Superoxide anion free radical activated indometacin-transretinoic acid prodrug and application thereof

Abstract

The invention provides a superoxide anion free radical activated indometacin-transretinoic acid prodrug, a preparation method of an albumin compound of the superoxide anion free radical activated indometacin-transretinoic acid prodrug and application of a composition prepared from the compound and an antitumor drug. The prodrug compound and an anti-tumor drug are prepared into the composition, the prodrug is activated by superoxide anion free radicals overexpressed in a tumor microenvironment, prototype drugs of indometacin and transretinoic acid are released, the damage effect of the transretinoic acid on a Golgi apparatus structure of a tumor cell is remarkably enhanced, and excellent broad-spectrum applicability is shown; and the device can be adapted to various drug delivery systems and disease models.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of based on superoxide anion radical activation indomethacin-trans retinoic acid prodrug, albumin complex and its preparation method and the application of anti-tumor drug combination, belong to anti-tumor drug technical field. BACKGROUND

[0002] Malignant tumor, as a major public health challenge to human health, its disease burden continues to rise globally. Although modern medicine has made significant progress in treatment and drug development, most cancer patients still face the clinical dilemma of low response rate and limited survival cycle. Research data shows that more than 60% of solid tumor patients still have drug resistance recurrence and metastasis after receiving standard chemotherapy. In this context, developing new treatment strategies and optimizing existing therapy synergistic means has important clinical value and social significance.

[0003] The use of cytotoxic anticancer drugs has the potential risk of promoting tumor metastasis, which may be closely related to drug mechanism, tumor microenvironment regulation and drug resistance. Cytotoxic anticancer drug doxorubicin can induce tumor cells to release pro-inflammatory factors (such as IL-6, TGF-β) and activate epithelial-mesenchymal transition (EMT), thereby enhancing the migration and invasion ability of tumor cells; and long-term use of cytotoxic anticancer drugs can aggravate the tumor hypoxic microenvironment, further promoting angiogenesis and metastasis; some existing studies suggest that cytotoxic drugs can promote tumor recurrence and metastasis by activating tumor stem cell-related pathways. To address the risk of cytotoxic drugs promoting metastasis, consider optimizing the targeted delivery system and reducing systemic exposure to avoid this risk.

[0004] Golgi apparatus, as an important membrane organelle in eukaryotic cells, plays a key role in post-translational modification of proteins, intracellular transport and exocytosis. Studies have shown that abnormalities in the structure and function of this organelle are closely related to various pathological processes, especially in neurodegenerative diseases and malignant tumors. Notably, abnormal expansion of Golgi structure is common in malignant tumor cells, and their secretion activity is significantly enhanced, affecting tumor progression by regulating the exocytosis of pro-cancer factors and metastasis-related proteins. Clinical data analysis shows that the expression levels of many Golgi-specific proteins are significantly correlated with the metastasis risk and survival prognosis of cancer patients. Based on these findings, by targeting the biological function of Golgi and interfering with its protein processing and transport system, the maturation and release of pro-cancer factors can be effectively blocked, thereby regulating key signaling networks in tumor development and affecting multiple signaling cascades in tumor progression, ultimately achieving the therapeutic goal of inhibiting cancer cell proliferation and metastasis. However, drugs based on this mechanism still have limited therapeutic effect against tumors and tumor metastasis.

[0005] At present, Golgi-targeted drug delivery systems are rarely used for disease treatment. Patent CN116570724A discloses a nano particle based on tumor microenvironment activated Golgi-targeted interference agent, the prodrug linker of which is an oxygen-responsive nitro structure. The invention is based on the research of Kumar R et al. that nitro groups can be reduced to amino groups by tumor cell overexpressed nitro reductase (Kumar R, Kim EJ, Han J, et al. Hypoxia-directed and activated theranostic agent: Imaging and treatment of solid tumor. Biomaterials.); and in the related research of Liu et al., triflate groups can be selectively removed by superoxide anion radical groups, so that triflate groups can be designed as superoxide anion radical group response groups (Liu Y, Li Q, Zheng Z, et al. Simultaneously detecting O2•⁻, polarity and viscosity with different fluorescence signals in Amiodarone-induced liver injury [J]. Sens. Actuators B Chem.). Superoxide anion radical is one of reactive oxygen species (ROS), and the ROS response mechanism has significant advantages over hypoxia response: ROS response does not depend on local oxygen concentration, significantly improving tissue applicability (Li JQ, Zhuang ZY, Zhao ZJ, et al. Type IAIE Photosensitizers: Mechanism and Application. View 2021); higher targeting specificity, faster reaction kinetics (seconds), and activation through direct oxidation-reduction mechanism without enzyme catalysis, significantly improving conversion efficiency (Xinyan Li, et al. Precise modulation and use of reactive oxygen species for immunotherapy. Sci. Adv. 10). SUMMARY

[0006] The purpose of the present application is to develop an indomethacin-trans retinoic acid prodrug and related albumin complex preparation with superoxide anion radical response release, simple preparation process and wider application range. By the ability of indomethacin to specifically bind to the overexpressed cyclooxygenase COX-2 on the Golgi body in tumor cells, the delivery efficiency of the Golgi interference agent trans retinoic acid is improved, thereby destroying the Golgi structure and function of tumor cells and blocking the production and secretion of tumor metastasis-related regulatory proteins, thereby improving the treatment effect on tumors and their metastasis.

[0007] One of the purposes of the present application is to provide a superoxide anion radical activated indomethacin-trans retinoic acid prodrug, which comprises a Golgi body targeting group indomethacin (IMC), an intermediate response group and a Golgi body interfering agent trans retinoic acid (RA), and the structural formula is (Formula I, ISR) One of the purposes of the present application is to provide a synthesis method of a superoxide anion radical activated indomethacin-trans retinoic acid prodrug, which comprises the following steps: (1) 2-hydroxy-5-methyl resorcinol reacts with tert-butyl dimethyl chlorosilane to generate intermediate compound 1, and the structural formula of intermediate compound 1 is: (2) Intermediate compound 1 reacts with trifluoromethyl sulfonic anhydride to generate superoxide anion radical responsive intermediate compound 2, and the structural formula of intermediate compound 2 is: (3) Under the action of p-toluenesulfonic acid, intermediate compound 2 is removed TBS protective group to generate intermediate compound 3, and the structural formula of intermediate compound 3 is: (4) Intermediate compound 3 reacts with tert-butyl dimethyl chlorosilane to generate single side silicon-based protected intermediate compound 4, and the structural formula of intermediate compound 4 is: (5) Indomethacin reacts with intermediate compound 4 by esterification to generate intermediate compound 5, and the structural formula of intermediate compound 5 is: (6) Intermediate compound 5 removes TBS protective group to generate intermediate compound 6, and the structural formula of intermediate compound 6 is: (7) Trans retinoic acid reacts with intermediate compound 6 by esterification to generate superoxide anion radical activated indomethacin-trans retinoic acid prodrug (Formula I, ISR).

[0008] The intermediate response group described in the present application can be activated by high concentration of superoxide anion radicals of tumor cells, further triggering a domino elimination reaction, so that the prototype drugs indomethacin and trans retinoic acid are released, and the purpose of precise anti-tumor treatment is achieved.

[0009] One of the purposes of the present application is to provide a superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug-albumin complex, comprising 1 part of superoxide anion radical-activated prodrug and 1-20 parts of albumin; preferably, 1 part of superoxide anion radical-activated prodrug and 5-20 parts of albumin.

[0010] The prodrug prepared in the present application can form a stable molecular complex with albumin. Since albumin has tumor cell targeting property, the prodrug-albumin complex can also target tumor cells and play a role.

[0011] Further, the prodrug-albumin complex of the present application can be made into a composition with an antitumor drug, wherein the antitumor drug used is selected from anthracyclines, taxanes, antimetabolites and other cytotoxic chemotherapeutics, the anthracycline antitumor drugs cover doxorubicin, daunorubicin, epirubicin and their derivatives, and specifically include but are not limited to pyran doxorubicin, aclacinomycin, amrubicin and other therapeutic agents, and can also be used as a synergistic component with zorubicin, plicamycin, idarubicin or mitoxantrone; the taxane antitumor drugs mainly include paclitaxel, docetaxel and the like; the antimetabolite antitumor drugs mainly include 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), gemcitabine and the like.

[0012] The superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug-albumin complex or the composition described in the present application can also add a surfactant with polyethylene glycol as a hydrophilic group and an amphiphilic compound as a surface modification material.

[0013] The surface modification material is selected from a surfactant with polyethylene glycol as a hydrophilic group and an amphiphilic compound, including but not limited to one or more of the surfactants Solutol HS15, polysorbate series (Tween), Lipoid, Labrasol, poloxamer, one or more of the amphiphilic polymers PEGylated phospholipid, polyethylene glycol-lactide-glycolide copolymer (PEG-PLGA), polyethylene glycol-polylactic acid (PEG-PLA) and polyethylene glycol-polycaprolactone (PEG-PCL); the preferred surface modification material is one or more of mPEG-DSPE, Solutol HS15. The surface modification material is added in an amount of 0-100 parts by weight, and the preferred implementation range is 1-10 parts. 2000

[0014] ​A pharmaceutical preparation comprising the above-mentioned composition and a pharmaceutically acceptable adjuvant selected from one or more of an osmotic pressure regulator, a stabilizer, a microbial inhibiting component or a buffer system; the buffer system achieves physiological compatibility regulation by a pH regulator; the osmotic pressure regulator component is selected from one or more of sodium chloride, glucose, glycerol, mannitol, sorbitol, potassium chloride, sodium lactate, amino acids, dextran, gelatin, povidone, starch derivatives and the like; the stabilizer is selected from one or more of glycerol, sucrose, polyethylene glycol and the like; the microbial inhibitor is selected from one or more of methyl phenol, phenol and the like; and the pH regulator is selected from one or more of hydrochloric acid, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium bicarbonate, sodium carbonate, lactic acid, sodium lactate and the like.

[0015] One of the purposes of the present application is to provide a preparation method of superoxide anion radical-activated prodrug-albumin complex, which is characterized by comprising the following steps: (1) dissolving the superoxide anion radical-activated prodrug in an organic solvent; (2) adding an albumin aqueous solution, and performing high-pressure homogenization or probe ultrasonic emulsification; (3) removing the organic solvent by rotary evaporation, and obtaining the product.

[0016] One of the purposes of the present application is to provide a preparation method of superoxide anion radical-activated prodrug-albumin complex, which is characterized by comprising the following steps: (1) dissolving the superoxide anion radical-activated prodrug in an organic solvent; (2) adding an albumin aqueous solution, and performing high-pressure homogenization or probe ultrasonic emulsification; (3) when the antitumor drug is a water-soluble antitumor drug, mixing (1) and (2), and performing high-pressure homogenization or probe ultrasonic emulsification; when the antitumor drug is a fat-soluble drug, mixing (1) and (2), adding an albumin aqueous solution, and performing high-pressure homogenization or probe ultrasonic emulsification; (4) removing the organic solvent by rotary evaporation, and obtaining the product.

[0017] Taking an anthracycline antitumor drug as an example, step (1) can adopt the method of directly dissolving the anthracycline antitumor drug in an organic solvent, or taking a pharmaceutically acceptable salt of the anthracycline antitumor drug, first neutralizing the corresponding acid with an alkaline substance, and then dissolving in an organic solvent. Among them, the alkaline substance in step (1) is mainly used to neutralize the acid radical in the pharmaceutically acceptable salt of the anthracycline antitumor drug, to obtain the drug molecule in the form of free base.

[0018] The basic system comprises inorganic alkali regulators and organic alkali regulators: the inorganic alkali is selected from sodium / potassium carbonates and hydroxides, including NaOH, KOH, Na2CO3, KHCO3 and derivatives thereof; the organic alkali comprises triethylamine, ammonia and other donor compounds; the basic substance in step (1) is preferably sodium bicarbonate, and the amount of the basic substance added can be determined according to the properties of the drug.

[0019] The organic solvent in steps (1) and (2) is selected from one or more of the following: halogenated hydrocarbons such as chloroform and dichloromethane, ethers / ketones such as tetrahydrofuran and acetone, and mixtures of one or more of ethyl acetate, ethanol, methanol, dioxane, acetonitrile, dimethyl sulfoxide, dimethylformamide, methylpyrrolidone and similar solvents, or a mixture of one or more of the above solvents; preferably chloroform.

[0020] The rotary evaporation temperature in step (4) is in the range of 20-70°C, preferably 30-40°C.

[0021] The superoxide anion radical-activated prodrug albumin complex also comprises a surface modification material that is soluble in an organic solvent and / or in deionized water, and / or is added directly after step (4).

[0022] One of the purposes of the present application is to provide a superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug albumin complex and an antitumor drug for treating tumors and tumor metastasis.

[0023] Invention benefits (1) The superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug of the present application can improve the targeting ability to the Golgi apparatus in tumor cells, thereby improving the treatment effect; (2) The superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug of the present application has a simple synthesis process, can be delivered by various drug delivery systems, and can be used to treat various tumor-related diseases, thus having a wide range of applications; (3) The superoxide anion radical-activated prodrug albumin complex of the present application has a simple preparation method, readily available raw materials and a large drug loading capacity, and can play a role in various situations; (4) The superoxide anion radical-activated prodrug albumin complex of the present application can significantly improve the anti-tumor and anti-tumor metastasis treatment effect when used in combination with an antitumor drug, and has good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 . Nuclear magnetic resonance hydrogen spectrum of superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug (ISR).

[0025] Figure 2 . Photographs of appearance of Blank@HSA-Dspe, ISR@HSA-Dspe, DOX@HSA-Dspe, DOX-ISR@HSA and DOX-ISR@HSA-Dspe.

[0026] Figure 3 . Uptake of doxorubicin by 4T1 cells (n=3). ****p<0.0001.

[0027] Figure 4 . Results of in vitro cytotoxicity experiment (n=6).

[0028] Figure 5 . Results of scratch test. Scale bar: 100 pm.

[0029] Figure 6 . Confocal microscope images of immunofluorescence labeling of Golgi-specific structural proteins after treatment of 4T1 tumor cells with free drug or different formulations. Scale bar: 10 pm.

[0030] Figure 7 . PGE2 concentration in 4T1 cell culture supernatant after administration of different groups (n=4). ***p<0.001, ****p<0.0001 Figure 8 . In vivo anti-tumor effect.

[0031] (A) Tumor growth curve of each group (n=6). **p<0.01, ***p<0.001, ****p<0.0001.

[0032] (B) Tumor weight on day 22 (n=6). *p<0.05, **p<0.01, ****p<0.0001.

[0033] (C) Tumor inhibition rate calculated according to tumor weight (n=6). *p<0.05, **p<0.01, ****p<0.0001. (D) Photographs of tumor tissues taken on day 22 (n=6).

[0034] (E) Kaplan-Meier survival curve of different treatment groups (n=10).

[0035] Figure 9 . In vivo anti-metastasis effect.

[0036] (A) Photographs of lung tissues fixed with Bouin's fixative.

[0037] (B) Number of lung metastatic nodules (n=3). **p<0.01, ***p<0.001, ****p<0.0001. Detailed Implementation

[0038] The following embodiments are further illustrations of the present invention, but are by no means limitations on the scope of the invention. The present invention is further described in detail below with reference to the embodiments; however, those skilled in the art should understand that the present invention is not limited to these embodiments and the preparation methods used. Furthermore, those skilled in the art can make equivalent substitutions, combinations, improvements, or modifications to the present invention based on the description thereof, but all such substitutions and modifications will be included within the scope of the present invention.

[0039] Example 1 The synthetic steps of the superoxide anion radical activated indomethacin-trans retinoic acid prodrug (ISR) are as follows: (1) Dissolve 10.1g of 2-hydroxy-5-methyl-isophthalic acid and 8.9g of imidazole in 40mL of ultra-dry DMF, cool to 0°C, dissolve 19.7g of tert-butyldimethylchlorosilane (TBSCl) in 90mL of ultra-dry DMF, add dropwise, stir at room temperature for 2h, and incubate overnight in an oil bath at 30°C to obtain crude product. Purify by column chromatography to obtain a yellow viscous liquid, which is intermediate compound 1; (2) Take 2.5g of intermediate compound 1 and 15ml of pyridine and dissolve them in 15ml of DCM. React at -78°C for 30min. Add 1.9ml of trifluoromethanesulfonic anhydride dropwise. After reacting at -78°C for 15min, react at room temperature for 3h to obtain crude product. Purify by column chromatography to obtain a light yellow viscous liquid, which is intermediate compound 2. (3) Take 4.2g of intermediate compound 2 and dissolve it in 20ml of methanol. Add 257.1mg of catalyst p-toluenesulfonic acid, stir at room temperature for 1h, and purify by rotary evaporation column chromatography to obtain a light yellow viscous liquid, which is intermediate compound 3; (4) Dissolve 1.6g of intermediate compound 3 and 1.1g of imidazole in 30ml of DMF, dissolve 0.9g of tert-butyldimethylchlorosilane (TBSCl) in 15ml of DMF, slowly add the solution, and incubate overnight in an oil bath at room temperature to obtain the crude product. Purify by column chromatography to obtain a pale yellow viscous liquid, which is intermediate compound 4. (5) Dissolve 990.1 mg of intermediate compound 4 and 350.4 mg of 4-dimethylaminopyridine (DMAP) in 40 ml of dichloromethane (DCM), add 1.0 g of indomethacin (IMC) and 591.8 g of dicyclohexylcarbodiimide (DCC), and incubate overnight in an oil bath at 30°C to obtain the crude product. Purify by column chromatography to obtain a pale yellow viscous liquid, which is intermediate compound 5; (6) Dissolve 1.6g of intermediate compound 5 in 20ml of methanol, dissolve 35.4mg of catalyst p-toluenesulfonic acid in 5ml of methanol, add dropwise, stir overnight at room temperature to obtain crude product, and purify by column chromatography to obtain a pale yellow viscous liquid, which is intermediate compound 6; (7) Take 835.0 mg of trans-retinoic acid (RA) and 339.6 mg of 4-dimethylaminopyridine (DMAP) and dissolve them in 5 mL of dichloromethane (DCM), activate for 15 min, add 1.1 g of intermediate compound 6 and 573.6 mg of dicyclohexyl carbodiimide (DCC), and then place in an oil bath at 30°C overnight to obtain a crude product. After drying the product in vacuum, a yellow solid powder is obtained, which is a tumor active oxygen-activated Golgi interferase (ISR).

[0040] Example 2 Take 4 mg of ISR and dissolve them in 0.4 mL of chloroform, take 4 mg of mPEG 2000 -DSPE and 60 mg of human serum albumin are dissolved in 4 mL of deionized water, and after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), and then rotary evaporation is performed at 37°C to remove chloroform, and thus ISR@HSA-Dspe is obtained.

[0041] Example 3 Take 2 mg of doxorubicin (DOX) and dissolve them in 0.4 mL of chloroform, take 4 mg of mPEG 2000 -DSPE and 60 mg of human serum albumin are dissolved in 4 mL of deionized water, and after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), and then rotary evaporation is performed at 37°C to remove chloroform, and thus DOX@HSA-Dspe is obtained.

[0042] Example 4 Take 2 mg of doxorubicin and 4 mg of ISR and dissolve them in 0.4 mL of chloroform, take 60 mg of human serum albumin and dissolve them in 4 mL of deionized water, and after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), and then rotary evaporation is performed at 37°C to remove chloroform, and thus DOX-ISR@HSA is obtained.

[0043] Example 5 Take 2 mg of doxorubicin and 4 mg of ISR and dissolve them in 0.4 mL of chloroform, take 4 mg of mPEG 2000 -DSPE and 60 mg of human serum albumin are dissolved in 4 mL of deionized water, and after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), and then rotary evaporation is performed at 37°C to remove chloroform, and thus DOX-ISR@HSA-Dspe is obtained.

[0044] Example 6 Take 2 mg of paclitaxel (PTX) and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 60 mg of human serum albumin dissolved in 4 mL of deionized water, after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to get PTX-ISR@HSA. After diluting PTX-ISR@HSA with deionized water to the appropriate concentration, the particle size of the complex is 90.44 nm and the PDI is 0.142, which is measured by a laser particle size analyzer.

[0045] Example 7 Take 2 mg of paclitaxel (PTX) and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 60 mg of human serum albumin dissolved in 4 mL of deionized water, after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to get PTX-ISR@HSA. After diluting PTX-ISR@HSA with deionized water to the appropriate concentration, the particle size of the complex is 90.44 nm and the PDI is 0.142, which is measured by a laser particle size analyzer.

[0046] Example 8 Take 2 mg of doxorubicin and 1 mg of prodrug ISR dissolved in 0.4 mL of chloroform, take 4 mg of mPEG 2000 -DSPE and 60 mg of human serum albumin dissolved in 4 mL of deionized water, after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to get DOX-ISR@HSA-Dspe.

[0047] Example 9 Take 2 mg of doxorubicin and 2 mg of prodrug ISR dissolved in 0.4 mL of chloroform, take 4 mg of mPEG 2000 -DSPE and 60 mg of human serum albumin dissolved in 4 mL of deionized water, after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to get DOX-ISR@HSA-Dspe.

[0048] Example 10 Take 2 mg of doxorubicin and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 4 mg of mPEG 2000 -DSPE and 80 mg of human serum albumin dissolved in 4 mL of deionized water, after mixing, the probe is ultrasonicated for 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to get DOX-ISR@HSA-Dspe.

[0049] Example 11 Take 2 mg of doxorubicin and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 4 mg of mPEG 2000- DSPE and 100 mg human serum albumin were dissolved in 4 mL deionized water, after mixing, the probe was ultrasonic 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to obtain DOX-ISR@HSA-Dspe.

[0050] Example 12 Take 2 mg of doxorubicin and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 4 mg of mPEG 2000 - DSPE and 100 mg human serum albumin were dissolved in 4 mL deionized water, after mixing, the probe was ultrasonic 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to obtain DOX-ISR@HSA-Dspe.

[0051] Example 13 Take 2 mg of doxorubicin and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 6 mg of mPEG 2000 - DSPE and 100 mg human serum albumin were dissolved in 4 mL deionized water, after mixing, the probe was ultrasonic 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to obtain DOX-ISR@HSA-Dspe.

[0052] Example 14 Take 2 mg of doxorubicin and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 8 mg of mPEG 2000 - DSPE and 100 mg human serum albumin were dissolved in 4 mL deionized water, after mixing, the probe was ultrasonic 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to obtain DOX-ISR@HSA-Dspe.

[0053] Example 15 Take 2 mg of doxorubicin and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 10 mg of mPEG 2000 - DSPE and 100 mg human serum albumin were dissolved in 4 mL deionized water, after mixing, the probe was ultrasonic 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to obtain DOX-ISR@HSA-Dspe.

[0054] Example 16 Take 2 mg of doxorubicin and 4 mg of ISR dissolved in 0.4 mL of chloroform, take 4 mg of Solutol HS15 and 60 mg of human serum albumin dissolved in 4 mL of deionized water, after mixing, the probe was ultrasonic 5 min (power 200 W, ultrasonic 5 s, rest 5 s), then 37 °C rotary evaporation to remove chloroform to obtain DOX-ISR@HSA-HS15.

[0055] Example 17 Dissolve 2 mg of doxorubicin and 4 mg of ISR in 0.4 mL of chloroform, dissolve 6 mg of Solutol HS 15 and 60 mg of human serum albumin in 4 mL of deionized water, mix them together and then sonicate for 5 min (power 200 W, sonicate for 5 s, stop for 5 s) with a probe, and then remove the chloroform by rotary evaporation at 37 °C to obtain DOX-ISR@HSA-HS 15.

[0056] Example 18 Dissolve 2 mg of doxorubicin and 4 mg of ISR in 0.4 mL of chloroform, dissolve 8 mg of Solutol HS 15 and 60 mg of human serum albumin in 4 mL of deionized water, mix them together and then sonicate for 5 min (power 200 W, sonicate for 5 s, stop for 5 s) with a probe, and then remove the chloroform by rotary evaporation at 37 °C to obtain DOX-ISR@HSA-HS 15.

[0057] Example 19 Dissolve 4 mg of ISR in 0.4 mL of chloroform, dissolve 2 mg of mitoxantrone hydrochloride, 4 mg of mPEG 2000 -DSPE and 60 mg of human serum albumin in 4 mL of deionized water, mix them together and then sonicate for 5 min (power 200 W, sonicate for 5 s, stop for 5 s) with a probe, and then remove the chloroform by rotary evaporation at 37 °C to obtain MTO-ISR@HSA-Dspe.

[0058] Experimental Example 1 The superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug (ISR) synthesized in Example 1 was dissolved in deuterated chloroform for nuclear magnetic resonance hydrogen spectrum analysis, and the results are shown in Figure 1 As can be seen, the superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug shown in Formula I was successfully synthesized.

[0059] Experimental Example 2 The complexing rate of the chemotherapeutic drug DOX and / or the prodrug ISR in the complexes obtained in Example 2, Example 4 and Example 5 was determined by ultrafiltration. 0.4 mL of ISR@HSA-Dspe, DOX-ISR@HSA and DOX-ISR@HSA-Dspe was taken into an ultrafiltration tube with a molecular cut-off of 10 kDa, centrifuged at 5000 rpm for 20 min, and 0.1 mL of the ultrafiltrate was appropriately diluted and then used to determine the concentration of DOX by fluorescence spectrophotometry and to determine the concentration of ISR by HPLC, and the complexing rate was calculated.

[0060] Table 1 Complexing rate of the complex It can be seen that in the complexes of Example 2, Example 4 and Example 5, human serum albumin has a high complexing rate with both the chemotherapeutic drug DOX and the prodrug ISR.

[0061] Experimental Example 3 The appearance of the complexes was observed in a Schlenk flask, wherein the blank Blank@HSA-Dspe was prepared as follows: 4 mg mPEG-DSPE and 60 mg human serum albumin were dissolved in 4 mL deionized water, and 0.4 mL chloroform was added thereto, and the mixture was subjected to probe sonication for 5 min (power 200 W, sonication for 5 s and rest for 5 s), and then rotary evaporation was performed at 37°C to remove the chloroform. 2000

[0062] The results are shown in Table 1. Figure 2 As shown in Table 1, the appearance of the preparations was clear and transparent.

[0063] Experimental Example 4 In-vitro cell uptake experiment. Mouse 4T1 breast cancer cells in good growth state were trypsinized, centrifuged to collect the cells, resuspended with complete culture medium, and inoculated into 12-well cell culture plates at an appropriate density, and cultured in a constant humidity incubator at 37°C and 5% CO2. After 24 h, the culture medium was discarded and the cells were washed with PBS, and then 1 mL of free DOX-ISR solution diluted with blood-free and antibody-free culture medium, and DOX-ISR@HSA-Dspe prepared in Example 5 were added to each well, respectively, and the drug concentration was 3 μg / mL calculated according to the DOX dose, and three replicate wells were set in each group. After 2 h and 4 h of incubation after drug administration, the drug solution was aspirated and washed with PBS three times, and then trypsinized and centrifuged at 2000 rpm for 3 min to collect the cells. After discarding the supernatant, the cells were washed with PBS three times, and finally resuspended in 300 μL of PBS. The cell uptake amount was detected by flow cytometry through the fluorescence of DOX. The free DOX-ISR solution was prepared as follows: DOX and ISR were dissolved in 400 μL of a mixed solvent containing DMSO, Tween 80 and PEG200, and water for injection was added to make the system 4 mL.

[0064] The experimental results are shown in Table 2. Figure 3 The uptake amount of 4T1 cells to DOX-ISR@HSA-Dspe was 1.96 times and 2.10 times that of free drug at 2 h and 4 h, respectively, indicating that the prodrug complex can significantly increase the uptake amount of 4T1 cells to the drug.

[0065] Experimental Example 5 ​Cytotoxicity test. 4T1 cells in good condition and in logarithmic growth phase were selected and inoculated in 96-well plates, and after 24 h of culture in a 37°C, 5% CO2 incubator, the upper culture medium was discarded, and each group of drugs or preparations: freeDOX-ISR, DOX@HSA-Dspe prepared in Example 3, DOX-ISR@HSA prepared in Example 4 and DOX-ISR@HSA-Dspe prepared in Example 5 were diluted into a series of concentration gradients with serum-free and antibiotic-free culture medium: 6 replicate wells were set for each concentration, and a group of blank control wells were added with blank culture medium. After adding the drug, the drug solution was discarded after 24 h of culture at 37°C, 5% CO2, 100 μL of 10% cck8 solution was added to each well, and the incubation was continued for 20 min at 37°C, 5% CO2, then the absorbance at 450 nm was measured using an enzyme-linked immunoassay instrument, and the results were recorded to calculate the cell survival rate.

[0066] According to Figure 4 As shown in FIG. 6, the cytotoxicity of the DOX-ISR@HSA-Dspe drug group was significantly higher than that of the other drug experimental groups, which may be due to its good stability and higher cell uptake. For the freeDOX-ISR group, the DOX@HSA-Dspe group and the DOX-ISR@HSA group, it may be due to the combined use of freeDOX and ISR, which is more easily diffused in the drug solution, which is beneficial to cell uptake.

[0067] Experimental Example 6 Cell migration experiment. 4T1 cells in good growth condition were inoculated in 12-well cell culture plates and cultured for 24 h until the cell density reached 80%, then the culture medium was discarded, and a sterile pipette tip was used to draw a wound in the center of the culture well along the diameter direction, and the cells were washed with sterile PBS for 3 times to remove excess cell residues, then 1 mL of freeDOX-ISR, DOX@HSA-Dspe prepared in Example 3, DOX-ISR@HSA prepared in Example 4 and DOX-ISR@HSA-Dspe prepared in Example 5 diluted with serum-free and antibiotic-free culture medium were added, and the drug dose was calculated as 100 ng / mL of DOX, and serum-free and antibiotic-free culture medium was used as a control group, 3 replicate wells were set for each group, and incubated in a 37°C, 5% CO2 incubator for 24 h, then the drug solution was discarded, and the cells were fixed with 4% paraformaldehyde after washing with PBS. The pictures at 0 h and 24 h were collected using an inverted fluorescence microscope.

[0068] Tumor cell migration is an important step in the process of tumor metastasis, such as Figure 5As shown, freeDOX-ISR, DOX@HSA-Dspe inhibited cell migration to a small extent, DOX-ISR@HSA inhibited cell migration significantly, and the DOX-ISR@HSA-Dspe group showed comparable anti-migration ability, and the effect was optimal.

[0069] Experimental Example 7 Golgi structure destruction characterization. Well-grown 4T1 cells were seeded in a glass-bottomed culture dish and incubated overnight at 37°C, 5% CO2 in a cell incubator, then washed the cells 3 times with PBS, then added PBS, freeDOX-ISR, DOX@HSA-Dspe prepared in Example 3, DOX-ISR@HSA prepared in Example 4 and DOX-ISR@HSA-Dspe prepared in Example 5, respectively, the drug concentration was 200 ng / mL according to THP, incubated in the cell incubator for 12 h, then discarded the drug solution, washed 3 times with PBS, fixed the cells with 4% paraformaldehyde at room temperature for 10 min, washed the cells 3 times with PBS, added 0.1% Triton X-100 and incubated at room temperature for 5 min, then washed 3 times with PBS, added 5% BSA and incubated at 37°C for 1 h, washed 3 times with PBS, added GM130 antibody and incubated at 4°C overnight, then washed 3 times with PBS, added CoraLite 647-labeled secondary antibody and incubated at room temperature for 1 h, washed 3 times with PBS, finally stained the cell nucleus with DAPI at room temperature for 10 min, washed 3 times with PBS, and observed the fluorescence distribution under a laser confocal microscope.

[0070] As shown in Figure 6 The traditional chemotherapeutic drug doxorubicin hydrochloride DOX did not produce significant changes to the Golgi space structure of eukaryotic cells, but after adding the prodrug ISR, the distribution pattern of the organelle Golgi changed from a nuclear periphery dense aggregation state to a whole cytoplasmic diffuse distribution, confirming that the prodrug can target the Golgi, making RA reach the Golgi more, significantly improving its effect. From the cell uptake experiment, it can be seen that DOX-ISR@HSA-Dspe can be taken up by cells more, and at the same time has good stability, and has the strongest destruction effect on the Golgi.

[0071] Experimental Example 8 The well-grown 4T1 cells were inoculated in a twelve-hole plate at a suitable cell density, cultured overnight at 37°C in a 5% CO2 cell incubator, washed with PBS for 3 times, and then added with PBS, freeDOX-ISR, ISR@HSA-Dspe prepared in Example 2, DOX@HSA-Dspe prepared in Example 3, DOX-ISR@HSA prepared in Example 4, and DOX-ISR@HSA-Dspe prepared in Example 5, respectively, with the drug concentration being 100 ng / mL of DOX. After 24 h of incubation in the cell incubator, the culture supernatant was collected and the PGE2 concentration in the cell culture supernatant was detected according to the operation instruction of the detection kit.

[0072] The selective COX-2 inhibitor IMC can significantly inhibit the proliferation and metastasis of tumor cells by regulating the biosynthesis pathway of prostaglandin E2 (PGE2). As shown in Figure 7 The PGE2 concentration in the cell culture supernatant of the treated DOX-ISR@HSA-Dspe group showed a downward trend, which was significantly related to the drug action mechanism.

[0073] Experimental Example 9 In vivo anti-tumor pharmacodynamic study. The well-grown 4T1 cells in the logarithmic growth phase were collected, resuspended with PBS and adjusted to a cell concentration of 1×10 7 The 4T1 breast cancer-bearing mouse model was established by injecting 0.1 mL of the cell suspension into the right axillary mammary fat pad of the mouse with a syringe. On the 5th day after inoculation of the 4T1 cells, the tumor-bearing mice successfully modeled were randomly divided into 6 groups, 16 in each group, and then injected with physiological saline, freeDOX-ISR, ISR@HSA-Dspe prepared in Example 2, DOX@HSA-Dspe prepared in Example 3, DOX-ISR@HSA prepared in Example 4, and DOX-ISR@HSA-Dspe prepared in Example 5, respectively, via the tail vein, with the drug dosage being 4 mg / kg of DOX and 8 mg / kg of ISR, and the drug was administered once every 3 days for 4 times. The long diameter and short diameter of the mouse tumor were measured every two days with a vernier caliper from the first administration, and the tumor volume was calculated. On the 22nd day after inoculation, 6 mice in each group were sacrificed by cervical dislocation, the tumor tissues were dissected, washed with physiological saline, and then dried with filter paper, photographed and weighed, and the tumor inhibition rate was calculated. The remaining mice were used for survival period recording.

[0074] As Figure 8As shown, the tumor volume of the saline group mice continuously increased after inoculation of 4T1 tumor cells, the freeDOX-ISR solution inhibited the growth of the tumor to some extent, but the body weight of the mice decreased significantly, the tumor growth rate of the mice in the combination administration group was significantly reduced, and the growth of the tumor volume of the DOX-ISR@HSA-Dspe group mice was most significantly inhibited. After the tumor tissues were weighed, the tumor inhibition rates of the freeDOX-ISR, ISR@HSA-Dspe, DOX@HSA-Dspe, DOX-ISR@HSA and DOX-ISR@HSA-Dspe groups were 61.72%, 19.36%, 11.71%, 50.15% and 89.67%, respectively, indicating that DOX-ISR@HSA-Dspe had the strongest tumor inhibition effect. In addition, the median survival time of each group was: 30 (saline), 43.5 (freeDOX-ISR), 33.5 (ISR@HSA-Dspe), 34.5 (DOX@HSA-Dspe), 35.5 (DOX-ISR@HSA) and 59.5 (DOX-ISR@HSA-Dspe) days, DOX-ISR@HSA-Dspe significantly prolonged the survival of the mice, and 20% of the mice survived for nearly three months with tumor disappearance, proving the good therapeutic effect of DOX-ISR@HSA-Dspe.

[0075] Experimental Example 10 Effect of treating orthotopic breast cancer lung metastasis. Collect 4T1 cells in good growth condition and in logarithmic growth phase, resuspend with PBS and adjust the cell concentration to 1×10 7 After 7 days of inoculation of 4T1 cells, the tumor-bearing mice that were successfully modeled were randomly divided into 6 groups, 3 mice in each group, and were injected with saline, freeDOX-ISR, ISR@HSA-Dspe prepared in Example 2, DOX@HSA-Dspe prepared in Example 3, DOX-ISR@HSA prepared in Example 4 and DOX-ISR@HSA-Dspe prepared in Example 5 via the tail vein, respectively, the administration dose was DOX 4 mg / kg and ISR 8 mg / kg, and the administration was performed once every 3 days, for 4 times in succession. The mice were sacrificed by cervical dislocation on the 30th day after tumor inoculation, the lung tissues were dissected and fixed with Bouin’s fixative for 24 h, and the number of tumor metastatic nodules on the lung tissues was observed and recorded.

[0076] The experimental results are as follows Figure 9As shown, after fixation with Bouin's fixative, normal lung tissue appeared yellowish-brown, while lung metastatic nodules appeared bright yellow, which could be distinguished from normal tissue and facilitate counting of metastatic nodules. The average number of lung metastatic nodules in the saline group reached 25, while the freeDOX-ISR, ISR@HSA-Dspe, DOX@HSA-Dspe and DOX-ISR@HSA groups were 18, 20, 11 and 8, respectively. No lung metastatic nodules were observed on the lung tissue of the DOX-ISR@HSA-Dspe group. The above results show that DOX-ISR@HSA-Dspe can effectively inhibit lung metastasis of orthotopic breast cancer.

Claims

1. A superoxide anion radical activated indomethacin-trans retinoic acid prodrug, characterized in that, include: Golgi targeting group indomethacin, intermediate reactive oxygen species responsive group and Golgi interference agent trans-retinoic acid; Its structural formula is: 。 2. A method for synthesizing a superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug, characterized in that, Includes the following steps: (1) 2-Hydroxy-5-methylisophthalic acid reacts with tert-butyldimethylchlorosilane to generate intermediate compound 1. The structural formula of intermediate compound 1 is: (2) Intermediate compound 1 reacts with trifluoromethanesulfonic anhydride to generate reactive oxygen species-responsive intermediate compound 2. The structural formula of intermediate compound 2 is: (3) Under the action of p-toluenesulfonic acid, intermediate compound 2 loses its hydroxyl protecting group to generate intermediate compound 3. The structural formula of intermediate compound 3 is as follows: (4) Intermediate compound 3 reacts with tert-butyldimethylchlorosilane to generate intermediate compound 4 protected by a single-sided silicon group. The structural formula of intermediate compound 4 is: (5) Indomethacin reacts with intermediate compound 4 via esterification to generate intermediate compound 5. The structural formula of intermediate compound 5 is as follows: (6) Intermediate compound 5 undergoes a TBS protecting group removal reaction to generate intermediate compound 6. The structural formula of intermediate compound 6 is: (7) Trans-retinoic acid and intermediate compound 6 undergo esterification to generate a superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug, with the following structural formula: 。 3. An albumin complex of a superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug, characterized in that, The complex comprises, by weight, 1 part of a superoxide anion radical-activated indomethacin-trans retinoic acid prodrug and 1 to 20 parts of albumin; comprising 1 part of the superoxide anion radical-activated indomethacin-trans retinoic acid prodrug as described in claim 1 and 15 to 20 parts of albumin; wherein the albumin is human serum albumin or bovine serum albumin.

4. A composition, characterized in that, The albumin complex comprising the superoxide anion radical activated indomethacin-trans retinoic acid prodrug as described in claim 3 and an antitumor drug.

5. The composition according to claim 4, characterized in that, The antitumor drugs mentioned are anthracyclines, taxanes, or antimetabolites. The anthracycline antitumor drugs are any one or a combination of at least two of doxorubicin, daunorubicin, epirubicin, pirarubicin, aclarubicin, amrubicin, zolrubicin, pentorubicin, idarubicin, and mitoxantrone. The taxane antitumor drugs are any one or a combination of at least two of paclitaxel, docetaxel, and cabazitaxel. The antimetabolites antitumor drugs are any one or a combination of at least two of 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), and gemcitabine.

6. The composition according to any one of claims 4 or 5, characterized in that, It also includes surface-modified materials prepared by surface modification techniques, specifically comprising at least one of the following technical features: (1) The surface modification material is selected from surfactants with polyethylene glycol as the hydrophilic group and amphiphilic compounds; (2) The surface modification material is selected from one or more of Solutol HS15, polysorbate series (Tween), meropenem, benzyl ether, and poloxamer; and one or more of the amphiphilic polymers PEGylated phospholipids, polyethylene glycol-lactide copolymer (PEG-PLGA), polyethylene glycol-polylactic acid (PEG-PLA), and polyethylene glycol-polycaprolactone (PEG-PCL). The surface modification material is selected from mPEG. 2000 -A combination of one or more of DSPE and SolutolHS15.

7. A pharmaceutical preparation, characterized in that, The composition comprising claim 6 further comprises: a pharmaceutically acceptable excipient selected from one or more of osmotic pressure regulators, stabilizers, microbial inhibitors, or buffer systems; the buffer system achieving physiological compatibility regulation via a pH adjuster; the osmotic pressure regulator being selected from one or more of sodium chloride, glucose, glycerol, mannitol, sorbitol, potassium chloride, sodium lactate, amino acids, dextran, gelatin, povidones, starch derivatives, etc.; the stabilizer being selected from one or more of glycerol, sucrose, polyethylene glycol, etc.; the microbial inhibitor being selected from one or more of cresol, phenol, etc.; and the pH adjuster being selected from one or more of hydrochloric acid, sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, acetic acid, sodium acetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, sodium bicarbonate, sodium carbonate, lactic acid, sodium lactate, etc.

8. A method for preparing an albumin complex of a superoxide anion radical-activated indomethacin-trans-retinoic acid prodrug, characterized in that, Includes the following steps: (1) Dissolve the superoxide anion radical-activated prodrug in an organic solvent; (2) Add albumin aqueous solution, and homogenize under high pressure or use probe ultrasonic emulsification; (3) The organic solvent was removed by rotary evaporation to obtain the albumin complex.

9. A method for preparing the composition according to claim 6, characterized in that, Includes the following steps: (1) Dissolve water-soluble antitumor drugs in albumin aqueous solution or dissolve lipid-soluble antitumor drugs in organic solvents; (2) Dissolve the prodrug in an organic solvent; (3) When the antitumor drug is a water-soluble antitumor drug, mix (1) and (2) and homogenize under high pressure or use a probe for ultrasonic emulsification; when the antitumor drug is a lipid-soluble drug, mix (1) and (2) and add an albumin aqueous solution, and homogenize under high pressure or use a probe for ultrasonic emulsification. (4) Remove the organic solvent by rotary evaporation to obtain the product; Wherein, the organic solvent mentioned in steps (1) and (2) is selected from one or more of chloroform, dichloromethane, tetrahydrofuran, acetone, ethyl acetate, ethanol, methanol, dioxane, acetonitrile, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone; the rotary evaporation temperature range is 20 to 70°C; The surface modification material is dissolved in an organic solvent and / or in water in step (1) or (2), and / or added directly after step (4).

10. The use of an albumin complex of the superoxide anion radical-activated indomethacin-trans retinoic acid prodrug of claim 1, the superoxide anion radical-activated indomethacin-trans retinoic acid prodrug of claim 3, the composition of claim 4, and the pharmaceutical preparation of claim 7 in a drug for tumor metastasis.

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