An ixazomib nanoparticle, and a preparation method and application thereof

Abstract

The application relates to the fields of chemistry and biological medicine, and particularly relates to an isazomide nanoparticle and a preparation method and application thereof. The preparation method comprises the following steps: dissolving an isazomide derivative and DSPE-PEG2000 in dimethyl sulfoxide, and then injecting the mixed solution into water to obtain the isazomide nanoparticle. In the application, the isazomide derivative is connected with the drug through a dynamic catechol-boric acid ester bond, the bond is unstable in a tumor acidic microenvironment (pH 6.5-6.8), active isazomide is released, and the bond is stable under normal physiological pH 7.4. The "anchoring-release" mechanism realizes tumor-specific activation and reduces off-target effects. The isazomide delivered by the nanoparticle can more effectively induce endoplasmic reticulum stress and mitochondrial ROS production, and further trigger CRT exposure, HMGB1 release and ATP secretion, and activate an anti-tumor immune response, which is a synergistic effect that cannot be achieved by free drugs.

Description

Technical Field

[0001] This application relates to the fields of chemistry and biomedicine, specifically to an enzazomi nanoparticle, its preparation method, and its application. Background Technology

[0002] Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive malignant tumor originating from the bile duct epithelium, accounting for 10-20% of primary liver cancers and 20-30% of cholangiocarcinomas. iCCA has an insidious onset and rapid progression; most patients are diagnosed at an advanced stage, resulting in a 5-year overall survival rate of only 7-20%. Surgical resection can potentially eradicate iCCA, but patients are highly likely to miss the optimal window for surgical treatment. Although some progress has been made in precision chemotherapy (such as using FGFR2 and IDH1 inhibitors), the clinical outcomes of these treatments are limited. Therefore, new treatment strategies are urgently needed to enhance the anti-tumor efficacy against iCCA.

[0003] The proteasome is a core regulatory complex involved in intracellular protein degradation, selectively removing abnormal or excess proteins through multiple catalytically active subunits. Proteasome inhibitors developed based on this mechanism have become important therapeutic tools for hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Bortezomib was the first proteasome inhibitor approved by the FDA in 2003, followed by esazomib (IXZ) and other compounds that have gradually entered clinical trials. However, their widespread use in oncology is severely limited by low bioavailability, systemic toxicity due to nonspecific distribution (e.g., hematologic toxicity and neuropathy), and lack of stability in the tumor microenvironment. Summary of the Invention

[0004] To address the limitations of proteasome inhibitors such as bortezomib and esazomib in oncology, this application provides esazomib nanoparticles, their preparation method, and their applications.

[0005] In a first aspect, this application provides a method for preparing an isazomib derivative, employing the following technical solution:

[0006] A method for preparing an isazomib derivative includes the following steps:

[0007] Activation: N-hydroxysuccinimide, unsaturated fatty acid, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed, dissolved in dichloromethane, heated and reacted, and then purified after cooling to obtain activated unsaturated fatty acid;

[0008] Synthetic excipients: Dopamine hydrochloride, activated unsaturated fatty acids and diisopropylethylamine are mixed and dissolved in N,N-dimethylformamide. The mixture is heated and reacted under inert gas protection, followed by rotary evaporation. The product is dissolved in dichloromethane, then washed, dried and purified to obtain the dopamine derivative.

[0009] Preparation: Dopamine derivative and esazolidinium were mixed, dissolved in pyridine, and heated under inert gas protection. The mixture was then dried by rotary evaporation to obtain the esazolidinium derivative.

[0010] By adopting the above technical solution, the above preparation method employs a three-step stepwise synthesis strategy, which has the following significant advantages:

[0011] First, the unsaturated fatty acids are activated to convert their carboxyl groups into activated esters, thereby increasing their reactivity with the amino groups in dopamine hydrochloride, avoiding side reactions, and improving coupling efficiency. The activation step uses an EDC and DMAP catalytic system, which enables efficient activation under mild conditions. The activated fatty acids obtained after purification exhibit good stability, facilitating subsequent reactions.

[0012] Secondly, activated fatty acids were reacted with dopamine hydrochloride to synthesize dopamine derivatives. The dopamine molecule contains both a catechol group and an amino group. The amino group undergoes an amidation reaction with the activated fatty acid, forming a stable amide bond; the catechol group retains its ability to form a dynamic covalent catechol-boronic acid ester bond with the boric acid group. This design allows dopamine to act as a bridge connecting fatty acids and isazorimi, ensuring both the stability of the connection and preserving the site for the formation of dynamic, reversible bonds.

[0013] Finally, the dopamine derivative was reacted with esazolidinium, utilizing the dynamic covalent catechol-boronic acid ester bond formed between the alkyl borate group in the esazolidinium molecule and the catechol group in the dopamine derivative to complete the construction of the entire derivative. This dynamic bond is stable at physiological pH and can be broken in the acidic tumor microenvironment, enabling controlled drug release.

[0014] In summary, the core advantages of this method are: (1) stepwise synthesis, easy purification and characterization of intermediate products, and high product purity; (2) introduction of dynamic covalent bonds to achieve reversible "lipid anchoring" of drugs, which can both self-assemble into nanoparticles and release active drugs in response at the tumor site; (3) avoidance of the disadvantages of traditional ester bonds or amide bonds being difficult to break, and improvement of the activation efficiency of prodrugs.

[0015] Preferably, the unsaturated fatty acid includes at least one of oleic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid.

[0016] Preferably, the unsaturated fatty acid is oleic acid.

[0017] By employing the above-mentioned technical solution, oleic acid, a monounsaturated C18 fatty acid, possesses a moderate carbon chain length and a single double bond structure that provides the derivative with an optimal hydrophilic-hydrophobic balance. Compared to polyunsaturated fatty acids (EPA, DHA), oleic acid is less prone to lipid peroxidation, providing a more stable hydrophobic core in nanoassemblies. Compared to linoleic acid, the single double bond in oleic acid avoids conformational distortion induced by additional double bonds, thereby enhancing the co-assembly efficiency with DSPE-PEG2000 and forming nanoparticles with more uniform particle size and higher colloidal stability. Therefore, oleic acid is a key structural choice for achieving efficient drug loading and tumor-specific release in "lipid anchoring" strategies.

[0018] Secondly, this application provides an isazomib derivative, which adopts the following technical solution:

[0019] An isazomib derivative prepared by the above-mentioned isazomib derivative preparation method.

[0020] The structural formulas of the isazomib derivatives include those shown in formulas I-IV:

[0021] Formula I;

[0022] Formula II;

[0023] Formula III;

[0024] Formula IV.

[0025] Thirdly, this application provides a method for preparing isazomib nanoparticles, using the following technical solution:

[0026] A method for preparing isazomi nanoparticles includes the following steps:

[0027] Mixing: The above-mentioned isazomib derivative and DSPE-PEG2000 were dissolved in dimethyl sulfoxide to obtain a mixture;

[0028] Injection: The mixture is injected into water to obtain esazomi nanoparticles.

[0029] By employing the above technical solution, the esazolidinyl derivative structure contains hydrophobic long chains and hydrophilic catechol-boron ester bond regions, exhibiting typical amphiphilicity. When the DMSO mixture is injected into water, the solvent polarity increases sharply, and the amphiphilic molecules spontaneously assemble into a core-shell structure through hydrophobic interactions and π-π stacking: DHA chains form the hydrophobic core, while catechol-boron ester bonds and PEG chains are distributed on the surface. DSPE-PEG2000 is further embedded in the lipid layer, and its PEG chains form a hydration layer, providing steric stabilization to prevent particle aggregation, thereby improving colloidal stability.

[0030] Nanoparticles are well-suited for passive targeting through the windows of tumor angiogenesis, while avoiding rapid renal clearance, thus enhancing tumor targeting and retention. Free esazolidinone, due to its non-specific distribution, leads to hematologic toxicity (leukopenia, erythrocyte reduction, thrombocytopenia), hepatotoxicity, nephrotoxicity, and neuropathy. After encapsulation with nanoparticles, the drug primarily accumulates in the tumor, reducing exposure to healthy organs. The nanoparticles link the drug via dynamic catechol-boron ester bonds, exhibiting instability in the acidic tumor microenvironment (pH 6.5-6.8), releasing active esazolidinone; however, it remains stable at the normal physiological pH of 7.4. This "anchor-release" mechanism achieves tumor-specific activation and reduces off-target effects. Nanoparticle-delivered esazolidinone more effectively induces endoplasmic reticulum stress and mitochondrial ROS production, thereby triggering CRT exposure, HMGB1 release, and ATP secretion, activating an anti-tumor immune response—a synergistic effect difficult to achieve with free drugs.

[0031] Fourthly, this application provides an isazomi nanoparticle, which adopts the following technical solution:

[0032] An esazolidone nanoparticle is prepared by the above-described method for preparing esazolidone nanoparticles.

[0033] Fifthly, this application provides an application of isazomib nanoparticles, employing the following technical solution:

[0034] One application of isazomib nanoparticles refers to their use in the treatment of intrahepatic cholangiocarcinoma.

[0035] By employing the above-mentioned technical approach, proteasome inhibitors are effective candidate drugs for intrahepatic cholangiocarcinoma cell lines, exhibiting high inhibition rates. Esazomib nanoparticles demonstrated stronger cytotoxicity, colony formation inhibition, G2 / M phase arrest, and apoptosis induction in vitro against CCLP1 and RBE cells, with effects superior to free esazomib.

[0036] Esazomib nanoparticles release esazomib, which selectively inhibits 20S proteasome activity, leading to the accumulation of misfolded proteins and the formation of aggregates. This protein accumulation triggers endoplasmic reticulum (ER) stress, manifested as upregulation of GRP78, CHOP, and phosphorylated IRE1, as well as ER morphological swelling, which in turn upregulates the DR5 receptor and initiates the extrinsic apoptosis pathway. ER stress intersects with mitochondrial function, resulting in the excessive production of mitochondrial superoxide, further amplifying cell damage.

[0037] Damaged tumor cells release ATP, expose CRT, and translocate intranuclear HMGB1 to the cytoplasm and extracellular space. These danger signals can activate antigen-presenting cells and induce anti-tumor immune responses, which is of breakthrough significance for the immunosuppressive intrahepatic cholangiocarcinoma microenvironment.

[0038] In summary, this application has the following beneficial effects:

[0039] Esazomib nanoparticles link the drug via dynamic catechol-boron ester bonds. These bonds are unstable in the acidic tumor microenvironment (pH 6.5-6.8), releasing active esazomib; however, they remain stable at the normal physiological pH of 7.4. This "anchor-release" mechanism achieves tumor-specific activation and reduces off-target effects. Esazomib delivered by nanoparticles more effectively induces endoplasmic reticulum stress and mitochondrial ROS production, thereby triggering CRT exposure, HMGB1 release, and ATP secretion, activating an anti-tumor immune response—a synergistic effect that is difficult to achieve with free drugs. Attached Figure Description

[0040] Figure 1 These are images of CCLP1 tumors in mice from Examples 1-4 and Comparative Examples 1-2 at 12 days.

[0041] Figure 2 These are graphs showing the changes in CCLP1 tumor volume in mice from Examples 1-4 and Comparative Examples 1-2.

[0042] Figure 3 This is a graph showing the changes in the number of survival days of mice in each group of Examples 1-4 and Comparative Examples 1-2.

[0043] Figure 4 This is a graph showing the volume changes of PDX tumor models in mice from Example 5 and Comparative Examples 3-4.

[0044] Figure 5 This is a graph showing the changes in the number of survival days of mice in each group of Examples 5 and Comparative Examples 3-4.

[0045] Figure 6 These are physical images of fluorescence changes in mice in Example 6 and Comparative Examples 5-6.

[0046] Figure 7These are fluorescence change images of mice in Example 6 and Comparative Examples 5-6, as well as a comparison image of liver morphology of mice in each group on day 40.

[0047] Figure 8 This is a bar chart showing the liver weight of mice in Example 6 and Comparative Examples 5-6 after 40 days.

[0048] Figure 9 This is a graph showing the weight changes of mice in each group of Examples 7 and Comparative Examples 7-8.

[0049] Figure 10 This is a graph showing the effect of Example 7 and Comparative Examples 7-8 on the activity of CCLP1 and RBE cell lines. Detailed Implementation

[0050] The raw materials in this application include the following:

[0051] Oleic acid: Uses commercially available product with CAS number 112-80-1;

[0052] Linoleic acid: Uses commercially available products with CAS number 60-33-3;

[0053] Eicosapentaenoic acid: Uses commercially available products with CAS number 10417-94-4;

[0054] Docosahexaenoic acid (DHA): Uses a commercially available product with CAS number 6217-54-5;

[0055] N-Hydroxysuccinimide: Uses a commercially available product with CAS number 6066-82-6;

[0056] 4-Dimethylaminopyridine: The commercially available product with CAS number 1122-58-3 is used;

[0057] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide: Use the commercially available product with CAS number 1892-57-5;

[0058] Dichloromethane: Use commercially available products with CAS number 75-09-2;

[0059] Dopamine hydrochloride: Uses a commercially available product with CAS number 62-31-7;

[0060] Diisopropylethylamine: Use the commercially available product with CAS number 7087-68-5;

[0061] N,N-Dimethylformamide: Use commercially available products with CAS number 68-12-2;

[0062] Pyridine: Use commercially available product with CAS number 110-86-1;

[0063] DSPE-PEG2000: Uses a commercially available product with CAS number 147867-65-0;

[0064] Dimethyl sulfoxide: Use commercially available products with CAS number 67-68-5;

[0065] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0066] Example 1

[0067] A method for preparing an isazomib derivative-1 includes the following steps:

[0068] Activation: 173.2 mg N-hydroxysuccinimide, 443.5 mg oleic acid, 207.5 mg 4-dimethylaminopyridine and 263.4 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed, dissolved in 3 mL dichloromethane, and reacted at 45 °C for 4 h. After cooling, the mixture was purified to obtain activated oleic acid.

[0069] Synthetic excipients: 106 mg dopamine hydrochloride, 212.2 mg activated oleic acid and 144.51 mg diisopropylethylamine were mixed and dissolved in 2 mL of N,N-dimethylformamide. The mixture was heated at 45 °C for 4 h under nitrogen protection, and then rotary evaporated. The product was dissolved in dichloromethane, washed, dried and purified to obtain dopamine derivative-1.

[0070] Preparation: 7.54 mg of dopamine derivative-1 and 6.28 mg of esazolidinyl were mixed and dissolved in 1.5 mL of pyridine. The mixture was heated at 75 °C for 1 h under nitrogen protection. Molecular sieves were added to remove water produced during the reaction. The mixture was then dried by rotary evaporation to obtain esazolidinyl derivative-1. (See Formula I)

[0071] .

[0072] A method for preparing isazomib nanoparticles-1 includes the following steps:

[0073] Mixing: Dissolve 18 mg of esazolamide derivative-1 and 2 mg of DSPE-PEG2000 in 1 mL of dimethyl sulfoxide to obtain a mixture;

[0074] Injection: The mixture was injected into 10 mL of water, and the organic solvent was removed by dialyzing to obtain esazolmi nanoparticles-1.

[0075] An application of isazomizomi nanoparticle-1 includes the following steps:

[0076] The application was performed in seven preclinical mouse models carrying subcutaneous CCLP1 tumors, when the tumor volume reached approximately 100 mm. 3At that time, esazolidone nanoparticles-1 (corresponding to an esazolidone dose of 2 mg / kg) were administered intravenously every 3 days for a total of four intravenous injections. The solvent in the esazolidone nanoparticles-1 and subsequent nanoparticles and free esazolidone injections was physiological saline.

[0077] Examples 2-4

[0078] Example 2 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0079] A method for preparing an isazomib derivative-2 includes the following steps:

[0080] Activation: 162.9 mg N-hydroxysuccinimide, 430 mg linoleic acid, 207.5 mg 4-dimethylaminopyridine and 263.4 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed and dissolved in 3 mL dichloromethane. The mixture was heated at 45 °C for 4 h, cooled, and purified to obtain activated linoleic acid.

[0081] Synthetic excipients: 106 mg dopamine hydrochloride, 209.6 mg activated linoleic acid and 144.51 mg diisopropylethylamine were mixed and dissolved in 2 mL of N,N-dimethylformamide. The mixture was heated at 45 °C for 4 h under nitrogen protection, and then rotary evaporated. The product was dissolved in dichloromethane, washed, dried and purified to obtain dopamine derivative-2.

[0082] Preparation: 7.33 mg of dopamine derivative-2 and 6.37 mg of esazolidinyl were mixed and dissolved in 1.5 mL of pyridine. The mixture was heated at 75 °C for 1 h under nitrogen protection. Molecular sieves were added to remove water produced during the reaction. The mixture was then dried by rotary evaporation to obtain esazolidinyl derivative-2. (See Formula II)

[0083] .

[0084] Subsequently, the esazomi nanoparticles were prepared by replacing esazomi derivative-1 with esazomi derivative-2.

[0085] Example 3 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0086] A method for preparing an isazomib derivative-3 includes the following steps:

[0087] Activation: 69.96 mg N-hydroxysuccinimide, 183.85 mg eicosapentaenoic acid, 89.12 mg 4-dimethylaminopyridine and 113.25 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed, dissolved in 3 mL dichloromethane, heated at 45 °C for 4 h, cooled and purified to obtain activated eicosapentaenoic acid;

[0088] Synthetic excipients: 106 mg dopamine hydrochloride, 223.16 mg activated eicosapentaenoic acid and 144.51 mg diisopropylethylamine were mixed and dissolved in 2 mL of N,N-dimethylformamide. The mixture was heated at 45 °C for 4 h under nitrogen protection, and then rotary evaporated. The product was dissolved in dichloromethane, washed, dried and purified to obtain dopamine derivative-3.

[0089] Preparation: 5.27 mg of dopamine derivative-3 and 4.35 mg of esazolidinyl were mixed and dissolved in 1.5 mL of pyridine. The mixture was heated at 75 °C for 1 h under nitrogen protection. Molecular sieves were added to remove water produced during the reaction. The mixture was then dried by rotary evaporation to obtain esazolidinyl derivative-3. (See Formula III)

[0090] .

[0091] Subsequently, the esazomi nanoparticles were also prepared by replacing esazomi derivative-1 with esazomi derivative-3.

[0092] Example 4 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0093] A method for preparing isazomib derivative-4 includes the following steps:

[0094] Activation: 69.96 mg N-hydroxysuccinimide, 199.7 mg docosahexaenoic acid, 89.12 mg 4-dimethylaminopyridine and 113.25 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide were mixed, dissolved in 3 mL of dichloromethane, heated at 45 °C for 4 h, cooled and purified to obtain activated docosahexaenoic acid;

[0095] Synthetic excipients: 106 mg dopamine hydrochloride, 237.7 mg activated docosahexaenoic acid and 144.51 mg diisopropylethylamine were mixed and dissolved in 2 mL of N,N-dimethylformamide. The mixture was heated at 45 °C for 4 h under nitrogen protection, and then rotary evaporated. The product was dissolved in dichloromethane, washed, dried and purified to obtain dopamine derivative-4.

[0096] Preparation: 5.54 mg of dopamine derivative-4 and 4.31 mg of esazolidinyl were mixed and dissolved in 1.5 mL of pyridine. The mixture was heated at 75 °C for 1 h under nitrogen protection. Molecular sieves were added to remove water produced during the reaction. The mixture was then dried by rotary evaporation to obtain esazolidinyl derivative-4. (See Formula IV)

[0097] .

[0098] Subsequently, the esazomi nanoparticles were prepared by replacing esazomi derivative-1 with esazomi derivative-4.

[0099] Comparative Examples 1-2

[0100] Comparative Example 1 modifies the preparation method of Example 1 as follows:

[0101] An application of free IXZ includes the following steps:

[0102] The application was performed in seven preclinical mouse models carrying subcutaneous CCLP1 tumors, when the tumor volume reached approximately 100 mm. 3 At that time, the free IXZ solution (corresponding to an esazolamide dose of 2 mg / kg) was administered intravenously once every 3 days for a total of four intravenous injections.

[0103] Comparative Example 2 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0104] The application of a saline solution includes the following steps:

[0105] The application was performed in seven preclinical mouse models carrying subcutaneous CCLP1 tumors, when the tumor volume reached approximately 100 mm. 3 At that time, normal saline was injected intravenously once every 3 days, for a total of 4 intravenous injections.

[0106] Performance testing

[0107] 1. To detect the tumor volume and survival status of the experimental mice in the examples and comparative examples.

[0108] Table 1 Performance test results of Examples 1-4 and Comparative Examples 1-2

[0109] <![CDATA[CCLP1 tumor volume at 20 days / mm 3 > 574 599 732 891 884 1095 Maximum survival days in CCLP1 tumor mice ＞40 ＞40 38 40 38 34

[0110] Referring to Table 1, comparing Examples 1-4 and Comparative Examples 1-2, it can be seen that tumors shrank in Examples 1 and 2, while Comparative Example 1 failed to stop tumor growth (see Table 1). Figure 1Tumor growth kinetics monitored every few days further supported this result, with no significant difference in tumor growth curves between Examples 3-4 and Comparative Examples 1-2. At low doses, Examples 1-2 both demonstrated excellent therapeutic effects (see...). Figure 2 The survival curves of mice also demonstrate this (see...). Figure 3 ).

[0111] Comparative Examples 1-4 and 1-2 show that the esazolidinyl nanoparticles prepared using the above method exhibit better performance. This is because the esazolidinyl derivative structure contains hydrophobic long chains and hydrophilic catechol-boron ester bond regions, exhibiting typical amphiphilicity. When the DMSO mixture is injected into water, the solvent polarity increases sharply, and the amphiphilic molecules spontaneously assemble into a core-shell structure through hydrophobic interactions and π-π stacking: DHA chains form the hydrophobic core, while catechol-boron ester bonds and PEG chains are distributed on the surface. DSPE-PEG2000 is further embedded in the lipid layer, and its PEG chains form a hydration layer, providing steric stabilization to prevent particle aggregation, thereby improving colloidal stability.

[0112] Nanoparticles are well-suited for passive targeting through the windows of tumor angiogenesis, while avoiding rapid renal clearance, thus enhancing tumor targeting and retention. Free esazolidinone, due to its non-specific distribution, leads to hematologic toxicity (leukopenia, erythrocyte reduction, thrombocytopenia), hepatotoxicity, nephrotoxicity, and neuropathy. After encapsulation with nanoparticles, the drug primarily accumulates in the tumor, reducing exposure to healthy organs. The nanoparticles link the drug via dynamic catechol-boron ester bonds, exhibiting instability in the acidic tumor microenvironment (pH 6.5-6.8), releasing active esazolidinone; however, it remains stable at the normal physiological pH of 7.4. This "anchor-release" mechanism achieves tumor-specific activation and reduces off-target effects. Nanoparticle-delivered esazolidinone more effectively induces endoplasmic reticulum stress and mitochondrial ROS production, thereby triggering CRT exposure, HMGB1 release, and ATP secretion, activating an anti-tumor immune response—a synergistic effect difficult to achieve with free drugs.

[0113] Example 5

[0114] Example 5 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0115] An application of isazomizomi nanoparticle-1 includes the following steps:

[0116] A PDX tumor mouse model was resuscitated from a patient diagnosed with iCCA and applied in five PDX tumor mouse models when the tumor volume reached approximately 50 mm. 3 At that time, intravenous injection of esazoli nanoparticle-1 (corresponding to esazoli dose of 8 mg / kg) was administered once every 3 days for a total of three intravenous injections.

[0117] Comparative Examples 3-4

[0118] Comparative Example 3 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0119] An application of free IXZ includes the following steps:

[0120] A PDX tumor mouse model was resuscitated from a patient diagnosed with iCCA and applied in five PDX tumor mouse models when the tumor volume reached approximately 50 mm. 3 At that time, the free IXZ solution (corresponding to the esazolidone dose of 8 mg / kg) was injected intravenously once every 3 days, for a total of three intravenous injections.

[0121] Comparative Example 4 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0122] The application of a saline solution includes the following steps:

[0123] A PDX tumor mouse model was resuscitated from a patient diagnosed with iCCA and applied in five PDX tumor mouse models when the tumor volume reached approximately 50 mm. 3 At that time, normal saline was injected intravenously once every 3 days, for a total of 3 intravenous injections.

[0124] The performance tests of Example 5 and Comparative Examples 3-4 were performed as described above, and the test results are shown in Table 2.

[0125] Performance testing

[0126] Table 2 Performance test results for Example 5 and Comparative Examples 3-4

[0127] <![CDATA[Tumor volume of PDX model at 27 days / mm 3 > 248 425 721 Maximum survival days in PDX model tumor mice ＞48 ＞48 39

[0128] Referring to Table 2, both Example 5 and Comparative Example 3 significantly inhibited tumor growth compared to Comparative Example 4. Notably, Example 5 exhibited superior activity in inhibiting tumor progression compared to Comparative Example 3 (see Table 2). Figure 4 Example 5 showed a significantly higher survival rate (see Example 5). Figure 5 ).

[0129] Example 6

[0130] Example 6 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0131] An application of isazomizomi nanoparticle-1 includes the following steps:

[0132] The study was conducted in five mice with stable luciferase-expressing CCLP1-luc orthotopic xenograft tumors. On day 12 after tumor inoculation, esazolamide nanoparticle-1 (corresponding to an esazolamide dose of 8 mg / kg) was injected intravenously every three days for a total of four intravenous injections.

[0133] Comparative Examples 5-6

[0134] Comparative Example 5 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0135] An application of free IXZ includes the following steps:

[0136] The drug was applied in five mice with stable luciferase-expressing CCLP1-luc orthotopic xenograft tumors. On day 12 after tumor inoculation, free IXZ solution (corresponding to esazolamide dose of 8 mg / kg) was injected intravenously every three days for a total of four intravenous injections.

[0137] Comparative Example 6 modifies the preparation method of Example 1 as follows:

[0138] The application of a saline solution includes the following steps:

[0139] The drug was applied in five mice with stable luciferase-expressing CCLP1-luc orthotopic xenograft tumors. On day 12 after tumor inoculation, the mice were given intravenous saline once every three days for a total of four intravenous injections.

[0140] Performance testing

[0141] 2. Detect the luminescence signal and liver weight of the experimental mice in the examples and comparative examples.

[0142] Table 3 Performance test results for Example 6 and Comparative Examples 5-6

[0143] Relative emission signal 6 12 31 / Liver weight / g 1.43 1.58 1.88 1.39

[0144] Referring to Table 3, a comparison between Example 6 and Comparative Examples 5-6 shows that Example 6 exhibited a good tumor-suppressive effect in the early stage of drug administration, delaying the trend of increased tumor bioluminescence signal (see Table 3). Figure 6 and Figure 7 Furthermore, at the monitoring endpoint (40 days), tumor-bearing liver samples were removed, and the results confirmed that the in vivo bioluminescence results were consistent with the luminescence signal performance in each group (see [link to relevant documentation]). Figure 7 The resected liver samples were subjected to weight analysis, i.e., tumor weight compared to the weight of a healthy liver, to provide feedback on the actual tumor burden in each group of mice at the monitoring endpoint. Compared with the saline and IXZ treatment groups, the PiNB-1 group had the lowest tumor burden, with the total liver weight almost indistinguishable from that of healthy mice (see [link to relevant documentation]). Figure 8).

[0145] Comparative Examples 5-6 and 3-6 demonstrate that esazolidinyl nanoparticles can be used to treat intrahepatic cholangiocarcinoma. This is because proteasome inhibitors are effective candidate drugs in intrahepatic cholangiocarcinoma cell lines, exhibiting high inhibition rates. Esazolidinyl nanoparticles demonstrate stronger cytotoxicity, colony formation inhibition, G2 / M phase arrest, and apoptosis induction in vitro against CCLP1 and RBE cells, with effects superior to free esazolidinyl.

[0146] Esazomib nanoparticles release esazomib, which selectively inhibits 20S proteasome activity, leading to the accumulation of misfolded proteins and the formation of aggregates. This protein accumulation triggers endoplasmic reticulum (ER) stress, manifested as upregulation of GRP78, CHOP, and phosphorylated IRE1, as well as ER morphological swelling, which in turn upregulates the DR5 receptor and initiates the extrinsic apoptosis pathway. ER stress intersects with mitochondrial function, resulting in the excessive production of mitochondrial superoxide, further amplifying cell damage.

[0147] Damaged tumor cells release ATP, expose CRT, and translocate intranuclear HMGB1 to the cytoplasm and extracellular space. These danger signals can activate antigen-presenting cells and induce anti-tumor immune responses, which is of breakthrough significance for the immunosuppressive intrahepatic cholangiocarcinoma microenvironment.

[0148] Example 7

[0149] Example 7 is based on the preparation method of Example 1, but the method is adjusted as follows:

[0150] An application of isazomizomi nanoparticle-1 includes the following steps:

[0151] The study was conducted in 5 normal mice. The mice were intravenously injected with esazoli nanoparticle-1 (corresponding to an esazoli dose of 12 mg / kg) every 3 days for a total of 4 intravenous injections.

[0152] Comparative Examples 7-8

[0153] Comparative Example 7 modifies the preparation method of Example 1 as follows:

[0154] An application of free IXZ includes the following steps:

[0155] The drug was administered to five normal mice by intravenous injection of free IXZ solution (corresponding to an esazolamide dose of 12 mg / kg) every three days for a total of four intravenous injections.

[0156] Comparative Example 8 modifies the preparation method of Example 1 as follows:

[0157] The application of a saline solution includes the following steps:

[0158] The drug was administered to five normal mice by intravenous injection of saline solution every three days for a total of four injections.

[0159] Cell activity was verified using the esazomi nanoparticles-1 in Example 7.

[0160] Cell viability was verified in the free IXZ in Example 7.

[0161] Cell viability was verified using the saline solution in Example 8.

[0162] Performance testing

[0163] 3. Detect the body weight of the experimental mice in the examples and comparative examples.

[0164] 4. The activities of esazoli nanoparticle-1, free IXZ and physiological saline on CCLP1 and RBE cell lines were evaluated using the CCK-8 assay.

[0165] Table 4 Performance test results for Examples 7 and Comparative Examples 7-8

[0166] Body weight / g at 9 days 28.78 25.65 33.73 CCLP1 cell percentage / % 21.89 44.78 100 RBE cell percentage / % 30.23 39.55 100

[0167] Referring to Table 4, comparing Example 7 and Comparative Examples 7-8, it can be seen that after administration, Comparative Example 7 showed a significant decrease in body weight. In contrast, Example 7 showed better weight loss, falling between the other two groups (see Table 4). Figure 9 ).

[0168] Both PiNB-1 and free IXZ treatments significantly reduced the colony-forming ability of both cell lines, with PiNB-1 showing a more pronounced inhibitory effect in CCLP1 (see [link to treatment]). Figure 10 ).

[0169] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for preparing an isazomib derivative, characterized in that, Includes the following steps: Activation: N-hydroxysuccinimide, unsaturated fatty acid, 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide are mixed, dissolved in dichloromethane, heated and reacted, cooled and purified to obtain activated unsaturated fatty acid; the unsaturated fatty acid is at least one of oleic acid and linoleic acid; Synthetic excipients: Dopamine hydrochloride, activated unsaturated fatty acids and diisopropylethylamine are mixed and dissolved in N,N-dimethylformamide. The mixture is heated and reacted under inert gas protection, followed by rotary evaporation. The product is dissolved in dichloromethane, then washed, dried and purified to obtain the dopamine derivative. Preparation: Dopamine derivative and esazolamide were mixed, dissolved in pyridine, and heated under inert gas protection. The mixture was then dried by rotary evaporation to obtain the esazolamide derivative. The structural formula of the isazomib derivative is shown in Formula I-II: Equation I; Formula II.

2. The method for preparing the isazomib derivative according to claim 1, characterized in that: The unsaturated fatty acid is oleic acid.

3. An isazomib derivative, characterized in that: It is prepared by the method for preparing the isazomib derivative as described in claim 1.

4. A method for preparing isazomib nanoparticles, characterized in that, Includes the following steps: Mixing: The isazomib derivative of claim 3 and DSPE-PEG2000 are dissolved in dimethyl sulfoxide to obtain a mixture; Injection: The mixture is injected into water, and the organic solvent is removed by dialysis to obtain isazomi nanoparticles.

5. An isazomizomi nanoparticle, characterized in that: It is prepared by the method for preparing isazomi nanoparticles as described in claim 4.

6. An application of the isazomi nanoparticles according to claim 5, characterized in that: Applications in the preparation of drugs for treating intrahepatic cholangiocarcinoma.

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