Regorafenib-entrapped albumin preparation, pharmaceutical composition as well as preparation method and application of regorafenib-entrapped albumin preparation

Abstract

The invention provides a regorafenib-entrapped albumin preparation, a pharmaceutical composition and a preparation method and application of the regorafenib-entrapped albumin preparation, the regorafenib-entrapped albumin preparation is prepared from regorafenib into an albumin nano preparation, the solubility of REG is enhanced, side effects are relieved, the biological half-life period is prolonged, long-acting circulation is achieved, and the regorafenib-entrapped albumin preparation has a good application prospect. And the curative effect is enhanced by increasing the tumor tissue drug accumulation amount. In addition, according to the pharmaceutical composition, the regorafenib component is added into the albumin paclitaxel preparation, so that the pharmaceutical composition has the capability of synergistically reprogramming macrophages. Cell experiments prove that the combined use of PTX and REG under certain proportion conditions can enhance the M1 type polarization of macrophages.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an albumin preparation containing regorafenib, a pharmaceutical composition, its preparation method, and its application. Background Technology

[0002] Regorafenib is an oral multi-kinase inhibitor that can effectively block multiple protein kinases involved in tumor angiogenesis, tumorigenesis, tumor metastasis, and tumor immunity. It has been approved by the FDA for hepatocellular carcinoma, metastatic colorectal cancer, and gastrointestinal stromal tumors.

[0003] From a physicochemical perspective, regorafenib belongs to Biopharmaceutics Class II, meaning it is a drug with low solubility and high permeability. Its solubility in water and physiological pH environments (pH 1.0–7.4) is extremely low (typically less than 10 μg / mL), and its solubility is significantly affected by pH; solubility is slightly higher in acidic environments and decreases sharply in neutral and alkaline environments. This physicochemical characteristic has become a bottleneck restricting its formulation development and clinical application. Currently, formulations based on conventional regorafenib technologies (such as ordinary tablets) generally suffer from low in vitro dissolution, slow and irregular in vivo absorption, and low oral bioavailability. This directly leads to unstable therapeutic effects and may affect medication safety due to large individual differences in absorption. At the same time, the development of dosage forms to improve its solubility also faces significant challenges.

[0004] In terms of treatment efficacy, in order to compensate for the low bioavailability, the existing regorafenib formulations are administered at relatively high clinical doses. High-dose administration not only further exacerbates the risk of adverse reactions, but also easily leads to drug resistance. Recent studies have shown that a considerable number of colorectal cancer patients have developed resistance to regorafenib. For colorectal cancer patients who cannot undergo radical surgery, drug resistance to targeted therapy has become an important issue affecting patient prognosis.

[0005] Therefore, developing a new formulation of regorafenib with high dissolution rate and good bioavailability of the active ingredient, and how to further improve the anti-tumor performance of regorafenib, are problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0006] To overcome the problems existing in the prior art, one objective of this invention is to provide an albumin formulation loaded with regorafenib. A second objective of this invention is to provide a method for preparing the aforementioned albumin formulation loaded with regorafenib. A third objective of this invention is to provide the application of the aforementioned albumin formulation loaded with regorafenib. A fourth objective of this invention is to provide a pharmaceutical composition. A fifth objective of this invention is to provide a method for preparing the aforementioned pharmaceutical composition. A sixth objective of this invention is to provide the application of the aforementioned pharmaceutical composition.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides an albumin formulation loaded with regorafenib, comprising the following components: regorafenib and albumin; wherein the albumin formulation is a nanoparticle formulation.

[0008] Preferably, the product contains the active ingredient regorafenib and the main carrier material albumin, wherein the active ingredient regorafenib is encapsulated in or adsorbed onto the carrier material.

[0009] Preferably, in the albumin formulation containing regorafenib, the weight ratio of regorafenib to the carrier material albumin is 1:(1~50).

[0010] Preferably, the albumin includes at least one of human serum albumin, bovine serum albumin, and mouse serum albumin.

[0011] Preferably, the albumin formulation loaded with regorafenib has an average particle size in the range of 100-500 nm.

[0012] More preferably, the average particle size is in the range of 150 to 350 nm. For example, it can be 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, or 340 nm.

[0013] Preferably, the albumin preparation is in the form of lyophilized powder.

[0014] More preferably, albumin formulations in lyophilized powder form do not include lyophilized excipients.

[0015] More preferably, albumin formulations in lyophilized powder form include pharmaceutically acceptable lyophilized excipients.

[0016] More preferably, the type of lyophilization excipient does not significantly affect the particle size and stability of the albumin formulation loaded with regorafenib according to the present invention. Therefore, it is not expected that the type of lyophilization excipient is particularly limited. In some embodiments, the pharmaceutically acceptable lyophilized excipient is selected from one or a mixture of two or more of the following: glucose, fructose, maltose, sorbitol, mannitol, xylitol, glycerol, sucrose, trehalose, lactose, alanine, cysteine, cystine, histidine, glutamic acid, phenylalanine, glycine, aspartic acid, lysine, leucine, arginine, serine, tryptophan, sodium chloride, zinc sulfate, EDTA, dextran, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium bicarbonate, sodium malate, sodium tartrate, sodium lactate, human serum albumin, hyaluronic acid, chitosan, alginate, hydroxyethyl starch, povidone, polyethylene glycol, polyvinyl alcohol, polylysine, polyaspartic acid, poloxamer, phospholipids, cholesterol, gelatin, collagen, protamine sulfate, etc.

[0017] The second aspect of the present invention provides a method for preparing the albumin formulation loaded with regorafenib as described in the first aspect, wherein the albumin formulation loaded with regorafenib is prepared by ultrasonic emulsification. Alternatively, the albumin formulation containing regorafenib can be prepared using a high-pressure homogenization method.

[0018] Preferably, the ultrasonic emulsification process includes: dissolving regorafenib in an organic solvent to form an oil phase solution, and dissolving albumin in a solvent to form an aqueous phase solution; adding the oil phase solution to the aqueous phase solution, and performing ultrasonic emulsification under ice bath conditions to form a nano-suspension, removing the organic solvent, to obtain the albumin formulation.

[0019] Preferably, the high-pressure homogenization process includes: dissolving regorafenib in an organic solvent to form an oil phase solution, and dissolving albumin in a solvent to form an aqueous phase solution; mixing the oil phase solution and the aqueous phase solution to form an emulsion; homogenizing the emulsion under high pressure using a homogenizer to form a nano-suspension, removing the organic solvent, and obtaining the albumin formulation.

[0020] More preferably, in the high-pressure homogenization method or the ultrasonic emulsification method, the organic solvent used to dissolve regorafenib is selected from one or more of the following: dimethyl sulfoxide, N,N-dimethylformamide, chloroform, dichloromethane, ethyl acetate, propyl acetate, butyl acetate, ethyl formate, methanol, ethanol, acetone, n-propanol, isopropanol, tert-butanol, methyl butyl ketone, and butanone. In some embodiments, the organic solvent used to dissolve regorafenib is a mixture of one or two of chloroform and dichloromethane with one or more of methanol, ethanol, tert-butanol, and acetone.

[0021] More preferably, the concentration of regorafenib in the oil phase solution is in the range of 0.1 to 50 mg / mL, for example, in the range of 0.1 to 20 mg / mL, and more preferably in the range of 0.1 to 10 mg / mL.

[0022] More preferably, in the high-pressure homogenization method or the ultrasonic emulsification method, the solvent used for the aqueous phase solution is pure water or a buffer solution with a pH of 7-9. Further, the buffer solution is selected from at least one of phosphate buffer, carbonate buffer, Tris buffer, HEPES buffer, borate buffer, or glycine buffer.

[0023] More preferably, the concentration of albumin in the aqueous solution is in the range of 1 to 100 mg / mL, for example, in the range of 0.1 to 20 mg / mL, and more preferably in the range of 0.1 to 10 mg / mL.

[0024] More preferably, the power of the ultrasonic emulsification is 100~1000 W. Further, the power of the ultrasonic emulsification is 200~400 W.

[0025] More preferably, the ultrasonic emulsification time is 2 to 8 minutes.

[0026] More preferably, the homogenizer homogenizes the nano-suspension under pressure conditions of 5000~30000 psi, for example, 10000~25000 psi.

[0027] More preferably, the method for removing organic solvents includes removing organic solvents from the nanosuspension by means of rotary evaporation, thin-film evaporation with reduced pressure, and freeze spray drying.

[0028] More preferably, it also includes freeze drying to obtain a freeze-dried powder form.

[0029] The third aspect of this invention provides the use of the albumin formulation containing regorafenib as described in the first aspect in any of the following aspects: A) Prepare drugs for activating the tumor immunosuppressive microenvironment; B) Preparation of anti-tumor drugs.

[0030] C) Prepare sensitizers for use in sensitizing chemotherapy drugs.

[0031] Preferably, the tumor is a solid tumor. More preferably, the solid tumor includes hepatocellular carcinoma, metastatic colorectal cancer, and gastrointestinal stromal tumor.

[0032] A fourth aspect of the present invention provides a pharmaceutical composition comprising the following components: regorafenib, paclitaxel, and albumin.

[0033] Preferably, it comprises the active ingredients regorafenib and paclitaxel, and the main carrier material albumin; the active ingredients regorafenib and paclitaxel are encapsulated in or adsorbed onto the carrier material.

[0034] Preferably, in the pharmaceutical composition, the weight ratio of regorafenib to the carrier material albumin is 1:(1~200).

[0035] More preferably, the weight ratio is 1:(1~50).

[0036] Preferably, in the pharmaceutical composition, the molar ratio of regorafenib to paclitaxel is 1:(0.05~20).

[0037] Preferably, the albumin includes at least one of human serum albumin, bovine serum albumin, and mouse serum albumin.

[0038] Preferably, the pharmaceutical composition is in the form of nanoparticles.

[0039] More preferably, the average particle size of the pharmaceutical composition is in the range of 100 to 300 nm.

[0040] More preferably, the average particle size is in the range of 150 to 250 nm. For example, it can be 160, 170, 180, 190, 200, 210, 220, 230, or 240 nm.

[0041] More preferably, the pharmaceutical composition is in the form of a lyophilized powder.

[0042] More preferably, the albumin preparation in lyophilized powder form does not include lyophilized excipients.

[0043] More preferably, the albumin formulation in lyophilized powder form includes pharmaceutically acceptable lyophilized excipients. The definition of lyophilized excipients is as shown in the first aspect.

[0044] The fifth aspect of the present invention provides a method for preparing the pharmaceutical composition described in the fourth aspect, wherein the pharmaceutical composition is prepared by ultrasonic emulsification. Alternatively, the pharmaceutical composition may be prepared using a high-pressure homogenization method.

[0045] Preferably, the ultrasonic emulsification process includes: dissolving regorafenib and paclitaxel in an organic solvent to form an oil phase solution, and dissolving albumin in a solvent to form an aqueous phase solution; adding the oil phase solution to the aqueous phase solution, and performing ultrasonic emulsification under ice bath conditions to form a nano-suspension, removing the organic solvent to obtain the drug composition.

[0046] Preferably, the high-pressure homogenization process includes: dissolving regorafenib and paclitaxel in an organic solvent to form an oil phase solution, and dissolving albumin in a solvent to form an aqueous phase solution; mixing the oil phase solution and the aqueous phase solution to form an emulsion; homogenizing the emulsion under high pressure using a homogenizer to form a nano-suspension, removing the organic solvent, and obtaining the pharmaceutical composition.

[0047] The process parameters for the ultrasonic emulsification method and the high-pressure homogenization method can be referred to the description in the second aspect.

[0048] The sixth aspect of this invention provides for the use of the pharmaceutical composition in any of the following aspects: A) Prepare drugs for activating the tumor immunosuppressive microenvironment; B) Preparation of anti-tumor drugs.

[0049] C) Prepare sensitizers for use in sensitizing chemotherapy drugs.

[0050] Preferably, the tumor is a solid tumor. More preferably, the solid tumor includes hepatocellular carcinoma, metastatic colorectal cancer, and gastrointestinal stromal tumor.

[0051] The beneficial effects of this invention are: This invention provides an albumin (Alb) formulation (Alb-REG) encapsulating regorafenib (REG). The REG is prepared into an albumin nanoparticle formulation, which enhances the solubility of REG, reduces side effects, prolongs the biological half-life to achieve long-term circulation, and increases the amount of drug accumulated in tumor tissue to enhance the therapeutic effect.

[0052] This invention also provides a pharmaceutical composition in which paclitaxel (PTX) and regorafenib synergistically reprogram macrophages. Cellular experiments have demonstrated that the combined use of PTX and REG under certain ratios enhances M1 polarization in macrophages. Furthermore, PTX and REG are further formulated into albumin nanoparticles (Alb-RP), which exhibit superior antitumor performance compared to both single-agent and combination therapies, contributing to improved efficacy of combination therapy. Attached Figure Description

[0053] Figure 1 A schematic diagram of the ultrasonic preparation method for albumin nanomedicine loaded with REG; Figure 2 A schematic diagram of a high-pressure homogenization method for preparing albumin nanomedicines loaded with REG; Figure 3 The physicochemical properties of albumin nanomedicines loaded with REG are tested, including (A) particle size distribution diagram; (B) zeta potential diagram; (C) particle size stability test diagram; and (D) dilution stability test diagram. Figure 4This is a scanning electron microscope image of albumin nanomedicine loaded with REG. Figure 5 The study investigated the cytotoxicity of REG and Alb-REG on tumor cells. The results show: (A) 24-hour in vitro cytotoxicity test on CT26 tumor cells; (B) 24-hour in vitro cytotoxicity test on MC38 tumor cells; (C) 48-hour in vitro cytotoxicity test on CT26 tumor cells; and (D) 48-hour in vitro cytotoxicity test on MC38 tumor cells. Figure 6 The relative gene expression of macrophages reprogrammed by Alb-REG; (A) relative mRNA expression level of M2 macrophage markers; (B) relative mRNA expression level of M1 macrophage markers; Figure 7 Protein expression levels in macrophages reprogrammed by Alb-REG; including (A) the proportion of CD206-positive cells (M2 marker); (B) the proportion of CD86-positive cells (M1 marker); and (C) the M1 / M2 ratio. Figure 8 This is a diagram illustrating the Alb-REG experimental method used in mice. Figure 9 Survival rate of Alb-REG in mouse experiments; Figure 10 A schematic diagram of the ultrasonic preparation method for albumin nanomedicines loaded with REG and PTX; Figure 11 Scanning electron microscopy image of albumin nanomedicine loaded with REG and PTX prepared by ultrasonication method; Figure 12 This is a schematic diagram of a high-pressure homogenization method for preparing albumin nanomedicines loaded with REG and PTX. Figure 13 The physicochemical properties of albumin nanomedicines loaded with REG and PTX are tested, including (A) particle size distribution diagram; (B) zeta potential diagram; (C) particle size stability test diagram; and (D) dilution stability test diagram. Figure 14 Scanning electron microscopy image of albumin nanomedicine loaded with REG and PTX prepared by high pressure homogenization method; Figure 15 Release curves of albumin nanomedicines loaded with REG and PTX; Figure 16 The relative gene expression of macrophages reprogrammed by Alb-RP; (A) relative mRNA expression levels of M2 macrophage markers; (B) relative mRNA expression levels of M1 macrophage markers; Figure 17Protein levels of macrophages were reprogrammed using Alb-RP; (A) the proportion of CD206-positive cells (M2 marker); (B) the proportion of CD86-positive cells (M1 marker); and (C) the M1 / M2 ratio. Figure 18 The experimental results show the enhancement of macrophage phagocytic capacity in each experimental group; Figure 19 To enhance the ability of macrophages to kill tumor cells in each experimental group; Figure 20 This is a diagram illustrating the Alb-RP experimental method used in mice. Figure 21 In vivo bioluminescence images of Alb-RP used in mouse experiments; Figure 22 Quantitative analysis of abdominal bioluminescent flux of Alb-RP in mouse experiments; Figure 23 The survival rate of Alb-RP in mouse experiments. Detailed Implementation

[0054] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0055] The albumin (Alb) used in the following examples can be at least one of human serum albumin, bovine serum albumin, and mouse serum albumin, without specific limitation, and was purchased from Sigma-Aldrich.

[0056] Example 1 This embodiment uses an ultrasonic method to prepare albumin nanomedicine (Alb-REG) encapsulated with REG. A schematic diagram of the preparation method is shown below. Figure 1 The specific preparation method is as follows: 1 mg of REG was weighed and dissolved in 300 μL of a chloroform-ethanol mixture to prepare an organic phase solution of REG, which was then kept in an ice bath for later use. Alb was weighed according to different feed ratios (see Table 1), dissolved in ultrapure water to a concentration of 5 mg / mL, and added to a 5 mL EP tube. The organic phase solution was then added vertically, and the tube was sonicated in an ice bath with the following parameters: 200–400 W power and 2–8 min. This embodiment can be implemented and achieve the relevant technical effects within this range, and the same range parameters are used in the following embodiments. After sonication, a milky white nano-suspension was obtained, which was quickly transferred to a round-bottom flask and rotary evaporated to remove the organic reagents, yielding albumin nanomedicine nanoparticles encapsulated with REG (Alb-REG), which were then lyophilized and stored.

[0057] Physicochemical properties of Alb-REG in Example 1 1. Testing method: (1) The particle size and polydispersity index (PDI) of Alb-REG were determined by dynamic light scattering analyzer. (2) High performance liquid chromatography was used to detect the encapsulation efficiency and drug loading of drugs in Alb-REG. Establishment of a high-performance liquid chromatography (HPLC) detection method: 2 mg of REG was weighed and dissolved in 1 mL of methanol. The solution was sonicated to ensure complete dissolution, yielding a REG stock solution with a final concentration of 2 mg / mL. The REG stock solution was diluted with methanol to obtain REG solutions of different concentrations (125 μg / mL, 62.5 μg / mL, 31.3 μg / mL, 15.6 μg / mL, 7.8 μg / mL, and 3.9 μg / mL). HPLC was used for detection. The instrument was a Shimadzu LC-16; the column specifications were Shimadzu Shim-pack Scepter C18-120 5 μm, 4.6 × 250 mm; the mobile phase was 0.1% acetic acid / water:acetonitrile = 30:70 (v / v); the flow rate was 1 mL / min; the column temperature was 25 ℃; the injection volume was 20 μL; and the detection wavelength was 260 nm. Inject REG solutions of different concentrations as described above, measure their peak areas, and plot regression curves.

[0058] The lyophilized Alb-REG particles were reconstituted with 1 mL of ultrapure water, centrifuged at 3000 rpm for 5 min to obtain free REG, dissolved thoroughly in methanol by sonication, filtered through a 220 nm filter membrane, and the free drug content was determined by HPLC. The encapsulation efficiency and drug loading of Alb-REG were calculated using the difference method. The formulas for calculating the drug encapsulation efficiency and drug loading are as follows: Encapsulation efficiency (%) = ×100% Drug loading (%) = ×100% 2. The experimental results are shown in Table 1: Table 1. Physicochemical properties of Alb-REG in Example 1

[0059] Results analysis: Nanoparticles with uniform particle size and high encapsulation efficiency were prepared by varying the mass ratio of Alb to REG from 1:1 to 50:1. However, the particle size of the particles prepared by ultrasonic emulsification was too large. Therefore, high-pressure homogenization was subsequently used to optimize the particles.

[0060] Example 2 This embodiment uses a high-pressure homogenization method to prepare albumin nanomedicine (Alb-REG) encapsulated with REG. A schematic diagram of the preparation method is shown below. Figure 2 The specific preparation method is as follows: Weigh 90 mg of albumin and dissolve it in 18 mL of ultrapure water. Keep the solution on ice. Weigh the corresponding mass of REG according to different feed ratios (see Table 2), dissolve it in 1.5 mL of chloroform-ethanol mixture to prepare the REG organic phase, and keep it on ice. Mix the albumin aqueous solution with the REG organic phase and disperse it using a high-speed shear mixer to form a primary emulsion. Pre-cool the material channel of the high-pressure homogenizer and transfer the primary emulsion to the pre-cooled feed inlet of the high-pressure homogenizer. Set the homogenization pressure to 10000~25000 psi and homogenize to obtain a milky white nano-suspension. Quickly transfer the nano-suspension to a 100 mL round-bottom flask, remove the organic reagents by rotary evaporation to obtain Alb-REG, which is then lyophilized and stored.

[0061] Physicochemical properties of Alb-REG in Example 2 1. Testing Method (1) The particle size, potential, particle size stability and dilution stability of Alb-REG were determined by dynamic light scattering analyzer; (2) Transmission electron microscopy was used to photograph the Alb-REG morphology; (3) High performance liquid chromatography was used to detect the encapsulation efficiency and drug loading of Alb-REG (same as above).

[0062] 2. Experimental Results: Encapsulation efficiency and drug loading are shown in Table 2: Table 2. Physicochemical properties of Alb-REG in Example 2

[0063] Figure 3 It is w Alb :w REG Physicochemical properties of Alb-REG prepared at a ratio of 16:1 (this Alb-REG was used in subsequent cell and animal experiments), including (A) particle size distribution diagram; (B) Zeta potential diagram; (C) particle size stability test diagram; (D) dilution stability test diagram; Figure 4 For w Alb :w REG Scanning electron microscope image of Alb-REG prepared at a ratio of 16:1; Results analysis: High-pressure homogenization can also be used to prepare nanoparticles with uniform particle size and high encapsulation efficiency from Alb and REG with different mass ratios. Moreover, compared with ultrasonic emulsification, the prepared particles have smaller and more uniform particle size.

[0064] Investigation of Alb-REG's toxicity to tumor cells 1. Experimental procedure: The in vitro cytotoxicity of REG and Alb-REG cells against CT26 and MC38 tumor cells was detected by the MTT assay. Tumor cells were digested and counted, and the cells were resuspended in culture medium to a density of 5 × 10⁻⁶ cells / mL. 4 Cells / mL, seeded into 96-well plates, 100 μL per well, and cultured overnight until cell adhesion. REG and Alb-REG were dispersed in preheated complete medium to prepare the drug delivery solution and co-cultured for 24 or 48 hours. Then, 10 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for 4 hours. The supernatant was discarded, and 150 μL of DMSO solution was added to each well. The plates were shaken for 10 min to fully dissolve the formazan crystals at the bottom. The absorbance of the sample wells was read at 490 nm using a multi-mode microplate reader.

[0065] 2. Experimental Results: Figure 5 To investigate the toxicity of REG and Alb-REG to tumor cells, after 24 hours of drug treatment, the toxicity of the particles was lower than that of the free drug; after 48 hours, as the drug in the particles was gradually released, the toxicity of the particles was comparable to that of the free drug.

[0066] An investigation into the effects of Alb-REG on macrophage reprogramming 1. Experimental procedure: (1) Macrophage extraction C57 mice were euthanized by cervical dislocation. Aseptic procedures were performed in a laminar flow hood. The hind limb muscles were dissected and washed three times with 1×PBS. The ends of the bone were cut to expose the red bone marrow. Using a 1 mL disposable sterile syringe, 1×PBS was drawn and rinsed from one end of the bone marrow cavity, repeating the process until the cavity was colorless. Muscle tissue was then removed by filtration through a sterile steel mesh. Bone marrow cells were collected by centrifugation at 450g for 5 min. 1 mL of erythrocyte lysis buffer was added, and the erythrocytes were fully lysed at 4°C. After 5 min, 10 times the volume of 1×PBS was added to terminate lysis, and the cells were collected by centrifugation. The bone marrow cells were resuspended and cultured in complete culture medium containing M-CSF cytokines at 37°C. Mature M0 macrophages were obtained on day 5.

[0067] (2) mRNA level Mature M0 macrophages were digested, counted, and seeded into 6-well plates at 800,000 cells / well. IL-4 was added for 24 hours to induce M0 macrophages to polarize into M2 macrophages, which were indicated by elongated strips. The culture medium was discarded, and the cells were treated with PBS, 2.5 μM REG, and Alb-REG (each particle contained an equal amount of 2.5 μM REG). After culturing for 12 hours, the supernatant of the 6-well plates was discarded, and the cells were washed twice with PBS. Cells were collected from the 6-well plates using Trizol, and RNA was extracted according to the RNA extraction procedure. The relative gene expression of macrophage M1 and M2 markers was detected by RT-qPCR.

[0068] (3) Protein level Mature M0 macrophages were digested, counted, and seeded into 24-well plates at 200,000 cells / well. IL-4 was added to induce M0 macrophages to polarize into M2 macrophages, which were indicated by their elongated shape. The culture medium was then discarded, and the cells were treated with PBS, 2.5 μM REG, and Alb-REG (each particle contained an equal amount of 2.5 μM REG). After 48 hours of culture, the cells in the 24-well plates were digested and collected. The M1 marker CD86 and the M2 marker CD206 were labeled with antibodies, and protein expression was detected by flow cytometry.

[0069] 2. Experimental Results: Figure 6 Reprogramming the relative gene expression of macrophages using Alb-REG; Figure 7 Reprogramming macrophage protein expression levels using Alb-REG; from Figure 6-7 It can be seen that, compared with the free drug REG, the particle Alb-REG more significantly upregulated the expression of M1-related genes; when the repolarization effect of the free drug was weak, the particle significantly downregulated the expression of M2 macrophage-related proteins and upregulated the expression of M1 macrophage-related proteins.

[0070] An investigation into the inhibitory effect of Alb-REG on the growth of CT26 peritoneal tumors: 1. The experimental procedure is as follows: Figure 8 The experimental method for mice is shown in the diagram below: (1) Construction of the CT26 peritoneal tumor-bearing mouse model: 0.5 mL of CT26-Luc cell suspension (500,000 cells / mouse) was injected into the peritoneal cavity of BALB / c mice, and the abdomen of the mice was massaged to ensure uniform cell distribution. On day 6 after cell implantation, bioluminescent images were captured using small animal in vivo imaging to detect the size of the peritoneal tumor.

[0071] (2) Study on the therapeutic effect of Alb-REG on the CT26 peritoneal tumor model: CT26 mice with peritoneal tumors were randomly divided into three groups, and each mouse was then intraperitoneally injected with 0.5 mL of a drug solution. The specific groups were: ①PBS; ②REG; ③Alb-REG. The ②REG group received a drug dose of 5 mg / kg REG, and the ③Alb-REG group also received a controlled dose of 5 mg / kg REG. Administered the drug every three days for a total of three doses. Mice survival was observed.

[0072] 2. Experimental Results: Table 3. Mouse survival status

[0073] Figure 9 The survival rate of Alb-REG in mouse experiments was analyzed, and the results showed that Alb-REG had a better tumor-suppressing effect than free REG and significantly prolonged the survival of mice.

[0074] Example 3 This embodiment uses an ultrasonic method to prepare albumin nanomedicine (Alb-RP) simultaneously encapsulating REG and PTX. A schematic diagram of the ultrasonic preparation method is shown below. Figure 10 As shown, the specific preparation method is as follows: Weigh 0.3 mg of REG and dissolve it in 150 μL of a chloroform-ethanol mixture to prepare a REG organic phase solution, which is then kept in an ice bath for later use. Weigh PTX according to different feed ratios and dissolve it in the above organic phase to prepare REG + PTX organic phase solutions with different ratios (1:0, 1:0.05, 1:0.25:1:0.5, 1:0.8, 1:1, 1:2, 1:4, 1:8, 1:20, 0:1). Weigh 18 mg of Alb and add it vertically to a 5 mL EP tube with 3.6 mL of ultrapure water. Then, sonicate the solution in an ice bath with the following parameters: 200–400 W power and 2–8 min sonication time. After sonication, a milky white nano-suspension was obtained, which was quickly transferred to a round-bottom flask and rotary evaporated to remove organic reagents, yielding albumin nanomedicine (Alb-RP) co-loaded with REG and PTX, which was then lyophilized and stored.

[0075] Physicochemical properties of Alb-RP in Example 3 1. Testing method: The particle size, polydispersity index (PDI), potential, and stability of Alb-RP were determined using a dynamic light scattering analyzer. The morphology of Alb-RP was captured by transmission electron microscopy.

[0076] 2. Experimental Results: The particle size, PDI, and potential of Alb-RP are shown in Table 4. Table 4. Particle size, PDI, and potential of Alb-RP in Example 3

[0077] Figure 11 This is a scanning electron microscope image of albumin nanomedicine loaded with REG and PTX prepared by an ultrasonic preparation method, combined with... Figure 11 Analysis of the results in Table 4 shows that the particle size of particles prepared with different feed ratios is relatively uniform.

[0078] The stability of Alb-RP is shown in Table 5: Table 5. Particle size (unit: nm) changes of Alb-RP in Example 3 at different times.

[0079] Note: " / " indicates that there is no data on uniform particle size.

[0080] Results analysis: The particles prepared with different feed ratios all showed good stability within 6 months.

[0081] Example 4 This embodiment uses a high-pressure homogenization method to prepare albumin nanomedicine (Alb-RP) co-loaded with PTX and REG. A schematic diagram of the high-pressure homogenization preparation method is shown below. Figure 12 The specific preparation method is as follows: Weigh 90 mg of albumin and dissolve it in 18 mL of ultrapure water. Keep it on ice. Weigh 1.5 mg of REG and different proportions of PTX (see Table 6), dissolve them in 750 μL of chloroform-ethanol mixture to prepare the organic phase. Keep it on ice. Mix the albumin aqueous solution with the organic phase and disperse it using a high-speed shear mixer to form a primary emulsion. Pre-cool the material channel of the high-pressure homogenizer and transfer the primary emulsion to the pre-cooled feed inlet of the high-pressure homogenizer. Homogenize at 10,000–25,000 psi to obtain a milky white nano-suspension. Quickly transfer the nano-suspension to a 100 mL round-bottom flask, remove the organic reagents by rotary evaporation, and obtain Alb-RP. Freeze-dry and store.

[0082] Physicochemical properties of Alb-RP in Example 4 1. Testing method: (1) The particle size, potential, particle size stability and dilution stability of Alb-RP were determined by dynamic light scattering analyzer; (2) Transmission electron microscopy was used to photograph the morphology of Alb-RP; (3) High performance liquid chromatography was used to detect the encapsulation efficiency, drug loading, and release curve of the drug in Alb-RP; Establishment of high-performance liquid chromatography (HPLC) detection method: Weigh 2 mg each of REG and PTX, dissolve them separately in 1 mL of methanol, and sonicate to ensure complete dissolution, obtaining a stock solution with a final concentration of 2 mg / mL. Dilute the stock solution with methanol to obtain solutions of different concentrations (250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.3 μg / mL, 15.6 μg / mL, 7.8 μg / mL, 3.9 μg / mL). Detection was performed using HPLC. Instrument model: Shimadzu LC-16; Column specifications: Shimadzu, Shim-pack Scepter C18-120 5 μm, 4.6 × 250 mm; Mobile phase: 0.1% acetic acid / water: acetonitrile = 30:70 (v / v); Flow rate: 1 mL / min; Column temperature: 25 ℃; Injection volume: 20 μL; Detection wavelength: 227 nm (PTX) and 260 nm (REG). Inject the above solutions of REG and PTX at different concentrations, measure their peak areas, and plot regression curves.

[0083] The lyophilized Alb-RP particles were reconstituted with 1 mL of ultrapure water, centrifuged at 3000 rpm for 5 min to obtain free REG and PTX, dissolved thoroughly in methanol by sonication, filtered through a 220 nm filter membrane, and the free drug content was determined by HPLC. The encapsulation efficiency and drug loading of Alb-RP were calculated using the difference method. The formulas for calculating the drug encapsulation efficiency and drug loading are as follows: Encapsulation efficiency (%) = ×100% Drug loading (%) = ×100% In vitro release studies were conducted using dialysis. A certain amount of Alb-RP was placed in a dialysis bag, immersed in 20 mL of PBS solution (containing 2% (v / v) Tween 80), and placed in a 37°C water bath with continuous shaking. At different time points (1, 2, 4, 8, 12, 24, 36, 48, and 72 hours), 1 mL of PBS was collected and replaced with an equal volume of fresh release medium. After filtration through a 220 nm filter, the peak area was determined by HPLC, and the drug concentration was calculated based on a standard curve.

[0084] 2. Experimental Results: (1) The results of encapsulation efficiency and drug loading are shown in Table 6: Table 6 Encapsulation efficiency and drug loading

[0085] Results analysis: High-pressure homogenization optimizes particle size to be smaller and more uniform, while maintaining high encapsulation efficiency and drug loading.

[0086] (2) Particle size, electric potential, and stability Figure 13 is n REG :n PTX Physicochemical properties of Alb-RP prepared at a ratio of 1:4 (this Alb-RP was used in subsequent cell and animal experiments), including (A) particle size distribution diagram; (B) Zeta potential diagram; (C) particle size stability test diagram; (D) dilution stability test diagram; Figure 14 For n REG :n PTX Scanning electron microscopy (SEM) images of albumin nanomedicines loaded with REG and PTX prepared by Alb-RP with a ratio of 1:4; it can be seen that the particles prepared by the high-pressure homogenization method are smaller and more uniform in size than those prepared by the ultrasonic emulsification method.

[0087] (3) Release curve Figure 15 For n REG :n PTX The release curve of Alb-RP prepared by a ratio of 1:4 shows that particles prepared by high-pressure homogenization can slowly release REG and PTX within 80 hours.

[0088] Alb-RP-regulated macrophage function verification 1. Promotes macrophage repolarization (1) Experimental procedure: C57 mice were euthanized by cervical dislocation. Aseptic procedures were performed in a laminar flow hood. The hind limb muscles were dissected and washed three times with 1×PBS. The ends of the bone were cut to expose the red bone marrow. Using a 1 mL disposable sterile syringe, 1×PBS was drawn and rinsed from one end of the bone marrow cavity, repeating the process until the cavity was colorless. Muscle tissue was then removed by filtration through a sterile steel mesh. Bone marrow cells were collected by centrifugation at 450g for 5 min. 1 mL of erythrocyte lysis buffer was added, and the erythrocytes were fully lysed at 4°C. After 5 min, 10 times the volume of 1×PBS was added to terminate lysis, and the cells were collected by centrifugation. The bone marrow cells were resuspended and cultured in complete culture medium containing M-CSF cytokines at 37°C. Mature M0 macrophages were obtained on day 5.

[0089] mRNA level: Mature M0 macrophages were digested, counted, and seeded into 6-well plates at 800,000 cells / well. IL-4 was added to induce M0 macrophages for 24 hours, which polarized M0 macrophages into M2 macrophages, indicating successful induction. The culture medium was discarded, and the macrophages were treated with PBS, REG, PTX, REG + PTX, or Alb-RP (5 μM for single-drug groups, and 1 μM REG and 4 μM PTX for combined and particulate groups). After culturing for 12 hours, the supernatant of the 6-well plates was discarded, and the macrophages were washed twice with PBS. Cells were collected from the 6-well plates using Trizol, and RNA was extracted according to the RNA extraction procedure. The relative gene expression of macrophage M1 and M2 markers was detected by RT-qPCR.

[0090] Protein levels: Mature M0 macrophages were digested, counted, and seeded into 24-well plates at 200,000 cells / well. IL-4 was added to induce M0 macrophages for 24 hours, which polarized M0 macrophages into M2 macrophages. The appearance of elongated strips indicated successful induction. The culture medium was discarded, and the cells were treated with the same drug as above. After 48 hours of culture, the cells in the 24-well plates were digested and collected. The M1 marker CD86 and the M2 marker CD206 were labeled with antibodies, and the protein expression was detected by flow cytometry.

[0091] (2) Experimental results: Figure 16 Alb-RP was used to reprogram the relative gene expression of macrophages. Figure 17 Alb-REG was used to reprogram the protein expression levels of macrophages. Compared with single-drug therapy, the combination of PTX and REG significantly upregulated the expression of genes and proteins related to M1 macrophages and downregulated the expression of genes and proteins related to M2 macrophages. Alb-RP showed better repolarization ability than free PTX+REG and could more significantly reprogram M2 macrophages into M1 macrophages.

[0092] 2. Enhance the phagocytic ability of macrophages (1) Experimental procedure: M0 macrophages were extracted in the same manner as described above; mature M0 macrophages were digested, counted, and seeded into 24-well plates at 200,000 cells / well. IL-4 was added to induce M0 macrophages for 24 hours until they polarized to M2 and formed elongated strips, indicating successful induction. The culture medium was discarded, and the cells were treated with the same drug as described above. After 24 hours of drug treatment, the cells were washed three times with PBS, and CFSE-labeled MC38 tumor cells were added at 50,000 cells / well. The cells were co-cultured for another 24 hours. The cells were collected and stained with antibody (APC-F4 / 80) and analyzed by flow cytometry.

[0093] (2) Experimental results: Figure 18The results of experiments on enhancing the phagocytic capacity of macrophages in each experimental group; compared with single drug, the combination of PTX and REG significantly enhanced the ability of macrophages to phagocytose MC38 tumor cells; Alb-RP has a similar ability to promote macrophage phagocytosis as free PTX+REG.

[0094] 3. Enhance the ability of macrophages to kill tumor cells. (1) Experimental procedure: M0 macrophages were extracted in the same manner as described above; mature M0 macrophages were digested, counted, and seeded into 96-well plates at 20,000 cells / well. IL-4 was added to induce M0 macrophages for 24 hours until they polarized to M2 and formed elongated strips, indicating successful induction. The culture medium was discarded, and the cells were treated with the drug as described above. After 48 hours of drug treatment, the culture medium was discarded, and the cells were washed twice with PBS. 5 × 10⁶ IL-4 was added to each well. 3 MC38 tumor cells were used, and a separate tumor cell group was set up. After 24 hours, the culture medium was discarded, 50 μL of cell lysis buffer was added, and 40 μL was transferred to a white plate. 40 μL of firefly luciferase assay reagent was added at a 1:1 ratio, and after mixing, the chemiluminescence signal value (RLU) was detected using a multi-functional microplate reader.

[0095] (2) Experimental results: Figure 19 To enhance the killing ability of macrophages against tumor cells in each experimental group; compared with single drug, the combination of PTX and REG significantly enhanced the ability of macrophages to kill MC38 tumor cells; Alb-RP has a similar ability to promote the killing of MC38 tumor cells by macrophages as free PTX+REG.

[0096] Investigation into the inhibitory effect of Alb-RP on the growth of MC38 peritoneal tumors: 1. The experimental procedure is as follows: Figure 20 As shown, the specific experimental method is as follows: Construction of MC38 peritoneal tumor-bearing mouse model 0.5 mL of MC38-Luc cell suspension (1 million cells / mouse) was injected into the peritoneal cavity of C57 mice, and the abdomen of the mice was massaged to ensure uniform cell distribution. On day 7 after cell implantation, bioluminescent images were captured using small animal in vivo imaging to detect the size of the peritoneal tumor.

[0097] Study on the therapeutic effect of Alb-RP on MC38 peritoneal tumor model MC38 mice with peritoneal tumors were randomly divided into groups, and each mouse was then intraperitoneally injected with 0.5 mL of the drug solution. The specific groups were: ①PBS; ②REG; ③PTX; ④REG+PTX; ⑤Alb-REG; ⑥Alb-PTX; ⑦Alb-RP. The drug dosage was 10 mg / kg PTX and 1.4 mg / kg REG. The drugs were administered every three days for a total of two doses. The growth of the peritoneal tumor in the mice was monitored weekly using small animal in vivo imaging.

[0098] 2. Experimental Results Figure 21 In vivo bioluminescence images of Alb-RP used in mouse experiments; Figure 22 Quantitative analysis of abdominal bioluminescent flux of Alb-RP in mouse experiments; Figure 23 The median survival rate of Alb-RP in mouse experiments is shown in Table 7. Table 7

[0099] Results analysis: Alb-RP showed better tumor-suppressing effects than the combination of free drugs PTX and REG, and was also significantly better than single-drug particles, significantly prolonging the survival time of mice.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An albumin formulation encapsulating regorafenib, characterized in that, It includes the following components: regorafenib and albumin; the albumin formulation is a nanoparticle formulation.

2. The method for preparing the albumin formulation loaded with regorafenib according to claim 1, characterized in that, The albumin formulation containing regorafenib was prepared using ultrasonic emulsification. Alternatively, the albumin formulation containing regorafenib can be prepared using a high-pressure homogenization method.

3. The use of the albumin formulation containing regorafenib as described in claim 1 in any of the following aspects: A) Prepare drugs for activating the tumor immunosuppressive microenvironment; B) Preparation of anti-tumor drugs. C) Prepare sensitizers for use in sensitizing chemotherapy drugs.

4. A pharmaceutical composition, characterized in that, It includes the following components: regorafenib, paclitaxel, and albumin.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is in nanoparticle form.

6. A method for preparing the pharmaceutical composition according to claim 4 or 5, characterized in that, The pharmaceutical composition was prepared by ultrasonic emulsification. Alternatively, the pharmaceutical composition may be prepared using a high-pressure homogenization method.

7. The method for preparing the pharmaceutical composition according to claim 6, characterized in that, The ultrasonic emulsification process includes: dissolving regorafenib and paclitaxel in an organic solvent to form an oil phase solution, and dissolving albumin in a solvent to form an aqueous phase solution; adding the oil phase solution to the aqueous phase solution, and performing ultrasonic emulsification under ice bath conditions to form a nano-suspension; removing the organic solvent to obtain the drug composition.

8. The method for preparing the pharmaceutical composition according to claim 6, characterized in that, The high-pressure homogenization process includes: dissolving regorafenib and paclitaxel in an organic solvent to form an oil phase solution, and dissolving albumin in a solvent to form an aqueous phase solution; mixing the oil phase solution and the aqueous phase solution to form an emulsion; homogenizing the emulsion under high pressure using a homogenizer to form a nano-suspension, removing the organic solvent, and obtaining the pharmaceutical composition.

9. The use of the pharmaceutical composition comprising regorafenib and paclitaxel as described in claim 4 or 5 in any of the following aspects: A) Prepare drugs for activating the tumor immunosuppressive microenvironment; B) Preparation of anti-tumor drugs. C) Prepare sensitizers for use in sensitizing chemotherapy drugs.

10. The application according to claim 9, characterized in that, The tumor is a solid tumor.

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