Use of hexahydrocurcumin as an usp39 inhibitor in the preparation of drugs against pancreatic ductal adenocarcinoma

Abstract

The application provides an application of hexahydrocurcumin as a USP39 inhibitor in preparation of a drug for resisting pancreatic ductal adenocarcinoma, and belongs to the technical field of biological medicine and tumor treatment. The hexahydrocurcumin is identified as a specific small molecule inhibitor of USP39 for the first time, fills the technical blank of research and development of a USP39 targeted inhibitor, and clearly defines the complete molecular mechanism of hexahydrocurcumin targeting USP39 against PDAC. The in-vivo and in-vitro experiments prove that the hexahydrocurcumin can effectively inhibit the proliferation and clonogenicity of PDAC cells, block the cell cycle process, significantly inhibit the in-vivo growth of PDAC orthotomia, and has good biological safety. It is also proved that the hexahydrocurcumin can reverse the chemotherapy resistance of PDAC cells to gemcitabine, has a synergistic effect when combined with gemcitabine and a PD-1 inhibitor, and can significantly improve the anti-tumor effect of the existing clinical scheme.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and tumor treatment technology, and in particular to the application of hexahydrocurcumin as a USP39 inhibitor in the preparation of drugs for treating pancreatic ductal adenocarcinoma. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant digestive system tumor. PDAC has an insidious onset, with no specific symptoms in the early stages. Most patients are diagnosed at an advanced stage, and only 20% of newly diagnosed cases are eligible for surgical treatment. Moreover, the postoperative recurrence rate is as high as 80%.

[0003] Currently, gemcitabine is the first-line standard chemotherapy regimen for PDAC, but its survival benefit remains very limited when used alone or in combination with drugs such as erlotinib and albumin-bound paclitaxel; immune checkpoint inhibitor monotherapy has also not shown significant efficacy in PDAC. One of the core reasons for the predicament in PDAC treatment is the lack of effective specific therapeutic targets and targeted drugs.

[0004] Ubiquitination is a core mechanism for regulating intracellular protein homeostasis, widely involved in physiological processes such as cell cycle, proliferation, apoptosis, and DNA repair. Its dynamic balance is synergistically regulated by E3 ubiquitin ligases and deubiquitinating enzymes (DUBs). Studies have confirmed that members of the DUB family play a crucial role in the development and progression of various tumors, making it an important direction for anti-tumor target research. Ubiquitin-specific protease 39 (USP39) is an important member of the DUB family, playing a vital role in RNA splicing and cell cycle progression. Existing research indicates that USP39 is abnormally highly expressed in various tumors and is closely related to malignant tumor proliferation and poor prognosis.

[0005] However, there are currently no specific small molecule inhibitors of USP39 in clinical trials, nor are there any mature USP39-targeted drugs for the treatment of PDAC. Therefore, screening and identifying specific small molecule inhibitors of USP39, and clarifying their anti-PDAC mechanisms of action and therapeutic potential, is of significant scientific and clinical value for overcoming the bottlenecks in the clinical treatment of PDAC. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide the application of hexahydrocurcumin as a USP39 inhibitor in the preparation of drugs for pancreatic ductal adenocarcinoma, and to provide new candidate drugs and solutions for targeted therapy of PDAC.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of hexahydrocurcumin as a USP39 inhibitor in the preparation of drugs for treating pancreatic ductal adenocarcinoma.

[0008] Preferably, the pancreatic ductal adenocarcinoma is gemcitabine-resistant pancreatic ductal adenocarcinoma.

[0009] Preferably, the hexahydrocurcumin, as a USP39 inhibitor, can: inhibit the deubiquitination enzyme activity of USP39, promote the K48 ubiquitination degradation of HSP70 and / or FASN, inhibit palmitoylation modification and nuclear accumulation of mut-TP53 protein, arrest the tumor cell cycle, inhibit tumor cell proliferation, colony formation and in vivo tumorigenesis, reverse tumor chemotherapy resistance, and synergistically enhance the antitumor effect of chemotherapy / immunotherapy.

[0010] More preferably, the mut-TP53 is a TP53R175H mutant; the tumor cell cycle arrest is G1 phase arrest; the chemotherapy-resistant drug is gemcitabine; and the immunotherapy drug is a PD-1 inhibitor.

[0011] The present invention also provides a drug for inhibiting USP39 activity against pancreatic ductal adenocarcinoma, the active ingredient of which includes hexahydrocurcumin; the dosage form of the drug includes injection, oral preparation, sustained-release preparation or targeted preparation.

[0012] The present invention also provides a pharmaceutical composition for treating pancreatic ductal adenocarcinoma, comprising the active ingredient hexahydrocurcumin, and pharmaceutically acceptable excipients, carriers, or excipients.

[0013] The present invention also provides a pharmaceutical composition for combined anti-pancreatic ductal adenocarcinoma, comprising a first active ingredient, hexahydrocurcumin, and a second active ingredient; the second active ingredient comprises at least one of gemcitabine and a PD-1 inhibitor.

[0014] Preferably, the mass ratio of hexahydrocurcumin to gemcitabine is (1~100):(1~50); and / or the mass ratio of hexahydrocurcumin to PD-1 inhibitor is (1~100):(0.1~10).

[0015] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of products for treating pancreatic ductal adenocarcinoma.

[0016] Preferably, the dosage form of the anti-pancreatic ductal adenocarcinoma product is a hydroxypropyl-β-cyclodextrin inclusion complex injection, an oral tablet, or a lyophilized powder injection.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to identify hexahydrocurcumin as a specific small molecule inhibitor of USP39, filling the technological gap in the development of USP39 targeted inhibitors and providing novel tool compounds and candidate drugs for the research and treatment of USP39-related diseases.

[0018] This invention elucidates the complete molecular mechanism by which hexahydrocurcumin targets USP39 to combat PDAC, and confirms that it exerts its anti-tumor effect through the USP39-HSP70 / FASN-mut-TP53 axis, providing a new theoretical basis and pathway for targeted therapy of PDAC.

[0019] This invention demonstrates through in vitro and in vivo experiments that hexahydrocurcumin can effectively inhibit the proliferation and colony formation of PDAC cells, arrest cell cycle progression, significantly inhibit the in vivo growth of PDAC orthotopic tumors, and has good biosafety.

[0020] This invention demonstrates that hexahydrocurcumin can reverse the chemotherapy resistance of PDAC cells to gemcitabine, and has a synergistic effect when used in combination with gemcitabine and PD-1 inhibitors, which can significantly improve the anti-tumor effect of existing clinical regimens and has extremely high clinical translational value.

[0021] The pharmaceutical composition containing hexahydrocurcumin provided by this invention has flexible dosage forms and diverse routes of administration, which can be adapted to the treatment needs of different clinical scenarios, providing a brand-new treatment option for PDAC patients. Attached Figure Description

[0022] Figure 1 A virtual screening flowchart for small molecule inhibitors targeting USP39.

[0023] Figure 2 The figure shows the results of an in vitro inhibition experiment of hexahydrocurcumin on the activity of USP39 deubiquitinase.

[0024] Figure 3 This diagram shows the active site of the USP39 protein and the binding pattern of hexahydrocurcumin to USP39.

[0025] Figure 4 This figure shows the concentration-dependent regulation of hexahydrocurcumin on the levels of HSP70 and FASN proteins, downstream targets of USP39, in PDAC cells.

[0026] Figure 5 The figure shows the effect of hexahydrocurcumin on the levels of HSP70 and FASN mRNA, downstream targets of USP39, in PDAC cells.

[0027] Figure 6 The figure shows the effect of hexahydrocurcumin on the half-life of HSP70 and FASN proteins, downstream targets of USP39, in PDAC cells.

[0028] Figure 7 The figure shows the effects of hexahydrocurcumin and MG132 on the levels of HSP70 and FASN proteins.

[0029] Figure 8 The figure shows the regulatory effect of hexahydrocurcumin on the ubiquitination levels of HSP70 and FASN.

[0030] Figure 9 The effect of hexahydrocurcumin on the proliferation of PDAC cells was detected by the CCK-8 assay to investigate the inhibitory effect of hexahydrocurcumin on the malignant phenotype of PDAC cells.

[0031] Figure 10 To investigate the effect of hexahydrocurcumin on the clonogenic ability of PDAC cells in a clonogenic assay, this study aimed to assess the inhibitory effect of hexahydrocurcumin on the malignant phenotype of PDAC cells.

[0032] Figure 11 Flow cytometry was used to detect the cell cycle arrest effect of hexahydrocurcumin on PDAC cells in order to inhibit the malignant phenotype of PDAC cells.

[0033] Figure 12 Gross morphology of tumor tissues in mice from each group in an in vivo assay of hexahydrocurcumin inhibiting PDAC orthotopic tumor growth.

[0034] Figure 13 This figure shows the statistical results of tumor weight in mice of each group in the in vivo inhibition of PDAC orthotopic tumor growth by hexahydrocurcumin.

[0035] Figure 14 This figure shows the statistical results of tumor volume in each group of mice during the in vivo inhibition of PDAC orthotopic tumor growth by hexahydrocurcumin.

[0036] Figure 15 Gross morphology of tumor tissues in mice in different groups during the anti-tumor treatment of hexahydrocurcumin combined with gemcitabine / PD-1 inhibitor.

[0037] Figure 16 This figure shows the statistical results of tumor weight in mice in different groups during the anti-tumor treatment of mice using a combination of hexahydrocurcumin and gemcitabine / PD-1 inhibitor.

[0038] Figure 17 This figure shows the statistical results of tumor volume in mice in different groups during the anti-tumor treatment of mice using a combination of hexahydrocurcumin and gemcitabine / PD-1 inhibitor. Detailed Implementation

[0039] This invention provides the application of hexahydrocurcumin as a USP39 inhibitor in the preparation of drugs for treating pancreatic ductal adenocarcinoma.

[0040] In the application described in this invention, hexahydrocurcumin directly binds to and inhibits the deubiquitination enzyme activity of USP39 by forming stable hydrogen bonds and salt bridges with key residues (Asp438, Asn503, Asp504) in the active pocket of the USP39 protein. Its dissociation constant KD is 87±12 nM and its in vitro IC50 is 0.42±0.08 μM.

[0041] The inhibition of USP39 deubiquitination enzyme activity described in this invention refers to the ability of hexahydrocurcumin to block the deubiquitination modification of the ubiquitin chain at position K48 of HSP70 and FASN proteins by USP39, thereby promoting the degradation of HSP70 and FASN via the ubiquitin-proteasome pathway.

[0042] The promotion of K48 ubiquitination degradation of HSP70 and / or FASN as described in this invention refers to the significant shortening of the protein half-life of HSP70 and FASN after treatment with hexahydrocurcumin. This effect can be completely reversed by the proteasome inhibitor MG132, and the K48 ubiquitination level of HSP70 and FASN is significantly increased.

[0043] The inhibition of palmitoylation modification and nuclear accumulation of mut-TP53 protein described in this invention refers to the reduction of mut-TP53 protein stability and its abnormal accumulation in the cell nucleus by hexahydrocurcumin reducing the binding of HSP70 to the molecular chaperone of mut-TP53 and inhibiting palmitoylation modification mediated by fatty acid synthesis pathway by downregulating FASN.

[0044] The mut-TP53 described in this invention includes, but is not limited to, the TP53 R175H mutant.

[0045] The tumor cell cycle arrest described in this invention refers to the arrest of PDAC cells in the G1 phase by hexahydrocurcumin downregulating cell cycle-related proteins downstream of mut-TP53 (including at least one of CCNA2, CCNB1, CDC25A, and CDK2), specifically manifested as an increase in the proportion of cells in the G1 phase and a decrease in the proportion of cells in the S phase.

[0046] The inhibition of tumor cell proliferation, colony formation, and in vivo tumorigenesis described in this invention refers to the time- and concentration-dependent inhibition of the proliferative activity and colony formation ability of PDAC cells by hexahydrocurcumin. In an in vivo pancreatic orthotopic xenograft model, it can significantly reduce tumor volume and weight in a dose-dependent manner.

[0047] The reversal of tumor chemotherapy resistance described in this invention refers to the ability of hexahydrocurcumin to restore the drug sensitivity of gemcitabine-resistant PDAC cells, with a combination index (CI) < 1.

[0048] The synergistic enhancement of the antitumor effect of chemotherapy / immunotherapy described in this invention refers to the fact that when hexahydrocurcumin is used in combination with gemcitabine and / or PD-1 inhibitors, it can significantly inhibit tumor growth and prolong the survival of tumor-bearing animals in an in vivo pancreatic cancer model, with effects superior to either single drug.

[0049] In this invention, the preferred pancreatic ductal adenocarcinoma is gemcitabine-resistant pancreatic ductal adenocarcinoma. Gemcitabine-resistant pancreatic ductal adenocarcinoma refers to pancreatic ductal adenocarcinoma in which the tumor cells exhibit a higher half-maximal inhibitory concentration (IC50) of gemcitabine compared to the parental cells, and the objective response rate to gemcitabine is stable disease (SD) or progressive disease (PD).

[0050] This invention also provides a drug for inhibiting USP39 activity in pancreatic ductal adenocarcinoma. The active ingredient of the drug is hexahydrocurcumin, and pharmaceutically acceptable dosage forms include, but are not limited to, injections (preferably hydroxypropyl-β-cyclodextrin inclusion complex injections), oral formulations (preferably tablets), sustained-release formulations, or targeted formulations.

[0051] In a further preferred embodiment of the injection, hexahydrocurcumin is solubilized using hydroxypropyl-β-cyclodextrin inclusion complexation technology, and each injection contains 10 mg of hexahydrocurcumin.

[0052] Further, the oral tablets are prepared with hexahydrocurcumin and conventional excipients such as microcrystalline cellulose and lactose, with each tablet containing 50 mg of hexahydrocurcumin.

[0053] This invention also provides a pharmaceutical composition for treating pancreatic ductal adenocarcinoma. The pharmaceutical composition comprises the active ingredient hexahydrocurcumin, and pharmaceutically acceptable excipients, carriers, or excipients. The excipients, carriers, or excipients are conventional excipients in the pharmaceutical formulation field, including but not limited to microcrystalline cellulose, lactose, crospovidone, magnesium stearate, hydroxypropyl-β-cyclodextrin, mannitol, sodium bicarbonate, etc.

[0054] This invention also provides a pharmaceutical composition for combined treatment of pancreatic ductal adenocarcinoma. The composition comprises a first active ingredient, hexahydrocurcumin, and a second active ingredient. The second active ingredient comprises at least one of gemcitabine and a PD-1 inhibitor. The PD-1 inhibitor includes, but is not limited to, clinically approved PD-1 monoclonal antibodies such as pembrolizumab and nivolumab.

[0055] The mass ratio of hexahydrocurcumin to gemcitabine according to the present invention is preferably (1~100):(1~50); the mass ratio of hexahydrocurcumin to the PD-1 inhibitor is preferably (1~100):(0.1~10). It is further preferred that the dosage form of the combined anti-pancreatic ductal adenocarcinoma pharmaceutical composition is preferably a lyophilized powder for injection, wherein hexahydrocurcumin and gemcitabine use mannitol as a lyophilization supporting agent, and sodium bicarbonate is used to adjust the pH value to 5.5~6.5.

[0056] The present invention further provides use of the above pharmaceutical composition in preparation of an anti-pancreatic ductal adenocarcinoma product. The dosage form of the anti-pancreatic ductal adenocarcinoma product is preferably a hydroxypropyl-β-cyclodextrin inclusion complex injection, an oral tablet or a lyophilized powder for injection.

[0057] The technical solution provided by the present invention will be described in detail with reference to the examples below, but they shall not be construed as limiting the protection scope of the present invention.

[0058] The instruments, reagents, cell lines and experimental animals used in the present invention are all commercially available ordinary products, all of which can be purchased from the market; wherein the sources, strains and batch numbers of cell lines and experimental animals shall be specified, and relevant experiments shall be approved by the ethics committee.

[0059] Example 1 Virtual Screening of USP39 Target and Screening of Candidate Inhibitors Compound library preparation: The compound library for this docking comes from commercial compound libraries such as MCE and Targetmol, with a total of 50081 compound entities; filtered according to the Lipinski's rule of five, compounds with molecular weight 100<MW<900 and lipophilicity -5<logP<8 are retained. The LigPre module of Schrödinger Maestro software is used to perform protonation and energy minimization processing on all compounds, and the force field selects OPLS3e.

[0060] USP39 target protein structure preparation: The USP39 protein structure is pretreated on the Maestro 11.9 platform, including removal of water and ions, protonation, addition of missing atoms, completion of missing groups, protein energy minimization and optimization, and the force field selects OPLS3e.

[0061] Active site determination and molecular docking: With reference to the USP2-Ub complex (PDB ID: 5XVE), USP39 and USP2-Ub protein are superimposed by Pymol 2.1 software to obtain the USP39-Ub complex, and the key interaction site is determined as the centroid of a 15Å×15Å×15Å docking box (x=195.71, y=245.74, z=170.34).

[0062] First, the compound library was initially screened using the SP docking mode, retaining 2964 compounds with an SPScore <-6.0 kcal / mol. Then, a secondary screening was conducted using the XP docking mode, retaining 1973 compounds with an XPScore <-6.0 kcal / mol. Subsequently, MMGBSA binding free energy assessment was performed, retaining 1330 compounds with an MMGBSA score <-40.0 kcal / mol. Finally, combining binding mode, key residue interactions, and druggability analysis, 12 candidate compounds were selected for further in vitro validation, such as... Figure 1 As shown.

[0063] In vitro activity validation of candidate compounds: An in vitro deubiquitination assay was conducted using a K48-linked diubiquitination chain as a substrate to examine the inhibitory effect of 12 candidate compounds on the deubiquitination activity of USP39. Results Figure 2 The results showed that hexahydrocurcumin exhibited the strongest inhibitory effect, with a docking fraction of -8.865511 kcal / mol and an MMGBSA binding free energy of -73.475143 kcal / mol. It could significantly block the cleavage of the K48 diubiquitin chain by USP39, thus identifying hexahydrocurcumin as the optimal candidate inhibitor of USP39.

[0064] Example 2 Verification of the direct binding and targeted inhibition of hexahydrocurcumin to USP39 Molecular binding mode validation: Surface plasmon resonance (SPR) experiments were simulated using molecular dynamics to detect the direct binding affinity between hexahydrocurcumin and recombinant USP39 protein. Results are as follows: Figure 3 The results showed that the dissociation constant KD of hexahydrocurcumin and USP39 protein was 87±12 nM, indicating that the two can bind directly. Protein-ligand interaction fingerprint analysis showed that hexahydrocurcumin forms stable hydrogen bonds with Asp438, Asn503 and Asp504 sites of USP39, and binds to key active site residues through salt bridges, stably occupying the active pocket of USP39.

[0065] Cellular-level targeting validation: PANC-1 and AsPC-1 human PDAC cell lines were selected and divided into wild-type (WT) and USP39 knockout (KO) groups, and treated with 0, 1, 5, 10, 20, and 50 μM hexahydrocurcumin for 72 h, respectively. CCK-8 assay results showed that the inhibitory effect of hexahydrocurcumin on the proliferation of WT PDAC cells was concentration-dependent, with IC50 values ​​of 0.42±0.08 μM (PANC-1) and 0.52±0.09 μM (AsPC-1); while in USP39-KO cells, the inhibitory effect of hexahydrocurcumin on proliferation completely disappeared, confirming that its antitumor effect is dependent on targeting USP39.

[0066] Cell-free deubiquitination assay: Recombinant USP39 protein was pre-incubated with different concentrations of hexahydrocurcumin at 4°C for 2 h, and then co-incubated with ubiquitinated HSP70 / FASN substrate proteins at 37°C for 4 h. Immunoblotting results showed that hexahydrocurcumin could, in a concentration-dependent manner, block the USP39-mediated HSP70 and FASN deubiquitination process, directly confirming the specific inhibitory effect of hexahydrocurcumin on the activity of USP39 deubiquitinizing enzymes.

[0067] Example 3 The regulatory effect of hexahydrocurcumin on the downstream pathway of USP39 in PDAC cells Effects on HSP70 and FASN protein expression: PANC-1 and AsPC-1 cells in logarithmic growth phase were treated with 0, 1, 5, 10, and 20 μM hexahydrocurcumin for 48 h, respectively. Total protein was extracted and analyzed by Western blotting. Results are as follows: Figures 4-5 The results showed that hexahydrocurcumin could downregulate the protein levels of HSP70 and FASN in a concentration-dependent manner, but qPCR detection confirmed that it had no significant effect on the mRNA levels of HSP70 and FASN, indicating that it regulates the expression of the two at the post-translational level.

[0068] Effects on the stability of HSP70 and FASN proteins: PANC-1 cells were divided into a control group and a 10 μM hexahydrocurcumin treatment group, with 100 μg / mL cyclohexylimide (CHX), a protein synthesis inhibitor, added. Cells were collected at 0, 4, 8, 12, and 24 h, and the levels of HSP70 and FASN proteins were detected by Western blotting. The results are as follows: Figures 6-7 The results showed that hexahydrocurcumin could significantly shorten the protein half-life of HSP70 and FASN and accelerate their degradation; while the addition of the proteasome inhibitor MG132 (10 μM) could completely reverse the downregulation effect of hexahydrocurcumin on HSP70 and FASN, confirming that it promotes the degradation of the two through the ubiquitin-proteasome pathway.

[0069] Effects on HSP70 and FASN ubiquitination levels: HEK293T cells were co-transfected with Flag-USP39, His-HSP70 / Myc-FASN, and HA-Ub plasmids, treated with 10 μM hexahydrocurcumin, and simultaneously treated with MG132 to inhibit protein degradation. Co-IP results are shown below. Figure 8 The results showed that treatment with hexahydrocurcumin significantly increased the ubiquitination level at the K48 position of HSP70 and FASN, consistent with the effect of USP39 knockdown, confirming that hexahydrocurcumin promotes the ubiquitination modification and degradation of HSP70 and FASN at the K48 position by inhibiting the deubiquitination activity of USP39.

[0070] Effects on mut-TP53 palmitoylation and protein accumulation: TP53R175H mutant PDAC cells were treated with 10 μM hexahydrocurcumin for 48 h. Results showed that hexahydrocurcumin significantly reduced the palmitoylation level of mut-TP53, decreased its nuclear protein accumulation, and downregulated the expression of cell cycle-related proteins CCNA2, CCNB1, CDC25A, and CDK2. Overexpression of USP39 reversed these effects of hexahydrocurcumin, confirming that hexahydrocurcumin regulates mut-TP53 stability and cell cycle progression by targeting USP39.

[0071] Example 4 Inhibitory effect of hexahydrocurcumin on malignant phenotype of PDAC cells Cell proliferation experiment: PANC-1 and AsPC-1 cells were cultured at 5 × 10⁻⁶ cells / year. 3 One sample per well was seeded into a 96-well plate and treated with 0, 1, 5, 10, 20, and 50 μM hexahydrocurcumin, respectively. CCK-8 reagent was added at 24, 48, 72, and 96 h, and the absorbance at 450 nm was measured after 2 h of incubation. Results are as follows: Figure 9 The results showed that hexahydrocurcumin could inhibit the proliferation of PDAC cells in a time- and concentration-dependent manner, with a statistically significant difference compared with the control group. p <0.05).

[0072] Colony formation assay: PANC-1 and AsPC-1 cells were seeded at 300 cells / well in 6-well plates and treated with 0, 5, and 10 μM hexahydrocurcumin, respectively. After 2 weeks of culture, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and the number of colonies formed was counted. Results are as follows: Figure 10 The results showed that hexahydrocurcumin significantly inhibited the colony formation ability of PDAC cells, with the colony formation rate in the 10 μM treatment group decreasing by approximately 37% compared to the control group. p <0.001).

[0073] Cell cycle detection: PANC-1 cells were treated with 0, 5, and 10 μM hexahydrocurcumin for 48 h, fixed overnight in pre-cooled 70% ethanol, and then subjected to flow cytometry after PI staining to detect cell cycle distribution. Results are as follows: Figure 11 The results showed that hexahydrocurcumin significantly arrested PDAC cells in the G1 phase. Compared with the control group, the proportion of cells in the G1 phase was significantly increased, while the proportion of cells in the S phase and G2 / M phase was significantly decreased in the treatment group. p <0.01), this effect disappears completely in USP39 gene knockout cells, confirming that it can inhibit PDAC cell proliferation by arresting the cell cycle and is strictly dependent on the deubiquitinating enzyme activity of USP39.

[0074] Example 5 Experimental study on the in vivo anti-PDAC effect of hexahydrocurcumin Animal model construction: Six-week-old male BALB / c nude mice were selected and housed in SPF condition to construct an orthotopic pancreatic xenograft model. PANC-1 cells were cultured at 5 × 10⁻⁶ cells / mL. 6 The hexahydrocurcumin was administered orally to the pancreas of nude mice at a density of 100 μL / mouse. The mice were randomly divided into a control group, a low-dose hexahydrocurcumin group (25 mg / kg / d), and a high-dose hexahydrocurcumin group (50 mg / kg / d), with 5 mice in each group. The administration was started by gavage on the 7th day after inoculation, while the control group was given an equal amount of solvent. The administration was continued for 6 weeks.

[0075] Evaluation of in vivo antitumor efficacy: Mouse body weight and tumor volume were measured weekly, and tumor growth curves were plotted; after drug administration, mice were euthanized, tumor tissue was dissected, and tumor weight was measured. Results are as follows: Figures 12-14 The results showed that, compared with the control group, the tumor volume and weight of mice in both the low- and high-dose hexahydrocurcumin groups were significantly reduced. p The dose was <0.01, and the effect was dose-dependent. During the administration period, the mice did not experience a significant decrease in body weight or obvious acute toxicity.

[0076] Histological examination: Tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (HE) and subjected to Ki-67 immunohistochemical staining. Results showed that in the control group, tumor tissues exhibited disordered cell arrangement, significant nuclear atypia, and a very high proportion of Ki-67 positive cells; in the hexahydrocurcumin-treated group, tumor tissues showed obvious necrotic areas, reduced inflammatory cell infiltration, and a significantly lower proportion of Ki-67 positive cells. p The result was <0.001, confirming that hexahydrocurcumin can significantly inhibit the proliferation of PDAC tumors in vivo.

[0077] In vivo mechanism verification: Western blot and qPCR were used to detect the expression of downstream pathway proteins of USP39 in tumor tissues. The results were consistent with the cell experiments. The expression of HSP70, FASN, mut-TP53 and cell cycle-related proteins in tumor tissues treated with hexahydrocurcumin was significantly downregulated, which confirmed that it exerts its anti-PDAC effect by targeting USP39 in vivo.

[0078] Example 6 Study on the antitumor effect of hexahydrocurcumin in combination therapy Experiment to reverse gemcitabine resistance: The gemcitabine-resistant PDAC cell line MIAPaCa-2 / Gem was constructed and treated with gemcitabine alone, hexahydrocurcumin alone, and a combination of both drugs. Cell proliferation was assessed using CCK-8 assay. Results showed that hexahydrocurcumin significantly reversed gemcitabine resistance in PDAC cells, and the combination of the two drugs significantly inhibited cell proliferation compared to the single-drug group. p The combined index (CI) <0.01 indicates that the two substances have a synergistic anti-tumor effect.

[0079] In vivo combination drug experiment: C57BL / 6 mice were randomly divided into a control group (CN), a gemcitabine group (Gem), a PD-1 inhibitor group (RMP1-14), a hexahydrocurcumin + gemcitabine group (Gem&HHC), and a hexahydrocurcumin + PD-1 inhibitor group (RMP1-14&HHC). A syngeneic mouse model of pancreatic cancer orthotopic xenograft was established, and drugs were administered at clinically equivalent doses. Results are as follows: Figures 15-17 The results showed that, compared with the single-drug group, the combination therapy group significantly inhibited tumor growth and significantly prolonged survival in mice. p <0.05), confirming that hexahydrocurcumin can synergistically enhance the anti-PDAC effect of chemotherapy and immunotherapy.

[0080] Example 7 A drug composition for treating pancreatic ductal adenocarcinoma containing hexahydrocurcumin, in the form of an oral tablet, is described below: Prescription: 50 g of hexahydrocurcumin, 120 g of microcrystalline cellulose, 80 g of lactose, 15 g of crospovidone, 2 g of magnesium stearate, and an appropriate amount of 5% hydroxypropyl methylcellulose aqueous solution, to make 1000 tablets.

[0081] Preparation process: The raw material of hexahydrocurcumin is pulverized and passed through a 100-mesh sieve, mixed evenly with microcrystalline cellulose, lactose and crospovidone, and hydroxypropyl methylcellulose aqueous solution is added to make a soft mass. The mass is granulated through a 20-mesh sieve, dried at 60℃, granulated through an 18-mesh sieve, and magnesium stearate is added and mixed evenly. The mass is then compressed into tablets to obtain oral tablets containing 50mg of hexahydrocurcumin per tablet.

[0082] Example 8 A drug composition for treating pancreatic ductal adenocarcinoma containing hexahydrocurcumin, in the form of an injection, is described below: Prescription: 10 g of hexahydrocurcumin, 200 g of hydroxypropyl-β-cyclodextrin, and water for injection to a final volume of 2000 mL, to make 1000 vials.

[0083] Preparation process: Hydroxypropyl-β-cyclodextrin is added to 80% water for injection and stirred until completely dissolved. Hexahydrocurcumin is added and stirred until the drug is completely dissolved. Water for injection is added to the total volume. The solution is then filtered through a 0.22 μm microporous membrane for sterilization and filled into 2 mL ampoules. The ampoules are then sterilized by moist heat at 121℃ for 15 min to obtain an injection containing 10 mg of hexahydrocurcumin per ampoule.

[0084] Example 9 A drug composition for treating pancreatic ductal adenocarcinoma using a combination of hexahydrocurcumin and gemcitabine, in the form of a lyophilized powder for injection, is described below: Prescription: 20 g of hexahydrocurcumin, 10 g of gemcitabine, 150 g of mannitol, appropriate amount of sodium bicarbonate, add water for injection to 2000 mL, and make a total of 1000 vials.

[0085] Preparation process: Weigh each component according to the prescription amount, add water for injection and stir to dissolve, adjust the pH value to 5.5~6.5 with sodium bicarbonate, filter sterilely through a 0.22 μm microporous membrane, fill into vials, freeze dry, and stopper and cap to obtain the combined drug lyophilized powder injection.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of hexahydrocurcumin as a USP39 inhibitor in the preparation of drugs for pancreatic ductal adenocarcinoma.

2. The application according to claim 1, characterized in that, The pancreatic ductal adenocarcinoma mentioned is gemcitabine-resistant pancreatic ductal adenocarcinoma.

3. The application according to claim 1, characterized in that, The hexahydrocurcumin, as a USP39 inhibitor, can: inhibit the deubiquitination enzyme activity of USP39, promote the K48 ubiquitination degradation of HSP70 and / or FASN, inhibit palmitoylation modification and nuclear accumulation of mut-TP53 protein, arrest the tumor cell cycle, inhibit tumor cell proliferation, colony formation and in vivo tumorigenesis, reverse tumor chemotherapy resistance, and synergistically enhance the anti-tumor effects of chemotherapy / immunotherapy.

4. The application according to claim 3, characterized in that, The mut-TP53 is a TP53R175H mutant; the cell cycle arrest is G1 phase arrest; the chemotherapy-resistant drug is gemcitabine; and the immunotherapy drug is a PD-1 inhibitor.

5. A drug for inhibiting USP39 activity in pancreatic ductal adenocarcinoma, characterized in that, Its active ingredient includes hexahydrocurcumin; the dosage forms of the drug include injections, oral preparations, sustained-release preparations, or targeted preparations.

6. A pharmaceutical composition for treating pancreatic ductal adenocarcinoma, characterized in that, It includes the active ingredient hexahydrocurcumin, as well as pharmaceutically acceptable excipients, carriers, or excipients.

7. A pharmaceutical composition for combined treatment of pancreatic ductal adenocarcinoma, characterized in that, It includes a first active ingredient, hexahydrocurcumin, and a second active ingredient; the second active ingredient includes at least one of gemcitabine and a PD-1 inhibitor.

8. The pharmaceutical composition according to claim 7, characterized in that, The mass ratio of hexahydrocurcumin to gemcitabine is (1~100):(1~50); and / or the mass ratio of hexahydrocurcumin to PD-1 inhibitor is (1~100):(0.1~10).

9. Use of the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of an anti-pancreatic ductal adenocarcinoma product.

10. The application according to claim 9, characterized in that, The dosage form of the anti-pancreatic ductal adenocarcinoma product is hydroxypropyl-β-cyclodextrin inclusion complex injection, oral tablets, or lyophilized powder injection.

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