A lenvatinib-polyethylene glycol conjugate compound, and a preparation method and application thereof

Abstract

The application discloses a lenvatinib-polyethylene glycol coupling compound and a preparation method and application thereof. The lenvatinib-polyethylene glycol coupling compound has a structure as shown in the formula (I): in the formula (I), n is greater than or equal to 2. The application further provides a nano preparation containing vinpocetine and the lenvatinib-polyethylene glycol coupling compound and used for manufacturing drugs, which can be applied to tumor treatment, effectively improves the limitation of a single drug treatment method on tumor inhibition, and shows a positive application potential in the tumor treatment field.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, in particular to a lenvatinib-polyethylene glycol conjugate compound and a preparation method and application thereof. BACKGROUND

[0002] Cancer is a disease caused by uncontrolled growth and division of abnormal cells, which seriously threatens human health. Drug therapy is the main anti-tumor treatment, but many drugs face problems such as large toxic side effects, poor targeting, drug resistance, etc. With the continuous development of nanoscience in the field of medicine, in the treatment of cancer, nanomedicines such as Doxil (PEGylated doxorubicin liposome), Abraxane (paclitaxel albumin nanosuspension), Lipusu (paclitaxel liposome) have been successfully listed. The latest clinical data analysis results show that, compared with traditional anti-tumor drugs, these nanomedicines show the advantages of safety and low toxicity, but cannot significantly improve the clinical efficacy. This limited single treatment often cannot meet the clinical needs, therefore, the combination of multiple drugs or multiple treatment methods will have better clinical practical application.

[0003] Lenvatinib is a multi-targeted tyrosine kinase inhibitor, mainly used for the treatment of thyroid cancer, colorectal cancer, kidney cancer and other cancers. It exerts anti-tumor effect by inhibiting multiple receptor tyrosine kinase pathways, including inhibition of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR) pathways. By simultaneously inhibiting these key signaling pathways, lenvatinib can block tumor growth and spread, hinder the formation of new blood vessels, and resist the occurrence and development of tumors. However, a large number of clinical practices show that lenvatinib also faces some problems in clinical use, including drug resistance and a series of adverse reactions. Therefore, developing lenvatinib nanomedicines to improve its clinical therapeutic effect is an effective and promising treatment strategy. For example, the patent specification with publication number CN108567735A discloses a nano-carrier drug for precise targeted therapy of liver cancer and a preparation method, including the following preparation process: (1) adding lenvatinib raw drug into methanol and urea to form a dispersion liquid; (2) adding soluble divalent metal salt, soluble trivalent metal salt, and low-concentration hydrogen peroxide solution into the dispersion liquid, stirring and heating for condensation reflux reaction; (3) using aqueous solution of sulfamic acid for anion intercalation modification, and after completion, washing and drying to obtain a nano-scale liver cancer precise targeted carrier drug.

[0004] Vinpocetine is a phosphodiesterase-1 (PDE1) inhibitor drug, which is widely used in the treatment of cardiovascular diseases. It has multiple pharmacological effects, including neuroprotection, promoting metabolism, improving cerebral microcirculation, etc. In clinical practice, it is mainly used to improve the symptoms of dizziness, tinnitus, headache, dizziness, numbness of limbs, etc. induced by sequelae of cerebral infarction, sequelae of cerebral hemorrhage, cerebral arteriosclerosis, etc. Existing studies have shown that vinpocetine can effectively inhibit the growth of breast cancer cells in vitro and in vivo, and combined treatment with drugs such as cisplatin can effectively increase drug treatment sensitivity and reduce acute kidney injury. Vinpocetine combined with multi-target kinase inhibitor sorafenib can exert synergistic anti-hepatoma activity by activating GSK-3β, and may reverse the sorafenib resistance signal axis through PI3K / protein kinase B / GSK-3β. These studies show that vinpocetine has unique advantages in the combination of anti-tumor drugs, especially considering the off-target effect of multi-target kinase inhibitors, the preparation of vinpocetine and lenvatinib nanometer preparation will help to achieve the targeted treatment of anti-tumor drugs.

[0005] In recent years, the self-assembly strategy without carriers has been used to prepare new types of nanomedicines, i.e. carrier-free nanomedicine delivery systems, which have attracted extensive interest from researchers. The assembly of such drug delivery systems is based on the interactions between drug molecules, including stacking, hydrogen bonding, hydrophobic interaction, electrostatic interaction, etc. Single or synergistic forces can drive drug molecules to self-assemble into stable, nano-drugs with specific structures. Such carrier-free nanomedicine delivery systems not only retain the excellent properties of traditional nanomedicines, but also have the advantages of mild preparation conditions, high drug loading capacity, and combination therapy. However, the stability and targeting of such carrier-free nanomedicine systems are usually poor, and further modification is needed to achieve targeted aggregation therapy in the lesion part. To solve this problem, it has been proven that the hydrophilic polymer coating on the surface of the carrier-free nanomedicine can further improve the stability of the nanoparticles, reduce non-specific adsorption of proteins, and prolong the blood circulation time. SUMMARY

[0006] Based on the above background, the present application constructs a lenvatinib-polyethylene glycol conjugate compound, and further constructs a new nanodelivery system based on lenvatinib and vinpocetine, i.e. a nanometer preparation containing vinpocetine and lenvatinib-polyethylene glycol conjugate compound for making drugs, which can be applied to tumor treatment. The nanometer preparation provided by the present application is a tumor-targeted release nanoparticle co-assembled by vinpocetine and lenvatinib-polyethylene glycol conjugate compound. The present application integrates the combination strategy and precise targeted therapy into the same drug delivery platform, effectively improves the limitations of single drug treatment on tumor inhibition, and shows positive application potential in the field of tumor treatment.

[0007] The specific technical solutions are as follows:

[0008] In a first aspect, the present application provides a lenvatinib-polyethylene glycol conjugate compound having a structure as shown in formula (I):

[0009]

[0010] In formula (I), n≥2, further optionally, n=2-300, and more further optionally, n=100-125.

[0011] In a second aspect, the present application provides a preparation method of the lenvatinib-polyethylene glycol conjugate compound of the first aspect, comprising:

[0012] adding an acetonitrile solution containing lenvatinib, sodium p-toluenesulfinate monohydrate, and biotin-polyethylene glycol-aldehyde having a structure as shown in formula (II) to trimethylchlorosilane (TMSCl) under ice-bath, performing a first room temperature reaction, removing the solvent under reduced pressure after the reaction is completed, washing the obtained product, dissolving the product in dichloromethane after vacuum drying, adding Cs2CO3 and Na2SO4, and performing a second room temperature reaction under inert atmosphere, adding hexane after the reaction is completed, filtering, collecting the filtrate, removing the solvent under reduced pressure, and obtaining the lenvatinib-polyethylene glycol conjugate compound;

[0013]

[0014] In formula (II), n≥2, further optionally, n=2-300, and more further optionally, n=100-125.

[0015] The biotin-polyethylene glycol-aldehyde described in the present application can be obtained by existing technology or commercial channels.

[0016] In the preparation method of the second aspect, the molar ratio of lenvatinib and biotin-polyethylene glycol-aldehyde can be 1-2:1.

[0017] In the preparation method of the second aspect, the molar ratio of sodium p-toluenesulfinate monohydrate and lenvatinib can be 1:1.

[0018] In the preparation method of the second aspect, the molar ratio of trimethylchlorosilane and biotin-polyethylene glycol-aldehyde can be 1.5-2.5:1.

[0019] In the preparation method of the second aspect, the time of the first room temperature reaction can be 60-84 hours.

[0020] In the preparation method of the second aspect, the washing agent used in the washing can be hexane and diethyl ether.

[0021] In the preparation method of the second aspect, the Cs2CO3, Na2SO4 and the product can be used in a molar ratio of 1:1:1.

[0022] In the preparation method of the second aspect, the inert atmosphere refers to a gas atmosphere that does not participate in the reaction, and can be nitrogen or the like.

[0023] In the preparation method of the second aspect, the second room temperature reaction can be performed for 4-6 hours.

[0024] In a third aspect, the present application provides a nano-preparation comprising vinpocetine and the lenvatinib-polyethylene glycol conjugate compound of the first aspect.

[0025] In the nano-preparation of the third aspect, the effective component can include nanoparticles formed by co-assembly of vinpocetine and the lenvatinib-polyethylene glycol conjugate compound. The nanoparticles can be nano-assemblies formed by non-covalent forces, π-π stacking, hydrophobic interaction, intermolecular hydrogen bonding, etc.

[0026] Further, the co-assembly process can be performed in an aqueous phase, i.e., the nanoparticles can be formed in an aqueous phase.

[0027] In a fourth aspect, the present application provides a preparation method of the nano-preparation of the third aspect, comprising: adding an organic solution containing vinpocetine and the lenvatinib-polyethylene glycol conjugate compound to water, and stirring to obtain the nano-preparation.

[0028] In the preparation method of the fourth aspect, the molar ratio of the vinpocetine and the lenvatinib-polyethylene glycol conjugate compound in the organic solution can be 1:1-10, for example, 1:4, etc. Vinpocetine and the lenvatinib-polyethylene glycol conjugate compound in the above-mentioned molar ratio range can form better co-assembly nanoparticles.

[0029] In the preparation method of the fourth aspect, the organic solvent in the organic solution can include one or a combination of the other of methanol and dimethyl sulfoxide. Further, the organic solvent can include methanol and dimethyl sulfoxide. Still further, in the organic solvent, the volume ratio of the methanol and the dimethyl sulfoxide is 1:1.

[0030] The preparation method of the fourth aspect can further include a post-treatment operation after the stirring. The post-treatment operation can include centrifuging and washing the nanoparticles spontaneously formed by stirring with water to obtain the nano-preparation.

[0031] The preparation method of the fourth aspect can be performed at -40-40°C (for example, room temperature, 25°C, etc.).

[0032] In a fifth aspect, the present application provides the use of the lenvatinib-polyethylene glycol conjugate compound of the first aspect or the nano-formulation of the third aspect in the preparation of a medicament for preventing and / or treating cancer.

[0033] The present application rationally designs the multi-target kinase inhibitor drug lenvatinib, covalently couples lenvatinib with biotin and aldehyde-terminated polyethylene glycol through an imine bond, constructs a pH-responsive lenvatinib prodrug (i.e., a lenvatinib-polyethylene glycol conjugate compound), which targets tumor tissues using biotin, and the hydrazone bond connecting lenvatinib and polyethylene glycol can be broken in the acidic microenvironment of the tumor, thereby achieving drug release and exerting anti-tumor activity, which effectively avoids systemic toxic side effects and enhances the anti-tumor therapeutic effect of the drug.

[0034] The present application also provides a nano-sized drug delivery system (i.e., the nano-formulation) simultaneously loaded with a multi-target kinase inhibitor and a PDE1 inhibitor, which achieves efficient targeted drug delivery, reduces the systemic exposure level of the drug and increases the effective drug accumulation concentration at the tumor site, and overcomes the problems of existing anti-tumor drugs, such as poor water solubility, short half-life, unstable molecular structure, complex in vivo metabolism, poor target organ selectivity, and obvious side effects, thereby comprehensively improving the tumor treatment effect. Compared with the prior art, the present application provides a new, safe and effective drug delivery strategy for combination therapy.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The prepared lenvatinib-polyethylene glycol conjugate compound and the nano-formulation containing vinpocetine and the lenvatinib-polyethylene glycol conjugate compound have a simple and efficient preparation process, integrate the combination strategy and precise targeted therapy into the same drug delivery platform, effectively improve the limitations of kinase inhibitors on tumor inhibition, reduce the systemic exposure level of the drug and increase the effective drug accumulation concentration at the tumor site, and show positive application potential in the field of anti-tumor therapy. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a nuclear magnetic resonance spectrum for the characterization of the lenvatinib-polyethylene glycol conjugate compound.

[0038] Figure 2 It is a characterization result graph of the vinpocetine-lenvatinib co-assembled nanoparticles (V-L NPs), in which: A is a transmission electron microscope (TEM) photo; B and C are ultraviolet absorption under different conditions.

[0039] Figure 3Figure 1 is a result chart for characterization of a nanoformulation (PEG-Len / Vin NPs) containing vinpocetine (Vin) and lenvatinib (Len)-polyethylene glycol conjugated compound, in which: Figure A is a transmission electron microscope (TEM) photo; Figure B is a scanning electron microscope (SEM) photo; and Figure C is a nano particle size analyzer particle size determination result chart.

[0040] Figure 4 Figure 2 is a result chart for in vitro stability test of the PEG-Len / Vin NPs nanoformulation, in which: Figure A is a hemolysis test result chart; and Figure B is a hemolysis experiment quantitative result chart.

[0041] Figure 5 Figure 3 is a release curve chart for Vin and Len, in which: Figure A is a release curve chart for Vin release from the PEG-Len / Vin NPs nanoformulation under different pH conditions; and Figure B is a release curve chart for Len release from the PEG-Len / Vin NPs nanoformulation under different pH conditions.

[0042] Figure 6 Figure 4 is a chart for in vitro effect of different drugs or formulations on human colorectal cancer HCT116 cell and RKO cell proliferation, in which: Figure A is a chart for effect of control (Control), Vin, Len and PEG-Len / Vin NPs on HCT116 cell and RKO cell proliferation detected by MTT method; and Figure B is a chart for effect of different drug or formulation treatment on long-term proliferation of HCT116 cell and RKO cell detected by colony cloning experiment.

[0043] Figure 7 Figure 5 is a chart for in vitro effect of drugs or formulations on HCT116 cell cycle and apoptosis, in which: Figure A is a chart for effect of Control, Vin, Len and PEG-Len / Vin NPs on HCT116 cell cycle detected by flow cytometry; and Figure B is a chart for effect of Control, Vin, Len and PEG-Len / Vin NPs on HCT116 cell apoptosis detected by flow cytometry.

[0044] Figure 8 Figure 6 is a chart for in vivo anti-tumor effect of drugs or formulations, in which: Figure A is a chart for volume measurement of mouse tumor for two consecutive weeks; Figure B is a chart for mouse body weight record for two consecutive weeks; Figure C is a chart for comparison of mouse dissected tumor mass; and Figure D is a chart for mouse dissected tumor photo. DETAILED DESCRIPTION

[0045] The application will be further described in conjunction with the accompanying drawings and specific examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. The operation methods in the following examples without specific conditions are generally according to the conventional conditions or the conditions suggested by the manufacturers. The raw materials used in the application can be obtained by commercial channels or synthesized according to the prior art, unless otherwise specified. The raw materials used in the application can be obtained by commercial channels or synthesized according to the prior art, unless otherwise specified.

[0046] Example 1 Preparation of co-assembled nanoparticles of vinpocetine and lenvatinib (V-L NPs)

[0047] A certain amount of vinpocetine and lenvatinib were respectively dissolved in a mixed solvent (methanol / dimethyl sulfoxide = 50 / 50, v / v) to prepare a 25 mM vinpocetine solution and a 25 mM lenvatinib solution.

[0048] The molar ratio of vinpocetine to lenvatinib was designed to be 1:1. The mixed solution (500 μL of vinpocetine solution and 500 μL of lenvatinib solution) was slowly added to 20 mL of deionized water under the condition of stirring speed 1000 rpm. After stirring at 25°C for 1 hour, the particles were separated by centrifugation at 12000 rpm for 15 minutes, and washed twice with double distilled water to remove excess organic solvent and free drugs, to obtain co-assembled nanoparticles.

[0049] Example 2 Preparation of co-assembled nanoparticles of vinpocetine and polyethylene glycol modified lenvatinib (PEG-Len / Vin NPs)

[0050] To synthesize the lenvatinib-polyethylene glycol conjugate compound with the structure shown in formula (I), first, lenvatinib (2.5 mmol) was dissolved in acetonitrile, and sodium p-toluenesulfinate monohydrate (2.5 mmol) and biotin-polyethylene glycol-aldehyde (i.e. biotin-PEG-aldehyde, 1.7 mmol, n = 100-125) with the structure shown in formula (II) were added. Then, the mixture was added dropwise with TMSCl (3.3 mmol) under ice bath, and reacted at room temperature for 72 hours. After the reaction was completed, the solvent was removed under reduced pressure, and washed with hexane and diethyl ether. After the obtained crude product was dried in vacuum, part of the crude product (1 mmol) was dissolved in dichloromethane, and dry Cs2CO3 (1 mmol) and Na2SO4 (1 mmol) were added. After stirring at room temperature for 5 hours under nitrogen protection, hexane was added, filtered, and the filtrate was concentrated under reduced pressure to obtain a white powder solid, i.e. the lenvatinib-polyethylene glycol conjugate compound. The nuclear magnetic resonance results are shown in formula (I): Figure 1 The aldehyde proton peak at 10.1 of the raw material biotin-PEG-aldehyde disappeared, and a single proton peak of N=CH appeared at 8.9, indicating that the aldehyde group in the raw material biotin-polyethylene glycol-aldehyde reacted with the amine group in lenvatinib.

[0051] To prepare the vinpocetine and PEG-modified lenvatinib co-assembled nanoparticles (PEG-Len / Vin NPs), a certain amount of vinpocetine and the lenvatinib-polyethylene glycol conjugate compound having the structure shown in formula (I) (wherein n is 100-125) synthesized according to the above method were dissolved in a mixed solvent (methanol / dimethyl sulfoxide = 50 / 50, v / v) to prepare a 25 mM vinpocetine solution and a 25 mM lenvatinib solution.

[0052] When the molar ratio of vinpocetine to lenvatinib-polyethylene glycol conjugate compound was designed to be 1:4, the mixed solution (200 μL of vinpocetine solution and 800 μL of lenvatinib solution) was slowly added to 20 mL of deionized water under the condition of stirring speed 1000 rpm. After stirring at 25°C for 1 hour, the particles were separated by centrifugation at 12000 rpm for 15 minutes, and washed twice with double distilled water to remove excess organic solvent and free drug, to obtain the co-assembled nanoparticles.

[0053] Example 3 Characterization of prodrugs

[0054] 3.1 Characterization of V-L NPs

[0055] To observe the morphological characteristics of the nanoparticles, the V-L NPs prepared in Example 1 were uniformly dispersed in water and added dropwise to a 300-mesh copper mesh coated with a special film to prepare a transmission electron microscopy sample. After several minutes of deposition, the solution was removed with filter paper, and the sample was dried at room temperature overnight. Transmission electron microscopy was used to record the electron microscopy images. The electron microscopy results show that the V-L NPs prepared in Example 1 have a near-spherical nanostructure (A). Figure 2

[0056] To further detect the composition and interaction of V-L NPs, ultraviolet absorption, infrared absorption, and X-ray diffraction under different conditions were carried out. The ultraviolet absorption spectrum shows (B) that when the solvent is dimethyl sulfoxide (DMSO), the absorption wavelength of part of Vin and Len is observed in the ultraviolet absorption of V-L NPs, indicating that V-L NPs are composed of Vin and Len, and when the solvent is a mixture of DMSO and water, the absorption wavelength at 262 nm in V-L NPs moves to the right, indicating that hydrogen bonds are involved in the self-assembly behavior, and the ultraviolet changes of V-L NPs under different concentrations of ZnCl2 are not obvious (C), indicating that ionic bonds are not involved in the self-assembly process of Vin and Len. Figure 2 Figure 2

[0057] 3.2 Characterization of PEG-Len / Vin NPs

[0058] ​​​To observe the morphology of the nanoparticles, the PEG-Len / Vin NPs prepared in Example 2 were uniformly dispersed in water, and then dropped onto a 300-mesh copper grid coated with a special film to prepare a transmission electron microscopy sample, and dropped onto a silicon wafer to prepare a scanning electron microscopy sample. After several minutes of deposition, the solution was removed with filter paper, and the sample was dried at room temperature overnight. Transmission electron microscopy and scanning electron microscopy were used to record the electron microscopy images, respectively. Both the transmission electron microscopy and the scanning electron microscopy results showed that the PEG-Len / Vin NPs were relatively uniform spherical nanostructures. Figure 3 A and Figure 3 B). In addition, the PEG-Len / Vin NPs had an average particle size of 121.8 nm and a particle size distribution PDI of 0.183 Figure 3 C) as determined by a nanoparticle size analyzer, indicating a relatively uniform particle size distribution.

[0059] 3.3 Drug in vitro stability test

[0060] Fresh blood of SD rats was taken into a 50-mL clean beaker, and fibrin was removed by stirring with a glass rod in the same direction. The blood was washed with physiological saline and centrifuged (1500 rpm, 15 min). The supernatant was removed and repeated multiple times until the supernatant was clear and transparent. The supernatant was discarded, and physiological saline was added to prepare a red blood cell suspension (2%, v / v) at the bottom. The PEG-Len / Vin NPs solution prepared in Example 2 was diluted to a series of different concentrations. Different concentrations of PEG-Len / Vin NPs physiological saline solution were added to each tube in the experimental group, and the prepared red blood cell suspension was mixed uniformly. The mixture (red blood cell suspension and distilled water) was used as a positive (Positive) control group, and the mixture (red blood cell suspension and physiological saline) was used as a negative (Negative) control group. Each group had three parallel samples. Each group of samples was incubated in a 37°C constant temperature water bath for 1.5 h. After incubation, all samples were centrifuged at 1500 rpm for 15 min, and the supernatant was collected. The absorbance of the samples at 540 nm was detected using a microplate reader, and the hemolysis rate (HR) of each sample was calculated.

[0061] To determine the stability of the drug in the blood, as shown in Figure 4 A, different amounts of PEG-Len / Vin NPs were added to the mouse whole blood, and the hemolysis was observed after 1.5 h. From left to right, the drug mass gradually increased (0, 10, 50, 100, 200 μg), and the rightmost was the positive control. Figure 4B is the hemolysis rate determined by absorbance. The hemolysis rate of PEG-Len / Vin NPs is less than 5% in the concentration range of 0-200 μg / mL, which shows very weak hemolytic ability and preliminarily proves that the PEG-Len / Vin NPs have good blood compatibility and are not easy to cause hemolysis.

[0062] 3.3 Drug release in vitro experiment

[0063] The PEG-Len / Vin NPs sample obtained in Example 2 (at a concentration of 2 mg / mL) was placed in a regenerated cellulose membrane dialysis bag (2000) prepared in advance, and then placed in different condition PBS buffer (pH = 7.4 and pH = 5.6) with a volume of 20 mL, and placed in a water bath (37°C) for shaking at a speed of 100 rpm. Finally, 1 mL of medium was taken for detection at 6 time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h), and 1 mL of new medium was supplemented in addition to the above operations. The content of Len and Vin was determined by ultraviolet absorption spectrum, and the corresponding release rate was calculated according to the data obtained according to the standard curve.

[0064] As shown in Figure 5 A, the PEG-Len / Vin NPs can stably release Vin drugs under acidic conditions of pH = 5.6, and the drug release reaches the highest concentration around 24 hours, while under neutral conditions of pH = 7.4, the drug release is less. As shown in Figure 5 B, the PEG-Len / Vin NPs can also stably release Len drugs under acidic conditions of pH = 5.6, and the drug release reaches the highest concentration around 24 hours, while under neutral conditions of pH = 7.4, the drug release is less.

[0065] Example 4 Drug in vitro anti-tumor test

[0066] 4.1 Cell proliferation ability detection

[0067] The drug in vitro anti-tumor cell proliferation experiment first evaluates the cytotoxic effect of the PEG-Len / Vin NPs prepared in Example 2 on two kinds of colon cancer cell lines (including human HCT116 cells and RKO cells) in vitro by MTT experiment. The drug treatment is divided into 4 groups: Control, Vin, Len, PEG-Len / Vin NPs, and the cell viability is evaluated after 48 hours of treatment with different drugs, and the IC 50 values of all groups are determined according to the dose-response curve. In addition, the long-term proliferation inhibition ability of different drug treatments on tumor cells is also detected by colony cloning experiment.

[0068] The results are shown in Figure 6As shown in Figure A, both types of colon cancer cells exhibited some degree of proliferation inhibition after treatment with the three drugs or preparations. Under conditions where Vin was used alone, the prepared PEG-Len / Vin NPs showed a higher proliferation inhibition effect at the same mass concentration. However, using Len as the base drug, at the same mass concentration (drug loading of 23.86% (Drug loading in Example 2), mass Len:PEG-Len / Vin NPs = 1:5), PEG-Len / Vin NPs showed higher antitumor activity than Len at the same mass concentration.

[0069] Through cloning experiments ( Figure 6 B) also confirmed similar results to MTT, showing that PEG-Len / Vin NPs significantly inhibited the clonal formation of both types of colon cancer cells, and the inhibitory effect of PEG-Len / Vin NPs was significantly stronger than that of monotherapy.

[0070] 4.2 Cell cycle distribution detection

[0071] After treating cells with different drugs for 48 hours, cells were collected, washed with PBS, and fixed overnight in pre-chilled 70% ethanol. After fixation, the cell pellet was collected by centrifugation at 1500 rpm and washed twice with PBS. The collected cells were treated with 25 μL of RNase stock solution (100 μg / mL) for 30 minutes, stained with 200 μL of PI (50 μg / mL stock solution) for 15 minutes, and then analyzed by flow cytometry.

[0072] The results are as follows Figure 7 As shown in Figure A, HCT116 colon cancer cells did not show significant cell cycle arrest after Vin treatment, but both Len and PEG-Len / Vin NPs treatments showed some cell cycle arrest. The proportion of cells arrested in the G1 phase increased significantly after PEG-Len / Vin NPs treatment. At near-monitory concentrations, PEG-Len / Vin NPs treatment induced more significant cell cycle arrest than Len treatment alone.

[0073] 4.3 Apoptosis Detection

[0074] After treating the cells with different drugs or preparations for 48 hours, the cell culture solution was aspirated into a centrifuge tube, the adherent cells were washed once with PBS, the cells were digested and collected into a centrifuge tube. The cells in the centrifuge tube were centrifuged at 1000g for 5 minutes, the supernatant was discarded, the cells were resuspended with PBS and counted. 5-10 million resuspended cells were centrifuged at 1000g for 5 minutes, the supernatant was discarded, 5 μL Annexin V-FITC and 10 μL propidium iodide staining solution were added, and the mixture was mixed gently. After incubation at room temperature for 15 minutes in the dark, it was used for flow cytometry detection. Annexin V-FITC is green fluorescence, and propidium iodide (PI) is red fluorescence.

[0075] The results are shown in Figure 7 As shown in B, the apoptosis of HCT116 colon cancer cells was not obvious after treatment with Vin, while both Len and PEG-Len / Vin NPs prepared in Example 2 showed certain apoptosis. The apoptosis of cells treated with PEG-Len / Vin NPs reached 19.95%, and the apoptosis induced by PEG-Len / Vin NPs was more significant than that induced by single Len at a similar drug mass concentration.

[0076] Example 5 In vitro anti-tumor test of drugs

[0077] An animal model of CT26 cell tumor-bearing mice was established, and CT26 cells (7 x 10 5 cells in 100 μL) were subcutaneously inoculated into female BALB / c mice. When the tumor volume reached about 100 mm 3 , the tumor-bearing mice were randomly divided into 4 groups (5 mice per group); normal saline, Vin solution (10 mg / kg), Len solution (10 mg / kg), and PEG-Len / Vin NPs (Len ~ 10 mg / kg) solution were administered by intraperitoneal injection, and the administration was continuous for two weeks. The tumor volume and body weight were measured every two days. As shown in Figure 8 A and Figure 8 C-8D, the tumor volume of the normal saline group increased rapidly, and Len alone showed better anti-tumor effect than Vin alone. In contrast, the PEG-Len / Vin NPs group showed the most significant inhibition of tumor growth. As shown in Figure 8 B, the body weight of each administration group did not change significantly, indicating that the PEG-modified Len-Vin co-assembled nanoparticles have strong anti-tumor effect and no obvious toxicity to the body, and are a safe and effective drug delivery system.

[0078] In addition, it should be understood that, after reading the above description of the present application, those skilled in the art can make various modifications or modifications to the present application, and these equivalent forms also fall within the scope defined by the claims attached hereto.

Claims

1. A lenvatinib-polyethylene glycol coupling compound, characterized in that, It has the structure shown in equation (I): In equation (I), n ≥ 2.

2. The lenvatinib-polyethylene glycol coupling compound according to claim 1, characterized in that, In formula (I), n = 2 to 300.

3. The lenvatinib-polyethylene glycol coupling compound according to claim 2, characterized in that, In formula (I), n = 100 to 125.

4. The method for preparing the lenvatinib-polyethylene glycol coupling compound according to any one of claims 1 to 3, characterized in that, include: A solution of lenvatinib, sodium p-toluenesulfinate monohydrate, and a biotin-polyethylene glycol-aldehyde group having the structure shown in formula (II) was subjected to a first room temperature reaction by adding trimethylchlorosilane dropwise under an ice bath. After the reaction was completed, the solvent was removed under reduced pressure. The product was washed, dried under vacuum, dissolved in dichloromethane, and Cs2CO3 and Na2SO4 were added. A second room temperature reaction was carried out under an inert atmosphere. After the reaction was completed, hexane was added, the mixture was filtered, the filtrate was collected, and the solvent was removed under reduced pressure to obtain the lenvatinib-polyethylene glycol coupling compound. In equation (II), n ≥ 2.

5. The preparation method according to claim 4, characterized in that, In equation (II), n = 2 to 300.

6. The preparation method according to claim 5, characterized in that, In equation (II), n = 100 to 125.

7. The preparation method according to any one of claims 4 to 6, characterized in that, The molar ratio of lenvatinib to biotin-polyethylene glycol-aldehyde is 1 to 2:1; The molar ratio of sodium p-toluenesulfinate monohydrate to lenvatinib is 1:1; The molar ratio of trimethylchlorosilane to biotin-polyethylene glycol-aldehyde is 1.5–2.5:1; The first room temperature reaction takes 60–84 hours; The washing process uses hexane and diethyl ether as detergents. The molar ratio of Cs2CO3, Na2SO4 and the product is 1:1:1; The inert atmosphere is nitrogen; The second room temperature reaction takes 4 to 6 hours.

8. A nano-formulation, characterized in that, It comprises vinpocetine and the lenvatinib-polyethylene glycol coupling compound as described in any one of claims 1 to 3.

9. The nano-formulation according to claim 8, characterized in that, The active ingredient in the nano-formulation includes nanoparticles assembled from vinpocetine and lenvatinib-polyethylene glycol coupling compounds; The co-assembly process is carried out in an aqueous phase, and the nanoparticles are formed in the aqueous phase.

10. The method for preparing nano-formulations according to claim 8 or 9, characterized in that, include: An organic solution containing vinpocetine and lenvatinib-polyethylene glycol coupling compound was added dropwise to water and stirred to obtain the nano-formulation.

11. The preparation method according to claim 10, characterized in that, In the organic solution, the molar ratio of vinpocetine to the lenvatinib-polyethylene glycol coupling compound is 1:1 to 10.

12. The preparation method according to claim 11, characterized in that, In the organic solution, the molar ratio of vinpocetine to the lenvatinib-polyethylene glycol coupling compound is 1:

4.

13. The preparation method according to claim 10, characterized in that, The organic solvent in the organic solution includes one or a combination of two of methanol and dimethyl sulfoxide. The preparation method further includes a post-processing operation after stirring, which includes centrifugation and washing with water to obtain the nanoparticles spontaneously formed by stirring, thereby obtaining the nano-formulation.

14. The preparation method according to claim 13, characterized in that, The organic solvent includes methanol and dimethyl sulfoxide; the volume ratio of methanol to dimethyl sulfoxide in the organic solvent is 1:

1.

15. The use of the lenvatinib-polyethylene glycol coupling compound according to any one of claims 1 to 3 or the nanoformulation according to claim 8 or 9 in the preparation of a medicament for the prevention and / or treatment of cancer.

16. The application according to claim 15, characterized in that, The cancers mentioned include liver cancer, breast cancer, and colon cancer.

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