A pharmaceutical preparation for inhibiting esophageal squamous cell carcinoma metastasis and a preparation method thereof

Abstract

This invention belongs to the field of pharmaceutical formulation technology, and particularly relates to a pharmaceutical formulation for inhibiting esophageal squamous cell carcinoma metastasis and its preparation method. The preparation method of the pharmaceutical formulation for inhibiting esophageal squamous cell carcinoma metastasis includes the following steps: S1. Dissolving paclitaxel, cyperene, and a glyceryl stearate derivative in an organic solvent to obtain an oil phase; S2. Dissolving soybean lecithin and Tween 80 in water to obtain an aqueous phase; S3. Mixing the oil phase and the aqueous phase at 70-75°C, stirring to emulsify, cooling to room temperature, and freeze-drying to obtain the final product. This invention loads paclitaxel and cyperene into a nanodelivery system, constructing a pharmaceutical formulation that can both enhance antitumor effects and achieve precise and efficient mucosal delivery via oral administration, which has significant clinical significance and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and particularly relates to a pharmaceutical formulation for inhibiting the metastasis of esophageal squamous cell carcinoma and its preparation method. Background Technology

[0002] Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors worldwide, with esophageal squamous cell carcinoma (ESCC) being the predominant pathological type. Numerous factors contribute to the development of ESCC, primarily related to alcohol consumption, smoking, and various chronic irritants that damage the esophagus. Currently, the standard clinical treatment for ESCC is a comprehensive approach based on surgical resection, combined with radiotherapy and chemotherapy. The lack of specific and effective drugs and treatment regimens makes ESCC highly susceptible to metastasis and recurrence, resulting in extremely low survival rates.

[0003] Paclitaxel, a classic microtubule stabilizer and cell cycle arrestor, is widely used in the treatment of various solid tumors by interfering with the normal function of the mitotic spindle in tumor cells. However, some patients exhibit primary or persistent resistance to paclitaxel monotherapy. Therefore, exploring combination therapy strategies that can enhance the efficacy of paclitaxel, overcome drug resistance, and reduce its toxic side effects is of great significance. Combination therapy is one of the effective strategies for improving anticancer efficacy and delaying the onset of drug resistance. In recent years, researchers have focused on combining paclitaxel with other natural products or synthetic drugs with different mechanisms of action or synergistic effects. Cyperene is a natural sesquiterpene compound extracted from the traditional Chinese medicine Cyperus rotundus. Modern pharmacological studies have shown that it possesses anti-inflammatory, antioxidant, anti-fibrotic, and potential antitumor activities. However, the specific role of cyperene in the treatment of esophageal squamous cell carcinoma remains unknown.

[0004] Oral anticancer drugs, especially hydrophobic drugs like paclitaxel, face significant challenges in achieving effective oral delivery: First, these drugs exhibit poor physicochemical stability and are prone to degradation in the complex gastrointestinal environment. Second, paclitaxel's extremely low water solubility and high molecular weight result in poor solubility and permeability, making it difficult to be effectively absorbed through the intestinal mucosa. Third, the digestive tract mucosa is covered by a dense and dynamic mucus layer composed of mucin, water, and electrolytes. This viscoelastic gel network constitutes the primary physical and diffusion barrier for drug absorption, rapidly capturing and removing foreign particles and molecules, significantly shortening the contact time between the drug and the absorptive epithelium, further reducing its bioavailability. Therefore, developing novel drug delivery systems capable of overcoming these challenges for efficient oral delivery is crucial for improving oral formulations of ESCC (Extracorporeal Cell Carrier Cancer). Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, one of the objectives of this invention is to provide a method for preparing a pharmaceutical formulation for inhibiting esophageal squamous cell carcinoma metastasis. This invention loads paclitaxel and cyperone into a nanodelivery system, constructing a pharmaceutical formulation that enhances antitumor effects and achieves precise and efficient mucosal delivery via oral administration, possessing significant clinical significance and application prospects.

[0006] One of the objectives of this invention is achieved through the following technical solution: A method for preparing a pharmaceutical formulation for inhibiting the metastasis of esophageal squamous cell carcinoma, the method comprising the following steps: S1. Paclitaxel, cyperone, and glyceryl stearate derivative are dissolved in an organic solvent to obtain an oil phase; S2. Dissolve soybean lecithin and Tween 80 in water to obtain an aqueous phase; S3. Mix the oil phase and the aqueous phase at 70-75℃, stir to emulsify, cool to room temperature, and freeze dry to obtain the final product; The chemical structural formula of the stearic acid glyceride derivative is: .

[0007] Furthermore, the preparation of the stearic acid glyceride derivative includes the following steps: (1) N-BOC-4-aminobenzoic acid, glyceryl stearate and 4-dimethylaminopyridine were added to dichloromethane, EDCI was added at 0°C, and the mixture was stirred until homogeneous and then reacted at room temperature; then trifluoroacetic acid was added and the reaction was continued to obtain intermediate 1. (2) Add intermediate 1 and succinic acid to anhydrous N,N-dimethylformamide and stir until homogeneous. Then add 2-(7-azabistriazol-1-yl)-N,N,N',N'-tetramethyluryl hexafluorophosphate and diisopropylethylamine and stir at room temperature to obtain intermediate 2. (3) Add intermediate 2, ammonium persulfate and dimethyl sulfoxide to anhydrous 1,4-dioxane and heat to react to obtain glyceryl stearate derivative.

[0008] Further, in step (1), the ratio of N-BOC-4-aminobenzoic acid, glyceryl stearate, 4-dimethylaminopyridine, dichloromethane, EDCI, and trifluoroacetic acid is 20-23 mmol: 10 mmol: 2-4 mmol: 50 mL: 10-12 mmol: 8-10 mL; the reaction time at room temperature is 8-16 h; and the reaction time after adding trifluoroacetic acid is 1-2 h.

[0009] Further, in step (2), the ratio of intermediate 1, succinic acid, anhydrous N,N-dimethylformamide, 2-(7-azabistriazol-1-yl)-N,N,N',N'-tetramethyluryl hexafluorophosphate and diisopropylethylamine is 10 mmol: 20-23 mmol: 60 mL: 20-25 mmol: 40-60 mmol; the stirring reaction time at room temperature is 6-12 h.

[0010] Further, in step (3), the ratio of intermediate 2, ammonium persulfate, dimethyl sulfoxide and anhydrous 1,4-dioxane is 10 mmol: 40-50 mmol: 40-50 mmol: 100 mL; the heating reaction temperature is 90-100 °C and the time is 8-12 h.

[0011] Further, in step S1, the mass ratio of paclitaxel, cyperone, and glyceryl stearate derivative is (2-4):1:(18-30), and the ratio of the sum of the masses of paclitaxel and cyperone to the amount of organic solvent is 1g:110-120mL; the organic solvent is prepared by mixing chloroform and methanol in a volume ratio of 1:1.

[0012] Furthermore, in step S2, the concentration of soybean lecithin in water is 1-2 wt%, and the concentration of Tween 80 in water is 0.5-1.5 wt%.

[0013] Furthermore, in step S3, the volume ratio of the organic phase to the aqueous phase is 1:2; the emulsification time is 2-3 hours.

[0014] A second objective of this invention is to provide a pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma.

[0015] The second objective of this invention is achieved by the following technical solution: A pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma was prepared using the above-described preparation method.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides a novel use of paclitaxel combined with cyperene in the preparation of a therapeutic drug for esophageal squamous cell carcinoma. The research results of this invention show that the combined use of paclitaxel and cyperene has a significant inhibitory effect on the proliferation of esophageal squamous cell carcinoma cells, and the effect is better than that of single drugs. In ESCC cell tumor-bearing mice, the combined use of paclitaxel and cyperene can significantly inhibit tumor growth and prolong the survival time of mice.

[0017] 2. The stearic acid glyceride derivative of the present invention is prepared by modifying stearic acid glyceride with a "benzene ring-succinimide-ester" end. With the multiple non-covalent synergistic effects of the benzene ring, imide and ester group, the encapsulation efficiency of aromatic small molecules is significantly improved in the formulation stage, achieving high loading and high retention. When the drug formulation reaches the mucosa, the benzene ring at the end of the material and the aromatic residues of mucin undergo π-π stacking, producing an anchoring effect to prolong the retention time. At the same time, the succinimide interacts with the mucin sugar chain through multiple hydrogen bonds, reducing interfacial resistance and locally loosening the network barrier, forming a slip to promote penetration. The two work together to complete the progressive process of first anchoring and then slipping, allowing more drugs to efficiently cross the mucus layer and approach the epithelial absorption surface. The effective drug concentration increases accordingly, the absorption is improved, and the oral bioavailability is ultimately improved. Attached Figure Description

[0018] Figure 1 This is a transmission electron microscope image of the pharmaceutical preparation obtained in Example 1 of the present invention. Detailed Implementation

[0019] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0020] Example 1 A method for preparing a pharmaceutical formulation for inhibiting the metastasis of esophageal squamous cell carcinoma includes the following steps: S1. Paclitaxel, cyperone, and glyceryl stearate derivative are dissolved in an organic solvent (prepared by mixing chloroform and methanol in a volume ratio of 1:1) to obtain an oil phase; the mass ratio of paclitaxel, cyperone, and glyceryl stearate derivative is 3:1:28, and the ratio of the sum of the masses of paclitaxel and cyperone to the amount of organic solvent is 1 g: 110 mL; S2. Dissolve soybean lecithin and Tween 80 in water to obtain an aqueous phase; wherein the concentration of soybean lecithin in water is 1.5 wt% and the concentration of Tween 80 in water is 1 wt%. S3. At 70°C, the oil phase and the water phase are mixed at a volume ratio of 1:2, stirred and emulsified at 600 rpm / min for 2.5 h, concentrated under reduced pressure to remove organic solvents, cooled to room temperature, and freeze-dried to obtain a drug formulation for inhibiting esophageal squamous cell carcinoma metastasis.

[0021] The preparation of the stearic acid glyceride derivative includes the following steps: (1) N-BOC-4-aminobenzoic acid, glyceryl stearate and 4-dimethylaminopyridine (DMAP) were added to dichloromethane in a ratio of 22 mmol: 10 mmol: 3 mmol: 50 mL. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was added at 0°C, and the mixture was stirred until homogeneous before being gradually heated to room temperature for reaction. 12 h; then add trifluoroacetic acid and react for 1.5 h to remove the BOC protecting group; wherein the ratio of stearic acid glyceride, EDCI, and trifluoroacetic acid is 10 mmol: 10 mmol: 9 mL; the reaction solution is washed successively with 5 wt% sodium bisulfate solution, saturated sodium bicarbonate solution, and saturated brine, then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (hexane-ethyl acetate volume ratio 6:1) to obtain intermediate 1; the characterization results of intermediate 1 are as follows: 1 H NMR (C 35 H 52 N2O6, 400 MHz, DMSO): δ 7.64 (d, 4H), 6.47 (d,4H), 5.84 (m, 1H), 5.49 (s, 4H), 4.76-4.38 (m, 4H), 2.31 (t, 2H), 1.67 (m,2H), 1.32-1.28 (m, 28H), 0.87 (m, 3H); HRMS(ESI+): [M+H] + The calculated value is 597.38; the value found is 597.38. (2) Intermediate 1 and succinic acid were added to anhydrous N,N-dimethylformamide and stirred until homogeneous. Then, 2-(7-azabistriazol-1-yl)-N,N,N',N'-tetramethyluryl hexafluorophosphate (HATU, CAS No.: 148893-10-1) and diisopropylethylamine (DIEA) were added to the mixture. The intermediate 1, succinic acid, anhydrous N,N-dimethylformamide, 2-(7-azabistriazol-1-yl)-N,N,N',N The molar ratio of '-tetramethyluryl hexafluorophosphate to diisopropylethylamine was 10 mmol: 22 mmol: 60 mL: 23 mmol: 50 mmol; the mixture was stirred at room temperature for 8 h; the reaction was quenched with water, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane-methanol volume ratio 10:1) to obtain intermediate 2. The characterization results of intermediate 2 are as follows: 1 H NMR (C 43 H 60 N2O 12,400 MHz, DMSO): δ 12.16 (s, 2H), 9.96 (s, 2H), 7.84-7.76 (m, 8H), 5.84 (m,1H), 4.76-4.38 (m, 4H), 2.72 (t, 4H), 2.43 (t, 4H), 2.31 (t, 2H), 1.67 (m,2H), 1.32-1.28 (m, 28H), 0.87 (m, 3H); HRMS(ESI+): [M+H] + The calculation yields 797.41, and the value found is 797.40; (3) Intermediate 2, ammonium persulfate, and dimethyl sulfoxide were added to anhydrous 1,4-dioxane, wherein the ratio of intermediate 2, ammonium persulfate, dimethyl sulfoxide, and anhydrous 1,4-dioxane was 10 mmol: 45 mmol: 45 mmol: 100 mL; the reaction mixture was reacted at 95 °C for 10 h; after the reaction was completed, the mixture was filtered while hot, the filtrate was concentrated and diluted with chloroform, and washed successively with 1 M dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution, and brine; the organic layer was dried with anhydrous sodium sulfate, and the concentrate was used to obtain the glyceryl stearate derivative. The characterization results of the glyceryl stearate derivative are as follows: 1 H NMR (C 43 H 56 N2O 10 , 400 MHz, DMSO): δ 7.89 (d, 4H), 7.28 (d, 4H), 5.84 (m, 1H), 4.76-4.38 (m, 4H), 2.92 (s, 8H), 2.31 (t, 2H), 1.67 (m, 2H), 1.32-1.28 (m, 28H), 0.87 (m, 3H); HRMS(ESI+): [M+H] + The calculation yields 761.39, and the result is 761.40.

[0022] This embodiment also provides a pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma, which is prepared using the above-described preparation method.

[0023] Example 2 A method for preparing a pharmaceutical formulation for inhibiting the metastasis of esophageal squamous cell carcinoma includes the following steps: S1. Paclitaxel, cyperone, and glyceryl stearate derivative are dissolved in an organic solvent (chloroform and methanol in a volume ratio of 1:1) to obtain an oil phase; the mass ratio of paclitaxel, cyperone, and glyceryl stearate derivative is 2:1:18, and the ratio of the sum of the masses of paclitaxel and cyperone to the amount of organic solvent is 1 g: 110 mL; S2. Dissolve soybean lecithin and Tween 80 in water to obtain an aqueous phase; wherein the concentration of soybean lecithin in water is 1 wt%, and the concentration of Tween 80 in water is 0.5 wt%. S3. At 70°C, the oil phase and the water phase are mixed at a volume ratio of 1:2, stirred and emulsified at 600 rpm / min for 3 hours, concentrated under reduced pressure to remove organic solvents, cooled to room temperature, and freeze-dried to obtain a drug formulation for inhibiting esophageal squamous cell carcinoma metastasis.

[0024] The preparation of the stearic acid glyceride derivative includes the following steps: (1) N-BOC-4-aminobenzoic acid, glyceryl stearate, and 4-dimethylaminopyridine (DMAP) were added to dichloromethane in a ratio of 20 mmol: 10 mmol: 2 mmol: 50 mL. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was added at 0°C, and after stirring until homogeneous, the temperature was gradually raised to room temperature. The reaction mixture was subjected to an 8-hour incubation period. Trifluoroacetic acid was then added and reacted for 1 hour to remove the BOC protecting group. The ratio of stearic acid glyceride, EDCI, and trifluoroacetic acid was 10 mmol:10 mmol:8 mL. The reaction solution was washed sequentially with 5 wt% sodium bisulfate solution, saturated sodium bicarbonate solution, and saturated brine. The solution was then dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (hexane-ethyl acetate volume ratio 6:1) to obtain intermediate 1. The characterization results of intermediate 1 were the same as in Example 1. (2) Add intermediate 1 and succinic acid to anhydrous N,N-dimethylformamide and stir until homogeneous. Then add 2-(7-azabistriazol-1-yl)-N,N,N',N'-tetramethyluryl hexafluorophosphate (HATU, CAS No.: 148893-10-1) and diisopropylethylamine (DIEA). The intermediate 1, succinic acid, anhydrous N,N-dimethylformamide, 2-(7-azabistriazol-1-yl)-N,N,N',N' The ratio of tetramethyluryl hexafluorophosphate to diisopropylethylamine was 10 mmol: 20 mmol: 60 mL: 20 mmol: 40 mmol; the mixture was stirred at room temperature for 6 h; the reaction was quenched with water, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane-methanol volume ratio 10:1) to obtain intermediate 2; the characterization results of intermediate 2 were the same as in Example 1. (3) Intermediate 2, ammonium persulfate, and dimethyl sulfoxide were added to anhydrous 1,4-dioxane, wherein the ratio of intermediate 2, ammonium persulfate, dimethyl sulfoxide, and anhydrous 1,4-dioxane was 10 mmol: 40 mmol: 40 mmol: 100 mL; the reaction mixture was reacted at 90 °C for 12 h; after the reaction was completed, the mixture was filtered while hot, the filtrate was concentrated and diluted with chloroform, and washed successively with 1 M dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution, and brine; the organic layer was dried with anhydrous sodium sulfate, and the concentrate was used to obtain the glyceryl stearate derivative. The characterization results of the glyceryl stearate derivative were the same as in Example 1.

[0025] This embodiment also provides a pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma, which is prepared using the above-described preparation method.

[0026] Example 3 A method for preparing a pharmaceutical formulation for inhibiting the metastasis of esophageal squamous cell carcinoma includes the following steps: S1. Paclitaxel, cyperone, and glyceryl stearate derivative are dissolved in an organic solvent (chloroform and methanol in a volume ratio of 1:1) to obtain an oil phase; the mass ratio of paclitaxel, cyperone, and glyceryl stearate derivative is 4:1:30, and the ratio of the sum of the masses of paclitaxel and cyperone to the amount of organic solvent is 1 g:120 mL. S2. Dissolve soybean lecithin and Tween 80 in water to obtain an aqueous phase; wherein the concentration of soybean lecithin in water is 2 wt%, and the concentration of Tween 80 in water is 1.5 wt%. S3. At 75°C, the oil phase and the water phase are mixed at a volume ratio of 1:2, stirred and emulsified at 600 rpm / min for 2 hours, concentrated under reduced pressure to remove organic solvents, cooled to room temperature, and freeze-dried to obtain a drug formulation for inhibiting esophageal squamous cell carcinoma metastasis.

[0027] The preparation of the stearic acid glyceride derivative includes the following steps: (1) N-BOC-4-aminobenzoic acid, glyceryl stearate, and 4-dimethylaminopyridine (DMAP) were added to dichloromethane in a ratio of 23 mmol:10 mmol:4 mmol:50 mL. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was added at 0°C, and after stirring until homogeneous, the mixture was gradually heated to room temperature and reacted for 16 minutes. h; add trifluoroacetic acid and react for 2 h to remove the BOC protecting group; wherein the ratio of glyceryl stearate, EDCI, and trifluoroacetic acid is 10 mmol: 12 mmol: 10 mL; wash the reaction solution sequentially with 5 wt% sodium bisulfate solution, saturated sodium bicarbonate solution, and saturated brine; the dichloromethane phase is dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (hexane-ethyl acetate volume ratio 6:1) to obtain intermediate 1; the characterization results of intermediate 1 are the same as in Example 1; (2) Intermediate 1 and succinic acid were added to anhydrous N,N-dimethylformamide and stirred until homogeneous. Then, 2-(7-azabistriazol-1-yl)-N,N,N',N'-tetramethyluryl hexafluorophosphate (HATU, CAS No.: 148893-10-1) and diisopropylethylamine (DIEA) were added to the mixture. The intermediate 1, succinic acid, anhydrous N,N-dimethylformamide, and 2-(7-azabistriazol-1-yl)-N,N,N',N'- The ratio of tetramethyluryl hexafluorophosphate to diisopropylethylamine was 10 mmol: 23 mmol: 60 mL: 25 mmol: 60 mmol; the mixture was stirred at room temperature for 12 h; the reaction was quenched with water, and the mixture was extracted with ethyl acetate. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane-methanol volume ratio 10:1) to obtain intermediate 2; the characterization results of intermediate 2 were the same as in Example 1. (3) Intermediate 2, ammonium persulfate, and dimethyl sulfoxide were added to anhydrous 1,4-dioxane, wherein the ratio of intermediate 2, ammonium persulfate, dimethyl sulfoxide, and anhydrous 1,4-dioxane was 10 mmol: 50 mmol: 50 mmol: 100 mL; the reaction mixture was reacted at 100 °C for 8 h; after the reaction was completed, the mixture was filtered while hot, the filtrate was concentrated and diluted with chloroform, and washed successively with 1 M dilute hydrochloric acid, saturated sodium bicarbonate aqueous solution, and brine; the organic layer was dried with anhydrous sodium sulfate, and the concentrate was used to obtain the glyceryl stearate derivative. The characterization results of the glyceryl stearate derivative were the same as in Example 1.

[0028] This embodiment also provides a pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma, which is prepared using the above-described preparation method.

[0029] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that cyperone is omitted in the preparation of the drug formulation for inhibiting esophageal squamous cell carcinoma metastasis; otherwise, it remains the same as Example 1.

[0030] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that, in the preparation of the drug formulation for inhibiting esophageal squamous cell carcinoma metastasis, the stearic acid glyceride derivative was replaced with stearic acid glyceride; otherwise, it remained the same as Example 1.

[0031] Experimental Example 1 The morphology of the pharmaceutical preparation obtained in Example 1 was observed using transmission electron microscopy, and the results are as follows: Figure 1 As shown.

[0032] Depend on Figure 1 As can be seen, the drug preparation obtained in Example 1 of the present invention is spherical with a particle size of nanometers under transmission electron microscopy, which is beneficial to increasing its release efficiency at the lesion site.

[0033] Experimental Example 2 To calculate the encapsulation efficiency of the pharmaceutical formulations obtained in Examples 1-3 and Comparative Examples 1-2, the following tests were performed. 1 mL of the emulsified product obtained in step S3 of Examples 1-3 and Comparative Examples 1-2 was transferred and centrifuged at 8000 r / min for 15 min. The supernatant was diluted to 50 mL with 75% methanol, filtered through a 0.45 μm filter, and 10 μL was injected by HPLC to determine the mass of free paclitaxel in the supernatant. 75% ethanol was added to the emulsified product obtained in step S3 of Examples 1-3 and Comparative Examples 1-2, and the mixture was sonicated for 10 min to demulsify and release the encapsulated drug. Then, 75% methanol was added to dissolve and dilute to 50 mL, filtered through a 0.45 μm filter, and 10 μL was injected by HPLC to determine the mass of paclitaxel in the pharmaceutical formulation. The mass of free cyperone in the supernatant and the mass of cyperone in the pharmaceutical formulation were determined according to the above test methods.

[0034] The formulas are as follows: Paclitaxel encapsulation rate (%) = (mass of paclitaxel in the drug formulation - mass of free paclitaxel in the supernatant) / mass of paclitaxel in the drug formulation × 100%; Cyperone encapsulation rate (%) = (mass of cyperone in the drug formulation - mass of free cyperone in the supernatant) / mass of cyperone in the drug formulation × 100%. The test results are shown in Table 1.

[0035] Table 1 As shown in Table 1, the encapsulation efficiency of the formulations obtained in Examples 1-3 of this invention is significantly higher than that in Comparative Example 2. This is because the stearic acid glyceride derivative synthesized in this invention exhibits excellent encapsulation effect on aromatic small molecules due to the multiple non-covalent synergistic interactions of the benzene ring, imide, and ester group. A higher encapsulation efficiency in the pharmaceutical formulations of this invention indicates more controllable drug release in vivo, which helps improve the bioavailability and therapeutic efficacy of paclitaxel and cyperone.

[0036] Experimental Example 3 Log-phase KYSE450 and KYSE510 cells were harvested and adjusted to a size of 5 × 10⁻⁶. 4 Cell suspension of 0.5 μg / mL was seeded into 96-well plates and cultured until cell adhesion was achieved. The culture medium was then replaced with different concentrations of the drug formulations from Examples 1-3 and Comparative Examples 1-2, with final concentrations of 0.5, 2.5, 5, 10, and 20 μg / mL, respectively, for 72 h. A blank group containing only culture medium and a control group containing KYSE450 and KYSE510 cell suspensions were also established. The original culture medium was removed, and 1 mg / mL MTT solution was added under light-protected conditions. After 4 h of further culture, DMSO was added, and the plates were incubated at 37°C for 10 min. The absorbance was quantitatively measured at 490 nm using a microplate reader. Cell viability was calculated as: Cell viability = (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance). The results are shown in Tables 2 and 3.

[0037] Table 2. Cell viability results of KYSE450 Table 3. KYSE510 cell viability results As shown in Tables 2 and 3, the drug formulations obtained in Examples 1-3 of this invention can effectively reduce cell viability in a dose-dependent manner within a certain range. Cell survival rate decreases with increasing drug concentration. The above drug formulations have good inhibitory effects on KYSE450 and KYSE510 cells. Comparative Example 1, which omitted cypermethrin, showed less inhibitory effect on cells than Example 1. Compared to using the drug alone, this indicates that the combined use of cypermethrin and paclitaxel can reduce cell viability and inhibit cell survival.

[0038] Test Example 4 To evaluate the oral bioavailability of the drug formulations obtained in Examples 1-3 and Comparative Example 2 of this invention, healthy SD rats weighing 160-180g were randomly divided into four groups: Example 1, Example 2, Example 3, and Comparative Example 2, with six rats in each group. The rats were fasted for 12 hours the night before the experiment but had free access to water. The drug formulations corresponding to Examples 1-3 and Comparative Example 2 were administered by gavage to the groups in Examples 1-3 and Comparative Example 2, respectively (gavage dose calculated as paclitaxel 20 mg / kg). Blood samples were collected via the orbital sinus at 0.5, 1, 2, 3, 4, 8, 12, and 24 hours after administration. After heparin anticoagulation, the plasma was centrifuged at 4000 rpm for 15 min to obtain plasma. The plasma concentration of paclitaxel was detected by HPLC (injection volume 20 μL), and the results are shown in Table 4.

[0039] Table 4 As can be seen from the test results in Table 4, the blood drug concentration of the drug preparations obtained in Examples 1-3 of the present invention is higher than that in Comparative Example 2, indicating that the drug preparations of the present invention can maintain an effective concentration in vivo for a longer period of time and have good oral bioavailability.

[0040] Experimental Example 5 To evaluate the pharmacodynamic behavior of the drug formulations obtained in Examples 1-3 and Comparative Examples 1-2 of this invention, the following experiments were conducted: A human esophageal squamous cell carcinoma subcutaneous xenograft model was established: 5-6 week old male BALB / c nude mice were selected and acclimatized for one week before the experiment. KYSE450 and KYSE510 cells in the logarithmic growth phase were introduced at a rate of 3 × 10⁻⁶ cells / year. 6 Two BALB / c athymic nude mice were subcutaneously injected into the right axilla, with 200 μL injected per mouse. Tumor growth and animal survival were monitored every 48 hours. Tumors were continued until they reached approximately 100 mm in volume. 3 The experimental animals were randomly divided into a blank control group (administered with an equal volume of physiological saline), Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, and Comparative Example 2 group, with 10 animals in each group. The Example 1-3 groups and Comparative Example 1-2 groups were administered the prepared drug formulation (20 mg / kg, every other day) by gavage for 2 weeks. The volume of subcutaneous tumors in nude mice was measured on day 21 after the first administration. The experimental results are recorded in Table 5.

[0041] Table 5 As shown in Table 5, the tumor volume of groups 1-3 of this invention was significantly reduced compared with the blank control group; indicating that the combined use of paclitaxel and cyperene in ESCC cell tumor-bearing mice can significantly inhibit tumor growth and thus prolong the survival time of mice.

[0042] In Comparative Example 1, omitting the addition of cyperone resulted in a smaller tumor volume compared to the blank control group, but the effect was worse than that of Example 1. In Comparative Example 2, replacing the stearic acid glyceride derivative with stearic acid glyceride resulted in an increase in tumor volume compared to Example 1, but the effect was not as good as that of Example 1.

[0043] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a pharmaceutical preparation for inhibiting metastasis of esophageal squamous cell carcinoma, characterized by, The preparation method includes the following steps: S1. Paclitaxel, cyperone, and glyceryl stearate derivative are dissolved in an organic solvent to obtain an oil phase; S2. Dissolve soybean lecithin and Tween 80 in water to obtain an aqueous phase; S3. Mix the oil phase and the aqueous phase at 70-75℃, stir to emulsify, cool to room temperature, and freeze dry to obtain the final product; The chemical structural formula of the stearic acid glyceride derivative is: 。 2.The method for preparing the pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma according to claim 1, characterized in that, The preparation of the stearic acid glyceride derivative includes the following steps: (1) N-BOC-4-aminobenzoic acid, glyceryl stearate and 4-dimethylaminopyridine were added to dichloromethane, EDCI was added at 0°C, and the mixture was stirred until homogeneous and then reacted at room temperature; then trifluoroacetic acid was added and the reaction was continued to obtain intermediate 1. (2) Add intermediate 1 and succinic acid to anhydrous N,N-dimethylformamide and stir until homogeneous. Then add 2-(7-azabistriazol-1-yl)-N,N,N',N'-tetramethyluryl hexafluorophosphate and diisopropylethylamine and stir at room temperature to obtain intermediate 2. (3) Add intermediate 2, ammonium persulfate and dimethyl sulfoxide to anhydrous 1,4-dioxane and heat to react to obtain glyceryl stearate derivative. 3.The method for preparing the pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma according to claim 2, characterized in that, In step (1), the ratio of N-BOC-4-aminobenzoic acid, glyceryl stearate, 4-dimethylaminopyridine, dichloromethane, EDCI, and trifluoroacetic acid is 20-23 mmol: 10 mmol: 2-4 mmol: 50 mL: 10-12 mmol: 8-10 mL; the reaction time at room temperature is 8-16 h; and the reaction time after adding trifluoroacetic acid is 1-2 h. 4.The method for preparing the pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma according to claim 2, characterized in that, In step (2), the ratio of intermediate 1, succinic acid, anhydrous N,N-dimethylformamide, 2-(7-azabistriazol-1-yl)-N,N,N',N'-tetramethyluryl hexafluorophosphate and diisopropylethylamine is 10 mmol: 20-23 mmol: 60 mL: 20-25 mmol: 40-60 mmol; the stirring time at room temperature is 6-12 h. 5.The method for preparing the pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma according to claim 2, characterized in that, In step (3), the ratio of intermediate 2, ammonium persulfate, dimethyl sulfoxide and anhydrous 1,4-dioxane is 10 mmol: 40-50 mmol: 40-50 mmol: 100 mL; the heating reaction temperature is 90-100 °C and the time is 8-12 h. 6.The method for preparing the pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma according to claim 1, characterized in that, In step S1, the mass ratio of paclitaxel, cyperone, and glyceryl stearate derivative is (2-4):1:(18-30), and the ratio of the sum of the masses of paclitaxel and cyperone to the amount of organic solvent is 1g:110-120mL; the organic solvent is prepared by mixing chloroform and methanol in a volume ratio of 1:

1.

7. The method for preparing the pharmaceutical formulation for inhibiting esophageal squamous cell carcinoma metastasis according to claim 1, characterized in that, In step S2, the concentration of soybean lecithin in water is 1-2 wt%, and the concentration of Tween 80 in water is 0.5-1.5 wt%.

8. The method for preparing the pharmaceutical formulation for inhibiting esophageal squamous cell carcinoma metastasis according to claim 1, characterized in that, In step S3, the volume ratio of the organic phase to the aqueous phase is 1:2; the emulsification time is 2-3 hours.

9. A pharmaceutical preparation for inhibiting the metastasis of esophageal squamous cell carcinoma, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

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