

[0037] A third aspect of the present invention provides the use of the mitoxantrone albumin formulation described above in the preparation of antitumor drugs.

[0038] In a fourth aspect, the present invention provides an antitumor drug, wherein the active ingredient of the antitumor drug comprises the mitoxantrone albumin formulation as described above.

[0039] In some embodiments, the dosage form of the drug is an injection, an oral preparation, or an implant.

[0040] In some embodiments, the injectable is an injection solution or a lyophilized powder.

[0041] In some embodiments, the oral preparation is a granule, solution, tablet, capsule, pill, or powder.

[0042] In some embodiments, the antitumor drug is in the form of a lyophilized powder.

[0043] In some embodiments, the lyophilized powder comprises the mitoxantrone albumin formulation and lyophilization protectant as described above.

[0044] In some embodiments, the freeze-drying protectant is selected from at least one of pH buffers, fillers, and excipients.

[0045] In some embodiments, the freeze-drying protectant is selected from at least one of glucose, sucrose, maltose, lactose, mannose, trehalose, glycine, and dextran.

[0046] A fifth aspect of the present invention provides a method for preparing the lyophilized powder as described above, comprising the following steps: adding the lyophilization protectant to the mitoxantrone albumin preparation, followed by freeze-drying, to obtain the powder.

[0047] This invention provides a method for preparing mitoxantrone albumin formulations. The inventors discovered that by first dissolving mitoxantrone or its pharmaceutically acceptable salt and albumin separately in a solvent to obtain a solution, and then mixing them, nanoparticles can be formed through self-assembly.

[0048] Furthermore, in the preparation of the mitoxantrone albumin formulation, the pH value of the solvent used to dissolve the albumin has a very important influence on the sustained-release effect of the prepared mitoxantrone albumin formulation. Within a suitable pH range, the initial release rate of the prepared mitoxantrone albumin formulation can be slower, resulting in a better sustained-release effect.

[0049] Furthermore, the size of the obtained nanoparticles can be adjusted by controlling the mass ratio of albumin to mitoxantrone or its pharmaceutically acceptable salt, and the nanoparticles can have both high encapsulation efficiency and drug loading, effectively targeting tumor tissue, improving drug utilization and efficacy, and reducing toxicity.

[0050] The mitoxantrone albumin formulation of this invention uses simple raw materials, requiring only the self-assembly of albumin and mitoxantrone or its pharmaceutically acceptable salt in a suitable ratio. The preparation process does not involve the use of organic reagents, exhibiting good safety. Furthermore, the preparation process is simple, easy to control, and readily suitable for large-scale production. It possesses advantages such as high efficiency, low toxicity, good stability, and good sustained-release effect. It can also be combined with other formulation technologies to improve its pharmacokinetics and drug distribution, showing broad clinical application prospects. Attached Figure Description

[0051] Figure 1 The nanoparticle size distribution and high-magnification transmission electron microscopy image of the mitoxantrone albumin particles prepared in Example 2 are shown.

[0052] Figure 2 The fluorescence emission spectra of MTO, BSA, and mitoxantrone albumin particles (MTO@BSA) in different mass ratios are shown in Example 11.

[0053] Figure 3 The in vitro drug release curve of mitoxantrone albumin nanoformulation is shown.

[0054] Figure 4 The results of stability monitoring for mitoxantrone albumin nanoformulation.

[0055] Figure 5 The results show the killing effect of mitoxantrone hydrochloride technical, mitoxantrone albumin nanoparticles, and mitoxantrone hydrochloride liposomes on tumor cells.

[0056] Figure 6 This is the result of the uptake of mitoxantrone hydrochloride technical and mitoxantrone albumin nanoparticles by tumor cells.

[0057] Figure 7 This study describes the in vivo drug distribution of mitoxantrone hydrochloride technical and mitoxantrone albumin nanoparticle formulation.

[0058] Figure 8 The plasma drug metabolism of mitoxantrone hydrochloride technical and mitoxantrone albumin nanoparticle formulation.

[0059] Figure 9 The change in body weight of mice in each group of the E0771 model.

[0060] Figure 10 The changes in tumor growth curves of mice in each group of the MDA-MB-231 model.

[0061] Figure 11 Lung tumor metastasis in mice of different groups in the MDA-MB-231 model. Detailed Implementation

[0062] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0063] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0064] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0065] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0066] In some embodiments of the present invention, a method for preparing a mitoxantrone albumin formulation is disclosed, comprising the following steps:

[0067] (1) Dissolve mitoxantrone or a pharmaceutically acceptable salt thereof in solvent 1 to obtain solution 1;

[0068] (2) Dissolve albumin in solvent 2 to obtain solution 2;

[0069] (3) Mix solution 1 and solution 2 to obtain the final product;

[0070] Solvent 2 is a buffer solution with a pH value of 7 to 9.5.

[0071] In some embodiments, the solvent 2 is a buffer solution with a pH value of 8 to 9.5.

[0072] This invention reveals that by first dissolving mitoxantrone or its pharmaceutically acceptable salt and albumin separately in a solvent to obtain solutions, and then mixing them, nanoparticles can be formed through self-assembly. Furthermore, the pH value of solvent 2 used to dissolve albumin has a significant impact on the sustained-release effect of the prepared mitoxantrone albumin formulation; a better sustained-release effect can be achieved within a suitable pH range (7-9.5). When the pH value of solvent 2 is 7-9.5, the cumulative release over 4 hours is approximately 30%-60%; when the pH value of solvent 2 is 8-9.5, the cumulative release over 4 hours is approximately 30%-40%.

[0073] In some embodiments, the buffer solution is selected from at least one of the following: disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, sodium carbonate-sodium bicarbonate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, Tris-hydrochloric acid buffer, borate buffer, borax-hydrochloric acid buffer, boric acid-borax buffer, glycine-sodium hydroxide buffer, disodium hydrogen phosphate-citric acid buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, sodium barbital-hydrochloric acid buffer, and phosphate-buffered saline.

[0074] In some embodiments, the buffer solution is selected from at least one of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, phosphate buffer, sodium carbonate-sodium bicarbonate buffer, and phosphate buffer saline.

[0075] In some embodiments, the mass ratio of albumin to mitoxantrone or a pharmaceutically acceptable salt thereof is (1-15):(1-5).

[0076] In some embodiments, the mass ratio of albumin to mitoxantrone or a pharmaceutically acceptable salt thereof is (1~10):(1~3), preferably (1~10):1, more preferably (4~10):1.

[0077] In some embodiments, the mass ratio of the albumin to mitoxantrone or a pharmaceutically acceptable salt thereof is (4-6):1, (4-7):1, (6-8):1, or (8-10):1.

[0078] In some embodiments, when the mass ratio of bovine serum albumin (BSA) to mitoxantrone hydrochloride (MTO) used in preparation is (1-5):1, the particle size of the mitoxantrone albumin preparation is approximately 80 nm. With the amount of mitoxantrone hydrochloride (MTO) remaining constant, increasing the amount of bovine serum albumin (BSA) to a certain extent increases the particle size of the mitoxantrone albumin preparation. When the mass ratio of bovine serum albumin (BSA) to mitoxantrone hydrochloride (MTO) used in preparation is 20:1, micron-sized particles appear, but some particles around 60 nm and free protein still exist.

[0079] Furthermore, mitoxantrone hydrochloride (MTO) has a molecular weight of 517.4, and bovine serum albumin (BSA) has a molecular weight of approximately 66 kDa. After particle preparation, ultrafiltration was performed using a 50 kDa ultrafiltration tube. When the mass ratio of BSA to MTO was less than 4:1, the filtrate in the lower chamber of the ultrafiltration tube was blue, indicating the presence of some free MTO that was not bound to BSA. The free MTO existed in the form of nanocrystals with a size of approximately 2-3 nm. When the mass ratio of BSA to MTO was greater than 4:1, almost no free MTO was present, indicating that the MTO added during preparation was completely bound to BSA to form particles. When the mass ratio of BSA to MTO was greater than 5:1, some free protein was present.

[0080] The present invention has discovered that the size of the obtained nanoparticles can be adjusted by controlling the mass ratio of albumin to mitoxantrone or its pharmaceutically acceptable salt, and the nanoparticles can have both high encapsulation efficiency and drug loading. When the mass ratio of albumin to mitoxantrone or its pharmaceutically acceptable salt is (1~15):(1~5), the prepared nanoparticles have a size of approximately 80~600 nm, a drug encapsulation efficiency of approximately 30%~100%, and a drug loading of approximately 5%~25%; when the mass ratio of albumin to mitoxantrone or its pharmaceutically acceptable salt is (1~10):(1~3), the prepared nanoparticles have a size of approximately 80~300 nm, a drug encapsulation efficiency of approximately 30%~100%, and a drug loading of approximately 10%~25%; when the mass ratio of albumin to mitoxantrone or its pharmaceutically acceptable salt is (4~10):1, the prepared nanoparticles have a size of approximately 80~300 nm, a drug encapsulation efficiency of approximately 95%~100%, and a drug loading of approximately 10%~25%.

[0081] In some embodiments, the preparation method includes the following steps: (1) dissolving mitoxantrone or a pharmaceutically acceptable salt thereof in solvent 1 to obtain solution 1; (2) dissolving albumin in solvent 2 to obtain solution 2; (3) ultrasonically mixing solution 1 and solution 2 and allowing them to stand to obtain mitoxantrone albumin preparation.

[0082] In some embodiments, the ultrasonic conditions can be conventional in the art, for example, the ultrasonic frequency can be 20 kHz to 70 kHz.

[0083] In some embodiments, the preparation method includes the following steps: (1) dissolving mitoxantrone or a pharmaceutically acceptable salt thereof in solvent 1 to obtain solution 1; (2) dissolving albumin in solvent 2 to obtain solution 2; (3) establishing a flash nanoprecipitation device system, including an injection pump, an injection tube, a microfluidic chip, and a sample receiving device; transferring solution 1 and solution 2 to the injection tube, running the instrument, and preparing mitoxantrone albumin preparations with different mass ratios by adjusting the flow rate ratio.

[0084] In some embodiments, the preparation method includes the following steps: (1) dissolving mitoxantrone or a pharmaceutically acceptable salt thereof in solvent 1 to obtain solution 1; (2) dissolving albumin in solvent 2 to obtain solution 2; (3) mixing solution 1 and solution 2, and subjecting the mixture to high-pressure homogenization to obtain mitoxantrone albumin preparation.

[0085] In some embodiments, the preparation method includes the following steps: (1) dissolving mitoxantrone or a pharmaceutically acceptable salt thereof in solvent 1 to obtain solution 1; (2) dissolving albumin in solvent 2 to obtain solution 2; (3) mixing solution 1 and solution 2 and magnetically stirring until the solution is clear and transparent to obtain mitoxantrone albumin preparation.

[0086] In some embodiments, the preparation method includes the following steps: (1) dissolving mitoxantrone or a pharmaceutically acceptable salt thereof in solvent 1 to obtain solution 1; (2) dissolving albumin in solvent 2 to obtain solution 2; (3) mixing solution 1 and solution 2 and allowing them to stand to obtain mitoxantrone albumin preparation.

[0087] In some embodiments, the solvent 1 is water, including water for injection, purified water, etc., preferably water for injection.

[0088] In some embodiments, the concentration of mitoxantrone or its pharmaceutically acceptable salt in solution 1 is 0.5 mg / mL to 30 mg / mL, preferably 1 mg / mL to 20 mg / mL, more preferably 1 mg / mL to 5 mg / mL; the concentration of albumin in solution 2 is 0.5 mg / mL to 30 mg / mL, preferably 1 mg / mL to 20 mg / mL, more preferably 1 mg / mL to 5 mg / mL. When mitoxantrone or its pharmaceutically acceptable salt is added to albumin at different mass ratios, mixing corresponding volumes of solutions 1 and 2 according to their concentrations yields mitoxantrone-albumin particles of different sizes. For example, solutions 1 and 2 of the same concentration can be prepared, in which case the mass ratio of mitoxantrone or its pharmaceutically acceptable salt to albumin is equal to the volume ratio of solutions 1 and 2.

[0089] In some embodiments of the present invention, a mitoxantrone albumin formulation is provided, which is prepared by the preparation method described above.

[0090] In some embodiments of the present invention, an antitumor drug is provided, wherein the active ingredient of the antitumor drug comprises mitoxantrone albumin preparation as described above.

[0091] The drug of the present invention can be administered via intravenous, oral, or local routes, preferably via intravenous administration.

[0092] In some embodiments, the antitumor drug further includes pharmaceutically acceptable excipients, which may be selected from conventional excipients in the art, including at least one of commonly used pharmaceutical excipients such as osmotic pressure regulators, suspending agents, surfactants, antioxidants, preservatives, and lyophilization protectants.

[0093] In some embodiments, the dosage form of the drug includes injections, oral preparations, implants, etc.

[0094] In some embodiments, the injectable includes an injection solution, lyophilized powder, etc.

[0095] In some embodiments, the oral formulation includes granules, solutions, tablets, capsules, pills, powders, etc.

[0096] In some embodiments, the drug is in the form of a lyophilized powder, obtained by freeze-drying the mitoxantrone albumin preparation.

[0097] In some embodiments, the lyophilized powder includes the mitoxantrone albumin formulation and a lyophilization protectant.

[0098] In some embodiments, the lyophilized powder comprises the following components in parts by weight: 6000 parts of the mitoxantrone albumin preparation and 0-300 parts of the lyophilization protectant.

[0099] In some embodiments, the freeze-drying protectant is selected from at least one of pH buffers, fillers, and excipients; preferably, the freeze-drying protectant is selected from at least one of glucose, sucrose, maltose, lactose, mannose, trehalose, glycine, and dextran.

[0100] In some embodiments, the method for preparing the lyophilized powder includes the following steps: adding the lyophilization protectant to the mitoxantrone albumin preparation, followed by freeze-drying, to obtain the final product.

[0101] The present invention will be further described in detail below with reference to specific embodiments. In the following embodiments, the ultrasound device is a Ningbo Xinzhi Biotechnology Co., Ltd. SB25-12 DTD model.

[0102] Example 1

[0103] Appearance observation of mitoxantrone albumin formulation. The procedure in this example is as follows:

[0104] Mitoxantrone hydrochloride was dissolved in water for injection to prepare a 1 mg / mL mitoxantrone hydrochloride solution; bovine serum albumin was dissolved in phosphate-buffered saline (PBS) at pH 7.4 to prepare a 1 mg / mL bovine serum albumin solution; the mitoxantrone hydrochloride solution was slowly added dropwise to the albumin solution while sonicating, and after sonicating and mixing, the mixture was allowed to stand to obtain the mitoxantrone albumin preparation, which was a clear, homogeneous blue solution.

[0105] Example 2

[0106] The preparation of mitoxantrone albumin particles with a bovine serum albumin to mitoxantrone hydrochloride mass ratio of 1:1 is described in this embodiment:

[0107] (1) Preparation process: Dissolve 1 mg of mitoxantrone hydrochloride in 1 mL of water for injection to prepare a mitoxantrone hydrochloride solution of 1 mg / mL; dissolve 1 mg of bovine serum albumin in 1 mL of phosphate buffered saline (PBS) at pH 7.4 to prepare a bovine serum albumin solution of 1 mg / mL; while sonicating, slowly add 1 mL of mitoxantrone hydrochloride solution to 1 mL of bovine serum albumin solution, with a mass ratio of BSA to MTO of 1:1. After sonicating and mixing, let stand to obtain mitoxantrone albumin preparation.

[0108] (2) Characterization: such as Figure 1 As shown in Figure A, the particle size of bovine serum albumin dissolved in phosphate-buffered saline (PBS) at pH 7.4 was less than 10 nm, as detected by a particle size analyzer. The size of mitoxantrone albumin particles was approximately 60–80 nm, indicating successful particle preparation. Morphological characterization by transmission electron microscopy revealed a smooth, spherical shape. Figure 1 As shown in Figure B. Under a high-magnification transmission electron microscope, the particles appear as... Figure 1 As shown in C, lattice fringes were observed on the particle surface (see Figure C). Figure 1 (D) and the ring-shaped spot after Fourier transform (see D) Figure 1 (E) indicates that a type of crystal particle may have been formed in this embodiment, such as Figure 1 As shown.

[0109] Example 3

[0110] Preparation of mitoxantrone albumin formulation with a bovine serum albumin to mitoxantrone hydrochloride mass ratio of 3:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 2 is the different bovine serum albumin to mitoxantrone hydrochloride mass ratio; all other aspects are the same as in Example 2. The operation in this embodiment is as follows:

[0111] (1) Preparation process: Dissolve 1 mg of mitoxantrone hydrochloride in 1 mL of water for injection to prepare a mitoxantrone hydrochloride solution of 1 mg / mL; dissolve 3 mg of bovine serum albumin in 3 mL of phosphate buffered saline (PBS) at pH 7.4 to prepare a bovine serum albumin solution of 1 mg / mL; while sonicating, slowly add 1 mL of mitoxantrone hydrochloride solution to 3 mL of bovine serum albumin solution, with a mass ratio of BSA to MTO of 3:1. After sonicating and mixing, let stand to obtain mitoxantrone albumin preparation.

[0112] (2) Characterization: The nanoparticles were characterized by a nanoparticle tracking analyzer (NTA) and a transmission electron microscope, and were found to be smooth and spherical.

[0113] Example 4

[0114] Preparation of mitoxantrone albumin formulation with a bovine serum albumin to mitoxantrone hydrochloride mass ratio of 4:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 2 is the different bovine serum albumin to mitoxantrone hydrochloride mass ratio; all other aspects are the same as in Example 2. The operation of this embodiment is as follows:

[0115] (1) Preparation process: Dissolve 1 mg of mitoxantrone hydrochloride in 1 mL of water for injection to prepare a mitoxantrone hydrochloride solution of 1 mg / mL; dissolve 4 mg of bovine serum albumin in 4 mL of phosphate buffered saline (PBS) with pH 7.4 to prepare a bovine serum albumin solution of 1 mg / mL; while sonicating, slowly add 1 mL of mitoxantrone hydrochloride solution to 4 mL of bovine serum albumin solution, that is, the mass ratio of BSA to MTO is 4:1. After sonicating and mixing, let stand to obtain mitoxantrone albumin preparation.

[0116] (2) Characterization: The nanoparticles were characterized by a nanoparticle tracking analyzer (NTA) and a transmission electron microscope, and were found to be smooth and spherical.

[0117] Example 5

[0118] Preparation of mitoxantrone albumin formulation with a bovine serum albumin to mitoxantrone hydrochloride mass ratio of 10:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 2 is the different bovine serum albumin to mitoxantrone hydrochloride mass ratio; all other aspects are the same as in Example 2. The operation of this embodiment is as follows:

[0119] (1) Preparation process: Dissolve 1 mg of mitoxantrone hydrochloride in 1 mL of water for injection to prepare a mitoxantrone hydrochloride solution of 1 mg / mL; dissolve 5 mg of bovine serum albumin in 5 mL of phosphate buffered saline (PBS) with pH 7.4 to prepare a bovine serum albumin solution of 1 mg / mL; while sonicating, slowly add 0.5 mL of mitoxantrone hydrochloride solution to 5 mL of bovine serum albumin solution, that is, the mass ratio of BSA to MTO is 10:1. After sonicating and mixing, let stand to obtain mitoxantrone albumin preparation.

[0120] (2) Characterization: The particles were characterized by nanoparticle tracking analyzer (NTA) and transmission electron microscopy. They were smooth, spherical, and the particle size was between 150 nm and 300 nm.

[0121] Example 6

[0122] Preparation of mitoxantrone albumin formulation with a bovine serum albumin to mitoxantrone hydrochloride mass ratio of 15:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 2 is the different bovine serum albumin to mitoxantrone hydrochloride mass ratio; all other aspects are the same as in Example 2. The operation of this embodiment is as follows:

[0123] (1) Preparation process: Dissolve 1 mg of mitoxantrone hydrochloride in 1 mL of water for injection to prepare a mitoxantrone hydrochloride solution of 1 mg / mL; dissolve 5 mg of bovine serum albumin in 5 mL of phosphate buffered saline (PBS) at pH 7.4 to prepare a bovine serum albumin solution of 1 mg / mL; while sonicating, slowly add 0.2 mL of mitoxantrone hydrochloride solution to 3 mL of bovine serum albumin solution, i.e., the mass ratio of BSA to MTO is 15:1. After sonicating and mixing, let stand to obtain the mitoxantrone albumin preparation.

[0124] (2) Characterization: The nanoparticles were characterized by a nanoparticle tracking analyzer (NTA) and transmission electron microscopy. They were smooth-surfaced spherical particles with a particle size of 500 nm to 800 nm. There were also particles of about 50 nm and free proteins of about 8 nm.

[0125] Example 7

[0126] Preparation of mitoxantrone albumin formulation with a bovine serum albumin to mitoxantrone hydrochloride mass ratio of 20:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 2 is the different bovine serum albumin to mitoxantrone hydrochloride mass ratio; all other aspects are the same as in Example 2. The operation in this embodiment is as follows:

[0127] (1) Preparation process: According to the above prescription, 1 mg of mitoxantrone hydrochloride was dissolved in 1 mL of water for injection to prepare a mitoxantrone hydrochloride solution of 1 mg / mL; 10 mg of bovine serum albumin was dissolved in 10 mL of phosphate buffer (PBS) with pH 7.4 to prepare a bovine serum albumin solution of 1 mg / mL; while sonicating, 0.5 mL of mitoxantrone hydrochloride solution was slowly added dropwise to 10 mL of bovine serum albumin solution, that is, the mass ratio of BSA to MTO was 20:1. After sonicating and mixing, the mixture was allowed to stand to obtain the mitoxantrone albumin preparation.

[0128] (2) Characterization: Morphological characterization was performed using transmission electron microscopy. The particles were smooth and spherical, with some particles increasing in size to the micrometer level. Particles around 60 nm and free proteins around 8 nm were still present. Micrometer-sized particles are not suitable for intravenous injection due to their large size and potential aggregation, which pose safety concerns. Furthermore, micrometer-sized particles cannot achieve enrichment in tumors through the permeation and retention (EPR) effect, leading to side effects and low drug utilization. Compared to nanoparticles, tumor cells have a poorer ability to take up micrometer-sized particles, and chemotherapeutic drugs in particles usually need to be taken up by cells to exert their effects.

[0129] Example 8

[0130] Preparation of mitoxantrone albumin formulation with bovine serum albumin and mitoxantrone hydrochloride in a mass ratio of 1:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 2 is the use of a different solvent; all other aspects are the same as in Example 2. The operation in this embodiment is as follows:

[0131] (1) Preparation process: Dissolve 1 mg of mitoxantrone hydrochloride in 1 mL of water for injection to prepare a mitoxantrone hydrochloride solution of 1 mg / mL; dissolve 1 mg of bovine serum albumin in 1 mL of NaHCO3 / Na2CO3 buffer solution with a pH of about 9.2 to prepare a bovine serum albumin solution of 1 mg / mL; while sonicating, slowly add 1 mL of mitoxantrone hydrochloride solution to 1 mL of bovine serum albumin solution, with a mass ratio of BSA to MTO of 1:1. After sonicating and mixing, let stand to obtain mitoxantrone albumin preparation.

[0132] (2) Characterization: The particles were characterized by nanoparticle tracking analyzer (NTA) and transmission electron microscopy. They were found to be smooth-surfaced spherical with a particle size of about 60~80 nm.

[0133] Example 9

[0134] Preparation of mitoxantrone albumin formulation with bovine serum albumin and mitoxantrone hydrochloride in a mass ratio of 2:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 2 is the use of a different solvent; all other aspects are the same as in Example 2. The operation of this embodiment is as follows:

[0135] (1) Preparation process: Dissolve 1 mg of mitoxantrone hydrochloride in 1 mL of water for injection to prepare a mitoxantrone hydrochloride solution of 1 mg / mL; dissolve 1 mg of bovine serum albumin in 1 mL of NaHCO3 / Na2CO3 buffer solution with pH 9.2 to prepare a bovine serum albumin solution of 1 mg / mL; while sonicating, slowly add 0.5 mL of mitoxantrone hydrochloride solution to 1 mL of bovine serum albumin solution, that is, the mass ratio of BSA to MTO is 2:1. After sonicating and mixing, let stand to obtain mitoxantrone albumin preparation.

[0136] (2) Characterization: The nanoparticles were characterized by a nanoparticle tracking analyzer (NTA) and a transmission electron microscope, and were found to be smooth and spherical.

[0137] Example 10

[0138] The encapsulation efficiency and drug loading of mitoxantrone albumin formulations with different mass ratios (the albumin solvent was phosphate buffer at pH 7.4, and the preparation method was the same as in Example 2 except for the mass ratio of BSA to MTO) were determined by ultrafiltration. Encapsulation efficiency = amount of drug loaded into the particles / amount of drug added × 100%, and drug loading = amount of drug loaded into the particles / particle weight × 100%.

[0139] Formulations prepared by ultrafiltration at different BSA to MTO mass ratios were tested. The results showed that when the mass ratio of bovine serum albumin (BSA) to mitoxantrone hydrochloride (MTO) was less than 4:1, the filtrate in the lower chamber of the ultrafiltration tube was blue, indicating that a large amount of free MTO was not bound to albumin. When the mass ratio of bovine serum albumin (BSA) to mitoxantrone hydrochloride (MTO) was greater than 4:1, the filtrate in the lower chamber of the ultrafiltration tube was almost colorless, indicating that MTO was almost completely bound to albumin to form particles.

[0140] The sample solution was ultrafiltered (molecular weight 50 kDa), and the filtrate from the lower chamber was collected and directly subjected to ultraviolet detection. The encapsulation efficiency and drug loading were calculated by the difference method. The results are shown in Table 1.

[0141] According to the test results, when the mass ratio of bovine serum albumin (BSA) to mitoxantrone hydrochloride (MTO) is less than 4:1, some free MTO is present but not bound to BSA; when the mass ratio of bovine serum albumin (BSA) to mitoxantrone hydrochloride (MTO) is greater than 4:1, almost no free MTO is present, indicating that the MTO and BSA added during preparation exist in the form of nanocrystals or in the form of particles adsorbed between the two.

[0142] Furthermore, a sample containing bovine serum albumin (BSA) and mitoxantrone hydrochloride (MTO) at a mass ratio of 3:1 was subjected to ultrafiltration (molecular weight 50 kDa). The filtrate from the lower chamber was examined by transmission electron microscopy, and it was observed that free MTO existed in the form of nanocrystals with a size of approximately 3 nm.

[0143] Table 1 Encapsulation efficiency and drug loading of samples with different BSA to MTO mass ratios

[0144]

[0145] The above results indicate that when the mass ratio of bovine serum albumin (BSA) to mitoxantrone hydrochloride (MTO) is 4:1, all BSA and MTO undergo self-assembly, resulting in almost no free BSA and MTO in the particles. When the mass ratio of BSA to MTO is greater than 4:1, the mitoxantrone albumin formulation exhibits good drug loading and encapsulation efficiency. However, as the mass ratio of BSA to MTO further increases, the drug loading decreases significantly; when the mass ratio is 20:1, the drug loading is only about 4%. Therefore, the preferred mass ratio of BSA to MTO is (4~10):1.

[0146] Example 11

[0147] Fluorescence properties of mitoxantrone hydrochloride and albumin after self-assembly into particles were studied. The procedure in this example is as follows:

[0148] Example 1: (1) Mitoxantrone hydrochloride was dissolved in water for injection to prepare a 0.5 mg / mL mitoxantrone hydrochloride solution; (2) Bovine serum albumin was dissolved in phosphate buffered saline (PBS) at pH 7.4 to prepare a 2 mg / mL bovine serum albumin solution; (3) 1 mL of mitoxantrone hydrochloride solution was slowly added dropwise to 1 mL of bovine serum albumin solution while sonicating, i.e., the mass ratio of BSA to MTO was 4:1, the final concentration of BSA was 1 mg / mL, and after sonication and mixing, the mixture was allowed to stand to obtain an albumin to mitoxantrone mass ratio of 4:1 (W BSA :W MTO Mitoxantrone albumin formulation (ratio 4:1).

[0149] Example 2: (1) Mitoxantrone hydrochloride was dissolved in water for injection to prepare a 0.286 mg / mL mitoxantrone hydrochloride solution; (2) Bovine serum albumin was dissolved in phosphate buffered saline (PBS) at pH 7.4 to prepare a 2 mg / mL bovine serum albumin solution; (3) 1 mL of mitoxantrone hydrochloride solution was slowly added dropwise to 1 mL of bovine serum albumin solution while sonicating, i.e., the mass ratio of BSA to MTO was 7:1, the final concentration of BSA was 1 mg / mL, and after sonication and mixing, the mixture was allowed to stand to obtain an albumin to mitoxantrone mass ratio of 7:1 (W BSA :W MTO Mitoxantrone albumin formulation (7:1 ratio).

[0150] Example 3: (1) Mitoxantrone hydrochloride was dissolved in water for injection to prepare a 0.2 mg / mL mitoxantrone hydrochloride solution; (2) Bovine serum albumin was dissolved in phosphate buffered saline (PBS) at pH 7.4 to prepare a 2 mg / mL bovine serum albumin solution; (3) 1 mL of mitoxantrone hydrochloride solution was slowly added dropwise to 1 mL of bovine serum albumin solution while sonicating, i.e., the mass ratio of BSA to MTO was 10:1, the final concentration of BSA was 1 mg / mL, and after sonication and mixing, the mixture was allowed to stand to obtain an albumin to mitoxantrone mass ratio of 10:1 (W BSA :W MTO Mitoxantrone albumin formulation (r=10:1).

[0151] Example 4: (1) Mitoxantrone hydrochloride was dissolved in water for injection to prepare a 0.1 mg / mL mitoxantrone hydrochloride solution; (2) Bovine serum albumin was dissolved in phosphate buffered saline (PBS) at pH 7.4 to prepare a 2 mg / mL bovine serum albumin solution; (3) 1 mL of mitoxantrone hydrochloride solution was slowly added dropwise to 1 mL of bovine serum albumin solution while sonicating, i.e., the mass ratio of BSA to MTO was 20:1, the final concentration of BSA was 1 mg / mL, and after sonication and mixing, the mixture was allowed to stand to obtain an albumin to mitoxantrone mass ratio of 20:1 (W BSA :W MTO Mitoxantrone albumin formulation (r=20:1).

[0152] Example 5: (1) Mitoxantrone hydrochloride was dissolved in water for injection to prepare a 0.033 mg / mL mitoxantrone hydrochloride solution; (2) Bovine serum albumin was dissolved in phosphate buffered saline (PBS) at pH 7.4 to prepare a 2 mg / mL bovine serum albumin solution; (3) 1 mL of mitoxantrone hydrochloride solution was slowly added dropwise to 1 mL of bovine serum albumin solution while sonicating, i.e., the mass ratio of BSA to MTO was 60:1, the final concentration of BSA was 1 mg / mL, and after sonication and mixing, the mixture was allowed to stand to obtain an albumin to mitoxantrone mass ratio of 60:1 (WBSA :W MTO Mitoxantrone albumin formulation (r=60:1).

[0153] Example 6: Bovine serum albumin was dissolved in phosphate-buffered saline (PBS) at pH 7.4 to prepare a 1 mg / mL bovine serum albumin solution.

[0154] Experimental Example 7: Mitoxantrone hydrochloride was dissolved in water for injection to prepare a 0.5 mg / mL mitoxantrone hydrochloride solution.

[0155] With the excitation light fixed at 280 nm, the fluorescence emission intensity of the above samples was scanned in the wavelength range of 300–500 nm. The results are as follows: Figure 2 As shown, when the excitation wavelength is 280 nm, BSA molecules exhibit a strong fluorescence peak at an emission wavelength of 340 nm, while MTO shows no fluorescence peak. Under a constant BSA concentration, as the MTO concentration increases, the intrinsic fluorescence intensity of BSA decreases systematically, resulting in fluorescence quenching, indicating a binding interaction between mitoxantrone and BSA. When the mass ratio of BSA to MTO is within a suitable range (4–10:1), the interaction is stronger, resulting in lower fluorescence intensity; while when the mass ratio of BSA to MTO is greater than 20:1, a large amount of free albumin unbound by MTO is present, leading to higher fluorescence intensity. Simultaneously, a slight blue shift occurs in the maximum emission wavelength of the MTO@BSA group with different mass ratios, indicating that MTO increases the hydrophobicity around tryptophan residues.

[0156] Example 12

[0157] Preparation of mitoxantrone albumin formulation with bovine serum albumin and mitoxantrone hydrochloride in a mass ratio of 4:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 4 is the use of a different solvent; all other aspects are the same as in Example 4. The operation in this embodiment is as follows:

[0158] (1) Preparation process: Mix mitoxantrone hydrochloride solution with a sodium carbonate-sodium bicarbonate buffer solution with a pH of approximately 9.2 of bovine serum albumin, sonicate, and let stand to obtain mitoxantrone albumin preparation.

[0159] (2) Characterization: Characterized by transmission electron microscopy, it appears as a smooth, spherical shape.

[0160] Example 13

[0161] The operation in this embodiment is as follows:

[0162] Preparation process: Dissolve 1 mg of mitoxantrone hydrochloride in 1 mL of water for injection to prepare a 1 mg / mL mitoxantrone hydrochloride solution; dissolve 4 mg of bovine serum albumin in 4 mL of phosphate-buffered saline (PBS) at pH 7.4 to prepare a 1 mg / mL bovine serum albumin solution; mix the mitoxantrone hydrochloride solution and the bovine serum albumin buffer solution and place them in the injection tube of a high-pressure homogenizer. The pressure can be set to 10~15 KSL, and the mixture is cycled 2~20 times to obtain the mitoxantrone albumin preparation.

[0163] Characterization: Morphological characterization was performed by transmission electron microscopy, revealing smooth, spherical particles with uniform particle size.

[0164] Example 14

[0165] Preparation of mitoxantrone albumin formulations with human serum albumin (HSA) to mitoxantrone hydrochloride mass ratios of 4:1, 7:1, 10:1, and 15:1. The difference between the preparation process of mitoxantrone albumin particles in this embodiment and that in Example 13 lies in the different mass ratios of albumin to mitoxantrone hydrochloride and the different types of proteins used. The operation in this embodiment is as follows:

[0166] (1) Preparation process: Mix mitoxantrone hydrochloride solution with human serum albumin buffer solution in different proportions, i.e., the mass ratio of HSA to MTO is 4:1, 7:1, 10:1 and 15:1 respectively. After ultrasonic mixing, let stand to obtain mitoxantrone albumin preparation.

[0167] (2) Characterization: The particles were characterized by nanoparticle tracking analyzer (NTA) and transmission electron microscopy. They were smooth and spherical. The particle size of the 4:1 and 7:1 particles was about 60~100 nm; the particle size of the 10:1 particles was about 120~200 nm; and the particle size of the 15:1 particles was about 600~800 nm. Small particles and free proteins of about 8 nm were also present.

[0168] Example 15

[0169] This embodiment describes the preparation of mitoxantrone albumin formulations with albumin fusion protein (albumin-anti-EGFR antibody) and mitoxantrone hydrochloride at mass ratios of 0.5:1, 1:1, 3:1, 4:1, 7:1, 10:1, 15:1, 20:1, 30:1, 50:1, and 100:1. The difference between the preparation process of the mitoxantrone fusion protein particles in this embodiment and that in Example 14 lies in the type of protein used. The operation in this embodiment is as follows:

[0170] (1) Preparation process: Mitoxantrone hydrochloride solution and albumin fusion protein solution are mixed in different proportions, i.e., the mass ratio of fusion protein to MTO is 0.5:1, 1:1, 3:1, 4:1, 7:1, 10:1, 15:1, 20:1, 30:1, 50:1, and 100:1, respectively. After ultrasonic mixing, the mixture is allowed to stand to obtain mitoxantrone fusion protein formulation. During preparation, a certain amount of HSA or BSA can also be mixed in according to the required mass ratio, both of which can form a stable formulation.

[0171] (2) Characterization: The nanoparticles were characterized by a nanoparticle tracking analyzer (NTA) and transmission electron microscopy. They were found to be smooth, spherical, with a particle size of 50-200 nm.

[0172] Example 16

[0173] Sustained-release formulations require slow and continuous release, avoiding premature plateauing and excessively low plateau values. This example demonstrates the in vitro drug release efficacy of MTO@BSA.

[0174] 1. Release effect of MTO@BSA

[0175] The release-sustaining effects of the three groups of MTO@BSA prepared were tested.

[0176] Group 1: MTO@BSA prepared in Example 4 (bovine serum albumin dissolved in phosphate buffer at pH 7.4).

[0177] Group 2: Preparation of mitoxantrone albumin formulation with a bovine serum albumin (BSA) to mitoxantrone hydrochloride (MTO) mass ratio of 4:1. The preparation process differs from Example 4 in that the solvent used is different; all other aspects are the same as in Example 4. The procedure is as follows: 1 mg of mitoxantrone hydrochloride was dissolved in 1 mL of water for injection to prepare a 1 mg / mL mitoxantrone hydrochloride solution; 4 mg of bovine serum albumin was dissolved in 4 mL of phosphate buffer (pH 8.0) to prepare a 1 mg / mL bovine serum albumin solution; while sonicating, 1 mL of the mitoxantrone hydrochloride solution was slowly added dropwise to the 4 mL bovine serum albumin solution, i.e., the BSA to MTO mass ratio was 4:1. After sonication and mixing, the mixture was allowed to stand to obtain the mitoxantrone albumin formulation.

[0178] Group 3: MTO@BSA prepared in Example 12 (bovine serum albumin dissolved in sodium carbonate-sodium bicarbonate buffer at pH 9.2).

[0179] Two mL of each group of mitoxantrone albumin nanoparticle formulation (MTO@BSA) was placed into a 2000 Da dialysis bag. The ends of the dialysis bag were clamped and placed in 20 mL of PBS solution. The container was sealed and shaken at a constant rate at 37 °C. One mL of release medium was collected at 0.25 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and one mL of fresh 37 °C release medium was added. The content of mitoxantrone in the release medium was determined using an established ultraviolet spectrophotometric method.

[0180] The results are as follows Figure 3 As shown in Figure A, the pH value of the buffer solution used to dissolve bovine serum albumin affects the sustained-release effect of the prepared mitoxantrone albumin nanoparticles. Compared with a buffer pH of 7.4, the nanoparticles prepared at a buffer pH of 9.2 exhibit a slower initial release rate and better sustained-release effect, with the release at pH 8.0 falling between the two. Specifically, the cumulative 4-hour release of MTO@BSA prepared at buffer pH values ​​of 7.4, 8.0, and 9.2 (solvent 2) was 54%, 42%, and 33%, respectively. This indicates that within the pH range of solvent 2 in this invention, the sustained-release effect is better with increasing pH value.

[0181] 2. Release of MTO@HSA

[0182] 2 mL of the mitoxantrone albumin nanoformulation (MTO@HSA, W) from Example 14 was added. HSA :W MTO The mitoxantrone (4:1 ratio) solution was placed in a 2000 Da dialysis bag, and both ends of the dialysis bag were clamped. The bag was then placed in 20 mL of PBS solution, the container was sealed, and the solution was shaken at a constant rate at 37°C. At 0.25 h, 1 h, 2 h, 4 h, 12 h, 24 h, and 48 h, 1 mL of the release medium was collected and 1 mL of fresh 37°C release medium was added. The content of mitoxantrone in the release medium was determined using the established ultraviolet spectrophotometric method.

[0183] Drug release curves are shown below. Figure 3 The release of mitoxantrone albumin nanoparticles from methyl methacrylate (MTB) is slow, and the release data can be fitted using a first-order equation for sustained-release formulations, Mt = 50.487(1-e^(-1 / 2)). -0.198t ), R 2 =0.933.

[0184] Example 17

[0185] Stability monitoring of nano-formulations

[0186] The mitoxantrone albumin nanoformulation (MTO@HSA, W) from Example 14 was used. HSA :W MTOThe nanoformulation (4:1 ratio) was stored in a 4°C refrigerator, and its stability was monitored using a nanoparticle tracking analyzer (NTA).

[0187] See results Figure 4 During the monitoring period, the particle size of the nano-formulation did not change significantly, and its stability was good.

[0188] Example 18

[0189] Drug killing test on tumor cells

[0190] The MTT assay was used to investigate the killing effect of mitoxantrone albumin nanoparticles on mouse breast cancer cells (4T1). 4T1 cells in the logarithmic growth phase and in good growth condition were used in a assay of 3 × 10⁻⁶ cells. 3 ~5×10 3 The concentrations of each well were seeded into 96-well plates. After incubation overnight, fresh culture medium containing different concentrations (0.015, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 5 μM) of mitoxantrone hydrochloride aqueous solution, the mitoxantrone albumin preparation prepared in Example 4, and the mitoxantrone hydrochloride liposome preparation (PL-MTO, purchased from CSPC Zhongnuo Pharmaceutical (Shijiazhuang) Co., Ltd., National Drug Approval Number H20220001) was added. A blank control group without drug administration was also set up. After drug administration, the plates were incubated at 37 °C for 24 h and 48 h. After incubation, 10 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated at 37 °C for another 4 h. The culture medium was discarded, and 100 μL of DMSO solution was added to each well. The plates were shaken for 10 min, and the absorbance was measured at 490 nm using a microplate reader. The cell survival rate of each group was calculated using the following formula: Cell survival rate (%) = (Absorbance of experimental group - Absorbance of blank well) / (Absorbance of control group - Absorbance of blank well) × 100%).

[0191] The results of cell killing are shown in Figure 5 After 24 h of incubation, compared with the aqueous solution of mitoxantrone hydrochloride, the killing effect of the mitoxantrone albumin preparation and the mitoxantrone hydrochloride liposome preparation of the present invention on 4T1 cells was reduced (see [link to article]). Figure 5 (A) This is because mitoxantrone needs to be released from the formulation to exert its effect, and the release process limits the killing effect of mitoxantrone. After 48 h of drug incubation, the mitoxantrone albumin formulation showed better killing effect on 4T1 cells than mitoxantrone hydrochloride aqueous solution and mitoxantrone hydrochloride liposomes (see A). Figure 5 (B)

[0192] Example 19

[0193] Drug uptake by tumor cells

[0194] 4T1 cells in good growth condition and in logarithmic growth phase were collected, digested with 0.25% trypsin solution, and digestion was terminated with DMEM medium containing 10% FBS. Single-cell suspensions were collected, centrifuged at 800 rpm for 3 min, and resuspended in culture medium at 5 × 10⁻⁶ cells / cell. 4 ~10×10 4 Cells were seeded into 24-well plates at a density determined by the well density. After incubation at 37 °C with 5% CO2 until complete cell adhesion, the culture medium was discarded, and the cells were washed with sterile PBS. Then, 500 μL of mitoxantrone hydrochloride aqueous solution (MTO) containing 0.5 μM of the drug and MTO@HSA (W2O3) prepared in Example 14 were added. HSA :W MTO Serum-free and antibiotic-free culture media (ratios of 4:1, 7:1, and 10:1) were prepared in three wells per group and incubated for 1, 4, and 8 h, respectively. The drug-containing medium was then discarded, and the cells were washed with sterile PBS, digested with trypsin, collected, centrifuged at 3000 rcf for 3 min, and resuspended in 200 μL PBS. The mean fluorescence intensity of mitoxantrone in the cells was then measured by flow cytometry.

[0195] like Figure 6 As shown, at different uptake times, the uptake of 4T1 cells from Experiment 14 was higher than that from mitoxantrone hydrochloride aqueous solution, indicating that the MTO@HSA of the present invention is easily taken up by tumor cells.

[0196] Example 20

[0197] Investigation of drug distribution in vivo

[0198] Construct a mouse orthotopic breast cancer model, using 5×10 5 4T1 cells were suspended in 50 μL of phosphate-buffered saline (PBS) at pH 7.4 and then implanted into the second mammary pad on the left side of BALB / c mice. The tumor volume was increased to 300–500 mm². 3 At that time, tumor-bearing mice were randomly divided into two groups, and were injected via tail vein with 4 mg / kg of mitoxantrone hydrochloride aqueous solution (MTO) and the mitoxantrone albumin preparation (MTO@HSA, W) prepared in Example 14, respectively. HSA :W MTO =4:1). Three mice were sacrificed at 2 h, 6 h, 24 h and 48 h after administration, and their heart, liver, spleen, lung, kidney and tumor tissue were dissected. The mouse tissues were photographed using a small animal in vivo imaging system to capture fluorescence images.

[0199] See results Figure 7At each time point, the enrichment of the mitoxantrone albumin preparation of the present invention at the tumor site was higher than that of the mitoxantrone hydrochloride injection at the tumor site, indicating that the mitoxantrone albumin preparation of the present invention can improve the tumor targeting of the drug.

[0200] Example 21

[0201] Investigation of drug plasma metabolism kinetics

[0202] Construct a mouse orthotopic breast cancer model, using 5×10 5 4T1 cells were suspended in 50 μL of phosphate-buffered saline (PBS) at pH 7.4 and then implanted into the second mammary pad on the left side of BALB / c mice. The tumor volume was increased to 300–500 mm². 3 At that time, tumor-bearing mice were randomly divided into two groups, and were injected via tail vein with 4 mg / kg of mitoxantrone hydrochloride aqueous solution (MTO) and the mitoxantrone albumin preparation (MTO@HSA, W) prepared in Example 14, respectively. HSA :W MTO =4:1). Three mice were sacrificed at 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h after drug administration. Blood was collected from the mice into anticoagulant tubes, centrifuged at 600 g for 10 min, and the supernatant was separated. 50 μL of plasma sample was taken, and 100 μL of precipitant (1 M hydrochloric acid: methanol: acetonitrile = 2:1:2) was added to the tube. Vortexed for 1 min, incubated at room temperature for 30 min, sonicated for 5 min, and centrifuged at 12000 rpm for 15 min. The supernatant was filtered through a membrane, and then 10 μL of the supernatant was injected into the HPLC system for analysis. Pharmacokinetic parameters were calculated using DAS 2.0 software (China Drug Evaluation Network).

[0203] See results Figure 8 Compared to mitoxantrone hydrochloride aqueous solution, the mitoxantrone albumin formulation of the present invention has a prolonged circulation time in the blood.

[0204] Example 22

[0205] In vivo safety studies

[0206] Construct a mouse orthotopic breast cancer model, using 2×10 6 E0771 cells were suspended in 50 μL of a 1:1 mixture of phosphate-buffered saline (PBS) and matrix gel at pH 7.4, and then implanted into the second mammary pad on the left side of C57 mice. The tumor volume was monitored until it reached 50–100 mm². 3At that time, tumor-bearing mice were randomly divided into six groups and injected via tail vein with phosphate-buffered saline (PBS) at pH 7.4, mitoxantrone hydrochloride aqueous solution (4 mg / kg), and MTO@HSA (W2O3) prepared in Example 14. HSA :W MTO Mice were given 4:1, 7:1 and 10:1 ratios (4 mg / kg) and mitoxantrone hydrochloride liposome solution (4 mg / kg) once every 4 days for 2 consecutive times, and weighed every 2 days.

[0207] The results are as follows Figure 9 As shown, mice in the mitoxantrone hydrochloride (MTO) group experienced a continuous decrease in body weight, with the decrease exceeding 20% ​​of their initial body weight, indicating severe toxic side effects. Other treatment groups did not show significant weight loss, suggesting good safety.

[0208] Example 23

[0209] In vivo efficacy experiments

[0210] Constructing a humanized breast cancer model in nude mice, using 1×10 7 MDA-MB-231 cells were suspended in 100 μL of phosphate-buffered saline (PBS) at pH 7.4 and then implanted into the second mammary pad on the left side of BALB / c nude mice. The tumor volume was increased to 100–150 mm². 3 At that time, the tumor-bearing mice were randomly divided into four groups and administered phosphate-buffered saline (PBS) at pH 7.4, mitoxantrone hydrochloride aqueous solution (4 mg / kg), mitoxantrone albumin preparation with HSA to MTO mass ratio of 4:1 in Example 14 (4 mg / kg), and mitoxantrone hydrochloride liposome solution (4 mg / kg) via tail vein injection, respectively. The long and short diameters of the tumor were measured with calipers every two days.

[0211] Figure 10 This image shows the antitumor effects of different treatment groups in a nude mouse humanized breast cancer model. Figure 10 The tumor growth curves showed that, compared with the control group, the treatment groups all had a certain inhibitory effect on tumor growth; compared with the mitoxantrone hydrochloride aqueous solution group, the mitoxantrone hydrochloride albumin preparation and mitoxantrone hydrochloride liposomes had better anti-tumor effects, and there was no significant difference between the two.

[0212] Example 24

[0213] In vivo anti-tumor in situ metastasis experiment

[0214] Constructing a humanized breast cancer model in nude mice, using 1×10 7MDA-MB-231 cells were suspended in 100 μL of phosphate-buffered saline (PBS) at pH 7.4 and then implanted into the second mammary pad on the left side of BALB / c nude mice. The tumor volume was increased to 100–150 mm². 3 At that time, tumor-bearing mice were randomly divided into four groups and administered mitoxantrone hydrochloride aqueous solution (4 mg / kg), mitoxantrone albumin preparation (4 mg / kg) with HSA to MTO mass ratio of 4:1 (Example 14), and mitoxantrone hydrochloride liposome solution (4 mg / kg) via tail vein injection once. On the 10th day after administration, the mice were sacrificed, and the lung tissue of the mice was quickly dissected, washed with physiological saline, fixed with Bouin's fixative, photographed, and the number of metastatic nodules in the lungs was counted.

[0215] like Figure 11 As shown, in the PBS group, metastatic tumors in the lung tissue of the MDA-MB-231 model were observed to be diffusely and punctately distributed, with a large number of metastatic tumor nodules, some of which were quite large, approximately 100-250 nodules; the mitoxantrone hydrochloride solution group had 20-50 punctate nodules; the mitoxantrone albumin preparation and mitoxantrone hydrochloride liposome group of this invention significantly inhibited lung metastasis of mouse tumor cells, with approximately 5-20 nodules. This indicates that the drug treatment has a strong anti-metastatic effect.

[0216] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a mitoxantrone albumin formulation, characterized in that, Includes the following steps: (1) Dissolve mitoxantrone or a pharmaceutically acceptable salt thereof in solvent 1 to obtain solution 1; (2) Dissolve albumin in solvent 2 to obtain solution 2; (3) Mix solution 1 and solution 2 to obtain the final product; Solvent 2 is a buffer solution with a pH value of 7 to 9.

5.

2. The preparation method according to claim 1, characterized in that, Solvent 2 is a buffer solution with a pH value of 8 to 9.

5.

3. The preparation method according to claim 1, characterized in that, The mass ratio of albumin to mitoxantrone or its pharmaceutically acceptable salt is (1~15):(1~5), preferably (1~10):(1~3).

4. The preparation method according to any one of claims 1 to 3, characterized in that, The pharmaceutically acceptable salts of mitoxantrone include at least one of mitoxantrone hydrochloride, mitoxantrone citrate, mitoxantrone oxalate, mitoxantrone sulfate, mitoxantrone acetate, and mitoxantrone phosphate; and / or, The albumin is at least one of human albumin, bovine albumin, mouse albumin, and albumin fusion protein; and / or, The solvent 1 is water; and / or, The buffer solution is selected from at least one of the following: disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, sodium carbonate-sodium bicarbonate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, Tris-hydrochloric acid buffer, borax-hydrochloric acid buffer, boric acid-borax buffer, glycine-sodium hydroxide buffer, disodium hydrogen phosphate-citric acid buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, sodium barbital-hydrochloric acid buffer, and phosphate-buffered saline.

5. The preparation method according to claim 1, characterized in that, The concentration of mitoxantrone or a pharmaceutically acceptable salt thereof in solution 1 is 0.5 mg / mL to 30 mg / mL; and / or, The concentration of albumin in solution 2 is 0.5 mg / mL to 30 mg / mL.

6. The preparation method according to claim 1, characterized in that, Step (3) is: ultrasonically mixing solution 1 and solution 2 to obtain the final product; or, The solution 1 and solution 2 are mixed using microfluidic technology to obtain the final product; or, Mix solution 1 and solution 2, and homogenize under high pressure to obtain the desired product; or, Mix solution 1 and solution 2, and stir magnetically to obtain the final product; or, Mix solution 1 and solution 2, and let stand to obtain the final product.

7. A mitoxantrone albumin preparation, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the mitoxantrone albumin formulation as described in claim 7 in the preparation of antitumor drugs.

9. An antitumor drug, characterized in that, The active ingredient of the antitumor drug includes the mitoxantrone albumin formulation as described in claim 7.

10. The antitumor drug as described in claim 9, characterized in that, The dosage forms of the drug are injections, oral preparations, and implants; Preferably, the injection is an injection solution or lyophilized powder; and / or, the oral preparation is a granule, solution, tablet, capsule, pill, or powder. More preferably, the dosage form of the drug is a lyophilized powder, which includes the mitoxantrone albumin preparation and a lyophilization protectant.

Images