Mitoxantrone hydrochloride and ibrutinib composition and application thereof

Abstract

The invention belongs to the technical field of medicines, relates to a composition of mitoxantrone hydrochloride and ibrutinib and application of the composition, and particularly relates to a composition of sialic acid modified mitoxantrone hydrochloride liposome and sialic acid modified ibrutinib phospholipid complex nanoparticles and an effect of the composition in preparation of antitumor drugs. The sialic acid modified mitoxantrone hydrochloride liposome and the sialic acid modified ibrutinib phospholipid compound nanoparticle can be respectively prepared into injections, injections, injections, injections, injections, injections, injections, injections, injections, injections and injections. Or the sialic acid-modified mitoxantrone hydrochloride liposome and the sialic acid-modified ibrutinib phospholipid complex nanoparticles are respectively prepared into injections, the injections are subpackaged in the same medicine box, and the medicine is administered in a sequential manner or is administered at the same time. The sialic acid modified mitoxantrone hydrochloride lipidosome and the sialic acid modified ibrutinib phospholipid compound nanoparticles are combined for use, so that the anti-tumor activity can be obviously improved, immune memory and anti-tumor immune response can be established, and the secondary attack of tumors can be resisted.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the composition of mitoxantrone hydrochloride and ibrutinib and its application, specifically to the composition of sialic acid-modified mitoxantrone hydrochloride liposomes and sialic acid-modified ibrutinib phospholipid complex nanoparticles and their role in the preparation of antitumor drugs. Background Technology

[0002] Tumors are a complex pathological condition, far more than a simple aggregation of malignant cells; they are a highly ordered and complex ecosystem composed of various cellular and non-cellular components. The various components of this ecosystem collectively constitute the tumor microenvironment (TME), which generally includes tumor cells, immune cells, stroma, blood vessels, and cytokines. The immune components within the tumor are called the tumor immune microenvironment (TIME), including immune cells, extracellular immune factors, and cell surface molecules. These components interact in complex ways, profoundly influencing the biological behavior of tumors.

[0003] Throughout the continuous process of tumor growth, spread, and metastasis, a delicate game is constantly taking place between tumor cells and the time-dependent immune system (TIME), potentially leading to three outcomes: tumor elimination, tumor equilibrium, or tumor immune escape. In 2013, Daniel proposed the concept of the tumor immune cycle (TIC). Figure 1 A) revealed the mechanism by which the immune system kills tumor cells, consisting of seven steps: tumor cell antigen release, tumor antigen presentation, T cell activation, T cell migration to tumor tissue, T cell infiltration into tumor tissue, T cell recognition of tumor cells, and elimination of tumor cells. Through this tumor immune cycle mechanism, the body can effectively kill tumor cells. Recent research suggests that immune memory participates in the tumor immune cycle; memory T cells can be directly activated and generate effector T cells upon re-exposure to tumor antigens, entering the next cycle (…). Figure 1 B).

[0004] However, as the interaction between tumors and immune cells intensifies, tumor cells gain the upper hand, domesticating immune cells through various mechanisms, leading to changes in their phenotype and function, and gradually evolving into tumor-associated immune cells (TANs). These TANs enhance the immunosuppressive effect of the tumor microenvironment, weakening or even inhibiting the function of the body's anti-tumor effector cells, thereby causing tumor immune escape and promoting tumor metastasis. In this process, cell populations such as tumor-associated macrophages (TAMs) and tumor-associated neutrophils (TANs) play crucial roles. They work synergistically in tumor sites, blood circulation, and lymphoid tissues, creating an environment conducive to tumor growth and spread through complex mechanisms such as promoting angiogenesis, altering the microenvironment matrix, and helping tumor cells evade immune surveillance. The formation of this environment is a key factor contributing to the difficulty in treating and high recurrence rates of tumors in clinical practice. With ongoing research, more and more scientists have realized that the immunosuppression caused by TIME (Time-Induced Immune Response) is one of the major obstacles facing anti-tumor immunotherapy. Therefore, improving the tumor microenvironment to promote the infiltration of immune cells into tumor tissue is crucial for restoring and enhancing the body's anti-tumor immune response, and this strategy can effectively improve the efficacy of drug therapy. The advent of tumor immunotherapy has provided a new treatment concept for cancer, shifting the focus from solely tumor cells to the body's immune system.

[0005] Mitoxantrone hydrochloride (MIT), a third-generation anthracycline antitumor drug, is a cell cycle nonspecific agent. MIT can act at any stage of the cell cycle, killing both proliferating and quiescent cancer cells. Topoisomerase II is its target; MIT's mechanism of action involves inhibiting topoisomerase II activity, intercalating between DNA bases to block DNA replication and transcription, leading to DNA strand breaks and interfering with DNA and RNA synthesis. MIT can eliminate tumor cells through the immunogenic cell death (ICD) pathway, exposing tumor antigens, initiating the tumor immune cycle, and thus activating an antitumor immune response.

[0006]

[0007] Ibrutinib (IBR) is a small molecule inhibitor of Bruton's tyrosine kinase (BTK) and has been clinically approved for the treatment of B-cell malignancies. IBR's mechanism of action involves selectively binding to the cysteine ​​residue (Cys481) at the BTK active site, achieving irreversible inhibition of BTK activity. The advent of IBR has brought revolutionary progress to the treatment of hematologic malignancies. However, its value extends far beyond this; recent studies have revealed its significant potential in treating solid tumors. IBR can regulate tumor growth and invasion by inhibiting BTK activation, reducing the activity of various tumor-associated immune cells (such as TAMs and T cells), and improving the immunosuppressive state in the tumor microenvironment.

[0008] Therefore, the cytotoxicity of MIT and the ICD effect of this drug can be utilized to initiate the tumor immune cycle and activate the body's anti-tumor immune response; simultaneously, it can be combined with IBR to alleviate the immunosuppression of the tumor microenvironment. The two-drug combination therapy strategy aims to simultaneously kill tumor cells and regulate the tumor immune environment, and is expected to activate the body's immune memory to achieve better anti-tumor effects.

[0009] Sialic acid (SA) is a nonacarbonic amino sugar with a pyranose structure, typically located at the ends of glycolipids or glycoprotein glycans on the cell surface. It plays a crucial role not only in cell membrane stability and intercellular interactions but also in the regulation of various physiological processes, making it essential for maintaining normal biological function.

[0010] Researchers have observed abnormally elevated SA expression levels in various tumor tissues. High SA expression promotes tumor cell evasion of apoptosis and enhances tumor invasiveness, while also helping tumor cells evade immune system surveillance, thus achieving immune escape. Furthermore, abnormally high SA expression on the surface of tumor cells "domesticates" surrounding tumor-associated immune cells, causing them to highly express SA-binding receptors, thereby exerting an immunosuppressive effect. SA-binding receptors mainly include SA-binding immunoglobulin-like lectins (Siglecs) and selectins belonging to the C-type lectin family. Studies have shown that high expression of Siglec-1 on the surface of tumor-associated antigens (TAMs) enables these cells to more effectively recognize and take up exosomes released by tumor cells. This process interferes with the transmission of tumor-associated antigens to dendritic cells (DCs) and lymphocytes, thereby suppressing the body's anti-tumor immune response. In this way, the interaction between tumor cells and immune cells transmits immunosuppressive signals, further enhancing the inhibitory nature of the tumor environment and assisting tumor growth.

[0011] In recent years, with a deeper understanding of the mechanisms by which SA ligands act in tumor immune responses, researchers have revealed how they promote tumor development through interactions with specific receptors. In particular, members of the Siglecs family, due to their crucial role in tumor development, have become important targets for designing anti-tumor drug delivery systems. Given the high expression of SA-binding receptors on tumor-associated immune cell populations, we modified a nanodrug delivery system (NDDS) using SA and its derivatives to achieve targeted tumor therapy through tumor-associated immune cell-mediated phagocytosis and delivery.

[0012]

[0013] There are no existing reports on the combined use of sialic acid-modified mitoxanone hydrochloride liposomes and sialic acid-modified ibrutinib phospholipid complex nanoparticles for antitumor purposes. Summary of the Invention

[0014] To overcome the shortcomings of the prior art, the technical problem solved by the present invention is to provide the application of sialic acid-modified mitoxanone hydrochloride liposomes combined with sialic acid-modified ibrutinib phospholipid complex nanoparticles in the preparation of antitumor drugs.

[0015] This invention is achieved through the following technical solution:

[0016] The present invention first provides a composition of mitoxantrone hydrochloride and ibrutinib.

[0017] In the composition, the mass ratio of mitoxantrone hydrochloride to ibrutinib is 1:1 to 1:8.

[0018] Furthermore, the mitoxantrone hydrochloride is a mitoxantrone hydrochloride formulation modified with sialic acid.

[0019] The ibrutinib mentioned is a sialic acid-modified ibrutinib formulation.

[0020] The formulation is a nanomedicine delivery system selected from liposomes, nanoparticles, microemulsions, nanocapsules, or polymer micelles.

[0021] Furthermore, the formulation is a liposome or nanoparticle.

[0022] Preferably,

[0023] The mitoxantrone hydrochloride is a mitoxantrone hydrochloride liposome modified with sialic acid.

[0024] The ibrutinib described is a sialic acid-modified ibrutinib phospholipid complex nanoparticle.

[0025] The sialic acid is a lipid derivative of sialic acid; preferably, it is a sialic acid-cholesterol conjugate (SA-CH).

[0026] The sialic acid-modified mitoxantrone hydrochloride liposomes described herein use SA-CH as a target head, which is modified on the surface of the liposomes to give them the ability to actively target tumor-associated immune cell populations. The mitoxantrone hydrochloride is encapsulated in the aqueous phase of the liposomes using an active drug delivery method to reduce drug toxicity and enhance therapeutic effects.

[0027] The sialic acid-modified mitoxanone hydrochloride liposomes described herein are prepared using phospholipids and cholesterol (CH) as liposome membrane materials, sialic acid-cholesterol conjugates as targeting groups, and citrate-sodium citrate buffer as the hydration medium.

[0028] On a molar ratio, phospholipid:cholesterol:sialic acid-cholesterol conjugate = (50-60):(35-45):(2-10), preferably (50-60):(35-45):(5-10).

[0029] Based on the mass ratio of drug lipids, mitoxantrone hydrochloride: phospholipids = 1:(5-20), preferably 1:(5-10).

[0030] The phospholipids include one or more of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and cardiac phospholipids. They can be natural, semi-synthetic, or fully synthetic, such as one or more of egg yolk phosphatidylglycerol (EPG), hydrogenated soybean lecithin (HSPC), hydrogenated egg yolk phosphatidylglycerol (HEPG), dioleoylphosphatidylglycerol (DOPG), distearyl phosphatidylglycerol (DSPG), distearyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylglycerol (DPPG), and dimyristoyl phosphatidylglycerol (DMPG), preferably phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol.

[0031] The mitoxantrone hydrochloride liposome formulation has an encapsulation efficiency greater than 90%, a particle size less than 150 nm, a polydispersity index of 0.01 to 0.04, and a zeta potential of -10 to -20 mV.

[0032] The sialic acid-modified ibrutinib phospholipid complex nanoparticles are prepared by modifying the surface of phospholipid materials with SA-CH to form IBR phospholipid complex nanoparticles, thereby improving the solubility and bioavailability of IBR. At the same time, by endowing them with active targeting ability, the dosage is reduced and the therapeutic effect is improved.

[0033] Phospholipid complex nanoparticles have become a hot topic in the field of drug delivery due to their unique advantages. They offer benefits such as improved drug solubility, enhanced bioavailability, and reduced adverse reactions. Furthermore, chemical modification of the surface of phospholipid complex nanoparticles can enable targeted drug delivery or prolong drug circulation time in vivo.

[0034] The sialic acid-modified ibrutinib phospholipid complex nanoparticles comprise ibrutinib, phospholipids, and sialic acid-cholesterol conjugates, wherein the molar ratio of ibrutinib to phospholipids is 1:1 to 1:5, and the molar ratio of sialic acid-cholesterol conjugates to phospholipids is 1:1 to 1:19.

[0035] Preferably, the molar ratio of ibrutinib to phospholipid is 1:1 to 1:2, and the molar ratio of sialic acid-cholesterol conjugate to phospholipid is 1:1.5 to 1:4.

[0036] The phospholipids include one or more of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and cardiac phospholipids. They can be natural, semi-synthetic, or fully synthetic, such as one or more of egg yolk phosphatidylglycerol (EPG), hydrogenated soybean lecithin (HSPC), hydrogenated egg yolk phosphatidylglycerol, dioleoyl phosphatidylglycerol, distearate phosphatidylglycerol (DSPG), dipalmitoyl phosphatidylglycerol (DPPG), and dimyristoyl phosphatidylglycerol (DMPG), preferably phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol.

[0037] The ibrutinib nanocomposite of the present invention has a composite efficiency of greater than 90%, a nanocomposite particle size of less than 50 nm, a small particle size distribution, a polydispersity index of less than 0.3, and a zeta potential of -10 to -25 mV.

[0038] The present invention also provides the use of the aforementioned mitoxantrone hydrochloride and ibrutinib composition in the preparation of antitumor drugs.

[0039] Furthermore, this invention provides the application of sialic acid-modified mitoxanone hydrochloride liposomes combined with sialic acid-modified ibrutinib phospholipid complex nanoparticles in the preparation of antitumor drugs.

[0040] The tumors mentioned include S180 sarcoma, breast cancer, melanoma, lung cancer, leukemia, etc.

[0041] In this invention, the combined use of mitoxantrone hydrochloride and ibrutinib exerts an anti-tumor effect by regulating the body's immunity, alleviating the tumor suppressor microenvironment, reducing the accumulation of tumor-associated macrophages in tumor tissue, and promoting CD8. + T cell infiltration within tumor tissue activates the body's anti-tumor immune response.

[0042] In this invention, the combined use of mitoxantrone hydrochloride and ibrutinib can induce tumor "shedding," enabling mice to survive for an extended period.

[0043] In this invention, the combined use of mitoxantrone hydrochloride and ibrutinib can induce long-lasting and effective protective immune memory in mice, resisting secondary tumor attacks.

[0044] In this invention, the combination of mitoxantrone hydrochloride and ibrutinib promotes secondary tumor regression by upregulating immune activating factors IL-2 and IFN-γ.

[0045] In this invention, the combined use of mitoxantrone hydrochloride and ibrutinib exerts an anti-tumor effect by increasing the proportion of memory T cells in the spleen.

[0046] The mitoxantrone hydrochloride and ibrutinib of the present invention can be used together or packaged in the same box.

[0047] The sialic acid-modified mitoxanone hydrochloride liposomes and sialic acid-modified ibrutinib phospholipid complex nanoparticles of the present invention can be prepared into injections, respectively.

[0048] Alternatively, sialic acid-modified mitoxanone hydrochloride liposomes and sialic acid-modified ibrutinib phospholipid complex nanoparticles can be separately prepared into injectable formulations and packaged in the same drug box for use.

[0049] The administration of mitoxantrone hydrochloride and ibrutinib in combination can be sequential or simultaneous, including mitoxantrone hydrochloride first, followed by ibrutinib, or ibrutinib first, followed by mitoxantrone hydrochloride, or both simultaneously, with the most preferred method being simultaneous administration of mitoxantrone hydrochloride and ibrutinib.

[0050] Based on mitoxantrone hydrochloride and ibrutinib, the dose ratio of sialic acid-modified mitoxantrone hydrochloride liposomes to sialic acid-modified ibrutinib phospholipid complex nanoparticles is 1:1-1:8, preferably 1:2-1:5.

[0051] Liposomes and phospholipid complexes are both lipid-based nanoscale delivery systems with good biocompatibility and a range of shared advantages in drug delivery. These include improved drug stability, reduced drug toxicity, enhanced drug bioavailability, increased dosage, and better therapeutic effects.

[0052] This invention utilizes SA-CH to modify NDDS, preparing SA-modified MIT liposomes (SA-MIT) and IBR phospholipid complex nanoparticles (SA-IBR). By leveraging the interaction between SA and its ligand, it targets and intervenes in tumor-associated immune cell populations, thereby regulating the tumor's suppressive environment and activating the body's anti-tumor immunity to achieve the goal of tumor treatment. In the S180 tumor-bearing mouse model, combined treatment with SA-MIT and SA-IBR resulted in tumor detachment. Tumor re-challenge experiments in these detached mice demonstrated that the combined application of SA-MIT and SA-IBR can stimulate tumor-specific immune memory in mice, induce the activation of tumor memory T cells, and generate a durable anti-tumor immune response. Attached Figure Description

[0053] Figure 1 This is a diagram of the tumor immune circulation.

[0054] A: Tumor immune cycle; B: Tumor immune cycle involving immune memory.

[0055] Figure 2 The in vitro release curves are for MIT solution (MIT-S) and SA-MIT liposomes.

[0056] Figure 3 The in vitro release curves of IBR solution (IBR-S) and SA-IBR phospholipid complex nanoparticles are shown.

[0057] Figure 4 The figures show the curves of changes in tumor volume, tumor inhibition rate, and body weight in mice in each group in Example 7.

[0058] Figure 5 This leads to the accumulation of toxins in the distal extremities of different MIT formulations.

[0059] Figure 6 Changes in tumor volume in different groups of mice after secondary tumor bearing in recovered mice following administration of PEG-modified MIT liposomes.

[0060] Figure 7 This is a schematic diagram of the pharmacodynamic experimental protocol for Example 10.

[0061] Figure 8 The tumor growth curves are for S180 tumor-bearing mice.

[0062] The red arrow indicates drug administration, ***P<0.001, n=6.

[0063] Figure 9 The curves showing the changes in body weight and net body weight of S180 tumor-bearing mice are shown.

[0064] A: Body weight change curve; B: Net body weight change curve.

[0065] Figure 10 The tumor inhibition rate curve of S180 tumor-bearing mice.

[0066] The red arrow indicates drug administration, **P<0.01, n=6.

[0067] Figure 11 The tumor inhibition index curve and the tumor inhibition index on day 25 are shown for S180 tumor-bearing mice.

[0068] A: Tumor inhibition index curve of S180 tumor-bearing mice B: Histogram of tumor inhibition index of S180 tumor-bearing mice on day 25.

[0069] The red arrow indicates the administration of the drug, ***p<0.001, n=2~6.

[0070] Figure 12 Images of tumors "shedding" over time in mice in the SA-MIT+SA-IBR group, and magnified images of local tumor areas.

[0071] Figure 13 For H&E staining histological examination of the heart, liver, spleen, and tumors.

[0072] Scale bar = 50μm.

[0073] Figure 14 Representative images of Ki-67 immunofluorescence staining of S180 tumor sections.

[0074] Scale bar = 100μm DAPI (blue) Ki67 (red).

[0075] Figure 15 Immunofluorescence images of tumor tissues from different treatment groups.

[0076] Scale bar = 100μm DAPI (blue) CD8 (red) CD206 (green).

[0077] Figure 16 For the S180 tumor re-challenge experiment.

[0078] A: S180 tumor re-challenge timeline; B: S180 tumor re-challenge mouse tumor growth curve; C: Images of tumor regression in tumor-bearing mice in two separate events; D: Tumor growth curves of mice in each group.

[0079] Figure 17 The levels of IFN-γ, TNF-α and IL-2 cytokines in mouse peripheral blood were measured by ELISA.

[0080] The data are expressed as mean ± SD, n = 3.

[0081] ns, P>0.05, *P<0.05, ***P<0.001 (compared to tumor rechallenge mice (7 days)).

[0082] Figure 18 This is a long-term immune memory effect triggered by SA-MIT+SA-IBR.

[0083] A: Flow cytometry analysis of the percentage of T cells in the spleen;

[0084] B: Flow cytometry analysis of effector memory T cells (T cells) in mouse spleen EM ) ratio (CD3) + CD8 + CD44 + CD62L - ).

[0085] C: Mouse spleen T EM Proportional data analysis. Detailed Implementation

[0086] Example 1: Preparation of sialic acid-cholesterol conjugate SA-CH

[0087] SA-CH was prepared according to the method in Example 16 or Example 17 of Chinese Patent CN106188169A.

[0088] Example 2: Preparation of sialic acid-modified mitoxantrone hydrochloride (SA-MIT) liposomes

[0089] 1. Solution preparation

[0090] MIT drug solution: Accurately weigh 20.0 mg MIT into a clean beaker, add 2 mL of distilled water and stir thoroughly to dissolve. Then transfer the solution to a 5 mL volumetric flask, and bring it to the mark with distilled water. Shake well, and filter through a 0.22 μm microporous membrane to obtain 4.0 mg / mL. -1 MIT drug solution.

[0091] Hydration medium: Accurately weigh 2.1 g of citric acid into a clean beaker, add 40 mL of distilled water and stir thoroughly to dissolve. Adjust the pH to 4.0 with NaOH solution. Then transfer the solution to a 50 mL volumetric flask and add distilled water to the mark. Shake well and filter through a 0.22 μm microporous membrane to obtain a 200 mM citric acid-sodium citrate buffer solution (pH 4.0).

[0092] pH adjuster for external aqueous phase: Accurately weigh 1.9g of sodium phosphate into a clean beaker, add 8mL of distilled water and stir thoroughly to dissolve. Then transfer the solution to a 10mL volumetric flask and add distilled water to the mark. Shake well and filter through a 0.22μm microporous membrane to obtain a 500mM sodium phosphate solution.

[0093] 2. Preparation of blank liposomes

[0094] The ratio of HSPC to CH was chosen to be 55:40 (n / n), and the modification ratio of SA-CH was 5 mol% (i.e., HSPC / CH / SA-CH = 55 / 40 / 5 (n / n)). The blank liposomes were prepared using a modified ethanol injection method. The required membrane material was accurately weighed and placed in a vial. Anhydrous ethanol, not exceeding 10% (v / v) of the final volume, was added. The mixture was stirred in a water bath at 65°C until the membrane material was completely dissolved. The ethanol was then evaporated until the mixture was nearly dry. A preheated hydration medium was added to the membrane material, and the mixture was stirred at 65°C for 20 min to obtain the initial blank liposome sample. The initial blank liposome sample was then passed through polycarbonate membranes at 400 nm, 200 nm, and 100 nm eight times each at 65°C to adjust the particle size, resulting in a blank liposome suspension (phospholipid concentration of 20 mg / mL). -1 ).

[0095] 3. Preparation of gradient liposomes

[0096] Add the pre-prepared sodium phosphate solution to the above blank liposome suspension and adjust the pH of the external aqueous phase to 7.0 to obtain pH gradient liposomes.

[0097] 4. Drug delivery

[0098] Take the above gradient liposomes into a vial, and add MIT drug solution (4.0 mg / mL) at a drug-liposome ratio of 1:10 (w / w). -1 Stir in a 65°C water bath for 20 minutes, then stop drug loading by incubating in an ice-water bath for 3 minutes to obtain the final phospholipid concentration of 10 mg / mL. -1 The concentration of MIT is 1 mg·mL -1 Store at 4℃ away from light for later use.

[0099] Example 3 Properties of SA-MIT prepared in Example 2

[0100] The particle size, dispersion index (PDI), and zeta potential of SA-MIT liposomes are shown in Table 1.

[0101] Table 1. Particle size, dispersion index, and Zeta potential of SA-MIT liposomes (n=3).

[0102]

[0103] Calculate the encapsulation efficiency and drug loading using the following formulas:

[0104] Encapsulation efficiency EE% = Amount of encapsulated mitoxantrone hydrochloride / Total amount of mitoxantrone hydrochloride × 100%

[0105] Drug loading DL (%) = Mass of encapsulated mitoxantrone hydrochloride / Total mass of mitoxantrone hydrochloride liposomes × 100%

[0106] Table 2 shows the encapsulation efficiency and drug loading of SA-MIT liposomes:

[0107] Table 2 Encapsulation efficiency and drug loading of SA-MIT liposomes (n=3).

[0108]

[0109] In vitro release of SA-MIT liposomes:

[0110] Release medium: PBS containing 80 mM ammonium chloride and 10 mM histidine buffer, pH adjusted to 7.4 with sodium hydroxide, and 100 μg / mL of sodium hydroxide solution added. -1 Penicillin and streptomycin.

[0111] In vitro drug release was studied using dialysis. 1.0 mL each of 1 mg / mL MIT solution (MIT-S) and MIT liposomes (SA-MIT) were placed in pre-treated dialysis bags with a molecular weight cutoff of 8–14 kDa. The dialysis bags were clamped at both ends to seal the solutions inside. The dialysis bags were placed in a dissolution vessel containing 100 mL of release medium, protected from light, and shaken at a constant speed of 100 rpm at 37 ± 1 °C. At predetermined time points, 1.0 mL of dialysate was aspirated and an equal volume of release medium at the same temperature was added. The ΔF of the dialysate was measured, and the cumulative release rate Rn was calculated using the following formula. Release curves were plotted, and the results are shown below. Figure 2 .

[0112]

[0113] The results showed that the in vitro release patterns of both MIT-S and SA-MIT conformed to a first-order equation. MIT-S permeated the dialysis bag rapidly, with a permeation rate approaching 100% within 8 hours. In contrast, the drug release rate within the MIT liposomes was relatively slow, exhibiting a significant sustained-release phenomenon. The cumulative release rate of SA-MIT within 72 hours was 32.6% ± 2.1%, indicating that liposomes significantly delayed drug release. Comparative Example 1: Preparation of ordinary MIT liposomes (MIT-CL)

[0114] Referring to Example 2, the membrane material formulation was HSPC / CH (55:45, n / n), and other conditions remained unchanged to prepare ordinary MIT liposomes.

[0115] Preparation of 2PEG-modified MIT liposomes (MIT-PL) as a comparative example

[0116] Referring to Example 2, the membrane material formulation was HSPC / CH / mPEG2000-DSPE (50:45:5, n / n), and other conditions remained unchanged to prepare PEG-modified MIT liposomes.

[0117] Example 4: Preparation of ibrutinib phospholipid complex nanoparticles (SA-IBR)

[0118] Preparation of IBR solution: Accurately weigh 12.5 mg IBR into a clean beaker, add 0.5 mL DMSO to dissolve it, then add 2 mL distilled water and stir thoroughly. Transfer the solution to a 5 mL volumetric flask, and bring it to the mark with distilled water. Shake well and filter through a 0.22 μm microporous membrane to obtain 2.5 mg / mL IBR solution. -1 IBR drug solution.

[0119] Example 5: Preparation of SA-IBR phospholipid complex nanoparticles:

[0120] Accurately weigh 10.0 mg of IBR, 24.8 mg of EPG, and 11.0 mg of SA-CH into a vial (i.e., IBR / EPG / SA-CH = 1 / 1.4 / 0.6 (n / n)). Then, add 0.2 mL of anhydrous ethanol and reflux the mixture at 60 °C for 0.5 h. After reflux, continue stirring the mixture to evaporate the ethanol until it reaches a near-dry state. Next, add 4.0 mL of preheated 5% glucose injection solution to the vial and stir for 10 min. Finally, filter through a 0.22 μm microporous membrane to remove unrecombined IBR particles. The resulting solution has a concentration of 2.5 mg / mL. -1 IBR phospholipid complex nanoparticles.

[0121] Example 6 Properties of ibrutinib phospholipid complex nanoparticles prepared in Example 4

[0122] The particle size, dispersion index (PDI), and zeta potential of the SA-IBR phospholipid complex nanoparticles are shown in Table 3.

[0123] Table 3. Particle size, dispersion index, and Zeta potential (n=3) of SA-IBR phospholipid complex nanoparticles.

[0124]

[0125] The drug consolidation rate and drug loading were calculated using the following formulas. The consolidation rate and drug loading of the SA-IBR phospholipid complex nanoparticles are shown in Table 4.

[0126] Compatibility rate (CE) = (Mass of IBR already combined / Amount of IBR administered) × 100%

[0127] Drug loading DL (%) = (Mass of IBR compounded with IBR / Total mass of IBR phospholipid complex) × 100%

[0128] Table 4. Composite rate and drug loading of SA-IBR phospholipid complex nanoparticles (n=3).

[0129]

[0130] In vitro release of IBR phospholipid complex nanoparticles:

[0131] In vitro drug release studies were conducted using dialysis. 1.0 mL each of the previously prepared IBR solution (IBR-S) and the prepared IBR phospholipid complex nanoparticles (SA-IBR) were placed separately into pretreated dialysis bags with a molecular weight cutoff between 8 and 14 kDa. The bags were clamped at both ends with dialysis clamps and placed in a dissolution vessel containing 100 mL of release medium (pH 7.4, containing 0.5% (v / v) Tween 80 PBS) in the dark. The bags were shaken at a constant speed of 100 rpm at 37 ± 1 °C. At specific time points, 1.0 mL of dialysis solution was aspirated, and an equal volume of release medium at the same temperature was added. The IBR content in the samples was detected by HPLC, the cumulative release rate Rn was calculated, and release curves were plotted. The results are shown in [Figure number missing]. Figure 3 .

[0132] The results showed that the in vitro release patterns of both IBR-S and SA-IBR conformed to a first-order equation. IBR-S permeated the dialysis bag more rapidly, with a permeation rate approaching 100% within 12 hours. In contrast, SA-IBR effectively delayed drug release, exhibiting a sustained-release effect, with a cumulative release rate of 57.4% ± 1.7% within 12 hours and near-complete release within 72 hours.

[0133] Example 7: Pharmacodynamic Study of Different Mitoxantrone Hydrochloride Liposomes in the Treatment of Tumors

[0134] Experimental plan:

[0135] A heterotopic S180 xenograft model was established using 24 Kunming mice. S180 cells (1.8 × 10⁻⁶) were implanted into the lateral abdomen. 7 cells·mL -1 Cell suspension (0.2 mL per animal) was randomly divided into 4 groups of 6 animals each: 5% glucose control group (Control), ordinary MIT liposome group (MIT-CL), PEG-modified MIT liposome group (MIT-PL), and SA-CH modified MIT liposome group (MIT-SAL). The MIT dosage was 4 mg / kg, administered every 3 days.

[0136] Tumor volume and body weight were measured in mice every other day. Changes in tumor volume, tumor inhibition rate, and body weight were recorded for each group. Results are shown below. Figure 4 .

[0137] The results showed that all MIT formulations exhibited certain antitumor effects, with the tumor inhibition rate in the order of MIT-PL > MIT-SAL > MIT-CL. The MIT-PL formulation showed a tumor inhibition rate close to 100%, and five mice in this group achieved complete tumor remission. The average body weight gain of mice in the MIT-PL group was slower than that of other groups, likely due to the toxicity of the long-circulating MIT-PL formulation. PEG modification resulted in longer circulation times for liposomes, and the faster tumor growth rate led to a significant EPR effect, contributing to the excellent antitumor effect of the MIT-PL formulation. However, many marketed PEG-modified liposomes have not shown ideal efficacy in clinical use. This is likely because the EPR effect is overemphasized in mouse models, while it is not significant in humans. Furthermore, the increasing expression of anti-PEG antibodies in humans has led to frequent severe allergic reactions and reduced efficacy. All of this suggests that we should have a more balanced view of PEG-modified nanoparticles.

[0138] Example 8: Accumulation and toxicity accumulation in distal extremities of different MIT formulations

[0139] The MIT-PL group mice in Example 7 were observed and photographed. Results are shown below. Figure 5 The results showed that the strong hydrophilicity of PEG caused liposomes to accumulate in the skin, especially in the palms and soles, leading to tissue necrosis in severe cases. Skin toxicity has become a new dose-limiting toxicity. In this experiment, the palms and soles of mice in the MIT-PL group turned significantly blue, indicating that PEG-modified MIT liposomes accumulated in large quantities there, leading to toxic accumulation.

[0140] Example 9: Secondary tumor-bearing rechallenge experiment of PEG-modified MIT liposomes

[0141] Since PEG-modified MIT liposomes are highly toxic to the body, this embodiment examines whether the body's own immune system can eliminate tumors without any intervention by administering PEG-modified MIT liposomes in the MIT-PL group mice of Example 7 after a second tumor-bearing rechallenge experiment.

[0142] Secondary tumor-bearing re-challenge experimental protocol:

[0143] Four recovered mice from the MIT-PL group were taken and, 50 days later, were re-inoculated with S180 cells (1.5 × 10⁻⁶) in the flank. 7 cells·mL -1 Cell suspension (0.2 mL per mouse) was collected, and four age-matched healthy mice were simultaneously implanted with tumors at the same location (lateral ventral region), designated as the Control group. No drug treatment was administered, and tumor growth was observed and tumor volume was recorded. Results are shown below. Figure 6 .

[0144] The results showed that the tumor growth curves of the MIT-PL group mice after secondary tumor bearing were not significantly different from those of the control group, and even showed a tendency to surpass the control group in the later stages. During the experiment, it was found that the survival status of the MIT-PL group mice after re-challenge was poor. The mice exhibited symptoms such as rough and frizzy fur, hardened skin with dandruff, and body stiffness. All mice died at 15 days (2 mice) and 17 days (2 mice), respectively. In other words, although PEG-modified MIT liposomes have a very good anti-tumor effect and even show tumor cure, the body is unable to resist disease recurrence.

[0145] As can be seen from Examples 7-9, although PEG-modified MIT liposomes have better anti-tumor effects, they are severely toxic and cannot withstand tumor re-challenge experiments. Therefore, this invention further selects SA-MIT liposomes, which have better anti-tumor effects and are non-toxic, for subsequent experimental studies.

[0146] Example 10: Pharmacodynamics of SA-MIT liposomes combined with SA-IBR phospholipid complex nanoparticles against S180 tumors

[0147] Cells and Animals:

[0148] S180 mouse sarcoma cells (Shanghai Cell Bank, Chinese Academy of Sciences). Animals were acclimatized to a rearing environment of 25°C and 55% humidity one week before the experiment.

[0149] Test drugs: 5% glucose, MIT-S, IBR-susp, SA-MIT liposomes (Example 2); SA-IBR phospholipid complex nanoparticles (Example 4).

[0150] Preparation of IBR oral suspension (IBR-susp): IBR suspension was prepared according to the formulation of marketed ibrutinib tablets. 10.0 mg of IBR active pharmaceutical ingredient was weighed, and 10.0 mg of croscarmellose sodium, 1.2 mg of magnesium stearate, 18.0 mg of microcrystalline cellulose, and 0.8 mg of sodium dodecyl sulfate were added. Then, 5% glucose injection was added and mixed to obtain an IBR concentration of 2.0 mg / mL. -1 Oral suspension.

[0151] Experimental plan:

[0152] Thirty-six S180 tumor-bearing mice were randomly divided into six groups of six each. The groups were: a 5% glucose control group (Control), an MIT solution group (MIT-S), an MIT liposome group (SA-MIT), an IBR suspension group (IBR-susp), an IBR phospholipid complex nanoparticle group (SA-IBR), and a group receiving both MIT liposomes and IBR phospholipid complex nanoparticles simultaneously (SA-MIT+SA-IBR). MIT-S and SA-MIT were administered via tail vein injection at a dose of 4 mg / kg. -1 IBR-susp is administered orally via gavage at a dose of 25 mg / kg. -1 SA-IBR is administered via tail vein injection at a dose of 10 mg / kg. -1 When the tumor grows to 100mm... 3 Treatment began around [time missing], with one treatment every 3 days, for a total of 5 treatments. See the experimental protocol below. Figure 7 .

[0153] The tumor volume and growth curve, mouse body weight and net body weight, tumor inhibition rate and tumor inhibition index, and tumor detachment phenomenon in mice were investigated. The experimental methods and results are as follows:

[0154] (1) Mouse tumor volume and growth curve

[0155] During the pharmacodynamic experiment, the major and minor axes of the tumor were measured every other day using vernier calipers. The tumor volume was calculated, and a tumor growth curve was plotted. The results are shown in […]. Figure 8 .

[0156] The experimental results showed that during the pharmacodynamic experiment 25 days after drug administration, the tumor volume in the Control group mice continued to increase over time. All treatment groups inhibited tumor growth to varying degrees, with the tumor growth rate in the following order: IBR-susp group > MIT-S group > SA-IBR group > SA-MIT group > SA-MIT+SA-IBR group. Among these, SA-MIT showed a more significant inhibitory effect on tumor growth compared to MIT-S (***p<0.001). The poor anti-tumor effect of IBR-susp may be due to its poor water solubility and low bioavailability, making it difficult to exhibit excellent anti-tumor efficacy at this dosage. Notably, compared to other groups, the SA-MIT+SA-IBR group showed the slowest tumor volume increase over time (***p<0.001), and the tumor volume also showed a decreasing trend, demonstrating that the combined treatment of SA-MIT and SA-IBR can maximally inhibit tumor growth.

[0157] (2) Mouse body weight and net body weight

[0158] Throughout the pharmacodynamic experiment, mouse body weight was measured every other day, and curves showing changes in body weight and net body weight were plotted. The net body weight of tumor-bearing mice was equal to their body weight minus the tumor mass (assuming a tumor density of 1 g / cm³ during the experimental period). -3 The experimental results are shown in Figure 9 .

[0159] Experimental results showed that during the entire pharmacodynamic experiment, the body weight and net body weight of mice in the MIT-S group fluctuated significantly, showing an overall downward trend. Mice exhibited lethargy, loss of appetite, and dull, wrinkled fur. Particularly on day 5 after drug administration, the body weight and net body weight of mice decreased sharply. This was attributed to the strong cytotoxicity of MIT solution and its non-selective distribution in the body, causing non-specific damage. The body weight and net body weight change curves showed that the body weight gain rate of mice in the Control group was greater than that of the other groups, but the net body weight gain trend was slower, even lower than that of the SA-IBR group in the later stages of the experiment. This indicates that the rapid increase in body weight in the Control group was related to tumor growth. Except for the MIT-S group, the body weight and net body weight of mice in all other treatment groups generally showed an upward trend. Throughout the experiment, the mice survived well, and no deaths occurred.

[0160] (3) Tumor inhibition rate and tumor inhibition index

[0161] The differences in antitumor effects among the various formulations were compared, and the tumor inhibition rate and index of each formulation group were further calculated. Tumor inhibition rate variation curves were then plotted. The tumor inhibition rate results are shown in Table 5, and the tumor inhibition rate variation curves are shown in [the table below]. Figure 10 .

[0162] The tumor suppression index can take into account both the inhibitory effect of various treatments on tumor growth and the non-specific damage to the body. The higher the tumor suppression index, the better the overall treatment effect.

[0163] Tumor inhibition rate by volume = (Tumor volume in control group - Tumor volume in treatment group) / Tumor volume in control group × 100%

[0164] Tumor suppression index = Body weight of tumor-bearing animal / Tumor weight

[0165] Table 5. Tumor inhibition rate of S180 tumor-bearing mice in each group on day 25 (n=6).

[0166]

[0167] The results in Table 5 show that on day 25 of the pharmacodynamic experiment, the tumor inhibition rate of each group, from highest to lowest, was: SA-MIT+SA-IBR group > SA-MIT group > SA-IBR group > MIT-S group > IBR-susp group. Among them, the tumor inhibition rate of the SA-MIT+SA-IBR group was significantly higher than that of the other groups (**p<0.01), with a tumor inhibition rate as high as 99.3±1.1%, which almost completely inhibited the growth of tumors.

[0168] Table 6 shows the tumor inhibition index of mice on day 25, and the curve of the change in the tumor inhibition index is shown in Table 6. Figure 11 .

[0169] Table 6. Tumor inhibition index of S180 tumor-bearing mice in each group on day 25 (n = 2-6).

[0170]

[0171]

[0172] On day 25, the tumor suppression index (TI) of each group, ranked from largest to smallest, was: SA-MIT+SA-IBR group > SA-MIT group > SA-IBR group > MIT-S group > IBR-susp group > Control group. The SA-MIT+SA-IBR group stood out with a TI index of 660.3±168.0, which was 11.7 times and 14.0 times higher than that of the SA-MIT group (56.2±17.5) and the SA-IBR group (47.3±5.3), respectively (***p<0.001). This indicates that the combined treatment of SA-MIT+SA-IBR not only has the best anti-tumor effect but also the least non-specific toxicity to the body, resulting in the best overall effect.

[0173] Figure 11 The results showed that, except for the SA-MIT+SA-IBR group, the tumor inhibition index of all other groups decreased rapidly over time, while the tumor inhibition index of the SA-MIT+SA-IBR group increased slowly over time, reaching its maximum at 25 days. This indicates that the combined application of SA-MIT+SA-IBR has a significantly better anti-tumor effect than SA-MIT alone, SA-IBR alone, or the sum of both, demonstrating a synergistic anti-tumor effect when used in combination.

[0174] (4) Tumor shedding phenomenon in the SA-MIT+SA-IBR group

[0175] Throughout the pharmacodynamic experiment, the tumor status of the tumor-bearing mice was observed daily, and tumor changes were recorded by photographs. Unexpectedly, a series of changes was observed in the SA-MIT+SA-IBR group mice: the tumors "scabbed over," "shed," and then the wound gradually "healed." This phenomenon was accompanied by the process of "scabbing," "shedding," and the gradual "healing" of the wound at the shed site. It is important to note that this "shedding" phenomenon is not tumor ulceration, but rather the tumor tissue, along with its overlying skin, "peeling" from the tumor site, followed by gradual wound healing and normal hair growth. Four out of six mice in this group exhibited tumor "shedding," a "shedding" rate of 66.7%, while this phenomenon did not occur in the other groups. Subsequently, after three months of continuous observation, no tumor recurrence was observed in the mice with "shedding" tumors, and the mice were completely cured. The experimental results are shown below. Figure 12 .

[0176] Example 11 Histopathological Examination

[0177] To observe organ damage in each group of mice, the safety of the combined use of the two drugs was evaluated using H&E staining. After the efficacy experiment, the heart, liver, spleen, and tumor tissues of tumor-bearing mice in each group were fixed with 4% paraformaldehyde solution and embedded in paraffin. Histopathological changes were observed using an inverted microscope after H&E staining. The results are shown in [Figure number missing]. Figure 13 .

[0178] Depend on Figure 13 H&E staining results showed that the MIT-S group mice exhibited slight myocardial rupture (green arrows), indicating that MIT-S has some cardiotoxicity. The organ tissues of mice in the other treatment groups remained structurally intact, with tightly packed cells and clear boundaries; no obvious organ damage was observed. Furthermore, the SA-MIT+SA-IBR group mice showed large areas of cell necrosis (black arrows) in tumor tissue sections, with numerous tumor cells fragmented or dissolved, and extensive apoptosis.

[0179] Example 12 Immunofluorescence Staining Experiment - Tumor Cell Proliferation Signal

[0180] After the pharmacodynamic experiments, tumor tissues from mice in each group were collected and immediately frozen into sections. Ki67 staining was performed to detect tumor cell proliferation, and the cell nuclei were counterstained with DAPI. The prepared immunofluorescent sections were observed under a fluorescence microscope to examine the effect of each treatment group on tumor cell proliferation. The results are shown in [Figure number missing]. Figure 14 .

[0181] Figure 14The results showed that the control group had higher Ki67 fluorescence intensity, indicating that the tumor was in a rapid growth phase. After drug treatment, the Ki67 fluorescence intensity decreased to varying degrees in all groups. Among them, the SA-MIT+SA-IBR group had the weakest Ki67 fluorescence intensity, almost no expression, indicating that tumor proliferation was significantly inhibited.

[0182] Example 13 Immunofluorescence staining experiment - Immune cell infiltration

[0183] After the pharmacodynamic experiment, tumor tissues from mice in each group were collected, immediately frozen, sectioned, and stained with CD206 and CD8. The sections were observed and images were acquired using a fluorescence microscope to explore the underlying mechanism of the superior antitumor effect of the SA-MIT+SA-IBR group. The infiltration levels of M2-type TAMs and CD8+ T cells within the tumor tissue were detected. The results are shown in [Figure number missing]. Figure 15 In this context, green fluorescence represents M2-type TAMs labeled with CD206 antibody, and red fluorescence represents CD8-type TAMs labeled with CD8 antibody. + T.

[0184] The results showed that the tumors in the Control group were filled with a large number of M2-type TAMs, which acted as a "moat" for the tumor tissue, while CD8+ was present in the tumor tissue. + T is almost undetectable. M2-type TAMs have both tumor-promoting and immunosuppressive effects, playing a crucial role in tumor development and progression. Their presence also inhibits CD8. + T cells infiltrate the tumor, leading to severe immunosuppression. After drug treatment, the infiltration of M2-type TAMs in the tumor tissues of mice in all groups decreased to varying degrees, with the SA-MIT+SA-IBR group showing the lowest positive expression rate of M2-type TAMs, almost none. Furthermore, compared with the single-drug treatment group, the SA-MIT+SA-IBR group showed significantly higher levels of CD8+ expression in the tumor tissue. + The highest infiltration of T cells indicates that the immunosuppression in the tumor microenvironment has been relieved, and the body has initiated a positive anti-tumor immune response. These results suggest that the dual-drug combination strategy can not only kill tumor cells but also alter the immunosuppressive microenvironment and regulate effector T cell function, thereby generating a better anti-tumor immune response in vivo.

[0185] Example 14 Tumor Re-challenge Experiment

[0186] Ninety days after the pharmacodynamic experiment, four recovered mice in this group were re-inoculated with S180 cells (1.0 × 10⁻⁶) at the same site (right abdomen). 7 cells·mL -1Cell suspension (0.2 mL per mouse) was used for tumor rechallenge experiments, designated as the tumor rechallenge group. Six age-matched healthy mice were simultaneously inoculated as a tumor-bearing control group. The study observed whether the tumor-rechallenge mice could eliminate tumors using their own immune system without any drug treatment. Results are shown in […]. Figure 16 .

[0187] Surprisingly, in the tumor re-challenge group of mice, tumors spontaneously regressed without drug administration. Tumor growth was slow from day 3 to 7 after tumor implantation, followed by gradual shrinkage. Three mice showed complete tumor regression on day 17 (1 mouse) and day 19 (2 mice), respectively, with smooth skin over the tumor site and no tumor detachment. In another mouse, the tumor gradually shrank after day 7 after tumor implantation, eventually reaching a volume of 47 mm². 3 .

[0188] Example 15 Tumor Immune Memory Analysis - ELISA Detection of Peripheral Blood Cytokine Levels

[0189] On day 7 of the tumor rechallenge experiment, peripheral blood (approximately 1 ml per mouse) was collected from healthy mice, tumor-bearing control mice, and tumor rechallenge mice using the orbital venous plexus sampling method. Mice survived healthy blood collection after the procedure. On day 30 of the tumor rechallenge experiment, peripheral blood was collected again from the tumor rechallenge mice. Blood samples were allowed to clot at room temperature for 1 hour, centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. The levels of IFN-γ, TNF-α, and IL-2 cytokines in the peripheral blood of mice were detected using an ELISA kit. Results are shown below. Figure 17 .

[0190] Data from the healthy mouse group reflect baseline levels of peripheral blood cytokines in mice at rest. Figure 17It was found that the levels of three cytokines, IFN-γ, TNF-α, and IL-2, were lower in the peripheral blood of healthy mice. The levels of cytokines (IL-2 and TNF-α) in the tumor-bearing control group were significantly upregulated compared to the healthy group, indicating that the body responds rapidly to tumor antigens under tumor-bearing conditions by upregulating immune activating factors to resist tumor invasion. On day 7 of tumor re-challenge, compared to healthy mice, the levels of immune activating factors IL-2 (***P<0.001), IFN-γ (***P<0.001), and TNF-α (*P<0.05) were all significantly upregulated in the tumor re-challenge mice. After 30 days, the tumor re-challenge mice were in a tumor-free state. Although the overall cytokine levels showed signs of decreasing compared to day 7, they were still higher than in the healthy group, indicating that the body could still release immune protective factors. Comparing the differences between tumor-challenged mice (7 days) and tumor-bearing control mice when faced with tumor antigen attack, significant differences were found between the two groups in cytokines IL-2 (*P<0.05) and IFN-γ (***P<0.001). Therefore, it is speculated that IL-2 and IFN-γ may be involved in regulating systemic immune memory response and play an important role in the process of tumor regression.

[0191] Example 16 Tumor Immune Memory Analysis - Flow Cytometry Detection of Splenic T Cell Subsets

[0192] On day 30 of the tumor rechallenge experiment (day 120 on the timeline), the spleen of mice was removed, and splenic lymphocytes were isolated using a mouse organ lymphocyte assay kit to detect effector memory T cells (T cells). EM CD3 + CD8 + CD44 + CD62L - Distribution of T lymphocyte subsets. Mouse spleens were cut into sections approximately 2 × 2 mm in size. 3 Small pieces of spleen were gently pressed and ground using a syringe plunger. After filtering the tissue through a 70 μm nylon sieve, a single-cell suspension was collected and centrifuged at 1200 rpm for 5 min. The resulting cell pellet was resuspended in erythrocyte lysis buffer to remove erythrocytes. After washing with PBS, monoclonal antibody was added, and the cells were stained in the dark for 30 min. The cells were then fixed with 4% paraformaldehyde and analyzed by flow cytometry to investigate the specific mechanism of spontaneous tumor regression in tumor-rechallenged mice. Results are shown below. Figure 18 .

[0193] The results showed that T in the spleen of mice that experienced two tumor regressions EMThe proportion of tumor-bearing cells (36.5 ± 1.15%) was 2.2 times that of the tumor-bearing control group (16.9 ± 0.46%) (**p < 0.01). In mice with two tumor regressions, the proportion of memory T cells was significantly increased, indicating that SA-MIT combined with SA-IBR treatment induced long-lasting and effective protective immune memory in the mice. Further analysis of CD4+ in the spleen was performed. + T cells and CD8 + The proportion of T cells was found to shift towards CD4+ in the spleen of mice that had undergone secondary tumor-bearing autologous healing. + T cell increase tilt.

[0194] The immune system's defense against secondary tumor attacks is a complex and intricate process. When mice are first exposed to tumor antigens, their immune system develops immune memories against these antigens. These immune memories are primarily composed of long-lived memory T cells, which store information about specific antigens and can be rapidly activated and expanded upon re-exposure to the same antigens, forming an immune response against the tumor antigens. This rapid immune response allows the immune system to act earlier, preventing tumor growth and spread. Numerous studies have shown that memory T cells play a crucial role in inhibiting tumor progression, promoting tumor regression, and achieving long-term patient survival. This invention also provides evidence for this claim, showing a significant increase in memory T cells in the spleen of mice bearing tumors a second time, mediating tumor regression and long-term survival. Furthermore, cured mice may produce more immune activators, such as cytokines and antibodies, to enhance the efficacy of the immune response. IL-2 and IFN-γ, as important cytokines in the immune response, play crucial roles in regulating T cell function, promoting macrophage activation and enhancing phagocytic capacity, and modulating systemic immunity. When faced with tumor re-attack, the levels of IL-2 and IFN-γ cytokines in the peripheral blood of mice were significantly upregulated, positively regulating the body's immunity and promoting anti-tumor effects. In summary, the mouse immune system was able to recognize and eliminate tumor cells more rapidly and effectively after recovery, achieved through the formation of immune memory and enhanced immune responses. This mechanism of immune memory and anti-tumor immune response provides long-term protection for mice, enabling them to resist secondary tumor attacks.

[0195] The spleen, as the largest secondary lymphatic organ in the human body, acts as an "immune reservoir," reflecting changes in the overall immune system. We further examined CD4 levels in the spleen. + T cells and CD8 + The proportion of T cells was found to shift towards CD4+ in the spleen of cured mice. + T-cell increase tilt. Because people prefer a more direct approach to treating tumors, researchers have explored CD8 for many years. +The enthusiasm for T-cell anti-tumor activity far exceeds that for CD4. + T cells, and the results of this experiment suggest that we should also pay attention to CD4. + The important role of T cells in the body's anti-tumor process. CD4 + T cells have many similarities to CD8. + T cells possess distinct anti-tumor advantages, enhancing the immune system's ability to kill tumors through various mechanisms such as helper cytokine production, regulation of the body's immune response, and memory generation. Therefore, both T cells and CD4+ play a complementary role in tumor immunity, working together to combat tumor growth. + Whether T cells significantly contribute to spontaneous healing in tumor-rechallenged mice warrants further investigation into the specific mechanisms underlying this process.

[0196] CD4 in the spleen + T and CD8 + Changes in the proportion of T cells also revealed the plasticity of the spleen, an immune organ, after effective treatment, thus predicting the plasticity of the systemic immune status. This remodeling may involve the rebalancing of immune cell subsets, the establishment of immune memory, and the optimization of immune regulatory mechanisms. These results confirm that effective tumor immunotherapy can not only induce improvements in the tumor microenvironment but also cause significant changes in the body's entire immune landscape.

[0197] Example 17: Pharmacodynamic experiments on the anti-S180 tumor activity of SA-MIT liposomes combined with SA-IBR phospholipid complex nanoparticles via different administration routes.

[0198] Twenty-four S180 tumor-bearing mice were randomly divided into four groups of six mice each: a 5% glucose control group, a group receiving simultaneous administration of MIT liposomes and IBR phospholipid complex nanoparticles (SA-MIT+SA-IBR), a group receiving MIT liposomes followed by IBR phospholipid complex nanoparticles (SA-MIT then SA-IBR), and a group receiving IBR phospholipid complex nanoparticles followed by MIT liposomes (SA-IBR then SA-MIT). SA-MIT was administered via tail vein injection at a dose of 4 mg / kg. -1 SA-IBR is administered via tail vein injection at a dose of 10 mg / kg. -1 When the tumor grows to 100mm... 3 Treatment began around 3:00 AM, with one treatment every 3 days for a total of 5 treatments. The combined injection group received treatment one day apart.

[0199] Example 18 Tumor volume inhibition rate in mice under different administration methods

[0200] The drug was administered according to the experimental method of Example 17. During the pharmacodynamic experiment, the long and short diameters of the tumor were measured with vernier calipers every other day to obtain the tumor volume. The volume inhibition rate and tumor inhibition index were calculated, and the results are shown in Table 7.

[0201] Table 7. Tumor inhibition rate and tumor inhibition index of S180 tumor-bearing mice in each group on day 25 (n = 2-6).

[0202]

[0203] Different administration methods of SA-MIT liposomes combined with SA-IBR phospholipid complex nanoparticles can produce good tumor-suppressing effects. However, the group that was simultaneously administered SA-MIT liposomes and SA-IBR phospholipid complex nanoparticles (SA-MIT+SA-IBR group) had the best tumor inhibition rate and tumor inhibition index. Moreover, only the SA-MIT+SA-IBR group showed tumor detachment. No tumor detachment occurred in the SA-MIT-then-SA-IBR and SA-IBR-then-SA-MIT administration methods.

[0204] Example 19: Anti-B16F10 Tumor Pharmacodynamics of SA-MIT Liposomes Combined with SA-IBR Phospholipid Complex Nanoparticles

[0205] Experimental methods:

[0206] Twelve B16F10 tumor-bearing mice were randomly divided into two groups of six each: a 5% glucose control group (Control) and a group receiving simultaneous administration of MIT liposomes combined with IBR phospholipid complex nanoparticles (SA-MIT+SA-IBR). SA-MIT was administered via tail vein injection at a dose of 4 mg / kg. -1 SA-IBR is administered via tail vein injection at a dose of 10 mg / kg. -1 When the tumor grows to 100mm... 3 Treatment begins around 3:00 AM, with one treatment every 3 days, for a total of 5 treatments.

[0207] Tumor volume inhibition rate in mice

[0208] During the pharmacodynamic experiment, the long and short diameters of the tumor were measured with vernier calipers every other day to calculate the tumor volume, and the volume inhibition rate and inhibition index were calculated. The results are shown in Table 8.

[0209] Table 8. Tumor volume, tumor inhibition rate, and tumor inhibition index of B16F10 tumor-bearing mice in each group on day 25 (n=6).

[0210]

[0211]

[0212] The simultaneous administration of MIT liposomes and IBR phospholipid complex nanoparticles (SA-MIT+SA-IBR group) still showed good tumor inhibition in B16F10 mice. Therefore, the combined use of MIT liposomes and IBR phospholipid complex nanoparticles has good tumor universality.

[0213] Example 20

[0214] The anti-S180 experiment was conducted according to the experimental protocol of Example 10. The dosage ratio of SA-MIT and SA-IBR was changed, and the tumor inhibition index after simultaneous administration of SA-MIT and SA-IBR under different dosage ratios was investigated. The results are shown in Table 9.

[0215] Table 9. Antitumor inhibition index of S180 under different dosage ratios

[0216]

[0217] The results showed that when the dosage ratio of SA-MIT to SA-IBR was 1:1 to 1:8, the tumor inhibition index was greater than 500, indicating a significant synergistic effect. When the dosage ratio was 1:2 to 2.5, the tumor inhibition index was greater than 600, and the synergistic effect was even stronger.

[0218] In summary, the drug MIT in this invention has dual effects of cell killing and immunomodulation. It can kill tumor cells through the ICD pathway, activate the body's anti-tumor immune response, initiate the tumor immune cycle, and induce more effector T cells to participate in the anti-tumor process. SA-MIT can also utilize the binding of sialic acid ligands to target and kill suppressive tumor-associated immune cells that highly express SA binding receptors.

[0219] The drug IBR mainly exerts its anti-tumor effect by regulating the function of immune cells (macrophages, neutrophils, T cells, mast cells, etc.). For example, IBR can significantly inhibit the phosphorylation of BTK in TAMs, promote the differentiation of TAMs in a direction unfavorable to tumor growth, and ultimately achieve the goal of effectively inhibiting tumor growth.

[0220] Therefore, the combination of these two agents simultaneously kills tumor cells and regulates the body's immune system, relieving the immunosuppressive state of the tumor microenvironment and mobilizing the body's powerful anti-tumor immunity. Ultimately, the combined group achieves excellent results in tumor regression and resistance to secondary tumor attacks. The anti-tumor effect of the SA-MIT+SA-IBR combined application of this invention is significantly better than that of the SA-MIT alone or the SA-IBR alone, and the two produce a synergistic anti-tumor effect.

Claims

1. A composition of mitoxantrone hydrochloride and ibrutinib, characterized in that, The mass ratio of mitoxantrone hydrochloride to ibrutinib is 1:1 to 1:

8.

2. The composition according to claim 1, characterized in that, The mitoxantrone hydrochloride is a formulation containing mitoxantrone hydrochloride modified with sialic acid, and the ibrutinib is a formulation containing ibrutinib modified with sialic acid. The formulation is a nanomedicine delivery system, and the sialic acid is a lipid derivative of sialic acid. Preferably, the mitoxantrone hydrochloride is a mitoxantrone hydrochloride liposome modified with sialic acid-cholesterol conjugate, and the ibrutinib is an ibrutinib phospholipid complex nanoparticle modified with sialic acid-cholesterol conjugate.

3. The composition according to claim 2, characterized in that, The mitoxantrone hydrochloride liposome modified with the sialic acid-cholesterol conjugate comprises mitoxantrone hydrochloride, phospholipids, cholesterol, and the sialic acid-cholesterol conjugate, wherein, on a molar ratio, phospholipids:cholesterol:sialic acid-cholesterol conjugate = (50-60):(35-45):(2-10), preferably (50-60):(35-45):(5-10); and on a drug-liposome mass ratio, mitoxantrone hydrochloride:phospholipids = 1:(5-20), preferably 1:(5-10).

4. The composition according to claim 2, characterized in that, The ibrutinib phospholipid complex nanoparticles modified with sialic acid-cholesterol conjugate comprise ibrutinib, phospholipids, and sialic acid-cholesterol conjugates. The molar ratio of ibrutinib to phospholipids is 1:1 to 1:5, and the molar ratio of sialic acid-cholesterol conjugates to phospholipids is 1:1 to 1:

19. Preferably, the molar ratio of ibrutinib to phospholipids is 1:1 to 1:2, and the molar ratio of sialic acid-cholesterol conjugates to phospholipids is 1:1.5 to 1:

4.

5. Use of the composition according to any one of claims 1-4 in the preparation of an antitumor drug.

6. The application according to claim 5, characterized in that, The tumor in question is a sarcoma, breast cancer, melanoma, or lung cancer.

7. The application according to claim 5, characterized in that, The mitoxantrone hydrochloride and ibrutinib in the composition are administered sequentially or simultaneously, preferably simultaneously.

8. The application according to any one of claims 5-7, characterized in that, When the composition comprises mitoxantrone hydrochloride liposomes modified with sialic acid-cholesterol conjugate and ibrutinib phospholipid complex nanoparticles modified with sialic acid-cholesterol conjugate, the dosage ratio of mitoxantrone hydrochloride liposomes modified with sialic acid to ibrutinib phospholipid complex nanoparticles is 1:1 to 1:8, preferably 1:2 to 1:

5.

9. The application according to any one of claims 5-8, characterized in that, Mitoxantrone hydrochloride and ibrutinib, when used in combination, modulate the body's immune system, alleviate the tumor suppressor microenvironment, reduce the accumulation of tumor-associated macrophages in tumor tissue, and promote CD8 activation. + T cell infiltration within tumor tissue activates the body's anti-tumor immune response, thereby exerting an anti-tumor effect.

10. The application according to any one of claims 5-8, characterized in that, Mitoxantrone hydrochloride and ibrutinib exert their anti-tumor effects by stimulating tumor-specific immune memory in vivo and inducing the activation of tumor memory T cells, thereby generating a durable anti-tumor immune response.

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