Medicinal preparation for inhibiting biliary duct cancer kinesiosis and preparation method thereof

Abstract

The invention belongs to the technical field of anti-tumor pharmaceutical preparations, and particularly relates to a pharmaceutical preparation for inhibiting biliary duct cancer kinesiology and a preparation method thereof.The pharmaceutical preparation is prepared from, by weight, 10 parts of a UBE2M inhibitor, 80-100 parts of PLGA, 15-20 parts of DSPE-PEG2000-T7, 5 parts of a stabilizer and 8 parts of a penetrant, the UBE2M inhibitor with high binding force to UBE2M is obtained through virtual screening of a computer, and the UBE2M inhibitor with high binding force to the biliary duct cancer is used for inhibiting biliary duct cancer kinesiology. The UBE2M inhibitor has good pharmacological activity, can obviously inhibit the activity of UBE2M through inhibition so as to influence the PHB2 protein level and the quasi-kinesization process and achieve the anti-bile duct cancer effect, meanwhile, the UBE2M inhibitor is prepared into a nano preparation through a modification material with hydrophilic and hydrophobic and targeting characteristics, the leakage situation of the medicine in the in-vivo circulation process can be effectively reduced, and the anti-bile duct cancer effect is achieved. The accumulation at the nidus part of the bile duct cancer is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antitumor drug preparations, and particularly relates to a drug preparation for inhibiting cholangiocarcinoma neformation and a preparation method thereof. BACKGROUND

[0002] Cholangiocarcinoma is a highly malignant digestive tract tumor, which is insidious in onset and rapidly progressive. Most patients have lost the opportunity for surgery at the time of diagnosis. Current clinical treatment mainly relies on gemcitabine-based chemotherapy regimens, but the overall efficacy is limited, and the five-year survival rate of patients is still at a low level. Traditional chemotherapy drugs inhibit tumor growth through non-specific cytotoxicity, which not only kills cancer cells but also causes significant damage to normal tissues, leading to bone marrow suppression, liver and kidney dysfunction and other serious adverse reactions. In addition, the multi-drug resistance of tumor cells to chemotherapy drugs further restricts the clinical treatment effect, and it is urgent to develop new targeted treatment strategies. In recent years, molecular biology research has revealed the key role of the ubiquitin-proteasome system in tumor occurrence and development, and neformation modification as an important branch of ubiquitin-like modification is closely related to the proliferation and invasion of various tumors. UBE2M (ubiquitin conjugase E2M) is abnormally highly expressed in cholangiocarcinoma tissues and promotes tumor malignant progression by mediating the neformation modification of PHB2 protein. The discovery of this pathway provides a potential new target for cholangiocarcinoma treatment, but there is still no specific inhibitor targeting this pathway in the prior art, and there is a lack of a preparation system that can achieve targeted delivery.

[0003] Therefore, there is an urgent need for a molecular inhibitor targeting UBE2M, which can inhibit the activity of UBE2M and block the neformation process of PHB2 protein, thereby achieving an antitumor effect on cholangiocarcinoma. This not only provides a new technical path for the treatment of cholangiocarcinoma, but also provides a new idea for the targeted treatment of other refractory tumors. SUMMARY

[0004] In view of the above, the application provides a drug preparation for inhibiting cholangiocarcinoma neformation and a preparation method thereof. UBE2M is used as a binding target, a small molecule inhibitor with high binding is screened through computer simulation, the pharmacodynamic activity thereof is investigated and verified, and a nano preparation with tumor targeting function is prepared, so as to achieve the purpose of inhibiting tumor neformation and achieving an antitumor effect. This provides a new direction with small side effects and long-lasting efficacy for the treatment of cholangiocarcinoma and promotes the development of tumor targeted therapy.

[0005] To achieve the above purpose, the technical solutions adopted by the application are as follows: The application provides a drug preparation for inhibiting cholangiocarcinoma de novo, which comprises the following raw materials in parts by weight: 10 parts of UBE2M (ubiquitin conjugating enzyme E2M) inhibitor, 80-100 parts of PLGA (poly lactic acid-glycolic acid copolymer), 15-20 parts of DSPE (distearoyl phosphatidyl ethanolamine)-PEG2000 (polyethylene glycol 2000)-T7 (transferrin receptor peptide), 5 parts of stabilizer and 8 parts of penetrant.

[0006] Further, the stabilizer is selected from any one of trehalose, mannitol and galactose.

[0007] Further, the penetrant is selected from TPGS (vitamin E polyethylene glycol succinate) or cell penetrating peptide.

[0008] Further, the cell penetrating peptide is TAT peptide, and the amino acid sequence is GRKKRRQRRRPQ.

[0009] Further, the UBE2M inhibitor is obtained based on computer molecular docking simulation screening.

[0010] Further, the screening method of the UBE2M inhibitor is as follows: S1: searching, determining and downloading the protein sequence of UBE2M in the PDB database, analyzing and optimizing the target structure to obtain a protein structure containing a virtual site; S2: determining the force field, energy, protonation and hydrogen bond of computer molecular docking simulation, and generating a grid file for subsequent virtual screening, based on the compounds included in the ChemDiv database, protonating and hydrogenating each compound to be simulated and energy minimization simulation to obtain docking compounds; S3: taking the virtual site of the protein structure in the grid file as the binding site, respectively performing molecular docking simulation on the docking compounds, scoring, sorting and analyzing the binding mode of the simulation results, and screening high-binding compounds; S4: performing pharmacodynamic investigation on the high-binding compounds to screen high-binding compounds with better inhibition of de novo, i.e. UBE2M inhibitors.

[0011] The application further provides a preparation method of the drug preparation for inhibiting cholangiocarcinoma de novo, and the specific steps are as follows: Step 1: dissolving the UBE2M inhibitor and PLGA in an organic solvent to obtain an organic phase, dissolving DSPE-PEG2000-T7 and the penetrant in PBS (phosphate buffer) to obtain an aqueous phase; Step 2: slowly injecting the organic phase into the aqueous phase, ultrasonically homogenizing and emulsifying to obtain nanoemulsion, removing the organic solvent from the nanoemulsion by rotary evaporation, and extruding and filtering to obtain a nanoparticle solution; Step 3: Add stabilizer to the nanoparticle solution, stir at room temperature, then ultracentrifuge, collect the precipitate, freeze-dry the precipitate, and obtain the inhibiting cholangiocarcinoma drug preparation.

[0012] The present application has the following beneficial effects: The inhibiting cholangiocarcinoma drug preparation prepared by the present application is based on the mechanism that UBE2M-mediated PHB2 ubiquitination promotes the occurrence and development of cholangiocarcinoma. Through computer molecular docking simulation technology, a small molecule inhibitor is virtually screened, a lead compound specifically combined with the UBE2M target is efficiently identified from a compound library, the blindness and high cost of traditional drug screening are avoided, the hit rate and reliability of candidate compounds are significantly improved, the drug development cycle is shortened, and a high-specificity molecular tool is provided for cholangiocarcinoma treatment. By inhibiting the activity of UBE2M, the ubiquitination modification of PHB2 is directly blocked, thereby interfering with the signal transduction pathway of cancer cells and inhibiting tumor proliferation and invasion. In terms of inhibiting cholangiocarcinoma ubiquitination, the present application realizes precise intervention in the key link of tumor occurrence and development through a small molecule inhibitor targeting UBE2M. Inhibiting ubiquitination behavior can not only directly induce cancer cell apoptosis and growth arrest, but also reverse the malignant phenotype and reduce the risk of metastasis. This mechanism-specific treatment avoids the broad-spectrum toxicity of traditional chemotherapy and improves the tolerance and quality of life of patients.

[0013] Through dosage form design of the screened UBE2M small molecule inhibitor, a DSPE-PEG2000-T7 cholangiocarcinoma targeted delivery system is constructed, and the effect of tumor site accumulation and deep penetration is further improved by combining with a penetrant. PLGA is used to encapsulate the small molecule inhibitor to maintain its in vitro and in vivo activity, DSPE-PEG2000-T7 forms a shell structure to prevent leakage of the small molecule inhibitor, and the penetrant has a tumor microenvironment targeting function to further improve the accumulation of the drug at the cholangiocarcinoma site. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 is the crystal structure of UBC12 protein; Figure 2 is the crystal structure with high binding performance in UBE2M protein; Figure 3 is the three-dimensional structure of UBE2M protein and the site region thereof for molecular docking virtual screening; Figure 4 is the investigation result of the influence of the three small molecule compounds screened in Example 3 on the protein expression level of HUCCT1 cells; Figure 5The investigation results of Rebaudioside A on the protein expression levels of different gene knockout types of HUCCT1 cells screened in Example 3; Figure 6 The investigation results of cell clones of Rebaudioside A screened in Example 3; Figure 7 The investigation results of cell migration of Rebaudioside A screened in Example 3; Figure 8 The investigation results of mouse modeling and tumor weight; Figure 9 The investigation results of the influence of the drug preparation for inhibiting cholangiocarcinoma prepared in Example 6 on tumor growth; Figure 10 The results of molecular docking of Rebaudioside A screened in Example 3 and the affinity characterization with UBE2M protein. DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to one skilled in the art. In addition, any method and material similar or equivalent to those described can be used in the present application. The preferred implementation methods and materials described herein are only used for demonstration, but cannot limit the content of the present application.

[0017] In the following examples, unless otherwise specified, all are conventional methods; the materials used in the following examples, unless otherwise specified, are new materials purchased on the market.

[0018] Example 1: Computer-aided virtual screening S1 (preparation of three-dimensional structure of target point): Through the uniprot database, the analysis structure of UBE2M protein (ID: P61081) is found. The target protein is composed of 183 amino acids, and the 1-57 segment is the action region with UBA3, and the 29-173 is the UBC core. The UBC12 protein crystal structure is shown in Figure 1 The crystal structure of the complex RBX1-UBC12-NEDD8-CUL1-DCN1 is downloaded from the PDB database, as shown in Figure 2The structure was optimized, and the pKa value of the residues was predicted by PROPKA 3.0 method under the condition of pH 7.0, the protonation state of ionizable residues such as histidine and glutamic acid was allocated, the hydrogen bond network was optimized by OPLS3 force field, the iteration step was set to 5000 steps, the missing side chain and loop region (residue gap ≤10 Å) were modeled by Prime module, and the number of residues in the core area of the optimized structure Ramachandran plot was set to 0.5 kcal / mol·Å 2 ; S2 (virtual screening site definition): through software prediction, the possible binding site of small molecules was found as Figure 3 The black circled part is shown, and the small molecule compound may affect the binding of UBE2M with RBX1, CUL1 and DCN1, thereby affecting its biological function. Based on the interaction interface of UBE2M and RBX1, CUL1 in the complex crystal structure, the geometric center coordinates of the binding pocket were defined as the reference point (X=15.2 Å, Y=-8.7 Å, Z=22.3 Å), and the grid file was generated. The size of the cubic boundary box in the grid file was set to 20 Å×20 Å×20 Å, the grid spacing was 0.8 Å, the van der Waals radius scaling factor was set to 1.0, the charge cutoff value was ±0.25 e, and the local electrostatic potential interference of the edge residues was excluded by using the eceptorGrid Generation function of Glide module; S3 (compound library preprocessing): based on the ChemDiv database containing about 20,000 compounds, small molecule compounds with high binding force to the three-dimensional structure of the protein target in the grid file were screened. Before molecular docking simulation, the compounds in the ChemDiv database were preprocessed, the compound molecules were protonated and hydrogenated, and the three-dimensional spatial conformation was obtained by energy minimization. The Maestro format three-dimensional structure of each compound was output; S4 (molecular docking virtual screening): Virtual Screening Workflow of Schrodinger software was used for virtual screening of compounds, and step-wise strategy (i.e. three-step screening mode of HTVS→SP→XP with increasing accuracy) was adopted. HTVS mode: preliminary screening retained compounds with score < -6.0, van der Waals radius scaling factor 0.8, and electrostatic potential cutoff value ±0.15 e; SP mode: fine docking of HTVS results, adding hydrophobic contact item to scoring function, retaining compounds with Glide Score < -7.0; XP mode: using induced fitting docking, setting side chain flexibility to allow conformation sampling of binding pocket residues (distance < 5 Å), retaining top 1000 compounds in scoring order; S5: In order to ensure diversity and smooth progress of subsequent experiments, the clustering information of small molecules and the action mode with the receptor are comprehensively considered, and the compounds which form hydrogen bond interaction, hydrophobic interaction, etc. with H88, D89, P91, K92, V93, R116, E117, W119 of UBE2M, K759, E760 of CUL1 and K171 of DCN1 are preferentially selected from each type of compound screened. Through the binding ability-based docking scoring of the RBX1-UBE2M-NEDD8-CUL1-DCN1 complex and the small molecule compound at the binding site, there are 779 molecules with docking scoring < -7.0. The target-small molecule complex structure information and the interaction mode of the representative compounds obtained by virtual screening are viewed, and finally 200 candidate molecules with binding free energy < -9.0 kcal / mol are screened.

[0019] Example 2: Investigation of binding energy based on SPR (surface plasmon resonance) M1 (instrument preparation): the experiment is performed using a Biacore T200 instrument (Cytiva), which is gently pushed into the instrument card slot in the arrow direction, the chip cabin door is closed, the running buffer (200 mL 1x PBS Buffer), water bottle and waste bottle are placed in the left and right trays respectively, and the corresponding liquid inlet pipes are inserted; M2 (channel activation): a mixed solution of EDC and NHS (each 0.1 M) is injected at a flow rate of 10 μL / min to activate the carboxyl matrix of channel 4 for 5-10 minutes to generate a reactive ester; M3 (protein immobilization): the UBE2M protein is diluted with sodium acetate buffer (pH 4.5) to 50 μg / mL, and injected into channel 4 at a flow rate of 10 μL / min for about 10 minutes, and the coupling level is monitored in real time by the sensor gram (target response value 2000-3000 RU); M4 (blocking): 1 M ethanolamine (pH 8.5) is injected at a flow rate of 10 μL / min for 10 minutes to quench the unreacted ester group, and the reference channel is set to channel 3, which repeats the above M2-M3 steps, but the protein-free acetic acid buffer is used in the immobilization stage for non-specific binding subtraction; M5 (solvent correction): the correction solution is prepared according to the preparation ratio shown in Table 1 below, the series of correction solutions are obtained by mixing different volumes of 4.5% and 5.8% DMSO (dimethyl sulfoxide) mother liquor, and the series of correction solutions are sequentially flowed through the chip at a flow rate of 30 μL / min, the response values at each point are recorded, and a correction curve is generated for subsequent data analysis; Table 1 Solvent correction solution configuration table

[0020] M6 (Interaction force detection): The candidate molecules screened in Example 1, i.e. the test substances, were gradient-diluted in a 96-well plate at concentrations of 0.3125 mM, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM and 10 mM, and coupled with the target protein by the chip from low concentration to high concentration. The flow rate was 30 mL / min, and the duration was 150 s. After each concentration point flowed through, the chip was regenerated for 5 min with a 10 mM glycine hydrochloride (pH 2.0) solution, and this process was repeated until all the corresponding concentrations of the test substances were run. The data were globally fitted to a 1:1 Langmuir binding model using Biacore Insight evaluation software (Cytiva, Marlborough, MA, USA) to obtain the binding and dissociation constants. The top 21 small molecule compounds were screened for binding strength, and numbered 1-21, respectively.

[0021] Example 3: In vitro pharmacodynamic activity investigation Cell proliferation experiment (CCK-8 method): Cholangiocarcinoma cell lines HUCCT1 and RBE were used as in vitro pharmacodynamic investigation objects. After the cells were cultured and plated, 1x10 4 6 replicate wells were set for each concentration of each compound. The CCK-8 method was used for cell proliferation detection and statistical analysis, and the IC50 (half-inhibitory concentration) of different small molecule compounds was calculated. The results are shown in Table 2. Eleven compounds with obvious inhibitory effect on cholangiocarcinoma cell lines were screened, numbered 4, 5, 7, 8, 9, 10, 14, 16, 17, 18 and 19, respectively. The structures and main functions of each compound were determined by literature review, and finally compounds 5, 8 and 19 were selected as small molecule inhibitor candidate drugs. The three compounds were Hexa-D-arginine, Kaempferitrin and Rebaudioside A, respectively.

[0022] Table 2 IC50 values and inhibition rates of small molecule compounds

[0023] WB (Western blot) experiment: The three compounds screened in the cell proliferation experiment were added to HUCCT1 cells for incubation for 12 h, and total cell protein was extracted for WB experiment. DMSO was used as a control group to observe the changes in PHB2, UBE2M and TUBULIN protein levels. The results are shown in Figure 4, it was found that the expression level of PHB2 protein in cells decreased after the addition of Rebaudioside A, which could inhibit the expression of PHB2 protein, which met the expected design. The cholangiocarcinoma mimetic inhibition effect of Rebaudioside A was further investigated. Rebaudioside A was added to HUCCT1 cells, UBE2M gene knockout HUCCT1 cells and UBE2M gene knockout HUCCT1 cells, respectively, and incubated, which were WT group, KO group and RESCUE group, respectively. Protein was extracted and WB was investigated, and the results are shown in Figure 5 , it was found that the PHB2 protein level of KO group did not change after the addition of Rebaudioside A, indicating that the action site was UBE2M, which was consistent with the computer simulation results. Finally, Rebaudioside A was determined as a small molecule inhibitor of UBE2M protein, which was used as an UBE2M inhibitor for subsequent research.

[0024] In this embodiment, a drug preparation for inhibiting cholangiocarcinoma mimetics is provided, which comprises the following raw materials by weight: UBE2M inhibitor 10 parts, PLGA 80 parts, DSPE-PEG2000-T7 20 parts, trehalose 5 parts and TPGS 8 parts.

[0025] This embodiment also provides a preparation method of a drug preparation for inhibiting cholangiocarcinoma mimetics, and the specific steps are as follows: Step 1: UBE2M inhibitor 10 parts and PLGA 80 parts were dissolved in 5 mL mixed solvent (dichloromethane: acetone, volume ratio 3:1) to obtain an organic phase. DSPE-PEG2000-T7 20 parts and TPGS 8 parts were dissolved in 35 mL PBS to obtain an aqueous phase; Step 2: The organic phase was slowly injected into the aqueous phase, and the nanoemulsion was obtained by ultrasonic homogenization under ice bath at 400 W for 5 min. The nanoemulsion was rotary evaporated at 40°C for 15 min to remove the organic solvent, and then extruded and filtered to obtain a nanoparticle solution; Step 3: Trehalose 5 parts was added to the nanoparticle solution, and after stirring and dissolving at room temperature, it was ultracentrifuged at 100000xg for 20 min. The precipitate was collected and freeze-dried to obtain a drug preparation for inhibiting cholangiocarcinoma mimetics.

[0026] In this embodiment, a drug preparation for inhibiting cholangiocarcinoma mimetics is provided, which comprises the following raw materials by weight: UBE2M inhibitor 10 parts, PLGA 80 parts, DSPE-PEG2000-T7 20 parts, trehalose 5 parts and TPGS 8 parts.

[0027] This embodiment also provides a preparation method of a drug preparation for inhibiting cholangiocarcinoma mimetics, and the specific steps are as follows: Step 1: Take 10 parts of UBE2M inhibitor and 90 parts of PLGA and dissolve them in 5 mL of mixed solvent (dichloromethane: acetone, volume ratio 3:1) to obtain an organic phase. Dissolve 15 parts of DSPE-PEG2000-T7 and 8 parts of TAT peptide in 35 mL of PBS to obtain an aqueous phase; Step 2: Slowly inject the organic phase into the aqueous phase, and ultrasonically homogenize under ice bath for 5 min at 400 W to obtain nanoemulsion. Rotate the nanoemulsion at 40°C for 15 min to remove the organic solvent, and then extrude and filter to obtain a nanoparticle solution; Step 3: Add 5 parts of mannitol to the nanoparticle solution, stir to dissolve at room temperature, and then centrifuge at 100000 x g for 20 min. Collect the precipitate and freeze-dry it to obtain a cholangiocarcinoma inhibiting drug preparation.

[0028] Example 6: This example provides a cholangiocarcinoma inhibiting drug preparation, which comprises the following raw materials by weight: 10 parts of UBE2M inhibitor, 100 parts of PLGA, 18 parts of DSPE-PEG2000-T7, 5 parts of galactose, and 8 parts of TAT peptide.

[0029] This example also provides a preparation method of a cholangiocarcinoma inhibiting drug preparation, and the specific steps are as follows: Step 1: Take 10 parts of UBE2M inhibitor and 100 parts of PLGA and dissolve them in 5 mL of mixed solvent (dichloromethane: acetone, volume ratio 3:1) to obtain an organic phase. Dissolve 18 parts of DSPE-PEG2000-T7 and 8 parts of TAT peptide in 35 mL of PBS to obtain an aqueous phase; Step 2: Slowly inject the organic phase into the aqueous phase, and ultrasonically homogenize under ice bath for 5 min at 400 W to obtain nanoemulsion. Rotate the nanoemulsion at 40°C for 15 min to remove the organic solvent, and then extrude and filter to obtain a nanoparticle solution; Step 3: Add 5 parts of mannitol to the nanoparticle solution, stir to dissolve at room temperature, and then centrifuge at 100000 x g for 20 min. Collect the precipitate and freeze-dry it to obtain a cholangiocarcinoma inhibiting drug preparation.

[0030] Cell clone experiment: HUCCT1 cells and RBE cells were inoculated in each well of a cell culture 6-well plate, the cell morphology and density of each well were observed under a microscope, and the cell culture box was placed in a cell culture box, the cell growth and cell clone number were observed daily, the culture medium was replaced every 3 days, and complete culture medium, DMSO and complete culture medium containing 5 μg / mL were added as the WT group, the DMSO group and the inhibitor group, respectively, the cells were cultured for 14 days, the single cell clone number was observed to be greater than or equal to 50, PBS was washed, 1 ml of paraformaldehyde was added to each well, and fixed for 30 min, the formaldehyde in the six-well plate was removed, PBS was washed twice, crystal violet solution was added, and stained for 20 min, the crystal violet solution in the six-well plate was removed, PBS was washed again for 2 times, the liquid in the six-well plate was absorbed, and the cell clone group stained blue was visible, and the photograph was taken, and counted by imageJ software, and the results are shown in Figure 6 .

[0031] Cell invasion experiment: serum-free medium, 24-well plate, transwell chamber, and matrigel were prepared, HUCCT1 and RBE cells were used as test cells, 10 mg / ml matrigel was diluted to 1 mg / ml, 60 μl was added to each transwell chamber, and attention was paid to avoid air bubbles, the chamber was placed in a 24-well plate, and the chamber was placed in a cell culture box for 1 h, the 24-well plate was taken out, the un-solidified matrigel was absorbed, 100 μl of serum-free medium was added, and the cell culture box was placed again for 30 min, and the hydration was performed, 500 μl of complete culture medium was added to the hole of the 24-well plate, the transwell chamber was added, and 2×10 5 Figure 7 .

[0032] Subcutaneous tumor experiment of nude mice: 16 5-8 week old female BALB / c-nu nude mice were used for subcutaneous tumor experiment, and were divided into control group and preparation group, each group of 8, well-grown cholangiocarcinoma HUCCT1 cells were used as inoculation cells, and were inoculated subcutaneously at the right thigh root of the mice, and the number of inoculated cells was 1.6×10 7 ​One, 7 days after measuring the size of the tumor, construct a mouse CDX (xenograft tumor) model, after the completion of the control group and the preparation of the drug preparation group, respectively, saline and prepared in Example 6, after the end of the experiment, the mouse was painlessly killed under anesthesia, the tumor tissue was completely stripped, weighed, photographed and recorded, the results of the mouse CDX model and the tumor weight are shown in Figure 8 , tumor growth curve and tumor size results are shown in Figure 9 .

[0033] Inhibitor affinity investigation: the molecular structure of the UBE2M inhibitor (Rebaudioside A) screened in Example 2, the molecular docking results with UBE2M protein and the affinity detection results are shown in Figure 10 .

[0034] Figure 4 The results show that compound 19, i.e. UBE2M inhibitor (Rebaudioside A), has a significant effect on the expression level of PHB2 protein, and can reduce the expression level of PHB2 protein compared with compounds 5 and 8, showing good activity in inhibiting the conjugation of cholangiocarcinoma.

[0035] Figure 5 The results show that after adding the UBE2M inhibitor (Rebaudioside A), the PHB2 protein expression level of the KO group has no significant change, indicating that the UBE2M inhibitor mainly affects the activity of PHB2 protein by binding with UBE2M protein, which is consistent with the design expectation.

[0036] Figure 6 and Figure 7 The results show that after adding the UBE2M inhibitor (Rebaudioside A), the invasion, migration and cell colony formation ability of cholangiocarcinoma cell lines are significantly reduced, showing high active inhibition performance on cholangiocarcinoma.

[0037] Figure 8 and Figure 9 The results show that the mouse ectopic tumor modeling is successful, the tumor growth is good, and the tumor growth of the control group is significantly higher than that of the preparation group, indicating that the drug preparation prepared in Example 6 has good anti-cholangiocarcinoma activity.

[0038] Figure 10 The results show that the UBE2M inhibitor can efficiently bind with UBE2M protein, with a KD value of 7.74 x 10 -6 , indicating that Rebaudioside A has high affinity with UBE2M protein, high specificity and inhibition activity.

[0039] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely divergences of the principles and spirit of the application and that numerous modifications, changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the application as defined by the appended claims and their equivalents.

[0040] The above description of the application and its embodiments is not restrictive, and the embodiments shown in the drawings are only one of the embodiments of the application, and the actual application is not limited thereto. In summary, if a person skilled in the art is inspired by it, without departing from the purpose of the invention, without creative design, similar ways and embodiments of the technical solution should belong to the protection scope of the application.

Claims

1. A pharmaceutical preparation for inhibiting cholangiocarcinoma carcinomatization, characterized by, The pharmaceutical preparation comprises the following raw materials by weight: 10 parts of UBE2M inhibitor, 80-100 parts of PLGA, 15-20 parts of DSPE-PEG2000-T7, 5 parts of stabilizer and 8 parts of penetrating agent; The UBE2M inhibitor is obtained through computer molecular docking simulation screening; The screening method of the UBE2M inhibitor is as follows: S1: determining the protein sequence of UBE2M, optimizing the structure to obtain a protein structure containing a virtual site; S2: determining the computer molecular docking simulation parameters, preparing the docking compounds in the database to obtain the docking compounds; S3: performing molecular docking simulation and analysis on the protein structure containing the virtual site and the docking compounds to screen high-binding compounds; S4: performing pharmacodynamic investigation on the high-binding compounds to screen the UBE2M inhibitor.

2. The pharmaceutical preparation for inhibiting cholangiocarcinoma carcinogenesis according to claim 1, characterized by, The stabilizer is selected from any one of trehalose, mannitol and galactose.

3. The pharmaceutical preparation for inhibiting cholangiocarcinoma carcinogenesis according to claim 1, wherein The penetrating agent is selected from TPGS or cell penetrating peptide; The cell penetrating peptide is TAT peptide, and the amino acid sequence is GRKKRRQRRRPQ.

4. The pharmaceutical preparation for inhibiting cholangiocarcinoma carcinogenesis according to claim 1, wherein In S1, the protein sequence of UBE2M is obtained by searching and downloading from the PDB database; In S2, the database is ChemDiv database.

5. A method for the preparation of a pharmaceutical preparation for inhibiting cholangiocarcinoma carcinogenesis according to any one of claims 1 to 4, characterized by, The specific method is as follows: Step 1: dissolving UBE2M inhibitor and PLGA together to obtain an organic phase, dissolving DSPE-PEG2000-T7 and penetrating agent together to obtain an aqueous phase; Step 2: slowly injecting the organic phase into the aqueous phase, emulsifying, rotary evaporation, and extruding to obtain a nanoparticle solution; Step 3: adding the stabilizer to the nanoparticle solution, centrifuging, collecting the precipitate, freeze-drying the precipitate to obtain the cholangiocarcinoma inhibiting drug preparation.

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