Mitoxantrone hydrochloride liposome as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug delivery, and particularly relates to a mitoxantrone hydrochloride liposome as well as a preparation method and application thereof. According to the mitoxantrone hydrochloride liposome provided by the invention, the monovalent sulfonate is used as the internal water phase, mitoxantrone hydrochloride is efficiently and stably encapsulated in the internal water phase of the liposome, and the prepared mitoxantrone hydrochloride liposome can obtain a controllable and rapid in-vivo drug release rate by adjusting the concentration of the sulfonate in the internal water phase; the concentration of mitoxantrone hydrochloride in tumor tissues is improved, and the anti-tumor effect is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery, in particular to a mitoxantrone hydrochloride liposome and a preparation method and application thereof. Background Art

[0002] Mitoxantrone dihydrochloride (MIT) is a synthetic, broad-spectrum anti-tumor drug with an anthracycline structure used to treat malignant lymphoma, breast cancer, and various acute leukemias. Mitoxantrone hydrochloride embeds itself into the DNA bases of tumor cells through the planar portion of its molecular structure to form a stable complex, thereby blocking the synthesis and transcription of its DNA, leading to cross-linking of DNA chains and destruction of the chain structure. In addition, mitoxantrone hydrochloride is also a potent inhibitor of topoisomerase II, which can interfere with RNA synthesis, leading to DNA chain breaks and inhibition of DNA repair. Mitoxantrone hydrochloride is a non-specific drug for the cell cycle and has no selectivity for tumor cells. It has severe toxic and side effects after intravenous administration, with common adverse reactions including bone marrow suppression and cardiotoxicity.

[0003] Currently available mitoxantrone hydrochloride products include mitoxantrone hydrochloride aqueous injection, powder injection, and liposome formulations. Mitoxantrone hydrochloride liposome formulations are a key development area for research institutions both domestically and internationally. Liposomes can increase the drug's chemical stability by encapsulating it in an internal aqueous phase, altering its pharmacokinetics and tissue distribution in the body and reducing toxic side effects. Howard J. Lim's research group at the University of British Columbia, Canada, and Neopharm Pharmaceuticals, Inc. in the United States, among others, have conducted early research and development. Major domestic institutions engaged in this research include the Changzhou Pacific Pharmaceutical Research Institute, Zhang Zhirong's research group at Sichuan University, and Zhongqi Pharmaceutical Technology Co., Ltd., a subsidiary of CSPC Pharmaceutical Group. These institutions have studied the formulation process, raw material and excipient ratios, and stability of mitoxantrone hydrochloride liposomes. In January 2022, CSPC Pharmaceutical Group's mitoxantrone hydrochloride liposome injection, Duonda (10ml:10mg), officially received drug registration approval from the National Medical Products Administration (NMPA). It is indicated for the treatment of relapsed or refractory peripheral T-cell lymphoma (PTCL), making it the world's first commercially available mitoxantrone hydrochloride liposome. CSPC Pharmaceutical Group's mitoxantrone hydrochloride liposomes are composed of hydrogenated soy lecithin (HSPC), cholesterol, and DSPE-PEG2000, and are loaded using an ammonium sulfate gradient method.

[0004] Currently, several domestic pharmaceutical companies and research institutes have applied for patents for the preparation of mitoxantrone hydrochloride liposomes. These include Nanjing Kanghai Pharmaceutical Co., Ltd. (CN1915220A), Changzhou Pacific Pharmaceutical Research Institute Co., Ltd. (CN1602844A), and CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. (CN101209243B). These patents employ either passive drug delivery (e.g., CN1915220A and CN1602844A) or the active drug delivery method of ammonium sulfate gradient (CN101209243B).

[0005] The release rate of mitoxantrone hydrochloride from liposomes is influenced by both the dissociation rate of mitoxantrone hydrochloride sulfate in the aqueous phase and membrane permeability (Li C, Cui J, Wang C, Li Y, Zhang H, Wang J, Li Y, Zhang L, Zhang L, Guo W, Wang Y. Encapsulation of mitoxantrone into pegylated SUVs senhances its antineoplastic efficacy. Eur J Pharm Biopharm. 2008 Oct; 70(2): 657-65. doi: 10.1016 / j.ejpb.2008.05.019. Epub 2008 Jun 6. PMID: 18582570). Studies have found that a faster release rate of mitoxantrone hydrochloride is beneficial for improving antitumor efficacy. Therefore, the average particle size of the mitoxantrone hydrochloride liposomes (Doenda) produced by CSPC is approximately 60 nm. Compared to doxorubicin liposomes with an average particle size of 80-100 nm, Doenda has a larger specific surface area, accelerating the release of mitoxantrone. However, producing liposomes with a small particle size of approximately 60 nm requires the use of high-pressure equipment such as microfluidizers. The homogenization process is very intense, potentially affecting the chemical stability of the lipids. Furthermore, microfluidizers have high maintenance costs.

[0006] In addition to liposome particle size (specific surface area), factors influencing the rate of drug release from the liposome's aqueous phase include the type of ions in the aqueous phase, the concentration of ions in the aqueous phase, and the drug-to-lipid ratio. Cryo-transmission electron microscopy revealed that the insoluble salt formed by mitoxantrone and sulfate in the aqueous phase of Doenda is primarily crystalline. Compared to the amorphous form, the rate at which mitoxantrone dissociates from the crystalline precipitate and subsequently diffuses and releases into the aqueous phase outside the liposome is significantly slower. Therefore, it is of great significance to develop a mitoxantrone hydrochloride liposome that can achieve rapid mitoxantrone release without requiring specialized equipment to reduce the liposome particle size. Summary of the Invention

[0007] In response to the shortcomings of the prior art, the present invention proposes a mitoxantrone hydrochloride liposome using a monovalent sulfonate rather than a divalent sulfate as the internal aqueous phase. The mitoxantrone sulfonate precipitate in the internal aqueous phase is stably encapsulated in the liposome in an amorphous form, thereby accelerating the release of mitoxantrone. Furthermore, by adjusting the concentration of the monovalent sulfonate in the internal aqueous phase, the release rate of mitoxantrone is regulated, thereby increasing the drug concentration in tumor tissue. Without the need to use special equipment to reduce the liposome particle size, the drug release rate is significantly accelerated, and the liposome has a high encapsulation rate and good storage stability.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a mitoxantrone hydrochloride liposome, comprising mitoxantrone hydrochloride, a liposome membrane having a bimolecular structure, an inner aqueous phase located within the liposome membrane, and an outer aqueous phase located outside the liposome membrane; the mitoxantrone hydrochloride is encapsulated in the inner aqueous phase; and a sulfonate gradient exists between the inner aqueous phase within the liposome membrane and the outer aqueous phase outside the membrane.

[0010] In the present invention, the mitoxantrone hydrochloride liposome is a small unilamellar liposome, and the liposome membrane with a bimolecular structure is similar to a biological membrane.

[0011] Mitoxantrone is a weakly basic drug with a symmetrical molecular structure. It contains two ionizable secondary amine groups with dissociation constants (pKa) of 5.99 and 8.13, respectively (refer to Figure 1 At pH 7.4, secondary amines with a pKa of 8.13 preferentially ionize to ammonium ions (NH4 + ), thereby giving mitoxantrone a positive charge (cationic). Mitoxantrone cations can combine with sulfate ions or sulfonate ions to form insoluble salts. Among them, the commercially available liposomes Donda are prepared using the ammonium sulfate gradient method. In the aqueous phase of Donda liposomes, mitoxantrone cations and divalent sulfate ions have multiple binding modes. These insoluble sulfate salts are formed through the π-π stacking effect of the aromatic ring in the mitoxantrone structure (reference Figure 1 b), forming short rod-shaped nanocrystals similar to doxorubicin sulfate (ref. Figure 2 (a) Due to the slow dissociation rate of the crystals, in order to accelerate the release rate of mitoxantrone, the particle size of Doenda is maintained at about 60 nm to increase the specific surface area for mitoxantrone to diffuse through the lipid membrane. Unlike Doenda's design idea of ​​accelerating the drug release rate by reducing the particle size, the present invention proposes to use a monovalent sulfonate, such as methanesulfonate, as an active drug loading gradient to prepare mitoxantrone hydrochloride liposomes. The basic principle is: methanesulfonic acid (refer to Figure 1c) has a sulfonate group, which can form an insoluble salt with the mitoxantrone cation in the internal aqueous phase to achieve stable drug encapsulation. The combination of the monovalent sulfonate group and mitoxantrone is not conducive to the stacking of mitoxantrone, making it difficult to form an ordered structure in the aqueous phase within the liposome (refer to Figure 2 b), which facilitates the rapid dissociation of mitoxantrone and its release from the liposomes. The results of small-angle X-ray scattering measurements show that the structure of mitoxantrone hydrochloride liposomes prepared with monovalent sulfonate as the internal aqueous phase is significantly different from that of Doenda. Crystal structure signals are visible in the internal aqueous phase of Doenda, while liposomes with sulfonate as the internal aqueous phase have no obvious crystal peaks (reference Figure 3 ).

[0012] In some embodiments of the present invention, the inner aqueous phase comprises an aqueous solution of a sulfonate, and the sulfonate is a monovalent sulfonate, such as methanesulfonate. In some embodiments of the present invention, the cation of the sulfonate is selected from ammonium ions or triethylamine ions, preferably ammonium ions. In some specific embodiments of the present invention, the inner aqueous phase is an aqueous solution of ammonium methanesulfonate. The present invention utilizes the principle of active drug loading: a sulfonate ion gradient is formed between the intra- and extra-liposome aqueous phases, and mitoxantrone molecules that diffuse from the external aqueous phase into the intra-liposome aqueous phase combine with hydrogen ions generated by the ionization of ammonium ions in the inner aqueous phase to form mitoxantrone ions, which then form amorphous insoluble salts with the sulfonate groups, thereby being encapsulated in the intra-liposome aqueous phase. At the same time, ammonia gas generated by the ionization of ammonium ions in the aqueous phase within the liposomes continuously escapes from the aqueous phase within the liposomes, thereby maintaining the concentration of hydrogen ions in the aqueous phase within the liposomes, causing the mitoxantrone molecules in the aqueous phase within the liposomes to continuously combine with hydrogen ions to form mitoxantrone ions and form precipitates with sulfonate groups until almost all of the mitoxantrone in the external aqueous phase is encapsulated in the aqueous phase within the liposomes, resulting in a high drug loading capacity, high stability, and a faster release rate of the mitoxantrone liposomes.

[0013] In some embodiments of the present invention, the concentration of sulfonate ions in the aqueous sulfonate solution is 100-800 mM, which may be 100-200 mM, 200-300 mM, 300-400 mM, 400-500 mM, 600-700 mM or 700-800 mM, preferably 200-700 mM.

[0014] In some embodiments of the present invention, the pH of the inner aqueous phase is 4.0-9.0, which may be 4.0-4.5, 4.5-5.0, 5.0-5.5, 5.5-6.0, 6.0-6.5, 6.5-7.0, 7.0-7.5, 7.5-8.0, 8.0-8.5 or 8.5-9.0, preferably 4.5-8.0.

[0015] In some embodiments of the present invention, the external aqueous phase is a physiological isotonic solution. In some specific embodiments of the present invention, the external aqueous phase is selected from a 5% (w / v) glucose aqueous solution, a 10% (w / v) sucrose aqueous solution, or a 0.9% (w / v) sodium chloride aqueous solution, wherein % (w / w) refers to mass / mass percentage concentration, i.e., the mass of the solute contained in every 100g of the solution; and % (w / v) refers to mass / volume percentage concentration, i.e., the mass of the solute contained in every 100ml of the solution.

[0016] In some embodiments of the present invention, the concentration of mitoxantrone hydrochloride in the inner aqueous phase is 0.1-4 mg / mL, and can be 0.1-0.5 mg / mL, 0.5-1 mg / mL, 1-2 mg / mL, 2-3 mg / mL, 3-4 mg / mL, etc.

[0017] In some embodiments of the present invention, the mitoxantrone hydrochloride encapsulation efficiency of the mitoxantrone hydrochloride liposome is greater than or equal to 85%, and can be 85% to 90%, 90 to 95%, or more than 95%.

[0018] In some embodiments of the present invention, the average particle size of the mitoxantrone hydrochloride liposomes is 75-85 nm, and can be 75-77 nm, 77-79 nm, 79-81 nm, 81-83 nm, or 83-85 nm.

[0019] In some embodiments of the present invention, the nanoliposomes include phospholipids, cholesterol (CHOL) and excipients. In the present invention, there is no special limitation on the type and content of the phospholipids, cholesterol and excipients, as long as they can form a stable, leak-free liposome membrane with a bilayer structure. In some embodiments of the present invention, the phospholipid is hydrogenated soy lecithin (HSPC); in some embodiments of the present invention, the excipient is distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG); preferably, the molecular weight of polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol is 50 to 10,000, preferably 2,000. Modification of the liposome surface with polyethylene glycol (PEG) can prolong the half-life of the liposome, improve its stability in the blood circulation, and change the biological distribution of the liposome. DSPE-PEG 2000 The long cycle effect is better.

[0020] In some embodiments of the present invention, the molar ratio of hydrogenated soy lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol in the liposome carrier is (30-80): (0.1-40): (0.1-30), preferably 55:40:5.

[0021] In some embodiments of the present invention, the molar ratio of mitoxantrone hydrochloride to lipid in the mitoxantrone hydrochloride liposomes is 0.01-0.4, specifically 0.01-0.05, 0.05-0.1, 0.1-0.15, 0.15-0.2, 0.2-0.25, 0.25-0.3, 0.3-0.35, 0.35-0.4, more specifically 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4.

[0022] A second aspect of the present invention provides a method for preparing mitoxantrone hydrochloride liposomes, the method comprising the following steps:

[0023] (1) providing blank liposomes containing a sulfonate aqueous solution in the inner aqueous phase;

[0024] (2) dialyzing the blank liposomes obtained in step (1) using an external aqueous phase solution as a dialysis medium to obtain blank liposomes having a pH and sulfonate gradient between the internal aqueous phase and the external aqueous phase;

[0025] (3) Mixing the mitoxantrone hydrochloride solution with the blank liposomes having the pH and sulfonate gradients of the inner aqueous phase and the outer aqueous phase obtained in step (2), and incubating the mixture to obtain mitoxantrone hydrochloride liposomes.

[0026] In some embodiments of the present invention, the particle size of the blank liposomes in step (1) is 75-85 nm.

[0027] In some embodiments of the present invention, the dialysis in step (2) is performed by placing the blank liposomes in step (1) in a dialysis bag with a molecular weight cut-off of 10,000.

[0028] In some embodiments of the present invention, the volume ratio of the blank liposomes to the dialysis medium in step (2) is 1:500 to 1:1000.

[0029] In some embodiments of the present invention, the solvent of the mitoxantrone hydrochloride solution in step (3) is the same as the external aqueous phase solution.

[0030] In some embodiments of the present invention, the required volumes of the mitoxantrone hydrochloride solution in step (3) and the blank liposomes with pH and sulfonate gradients in the inner and outer aqueous phases obtained in step (2) are calculated according to the drug concentration, blank liposome concentration and drug-lipid ratio and mixed.

[0031] In some embodiments of the present invention, the incubation in step (3) is carried out in a water bath at a temperature of 50-60°C, which may be 50-52°C, 52-54°C, 54-56°C, 56-58°C or 58-60°C, and the time is 10-30 min, which may be 10-15 min, 15-20 min, 20-25 min or 25-30 min.

[0032] In some embodiments of the present invention, the method for preparing the blank liposomes in step 1 comprises the following steps:

[0033] S1. First, cholesterol, phospholipids and auxiliary materials are taken according to the ratio, ethanol is added, and they are dissolved and mixed to obtain an ethanol mixed solution;

[0034] S2, preparing a sulfonate buffer solution using a monovalent sulfonate solution and a cationic solution;

[0035] S3, mixing the ethanol mixed solution obtained in step S1 with the sulfonate buffer obtained in S2, and stirring in a water bath to obtain a liposome suspension;

[0036] S4. The liposome suspension obtained in step S3 is passed through a liposome extruder to obtain blank liposomes whose inner aqueous phase contains sulfonate.

[0037] Furthermore, the pH of the sulfonate buffer in step S2 is 4.0-6.0, and may be 4.0-4.5, 4.5-5.0, 5.0-5.5 or 5.5-6.0.

[0038] In some embodiments of the present invention, the water bath temperature in step S3 is 55-65°C, which can be 55-60°C or 60-65°C; the water bath time is 20-40 min, which can be 20-25 min, 25-30 min, 30-35 min or 35-40 min.

[0039] In some embodiments of the present invention, the liposome extruder in step S4 is specifically extruded through polycarbonate membranes with pore sizes of 200 nm, 100 nm, 80 nm, and 50 nm 10 times each.

[0040] The third aspect of the present invention provides a use of the above-mentioned mitoxantrone hydrochloride liposome in the preparation of anti-tumor drugs.

[0041] In simulated plasma, the release rate of mitoxantrone hydrochloride liposomes prepared by the present invention using monovalent sulfonate as the internal aqueous phase was significantly accelerated, significantly faster than that of the commercially available preparation Doenda (reference Figure 4Furthermore, by changing the concentration of monovalent sulfonate in the aqueous phase within the liposome, different numbers of sulfonate groups are bound to each mitoxantrone (according to the structure of mitoxantrone, the number of sulfonate groups bound may be 1 to 2), and mitoxantrone hydrochloride liposomes with different release rates can be obtained (refer to Figure 4 ). The pharmacokinetic results of the mouse pharmacokinetics test were obtained by intravenous injection (refer to Figure 5 ) also showed that the mitoxantrone hydrochloride liposomes proposed by the present invention with monovalent sulfonate as the active drug loading gradient have a faster in vivo release rate, which is faster than the commercially available preparation Doenda with a particle size of about 60 nm prepared by the ammonium sulfate gradient method.

[0042] In the tissue distribution experiment of tumor-bearing mice, the mitoxantrone hydrochloride liposomes proposed by the present invention with monovalent sulfonate as the active drug loading gradient significantly increased the drug concentration in the tumor tissue. After 24 hours, the drug concentration accumulated in the tumor tissue by the monovalent sulfonate-mitoxantrone hydrochloride liposomes was almost twice that of the commercially available preparation Doenda (reference Figure 6 ); Compared with mitoxantrone hydrochloride solution, monovalent sulfonate-mitoxantrone hydrochloride liposomes are mainly concentrated in the tumor area, rather than normal tissues. Among them, the drug concentration in heart, lung, and kidney tissues (C max ) compared with the drug solution of the same dose, respectively reduced by 47.4%, 30.5% and 169.4% (taking mitoxantrone hydrochloride liposomes with 400mM ammonium methylsulfonate as the internal aqueous phase as an example), and has good application prospects in the preparation of anti-tumor drugs.

[0043] In some embodiments of the present invention, the anti-tumor drug is an injectable preparation; preferably, the injectable preparation is selected from a subcutaneous injection formulation, an intravenous injection formulation, an intramuscular injection formulation or a pelvic injection formulation.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. The mitoxantrone hydrochloride liposomes provided by the present invention utilize the active drug loading principle to form a sulfonate ion gradient between the aqueous phase inside and outside the liposomes, so that mitoxantrone and sulfonate groups form an amorphous insoluble salt, which is then encapsulated in the aqueous phase within the liposomes. This has high drug loading capacity, high stability, and a faster release rate.

[0046] 2. The mitoxantrone hydrochloride liposomes provided by the present invention exhibit significantly accelerated drug release in simulated plasma, significantly faster than the commercially available formulation, Doenda, which has a particle size of approximately 60 nm and is prepared using an ammonium sulfate gradient method. Furthermore, by varying the concentration of monovalent sulfonates in the aqueous phase within the liposomes, mitoxantrone hydrochloride liposomes with varying release rates can be obtained. In vivo pharmacokinetic experiments in mice also demonstrated that mitoxantrone hydrochloride liposomes loaded with monovalent sulfonates exhibited a faster in vivo release rate than Doenda.

[0047] 3. In a tissue distribution experiment in S180 tumor-bearing mice, the mitoxantrone hydrochloride liposomes provided by the present invention significantly increased the drug concentration in tumor tissue. The mitoxantrone liposomes with 400mM and 500mM ammonium methanesulfonate as the internal aqueous phase accumulated more than twice the concentration in tumor tissue of the commercially available formulation, Doenda, over 24 hours. Compared with mitoxantrone hydrochloride solution, the monovalent sulfonate-mitoxantrone hydrochloride liposomes were mainly concentrated in the tumor area, rather than in normal tissues. Compared with the drug solution of the same dose, the drug concentration (C max ) were significantly reduced.

[0048] 4. The mitoxantrone hydrochloride liposomes provided by the present invention showed good therapeutic effects in the DBA / 2 mouse L1210 ascites tumor model. At a low dose (2 mg / kg), the mitoxantrone hydrochloride liposomes with 400 mM ammonium methylsulfonate as the internal aqueous phase had a therapeutic effect comparable to that of Doenda; at a high dose (4 mg / kg), over a 60-day observation period, the mitoxantrone hydrochloride liposomes with 400 mM ammonium methylsulfonate as the internal aqueous phase had a therapeutic effect on model mice that was superior to the commercially available formulation Doenda. Compared with Doenda, it extended the mean and median survival times of tumor-bearing mice by more than 5 days. In summary, the mitoxantrone hydrochloride liposomes provided by the present invention have good application prospects in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the effect of the salt-forming method of mitoxantrone on the precipitated crystal structure; wherein, a is the chemical structure of mitoxantrone hydrochloride and monovalent sulfonic acid (methylsulfonic acid); b is a schematic diagram of the precipitated crystal structure of mitoxantrone salted with divalent sulfate ion; c is a schematic diagram of the precipitated crystal structure of mitoxantrone salted with monovalent sulfonate ion.

[0050] Figure 2 Cryo-transmission electron microscopy images of Doenda (a) and mitoxantrone hydrochloride liposomes prepared with 600 mM methanesulfonic acid as the internal aqueous phase (b).

[0051] Figure 3The following are small-angle X-ray scattering patterns of mitoxantrone hydrochloride liposomes and Doenda prepared with methanesulfonic acid solution of different concentrations as the internal aqueous phase, where MS: methanesulfonic acid; AS: ammonium sulfate; blank: blank liposomes; Lipo: mitoxantrone hydrochloride liposomes; DED: Doenda. Among them, 250 mM ammonium sulfate mitoxantrone hydrochloride liposomes and DED were used for comparison.

[0052] Figure 4 The cumulative release rate of mitoxantrone hydrochloride liposomes (MS) and Doenda prepared with different concentrations of methanesulfonic acid solution as the internal aqueous phase in simulated plasma. The release medium was 100mM ammonium chloride-40mg / mL albumin saline (pH 7.4).

[0053] Figure 5 The PK results of mitoxantrone hydrochloride liposomes (MS), Doenda, and mitoxantrone hydrochloride solution prepared with different concentrations of methanesulfonic acid solution as the internal aqueous phase in mice after intravenous administration; including: (a) total mitoxantrone concentration-time graph in plasma; (b) mitoxantrone concentration-time graph encapsulated in liposomes in plasma; (c) percentage of mitoxantrone encapsulated in liposomes to the total drug amount at each time point.

[0054] Figure 6 This is a diagram of the tissue distribution kinetics in tumor-bearing mice after intravenous administration of mitoxantrone hydrochloride liposomes (MS), Doenda, and mitoxantrone hydrochloride solution prepared with different concentrations of methanesulfonic acid solution as the internal aqueous phase (a. heart; b. liver; c. spleen; d. lung; e. kidney; f. tumor).

[0055] Figure 7 This is the standard curve of mitoxantrone hydrochloride sucrose solution.

[0056] Figure 8 Survival curves of mice bearing L1210 ascites tumor in blank group and low-dose group (2 mg / kg); a. blank group; b. mitoxantrone hydrochloride solution group; c. Doenda group; d. 400 mM ammonium methanesulfonate mitoxantrone hydrochloride liposome group; e. 500 mM ammonium methanesulfonate mitoxantrone hydrochloride liposome group; f. 600 mM ammonium methanesulfonate mitoxantrone hydrochloride liposome group.

[0057] Figure 9 Survival curves of L1210 ascites tumor mice in blank group and high-dose group (4 mg / kg); a. blank group; b. Doenda group; c. 400 mM ammonium methanesulfonate mitoxantrone hydrochloride liposome group; d. 500 mM ammonium methanesulfonate mitoxantrone hydrochloride liposome group; e. 600 mM ammonium methanesulfonate mitoxantrone hydrochloride liposome group; f. mitoxantrone hydrochloride solution group. DETAILED DESCRIPTION

[0058] Before further describing the specific embodiments of the present invention, it should be understood that the scope of the present invention is not limited to the specific embodiments described below. It should also be understood that the terminology used in the examples is intended to describe specific embodiments and is not intended to limit the scope of the present invention. The experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers.

[0059] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0060] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0061] The phospholipids used in the examples of the present invention were purchased from Lipoid, Germany, specifically hydrogenated soy lecithin (HSPC, molecular weight 783.8); the PEGylated phospholipid was distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000); the cholesterol used was Lipoid's cholesterol (CHOL, molecular weight 386.7); mitoxantrone hydrochloride was purchased from Aladdin; methanesulfonic acid was purchased from Sigma-Aldrich; and ammonia was purchased from Aladdin. Bovine serum albumin (BSA) used for in vitro release studies was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; Dowex resin was purchased from Sigma-Aldrich. Ammonium chloride (99.5%) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0062] The terms involved are defined as follows:

[0063] Encapsulation efficiency: the ratio of the amount of drug encapsulated in liposomes to the total amount of drug put into the liposome solution.

[0064] Drug-to-lipid ratio: the molar ratio of drug to lipid in liposomes.

[0065] Example 1: Detection method of mitoxantrone hydrochloride

[0066] This example establishes a detection method for mitoxantrone hydrochloride, which is as follows:

[0067] UV-visible spectrophotometry was used for detection and analysis: the detection instrument was TECAN 200PRO; detection wavelength is 610nm; detection temperature is 24℃; detection plate is 96-well plates; detection volume is 200 μl.

[0068] 1.1. Establishment of the standard curve of mitoxantrone hydrochloride solution

[0069] Accurately weigh 2.5001 mg of mitoxantrone hydrochloride and dissolve it in a 25 ml volumetric flask to obtain a 0.1000 mg / ml mitoxantrone hydrochloride-sucrose solution. Then, dilute the mitoxantrone hydrochloride-sucrose solution in a gradient fashion to obtain mitoxantrone hydrochloride standard solutions at concentrations of 40 μg / ml, 30 μg / ml, 25 μg / ml, 20 μg / ml, 15 μg / ml, and 10 μg / ml. Measure the absorbance of the mitoxantrone hydrochloride standard solutions at the aforementioned concentrations at 610 nm.

[0070] Test results such as Figure 7 As shown, the standard curve of mitoxantrone hydrochloride sucrose solution is Y=0.01729X-0.00374, the linear range is 10-50μg / ml, R 2 =0.9995, which meets the requirements of quantitative detection.

[0071] 1.2 Recovery rate

[0072] Prepare mitoxantrone hydrochloride-sucrose standard solutions at low, medium, and high concentrations (10, 20, and 40 μg / ml) using the method described in 1.1 above. Prepare triplicate solutions for each concentration and measure their absorbance. Calculate the average recovery and relative standard deviation (RSD).

[0073] The results are shown in Table 1. The average recoveries of low, medium and high concentrations were 104.35%, 109.78% and 101.26%, respectively, and the RSDs were 1.45%, 2.41% and 1.18%, respectively, which met the requirements for quantitative detection.

[0074] Table 1 Recovery of Mitoxantrone Hydrochloride by UV Spectrophotometry

[0075]

[0076] 1.3 Precision

[0077] According to the method in 1.1, three concentrations of mitoxantrone hydrochloride sucrose standard solutions (10, 20, and 40 μg / ml) were prepared and their absorbance values ​​were measured. Each solution was measured in parallel three times. The average concentration and relative standard deviation (RSD) of mitoxantrone hydrochloride were calculated to investigate the precision of the method.

[0078] The results are shown in Table 2. The RSDs of the low, medium and high concentrations were 1.20%, 0.71% and 0.92%, respectively, indicating that the method had good precision.

[0079] Table 2 Precision study of mitoxantrone hydrochloride UV spectrophotometry

[0080]

[0081] Example 2: Preparation and characterization of mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase

[0082] 2.1 Preparation of Mitoxantrone Hydrochloride Liposomes

[0083] The specific preparation process is as follows:

[0084] Step (1), accurately weighing hydrogenated soybean lecithin, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000 in a mass ratio of 3:1:1, adding an appropriate amount of ethanol to fully dissolve and mix, to obtain a lipid ethanol mixed solution;

[0085] Step (2), accurately measuring an appropriate volume of methanesulfonic acid solution, adding ammonia water to a pH of 4.0 to 6.0, and saturating to volume with double distilled water (ddH2O) to obtain ammonium methanesulfonate buffer solutions with a pH of 4.0 to 6.0 and concentrations of 400 mM, 500 mM, and 600 mM, respectively;

[0086] Step (3), mixing the ethanol mixed solution obtained in step (1) with 400 mM, 500 mM and 600 mM ammonium methanesulfonate buffer solutions with a pH of 4.0 to 6.0 obtained in step (2), respectively, and placing the mixture in a stirring water bath at 60 to 70° C. for 30 minutes to obtain a relatively uniform liposome suspension;

[0087] Step (4), using a liposome extruder, sequentially extruding the liposome suspension obtained in step (3) through polycarbonate membranes with pore sizes of 200 nm, 100 nm, 80 nm, and 50 nm 10 times each, to finally obtain blank liposomes with an average particle size of about 75 to 85 nm, a uniform particle size distribution, and an inner aqueous phase containing different concentrations of sulfonate;

[0088] Step (5) is to place the blank liposomes prepared in step (4) with different concentrations of sulfonate in the inner aqueous phase in a dialysis bag with a molecular weight cut-off of 10,000, and dialyze overnight at 4°C using a 10% sucrose aqueous solution as the dialysis medium. The sample volume ratio to the dialysis medium is 1:1000, and the dialysate is changed three times during the dialysis to completely remove the ammonium methanesulfonate in the outer aqueous phase of the liposomes, thereby obtaining an outer aqueous phase composed of 10% sucrose, an ammonium methanesulfonate solution as the inner aqueous phase, and blank liposomes composed of a phospholipid bilayer (i.e., blank liposomes with inner and outer aqueous phases having a pH and sulfonate gradient, wherein the inner and outer aqueous phases of the liposomes have a certain pH and ammonium methanesulfonate concentration gradient. Specifically, the inner aqueous phase of the blank liposomes with inner and outer aqueous phases having a pH and sulfonate gradient is 400mM, 500mM, or 600mM ammonium methanesulfonate aqueous solution (pH 4.0-6.0), and the outer aqueous phase is a 10% sucrose aqueous solution by mass.

[0089] In step (6), the mitoxantrone hydrochloride sucrose solution is added to the blank liposome suspension with pH and sulfonate gradients in the inner and outer aqueous phases obtained in step (5) according to the desired drug loading amount (drug-to-lipid ratio), and the mixture is incubated in a 50-60° C. water bath for 10-30 min to obtain mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the inner aqueous phase.

[0090] 2.2 Characterization of Mitoxantrone Hydrochloride Liposomes

[0091] 2.2.1. Particle size determination of mitoxantrone hydrochloride liposomes

[0092] The mitoxantrone hydrochloride liposomes prepared in 2.1 above were diluted 100-fold with ultrapure water, and the average particle size and particle size distribution were determined using dynamic light scattering. Specifically, the diluted liposome sample was placed in a Zetasizer ZS90 (Malvern) at a laser angle of 633 nm, a temperature of 25°C, and a temperature equilibration time of 120 seconds. The viscosity and refractive index of the water were measured in triplicate for each sample.

[0093] The specific results are shown in Table 3. The particle size of the drug-loaded mitoxantrone hydrochloride liposomes was approximately 75-85 nm, with a particle size distribution (PDI) of less than 0.1. In addition, the particle size of the blank liposomes before drug loading was also approximately 75-85 nm, with a particle size distribution (PDI) of less than 0.1.

[0094] 2.2.2 Encapsulation efficiency determination

[0095] An appropriate amount of Dowex resin (Sigma-Aldrich) was added to the mitoxantrone hydrochloride liposomes prepared in 2.1 above and shaken thoroughly to adsorb unencapsulated mitoxantrone hydrochloride. After standing, 200 μl of the supernatant was collected and the mitoxantrone hydrochloride content in the liposomes before and after resin addition was determined using UV-visible spectrophotometry (same as in Example 1).

[0096] The encapsulation efficiency (EE) of mitoxantrone hydrochloride liposomes was calculated according to the following formula:

[0097]

[0098] Among them, M inter M is the content of mitoxantrone hydrochloride in the liposome preparation after the resin adsorbs the free drug, that is, the content of mitoxantrone hydrochloride encapsulated by the liposome; total It is the content of mitoxantrone hydrochloride in the mitoxantrone hydrochloride liposome preparation before resin adsorption, that is, the dosage of mitoxantrone hydrochloride.

[0099] The results are shown in Table 3. Table 3 shows that the mitoxantrone hydrochloride liposomes prepared according to the method in 2.1 above have good encapsulation efficiency and can achieve a high drug-to-lipid ratio of drug loading.

[0100] Table 3 Particle size and encapsulation efficiency of mitoxantrone hydrochloride liposomes with ammonium methanesulfonate solution as the internal aqueous phase

[0101]

[0102] 2.2.3 Morphological Characterization of Mitoxantrone Hydrochloride Liposomes

[0103] The morphology of mitoxantrone hydrochloride liposomes and the commercially available formulation of Doenda, using ammonium methanesulfonate as the internal aqueous phase, was observed using a cryo-transmission electron microscope (Talos F200S G2, 200 kV). Specifically, 3–5 μl of mitoxantrone hydrochloride liposomes or Doenda was dripped onto a copper mesh (Quantifil, TedPella, USA) using a Vitrobot Mark IV cryo-sample preparation system. Excess solution was removed with filter paper, and the sample was quickly immersed in liquid ethane. The liposome morphology was observed at a voltage of 200 kV, low-dose mode, and a magnification range of 115–480,000.

[0104] Doenda liposomes ( Figure 2 Middle a) is a perfect sphere under cryo-TEM, with a diameter of about 40-45 nm, and short rod-shaped nanocrystals formed by mitoxantrone and sulfate groups are clearly visible inside. Mitoxantrone hydrochloride liposomes with 600 mM ammonium methylsulfonate as the internal aqueous phase ( Figure 2(b) in the middle represents small unilamellar liposomes with a diameter of approximately 65-70 nm. (Cryo-TEM cannot directly observe the PEG hydration layer on the liposome surface (approximately 4-5 nm). Therefore, the liposome diameter observed by TEM is 8-10 nm smaller than the diameter determined by dynamic light scattering.) These liposomes are spherical, with an internal electron density higher than that of the extraliposomal aqueous phase (the internal aqueous phase is darker in color). However, they lack a distinct regular structure and are likely to be a non-crystalline, insoluble salt (precipitate) formed by mitoxantrone and mesylate.

[0105] 2.2.4 Small-angle X-ray scattering characterization of mitoxantrone hydrochloride liposomes

[0106] To investigate the microstructure formed by different concentrations of monovalent sulfonates in the liposome aqueous phase with mitoxantrone hydrochloride, small-angle X-ray scattering (SAXS) was used to characterize the structure of the mitoxantrone hydrochloride liposomes prepared in 2.1 above, which used ammonium methanesulfonate as the internal aqueous phase. The structures were compared with commercially available Doenda, which uses ammonium sulfate as the internal aqueous phase. All homemade mitoxantrone hydrochloride liposomes characterized by SAXS had a drug-to-lipid ratio of 0.1, a lipid concentration of 19.33 mM, an average particle size of approximately 80 nm, and a PDI of less than 0.1.

[0107] Small-angle X-ray scattering experiments were performed at the BL19U2 beamline of the Shanghai National Center for Protein Science (NFPS) at the Shanghai Synchrotron Radiation Facility (SSRF). The specific steps were as follows: the incident X-ray beam energy was set to 12 keV (wavelength λ = 0.103 nm), and a PILATUS1M detector (Decortes, Switzerland) was used to record the two-dimensional scattering images. The sample-to-detector distance was set to 2.6 m, so that the detection range of the scattering vector q = (4π / λ)sin(θ) was 0.1–4 nm. -1 (where 2θ is the scattering angle). 60 μl of each liposome sample was taken and 20 two-dimensional images were collected at room temperature using an automatic loading system. The exposure time was set to 1 s to obtain a good signal-to-noise ratio. The data were restored and preliminarily processed using the BioXTAS raw program, and the absolute scattering intensity was calibrated using purified water (Millipore, USA). The processed small-angle X-ray scattering curves are shown in Figure 3 .

[0108] Depend on Figure 3As shown in the small-angle X-ray scattering experimental results, no obvious crystal structure peak is found in the aqueous phase of the mitoxantrone hydrochloride liposomes with ammonium methylsulfonate as the inner aqueous phase of the three different concentrations, while in the aqueous phase of the commercially available preparation Doenda and the homemade mitoxantrone hydrochloride liposomes with 250mM ammonium sulfate as the inner aqueous phase, obvious short rod-shaped crystal signal peaks are presented, and the two structures are significantly different. The small-angle X-ray scattering results are consistent with the cryo-transmission electron microscopy observation results, that is, there is no obvious crystal structure in the aqueous phase of the mitoxantrone hydrochloride liposomes with ammonium methylsulfonate as the inner aqueous phase prepared in this embodiment. This structural feature is conducive to accelerating the release rate of mitoxantrone hydrochloride while ensuring high encapsulation efficiency and good storage stability of mitoxantrone hydrochloride, thereby obtaining better anti-tumor effect.

[0109] Example 3: In vitro release of mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase

[0110] The release of mitoxantrone hydrochloride from the aqueous phase within liposomes involves two steps: first, dissociation of mitoxantrone from its insoluble salt formed with the sulfonate group; and second, ionization of the dissociated mitoxantrone, where the molecular drug crosses the lipid membrane and diffuses outside the liposome. Therefore, by varying the concentration of ammonium methanesulfonate in the aqueous phase within the liposomes, the ratio of mitoxantrone hydrochloride to the sulfonate group can be adjusted, resulting in the formation of insoluble salts with varying structures and, consequently, varying drug release rates. To compare the in vitro release rates of mitoxantrone hydrochloride liposomes prepared with varying concentrations of sulfonate as the internal aqueous phase, in vitro release studies were conducted in this example. The drug-to-lipid ratio for all samples was fixed at 0.1, the drug loading concentration was 1 mg / ml, and the lipid concentration was 19.33 mM. The release medium consisted of 4% (w / v) bovine serum albumin in saline to simulate the protein environment of plasma. 100 mM ammonium chloride was added to the release medium as a release enhancer.

[0111] The specific process of in vitro release experiment is as follows:

[0112] (1) Accurately weigh 4.0152 g of bovine serum albumin (BSA) powder and 534.9 mg of ammonium chloride and dissolve them in 100 ml of normal saline to prepare a 100 mM ammonium chloride-40 mg / ml albumin solution in normal saline (the protein concentration is equivalent to 50% of the protein concentration in plasma);

[0113] (2) Mitoxantrone hydrochloride liposomes were prepared using three different concentrations of monovalent sulfonate solutions (pH 4.0-6.0) in Example 2 as the inner aqueous phase; wherein the lipid concentration was 19.33 mM and the drug-to-lipid ratio (molar ratio of drug to lipid) was 0.1.

[0114] (3) The liposomes were diluted 25-fold with a 100 mM ammonium chloride-40 mg / ml albumin saline solution. Sufficient Dowex resin was added to adsorb the free drug to create a sink condition. The mixture was shaken at 37°C and 100 rpm in a shaker (THZ-C constant temperature oscillator). Samples were taken at different time points (0 hour, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, and 48 hours). The content of mitoxantrone hydrochloride encapsulated in the liposomes was determined by visible spectrophotometry, and the cumulative release rate of mitoxantrone hydrochloride at different time points was calculated.

[0115] According to the above method, the in vitro release rate of the commercially available formulation Doenda, i.e., mitoxantrone hydrochloride liposomes with ammonium sulfate as the internal aqueous phase, in 100 mM ammonium chloride-albumin saline was measured in parallel, and compared with the release rate of the mitoxantrone hydrochloride liposome preparation provided in Example 2 of the present invention with sulfonate as the internal aqueous phase.

[0116] In vitro cumulative release curves of mitoxantrone hydrochloride liposomes in each group are as follows Figure 4 shown. Figure 4 The results showed that in a 100mM ammonium chloride-albumin solution, mitoxantrone hydrochloride liposomes containing three different concentrations of ammonium methanesulfonate as the internal aqueous phase all exhibited rapid release rates. Specifically, the 24-hour cumulative release percentage for mitoxantrone hydrochloride liposomes containing 400mM ammonium methanesulfonate reached 63.57%, for 500mM ammonium methanesulfonate as the internal aqueous phase reached 48.02%, and for 600mM ammonium methanesulfonate as the internal aqueous phase reached 39.3%. The 24-hour cumulative release percentage for the commercially available formulation, Doenda, measured in parallel, was 27.72%. The in vitro release rate of mitoxantrone hydrochloride liposomes containing monovalent sulfonate as the internal aqueous phase was significantly faster than that of the commercially available formulation, Doenda, containing ammonium sulfate as the internal aqueous phase.

[0117] Example 4: Determination of the total concentration of mitoxantrone hydrochloride in plasma and the concentration of mitoxantrone hydrochloride encapsulated in liposomes

[0118] The total concentration of mitoxantrone hydrochloride in plasma and the concentration of mitoxantrone hydrochloride encapsulated in liposomes were determined by fluorescence method.

[0119] 4.1 Working curve drawing

[0120] (1) Place mouse whole blood in a heparin anticoagulant tube, centrifuge at 3000 rpm for 10 min, and take the upper layer for later use.

[0121] (2) A 100 μg / ml mitoxantrone hydrochloride sucrose solution was diluted with ultrapure water in a gradient manner to prepare mitoxantrone hydrochloride stock solutions with concentrations of 4.0, 2.0, 1.0, 0.4, 0.2, and 0.1 μg / ml.

[0122] (3) Take 50 μl of blank plasma, add different concentrations of mitoxantrone hydrochloride stock solution, and mix well; add water / isopropanol (3:7, v / v) mixed solvent according to the volume ratio of 1:9, vortex for about 30 seconds to precipitate the protein, and prepare mitoxantrone hydrochloride working solutions with concentrations of 2.0, 1.0, 0.5, 0.2, 0.1, and 0.05 μg / ml.

[0123] (4) The mitoxantrone hydrochloride working solution in (3) was shaken at 4°C for 1 hour, centrifuged at 4°C and 10,000 rpm for 10 minutes, and 200 μl of the supernatant was collected. The fluorescence intensity was measured using a microplate reader at an excitation wavelength of 610 nm and an emission wavelength of 683 nm to establish a fluorescence working curve for the determination of mitoxantrone hydrochloride concentration in plasma. The results are shown in Table 4. The standard curve of mitoxantrone hydrochloride concentration in plasma and fluorescence intensity is Y = 14937X - 48.23, with a linear range of 0.05-2.0 μg / ml and R 2 =0.9996, which meets the requirements of quantitative detection.

[0124] Table 4 Fluorescence standard curve of mitoxantrone hydrochloride concentration in plasma

[0125]

[0126] 4.2 Method precision and recovery

[0127] The mitoxantrone hydrochloride stock solution in 4.1(2) was added to the blank plasma of mice, and three portions of mitoxantrone hydrochloride-plasma solutions of high, medium and low concentrations (2.0 μg / ml, 1.0 μg / ml and 0.1 μg / ml) were prepared. Protein was precipitated, shaken and centrifuged according to the method described in 4.1, and the supernatant was taken to detect the drug concentration. The recovery rate and intra-day precision of the method were investigated. The recovery rate results are shown in Table 5. The average recovery rates of the low, medium and high concentrations were 108.49%, 97.10% and 97.86%, respectively, and the RSDs were 0.71%, 2.78% and 2.52%, respectively, which met the quantitative detection requirements. The precision results are shown in Table 6. The RSDs of the low, medium and high concentrations were 1.87%, 2.18% and 3.64%, respectively, indicating that the method has good precision.

[0128] Table 5 Fluorometric determination of mitoxantrone concentration in plasma recovery

[0129]

[0130] Table 6 Precision of the method for determining mitoxantrone concentration in plasma by fluorescence method

[0131]

[0132] 4.3 Determination of the total concentration of mitoxantrone hydrochloride in mouse plasma and the concentration of liposome-encapsulated mitoxantrone hydrochloride

[0133] 4.3.1 Plasma separation

[0134] After the mouse eyeballs were removed and blood was collected, the anticoagulant tube was centrifuged at 4°C, 3000 rpm for 10 min, and the supernatant plasma was collected and stored at -20°C for later use.

[0135] 4.3.2 Determination of total mitoxantrone hydrochloride concentration in plasma

[0136] Take 100 μl of mouse plasma and dilute it with 400 μl of normal saline; take 50 μl of the diluted plasma and determine the total concentration of mitoxantrone hydrochloride in the plasma according to the method of drawing the working curve of the fluorescence method in 4.1.

[0137] 4.3.3 Determination of the concentration of liposome-encapsulated mitoxantrone hydrochloride in plasma

[0138] Add an appropriate amount of Dowex resin to the plasma diluted in 4.3.2. Shake for approximately 10 seconds and allow the resin to adsorb the free mitoxantrone hydrochloride in the plasma. Allow to stand. Collect 50 μl of the supernatant and, following the procedure in 4.1, add a protein precipitant to precipitate protein and dissolve the liposomes. Measure the fluorescence intensity and calculate the concentration of liposome-encapsulated mitoxantrone hydrochloride in the plasma.

[0139] Example 5: In vivo pharmacokinetics of mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase

[0140] In in vitro release experiments, three different concentrations of mitoxantrone hydrochloride liposomes containing ammonium methanesulfonate as the internal aqueous phase all showed rapid release rates, significantly faster than the commercially available formulation, Doenda. Therefore, we further determined the pharmacokinetic parameters of these three mitoxantrone hydrochloride liposomes containing ammonium methanesulfonate as the internal aqueous phase and the commercially available formulation, Doenda, in mice.

[0141] Since both liposome-encapsulated drugs and released drugs (free drugs) exist in plasma, and their distribution and clearance rates in the body are different, which directly affects the anti-tumor effect of mitoxantrone hydrochloride, therefore, in the pharmacokinetics study of mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase, we measured the total drug concentration of mitoxantrone in plasma and the drug concentration encapsulated by liposomes (the difference between the two can reflect the in vivo release rate of liposomes), and compared them with the results of Doenda.

[0142] The specific implementation method is as follows: Take the three mitoxantrone hydrochloride liposomes (MTO-Lipo) prepared with different concentrations of monovalent sulfonate solution as the inner aqueous phase in Example 2 and the commercially available preparation Doenda, a total of four groups of liposomes. Take 24 KM mice, 18-22g, half male and half female, in each group, and randomly divide them into 8 groups. According to the dosage of 2mg / kg, the four liposomes are injected into the tail vein respectively. A group of mice are randomly selected at 0.083, 0.25, 0.5, 1, 4, 8, 24, and 48 hours after administration, and the eyeballs are removed after anesthesia to collect blood. The mouse blood is placed in a heparin anticoagulant tube. According to the method of Example 4, the total concentration of mitoxantrone in the mouse plasma at different time points and the concentration of mitoxantrone encapsulated in the liposomes are determined, and the drug-time curve is drawn. The results are shown in FIG. Figure 5 shown.

[0143] Twenty-one KM mice were randomly divided into seven groups. Mitoxantrone hydrochloride solution (2 mg / kg) was injected intravenously into the tail vein. After administration, mice were randomly selected from each group at 0.083, 0.25, 0.5, 1, 2, 4, and 8 hours. Eyeballs were removed and blood was collected after anesthesia. The total mitoxantrone concentration in plasma was determined using the same method as above, and a drug-time curve was plotted. (Since the plasma concentration of mitoxantrone hydrochloride solution was below the limit of quantification 24 hours after injection, an additional sampling point of 2 hours was added.)

[0144] The blood drug concentration data were quantitatively analyzed using DAS (ver2.0) pharmacokinetic software to obtain the main pharmacokinetic parameters. The results are shown in Tables 7 and 8.

[0145] Table 7 Main pharmacokinetic parameters of mitoxantrone hydrochloride liposomes and Doenda prepared with different gradients (calculated based on the total concentration of mitoxantrone in plasma)

[0146]

[0147]

[0148] The pharmacokinetic parameters calculated based on the total plasma concentration of mitoxantrone (Table 7) showed that, compared with mitoxantrone hydrochloride solution, liposomes (including three mitoxantrone hydrochloride liposomes prepared with different concentrations of monovalent sulfonate solution as the internal aqueous phase and the commercial preparation Doenda) significantly improved the bioavailability of the drug (higher AUC) and prolonged the half-life of mitoxantrone (t 1 / 2 ), while the clearance rate (CLz) was significantly reduced. Similar to Doenda, mitoxantrone hydrochloride liposomes prepared using monovalent sulfonates also exhibit long circulation in vivo. Specifically, mitoxantrone hydrochloride liposomes prepared with 400mM ammonium methanesulfonate as the internal aqueous phase exhibited significantly shorter half-life and mean residence time (MRT) than the commercially available formulation Doenda, indicating a faster release rate.

[0149] Table 8 Main pharmacokinetic parameters of mitoxantrone hydrochloride liposomes and Doenda prepared with different gradients (calculated based on the concentration of mitoxantrone encapsulated in liposomes in plasma)

[0150]

[0151] The release rates of monovalent sulfonated mitoxantrone liposomes and Doenda were compared in mice based on the plasma concentration of liposome-encapsulated mitoxantrone. As shown in Table 8, the half-life and mean residence time (MRT) of mitoxantrone hydrochloride liposomes prepared with 400 mM ammonium methanesulfonate as the internal aqueous phase were significantly shorter than those of the commercially available formulation, Doenda, indicating a faster release rate.

[0152] Combined with the in vitro release results of Example 4, it can be concluded that the mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase have a faster in vitro release rate, especially the mitoxantrone hydrochloride liposomes with 400 mM ammonium methanesulfonate as the internal aqueous phase, the in vitro and in vivo release rates of which are significantly faster than those of the commercially available preparation Doenda.

[0153] Example 6: Determination of Mitoxantrone Concentration in Tumor-Bearing Mouse Tissues

[0154] The total concentration of mitoxantrone in tissues of S180 tumor-bearing mice was determined by fluorescence method.

[0155] The specific method is as follows: take S180 cells cultured in vitro and dilute them with sterile PBS to a concentration of 1×10 7 The solution of 100 / ml was inoculated subcutaneously in the right axilla of male KM mice, with 0.2 ml inoculated per mouse to establish a mouse sarcoma S180 subcutaneous inoculation model. Seven days after inoculation, the same batch of tumors with clear boundaries and a tumor volume of approximately 100 mm were selected. 3 The experiment was conducted on tumor-bearing mice.

[0156] 6.1 Drawing of the standard curve

[0157] (1) Tumor-bearing mice were killed by cervical dislocation and rapidly dissected. The heart, liver, spleen, lungs, kidneys, and tumor tissues were removed. Blood and residues were rinsed with saline, dried with filter paper, and weighed.

[0158] (2) 0.05 g each of heart, liver, spleen, lung, and kidney tissue samples and 0.1 g of tumor tissue sample were collected from tumor-bearing mice and placed in a 2 ml grinding tube. 0.4 ml of 20% ascorbic acid solution was added and the samples were ground using a tissue homogenizer to prepare tissue homogenates. 0.3 ml of the tissue homogenate was added to a certain volume of mitoxantrone hydrochloride sucrose stock solution to prepare mitoxantrone hydrochloride-tissue homogenates with concentrations of 5.0, 2.0, 1.0, 0.5, 0.2, and 0.1 μg / ml (for kidney and tumor tissues) and 2.0, 1.0, 0.5, 0.2, and 0.1 μg / ml (for heart, liver, spleen, and lung tissues).

[0159] (3) Protein precipitant (acetonitrile / methanol 10:90, v / v) was added to the mitoxantrone hydrochloride-tissue homogenate at a volume ratio of 1:1. The mixture was vortexed for 30 seconds, allowed to stand at room temperature for 30 minutes, and centrifuged at 11,000 rpm for 15 minutes. 200 μl of the supernatant was collected and the fluorescence intensity was measured using a microplate reader at an excitation wavelength of 610 nm and an emission wavelength of 683 nm to establish a mitoxantrone hydrochloride-tissue fluorescence working curve.

[0160] The results are shown in Table 9. The working curves of all tissues were linear, with correlation coefficients R 2 Above 0.99, it meets the requirements of quantitative detection.

[0161] Table 9 Working curve of fluorescence determination of mitoxantrone hydrochloride concentration in different tissues of tumor-bearing mice

[0162]

[0163] 6.2 Method precision and recovery

[0164] Add mitoxantrone hydrochloride solution to the tissue homogenate of tumor-bearing mice. Prepare three aliquots of mitoxantrone hydrochloride-tissue homogenate solutions at high, medium, and low concentrations (5.0 μg / ml, 1.0 μg / ml, and 0.1 μg / ml) according to the method described in 6.1. Take the supernatant and test its concentration. Investigate the recovery and intra-day precision of the method (applicable to kidney and tumor tissues).

[0165] Add mitoxantrone hydrochloride solution to the tissue homogenate of tumor-bearing mice. Prepare three aliquots of mitoxantrone hydrochloride-tissue homogenate solutions at high, medium, and low concentrations (2.0 μg / ml, 1.0 μg / ml, and 0.1 μg / ml) according to the method described in 6.1. Take the supernatant and test its concentration. Investigate the recovery and intra-day precision of the method (applicable to heart, liver, spleen, and lung tissues).

[0166] The results are shown in Tables 10 and 11. The recoveries of all tissue samples were between 85% and 115%, and the RSDs of recovery and precision were less than 10%, indicating that the method was accurate and reliable and met the quantitative standards.

[0167] Table 10 Fluorometric determination of the recovery of mitoxantrone hydrochloride concentration in different tissues of tumor-bearing mice

[0168]

[0169] Table 11 Precision of the method for determining the concentration of mitoxantrone hydrochloride in different tissues of tumor-bearing mice by fluorescence method

[0170]

[0171]

[0172] Example 7: Tissue distribution of mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase in tumor-bearing mice

[0173] S180 tumor-bearing mice were constructed according to the method described in Example 6. Mitoxantrone hydrochloride liposomes containing ammonium methanesulfonate solution as the aqueous phase, the commercially available formulation Doenda, and a mitoxantrone hydrochloride solution were injected intravenously at a dose of 4 mg / kg. At 0.083, 1, 2, 4, 8, and 24 hours after dosing, mice were sacrificed by cervical dislocation. Heart, liver, spleen, lung, kidney, and tumor tissue were rapidly dissected and removed. Blood and residues were rinsed with saline, dried with filter paper, weighed, and stored at -20°C until testing. Three mice were randomly selected at each time point.

[0174] The method in Example 6 was used to determine the concentration of mitoxantrone in various tissues of tumor-bearing mice at different time points, and the tissue distribution kinetics of mitoxantrone in various tissues were plotted ( Figure 6 ) and tissue distribution table (Table 12, using the highest concentration of mitoxantrone in each tissue for comparison).

[0175] Table 12 The highest concentrations of mitoxantrone hydrochloride liposomes, Doenda and mitoxantrone hydrochloride solution prepared with different gradients in various tissues of tumor-bearing mice

[0176]

[0177] Compared with free mitoxantrone hydrochloride, equal doses of mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase and the commercially available formulation Doenda were mainly concentrated in the tumor area, while the distribution in normal tissues was significantly reduced, indicating that the liposome preparation has significant targeting for tumor tissue. Taking mitoxantrone hydrochloride liposomes with 400mM ammonium methanesulfonate as the internal aqueous phase as an example, the maximum drug concentration (C max) were reduced by 47.4%, 30.5% and 169.4% respectively compared with the free drug at the same dose, that is, the liposome preparation significantly reduced the distribution and potential toxicity of the drug to the heart, lungs and kidneys.

[0178] Compared with Doenda, the drug concentration of mitoxantrone hydrochloride liposomes with monovalent sulfonate as the active drug loading gradient in tumor tissue was significantly higher than that of the commercially available preparation Doenda at the same time. Among them, the concentration of mitoxantrone accumulated in tumor tissue by mitoxantrone liposomes with 400mM and 500mM ammonium methylsulfonate as the internal aqueous phase was more than twice that of the commercially available preparation Doenda at the 24th hour (Table 13).

[0179] Table 13 Distribution dynamics of mitoxantrone hydrochloride liposomes and Doenda prepared with different gradients in tumor tissues of tumor-bearing mice

[0180]

[0181] Example 8: Pharmacodynamic Study of Mitoxantrone Hydrochloride Liposomes with Ammonium Methanesulfonate as the Internal Aqueous Phase

[0182] Mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase were observed to have a faster release rate in both in vitro release and in vivo pharmacokinetics, and significantly increased the drug concentration in the tumor tissue of tumor-bearing mice. Therefore, the pharmacodynamics of mitoxantrone hydrochloride liposomes with ammonium methanesulfonate as the internal aqueous phase were investigated using the L1210 ascites tumor model of DBA / 2 mice.

[0183] The specific method is as follows: L1210 cells cultured in vitro were taken and diluted with sterile PBS to a concentration of 5×10 6 The solution was intraperitoneally inoculated into DBA / 2 mice, with each mouse inoculated with 0.1 ml, containing approximately 5×10 tumor cells. 5A mouse L1210 ascites tumor model was established. 24 hours later, mice were randomly divided according to body weight into groups receiving mitoxantrone hydrochloride solution (2.0 mg / kg), mitoxantrone hydrochloride solution (4.0 mg / kg), 400 mM MS (2.0 mg / kg), 400 mM MS (4.0 mg / kg), 500 mM MS (2.0 mg / kg), 500 mM MS (4.0 mg / kg), 600 mM MS (4.0 mg / kg), 600 mM MS (4.0 mg / kg), Doenda (2.0 mg / kg), Doenda (4.0 mg / kg), and a blank control group. Each group received a single tail vein injection of 6-8 mice. Following administration, the animals were housed normally, with body weight measured three times per week. The animals' general condition was observed daily, and survival was calculated. Mean and median survival times were used to evaluate group survival. The experimental observation lasted for 60 days after inoculation. (See Tardi P, Choice E, Masin D, Redelmeier T, Bally M, Madden TD. Liposomal encapsulation of topotecan enhances anticancer efficacy inmurine and human xenograft models. Cancer Res. 2000 Jul 1; 60(13): 3389-93. PMID: 10910044.) The experimental data were statistically analyzed using SPSS 24.0.0.0 statistical software and the Kaplan-Meier method of survival analysis. Pairwise comparisons between multiple groups were performed using one-way analysis of variance, and P < 0.05 was considered significant.

[0184] The results of the pharmacodynamic experiments are shown in Tables 14 and 15. The survival curves of low-dose and high-dose animals are shown in Figure 8 、 Figure 9As shown in Table 14, the blank control group died about 19 days after inoculation of L1210 cells, and drug treatment can significantly prolong the survival time of animals. The survival time of the mitoxantrone hydrochloride liposome group was significantly higher than that of the equal-dose drug solution group. At low dose (2mg / kg), the average survival time of the mitoxantrone hydrochloride liposome group animals with 400mM ammonium methylsulfonate as the internal aqueous phase and the commercially available preparation Doenda group animals was 29.63 days. The low-dose Doenda group and the low-dose 400mM MS group survival rate were statistically analyzed, and the result showed that there was no significant difference between the two. As shown in Table 15, at high dose (4 mg / kg), the mean survival time and median survival time of the mitoxantrone hydrochloride liposome group animals with 400 mM ammonium methylsulfonate as the internal aqueous phase were significantly longer than those of the commercially available preparation Doenda group, being 50.83 ± 3.83 days and 50.00 ± 8.57 days (400 mM MS group) and 45.00 ± 5.24 days and 42.00 ± 7.35 days (Doenda group), respectively. There was a significant difference between the two. After 60 days of experimental observation, the mitoxantrone hydrochloride liposome group with 400 mM ammonium methylsulfonate as the internal aqueous phase, the Doenda group, and the mitoxantrone hydrochloride liposome group with 600 mM ammonium methylsulfonate as the internal aqueous phase still had 2 animals each to survive, and no changes in the disease state were observed in the surviving animals, and their outward appearance and physical condition were the same as those of healthy animals. In summary, the results of pharmacodynamic experiments showed that at a low dose (2 mg / kg), the therapeutic effects of mitoxantrone hydrochloride liposomes with 400 mM ammonium methanesulfonate as the internal aqueous phase and Doenda on the L1210 ascites tumor model of DBA / 2 mice were comparable. At a high dose (4 mg / kg), the therapeutic effect of mitoxantrone liposomes with 400 mM ammonium methanesulfonate as the internal aqueous phase was superior to that of the commercially available preparation Doenda on the L1210 ascites tumor model of DBA / 2 mice. Compared with Doenda, it extended the average and median survival times of tumor-bearing mice by more than 5 days.

[0185] Table 14 Effects of mitoxantrone hydrochloride liposomes, Doenda and mitoxantrone hydrochloride solution prepared with different gradients on the survival time of L1210 ascites tumor DBA / 2 mice at low doses (2 mg / kg)

[0186]

[0187] Table 15 Effects of mitoxantrone hydrochloride liposomes, Doenda and mitoxantrone hydrochloride solution prepared with different gradients on the survival time of L1210 ascites tumor DBA / 2 mice at high dose (4 mg / kg)

[0188]

[0189]

[0190] In summary, the release rate and in vivo tissue distribution behavior of the mitoxantrone hydrochloride liposomes proposed in the present invention, using a monovalent sulfonate as an active drug loading gradient, are significantly different from those of the commercially available formulation, Doenda. Their increased accumulation in tumor tissues helps further enhance the drug's therapeutic efficacy, and they significantly prolong the mean and median survival times of tumor-bearing mice, surpassing the commercially available formulation, Doenda. These research results suggest that the rapid-release mitoxantrone hydrochloride liposome injection provided by the present invention may provide a clinically effective anti-tumor drug.

[0191] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A mitoxantrone hydrochloride liposome, characterized in that: The invention comprises mitoxantrone hydrochloride, a liposome membrane with a bimolecular structure, an inner water phase located in the liposome membrane and an outer water phase located outside the liposome membrane; the mitoxantrone hydrochloride is encapsulated in the inner water phase; and a sulfonate gradient exists between the inner water phase in the liposome membrane and the outer water phase outside the membrane.

2. The mitoxantrone hydrochloride liposome according to claim 1, wherein Also includes one or more of the following features: (1) The internal aqueous phase contains a sulfonate aqueous solution, and the sulfonate is a monovalent sulfonate; (2) the cation of the sulfonate is selected from ammonium ion or triethylamine ion; (3) The external aqueous phase is a physiological isotonic solution; (4) The pH of the inner aqueous phase is 4.0 to 9.0, preferably 4.5 to 8.0; (5) the mitoxantrone hydrochloride encapsulation efficiency of the mitoxantrone hydrochloride liposome is greater than or equal to 85%; (6) The liposome carrier includes phospholipids, cholesterol and excipients.

3. The mitoxantrone hydrochloride liposome according to claim 2, wherein Also includes one or more of the following features: 1) In the inner aqueous phase, the cation of mitoxantrone hydrochloride and the sulfonate group form an insoluble salt, and the insoluble salt is amorphous; 2) the concentration of sulfonate ions in the sulfonate aqueous solution is 100 to 800 mM, preferably 200 to 700 mM; 3) The physiological isotonic solution is selected from a 5% (w / v) glucose aqueous solution, a 10% (w / v) sucrose aqueous solution, or a 0.9% (w / v) sodium chloride aqueous solution; 4) The phospholipid is hydrogenated soybean lecithin; 5) The auxiliary material is distearoylphosphatidylethanolamine-polyethylene glycol.

4. The mitoxantrone hydrochloride liposome according to claim 3, wherein Also includes one or more of the following characteristics: (i) the molar ratio of hydrogenated soybean lecithin, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol in the liposome carrier is (30-80): (0.1-40): (0.1-30); (ii) The molecular weight of the polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol is 50 to 10,000.

5. A method for preparing mitoxantrone hydrochloride liposomes, characterized in that: The steps include: (1) providing blank liposomes containing a sulfonate aqueous solution in the inner aqueous phase; (2) dialyzing the blank liposomes obtained in step (1) using an external aqueous phase solution as a dialysis medium to obtain blank liposomes having a pH and sulfonate gradient between the internal aqueous phase and the external aqueous phase; (3) Mixing the mitoxantrone hydrochloride solution with the blank liposomes having the pH and sulfonate gradients of the inner aqueous phase and the outer aqueous phase obtained in step (2), and incubating the mixture to obtain mitoxantrone hydrochloride liposomes.

6. The method for preparing mitoxantrone hydrochloride liposomes according to claim 5, wherein: Also includes one or more of the following characteristics: (a) The dialysis in step (2) is performed by placing the blank liposomes in step (1) in a dialysis bag with a molecular weight cut-off of 10,000; (b) the volume ratio of the blank liposomes to the dialysis medium in step (2) is 1:500 to 1:1000; (c) the solvent of the mitoxantrone hydrochloride solution in step (3) is the same as the external aqueous phase solution; (d) The incubation in step (3) is carried out in a water bath at a temperature of 50 to 60° C. for 10 to 30 minutes.

7. The method for preparing mitoxantrone hydrochloride liposomes according to claim 5, wherein: The method for preparing the blank liposomes in step 1 comprises the following steps: S1. First, cholesterol, phospholipids and auxiliary materials are taken according to the ratio, ethanol is added, and they are dissolved and mixed to obtain an ethanol mixed solution; S2, preparing a sulfonate buffer solution using a monovalent sulfonate solution and a cationic solution; S3, mixing the ethanol mixed solution obtained in step S1 with the sulfonate buffer obtained in S2, and stirring in a water bath to obtain a liposome suspension; S4. The liposome suspension obtained in step S3 is passed through a liposome extruder to obtain blank liposomes whose inner aqueous phase contains sulfonate.

8. The method for preparing mitoxantrone hydrochloride liposomes according to claim 7, wherein: Also includes one or more of the following characteristics: 1) The pH of the sulfonate buffer in step S2 is 4.0 to 6.0; 2) The water bath temperature in step S3 is 55-65° C., and the water bath time is 20-40 min; 3) In step S4, the liposome is extruded through pores of 200 nm, 100 nm, 80 nm, 50nm polycarbonate film 10 times each.

9. Use of the mitoxantrone hydrochloride liposome according to any one of claims 1 to 4 or the mitoxantrone hydrochloride liposome prepared by the preparation method according to any one of claims 5 to 8 in the preparation of antitumor drugs.

10. The use according to claim 9, wherein the anti-tumor drug is an injectable preparation; preferably, the injectable preparation is selected from a subcutaneous injection form, an intravenous injection form, an intramuscular injection form or a pelvic injection form.

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