Pharmaceutical compositions of a usp7 inhibitor with fenofibrate and their antitumor applications

Abstract

This invention belongs to the field of pharmaceutical technology and discloses a pharmaceutical composition of a USP7 inhibitor and fenofibrate and its antitumor application. The pharmaceutical composition uses a USP7 inhibitor and fenofibrate as the core active ingredients, with a molar ratio of 0.015625 to 0.625. The USP7 inhibitor is selected from at least one of LX04-104 and LML-17-133. This invention is the first to discover that fenofibrate can significantly enhance the antitumor effect of the USP7 inhibitor, and the combination of the two has a synergistic effect. This synergistic effect is cell type specific, particularly significant for nasopharyngeal carcinoma and pancreatic cancer, and the synergistic effect of fenofibrate is independent of the PPARα pathway. This composition can be prepared as an oral or injectable formulation for the treatment of nasopharyngeal carcinoma and pancreatic cancer, and has good prospects for clinical translation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a pharmaceutical composition of a USP7 inhibitor and fenofibrate, and the application of this composition in the preparation of drugs for treating nasopharyngeal carcinoma and pancreatic cancer. Background Technology

[0002] (1) Current status of research on USP7 target and inhibitors USP7 is an important deubiquitinating enzyme that plays a crucial role in tumorigenesis and development by stabilizing various oncogenic proteins such as MDM2 and DNMT1. Clinical studies have shown that USP7 is highly expressed in various tumors, including non-small cell lung cancer, colorectal cancer, and head and neck squamous cell carcinoma, and is closely associated with poor patient prognosis. In recent years, several selective small molecule inhibitors of USP7 have been reported, such as U-20, X21, ALM4, and OAT-4828. The inventors have also previously reported a series of novel and highly active USP7 inhibitors, including LX04-104 and LML-17-133.

[0003] (2) Limitations of USP7 inhibitors The antitumor activity of USP7 inhibitors is significantly cell type dependent. Only a very small number of tumor cells (such as RS4;11, OCI-ly10, LNCaP, etc.) are extremely sensitive to them (IC50). 50 <50 nM), while most tumor cells (including many solid tumors) are insensitive to it (IC50). 50 >1 μM). USP7 is also expressed and plays an important physiological role in normal cells, leading to challenges such as a narrow therapeutic window for monotherapy. To date, no USP7 inhibitors have entered clinical trials.

[0004] (3) Existing research on fenofibrate Fenofibrate (FF) is a widely used lipid-lowering drug in clinical practice and belongs to the PPARα agonist class. Recent studies have found that it possesses potential anti-tumor activity, with some effects independent of the PPARα pathway (such as mitochondrial function inhibition, NF-κB downregulation, and AKT signaling inhibition). However, fenofibrate monotherapy exhibits weak anti-tumor activity, requiring relatively high concentrations to produce an effect, thus limiting its feasibility for use as a standalone treatment for cancer.

[0005] (4) Combination therapy for tumors is an important strategy to overcome monotherapy resistance and broaden the treatment window. However, the effects of combination therapy are unpredictable: the combination of two drugs may produce synergistic, additive or antagonistic effects, and the synergistic effect of a specific combination is often cell type specific and cannot be simply deduced from the mechanism of monotherapy.

[0006] Based on the above-mentioned existing technologies, the following technical defects exist in this field: (1) USP7 inhibitor monotherapy has limited efficacy against solid tumors such as pancreatic cancer and nasopharyngeal carcinoma; (2) Fenofibrate monotherapy has weak antitumor activity and is not suitable for use alone.

[0007] (3) There are currently no research reports on the combination of USP7 inhibitors and fenofibrate for anti-tumor treatment. Summary of the Invention

[0008] To address the shortcomings of the existing technology, this invention provides a pharmaceutical composition of a USP7 inhibitor and fenofibrate. The combination of the two drugs produces a synergistic anti-tumor effect against nasopharyngeal carcinoma and pancreatic cancer, overcoming the limitation of the limited efficacy of USP7 inhibitor monotherapy against solid tumors. This provides a new strategy for the repurposing of fenofibrate and enhancing the clinical efficacy of USP7 inhibitors.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a pharmaceutical composition comprising a USP7 inhibitor and fenofibrate as active ingredients, wherein the USP7 inhibitor is selected from at least one of LX04-104 and LML-17-133, and the molar ratio of the USP7 inhibitor to fenofibrate is 0.015625 to 0.625:1.

[0010] Preferably, the USP7 inhibitor is LX04-104, and the molar ratio of LX04-104 to fenofibrate is 0.0625 to 0.25:1.

[0011] Preferably, the molar ratio of LX04-104 to fenofibrate is 0.125:1.

[0012] Preferably, the USP7 inhibitor is LML-17-133, and the molar ratio of LML-17-133 to fenofibrate is 0.31 to 0.625:1.

[0013] Preferably, the molar ratio of LML-17-133 to fenofibrate is 0.31 to 0.5:1.

[0014] The dosage form of the finished pharmaceutical composition of the present invention is an acceptable dosage form prepared by conventional pharmaceutical techniques, such as oral preparations and injectable preparations.

[0015] Various dosage forms of the pharmaceutical compositions described in this invention can be prepared using conventional pharmaceutical manufacturing methods. For example, the composition can be mixed with one or more carriers and then formulated into the desired dosage form.

[0016] The pharmaceutical composition is prepared by mixing a USP7 inhibitor with fenofibrate and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is selected from one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, absorbent carriers, surfactants, and lubricants.

[0017] When the drug is used as a clinical human preparation, the dosage is calculated based on the molar ratio of USP7 inhibitor to fenofibrate of 0.015625 to 0.5:1, with the effective antitumor dose as the basis for administration.

[0018] The pharmaceutical composition exists in the form of oral or injectable formulations, wherein oral formulations include tablets, powders, capsules, and granules.

[0019] Possible formulation options include: Each tablet contains the USP7 inhibitor LX04-104 and fenofibrate in a molar ratio of 0.125:1. The excipients are lactose, microcrystalline cellulose, sodium carboxymethyl starch, and magnesium stearate. Each capsule contains the USP7 inhibitor LML-17-133 and fenofibrate in a molar ratio of 0.31 to 0.5:1. The excipients are lactose, pregelatinized starch, and purified water. Each injection contains a USP7 inhibitor and fenofibrate in an effective molar ratio. The excipients are water for injection, mannitol, and Tween-80.

[0020] Secondly, the present invention provides the use of the pharmaceutical composition in the preparation of a drug for treating tumors.

[0021] Specifically, this refers to the application of the pharmaceutical composition in the preparation of drugs for treating nasopharyngeal carcinoma and pancreatic cancer. The nasopharyngeal carcinoma mentioned is EBV-related nasopharyngeal carcinoma.

[0022] The pancreatic cancers mentioned include pancreatic cancers derived from KPIC cells and pancreatic cancers derived from SW1990 cells.

[0023] This invention provides a synergistic antitumor drug composition of USP7 inhibitor and fenofibrate, its preparation method and application. The composition uses USP7 inhibitor and fenofibrate as the core active ingredients. The two are combined in a specific molar ratio to achieve a synergistic antitumor effect, which can effectively treat nasopharyngeal carcinoma and pancreatic cancer. The formulation forms cover pharmaceutically acceptable oral and injectable formulations.

[0024] The present invention has the following beneficial effects: (1) The synergistic anti-tumor effect of fenofibrate and USP7 inhibitors was discovered for the first time, solving the technical problem of the limited efficacy of USP7 inhibitors in solid tumors.

[0025] (2) The synergistic effect is cell type specific, and is particularly significant for nasopharyngeal carcinoma (C666-1) and pancreatic cancer (KPIC, SW1990).

[0026] (3) The mechanism of action of fenofibrate does not depend on the PPARα pathway and is its unique off-target effect (verified by comparison screening).

[0027] (4) The synergistic effect is dose-dependent, and USP7 inhibitors need to reach a specific concentration window that "partially inhibits cell viability".

[0028] (5) It broadened the therapeutic window of USP7 inhibitors and improved clinical safety.

[0029] (6) It realizes the repurposing of fenofibrate and has good prospects for clinical translation. Attached Figure Description

[0030] Figure 1 Screening of USP7 inhibitors in combination with antitumor drugs on C666-1 cells. (A) Efficacy and CI value of USP7 inhibitor LX04-104 combined with PPARα agonist Fenofibrate; (B) No synergistic effect between USP7 inhibitor LX04-104 and PPARδ agonist MBX8025; (C) No synergistic effect between USP7 inhibitor LX04-104 and PPARγ agonist Rosiglitazone; (D) USP7 inhibitor LX04-104 and PPARδ / PPARβ agonist GW501516 showed a certain synergistic effect only at LX04-104 40 μM and GW501516 90 μM. Since this concentration is far beyond the conventional pharmacological activity range, this combination was not used as a target combination for subsequent CI value calculation and experimental verification. * indicates P≤0.05, ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001; CI<1 indicates synergistic effect, CI=1 indicates additive effect, CI>1 indicates antagonistic effect.

[0031] Figure 2 The combined use of USP7 inhibitors and fenofibrate (FF) in C666-1 cells showed the following effects: (A) Fenofibrate increased the inhibitory effect of USP7 inhibitor LX04-104 on C666-1 cells and its CI value; (B) Fenofibrate increased the inhibitory effect of USP7 inhibitor LML-17-133 on C666-1 cells and its CI value; (C) Fenofibrate did not enhance the antitumor effect of USP7 inhibitor LX04-104 by activating PPARα. CI < 1 indicates synergistic effect, CI = 1 indicates additive effect, and CI > 1 indicates antagonistic effect.

[0032] Figure 3 Cell morphology diagram of Fenofibrate in combination with LX04-104 on C666-1 cells.

[0033] Figure 4 The combined use of USP7 inhibitors and fenofibrate (FF) in SW1990 cells showed a synergistic effect. (A) Fenofibrate increased the inhibitory effect of USP7 inhibitor LX04-104 on SW1990 cells and its CI value; (B) Fenofibrate increased the inhibitory effect of USP7 inhibitor LML-17-133 on SW1990 cells and its CI value. CI < 1 indicates synergistic effect, CI = 1 indicates additive effect, and CI > 1 indicates antagonistic effect.

[0034] Figure 5 The combined use of USP7 inhibitors and fenofibrate (FF) on KPIC cells showed a synergistic effect. (A) Fenofibrate increased the inhibitory effect of USP7 inhibitor LX04-104 on KPIC and its CI value; (B) Fenofibrate increased the inhibitory effect of USP7 inhibitor LML-17-133 on KPIC and its CI value. CI < 1 indicates synergistic effect, CI = 1 indicates additive effect, and CI > 1 indicates antagonistic effect.

[0035] Figure 6 Fenofibrate, in synergy with the USP7 inhibitor LX04-104, promotes apoptosis and inhibits proliferation of C666-1 cells. (A) Flow cytometry was used to detect the apoptosis rate; (B) Flow cytometry was used to detect cell proliferation.

[0036] Figure 7 Fenofibrate in combination with LX04-104 significantly inhibited tumor growth in mice. P ≤ 0.05 was considered statistically significant, * indicates P ≤ 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Detailed Implementation

[0037] Unless otherwise specified, the reagents, methods and equipment used in this invention are conventional reagents, methods and equipment in this technical field.

[0038] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0039] This invention explores drug screening using nasopharyngeal carcinoma cell line C666-1 and pancreatic carcinoma cell line KPIC as experimental subjects in vitro. Cell viability and apoptosis rates were used as indicators to verify the enhancing effect of fenofibrate, an FDA-approved drug, on the antitumor activity of USP7 inhibitors. The synergistic effect of the combination therapy requires the USP7 inhibitor to reach a specific concentration window that "partially inhibits cell viability." The specific steps are as follows: 1. Drugs: The structures, preparation methods, and bioactivity data of compounds LX04-104 and LML-17-133 were obtained from the literature: Discovery of Orally Bioavailable N-Benzylpiperidinol Derivatives as Potent and Selective USP7 Inhibitors with In Vivo Antitumor Immunity Activity against Colon Cancer, J. Med. Chem. 2022, 65, 16622−16639. Fenofibrate, GW501516, and Rosiglitazone were purchased from Shanghai Dibai Chemical Technology Co., Ltd., and MBX8025 and GW6471 were purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0040] 2. Cell lines: Human nasopharyngeal carcinoma cell line C666-1 was cultured in 1640 complete medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin, and placed in a CO2 incubator with saturated humidity at 37°C, 95% air, and 5% CO2. Cells grew in an adherent manner and were passaged every 3 days.

[0041] Mouse pancreatic cancer cell line KPIC and human pancreatic cancer cell line SW1990 were cultured in DMEM complete medium containing 10% fetal bovine serum, 1% penicillin, and streptomycin. The cells were cultured in a CO2 incubator at 37°C, 95% air, and 5% CO2 saturated humidity. Cells grew in an adherent manner and were passaged every two days.

[0042] 3. Laboratory animals and their care Male C57BL / 6J mice, 6-8 weeks old (weighing 16-20g).

[0043] 4. Experimental methods and results 4.1 CTG method for detecting cell viability, screening for combination drugs and cancer types suitable for combination drugs. (1) Experimental steps: ① Screening experiment of USP7 inhibitor combination drugs Collect C666-1 cells in the logarithmic growth phase, seed them in 96-well plates at 7000 cells / well and 100 μL of cell culture. After 24 hours of incubation, the drugs were added. The blank group was given DMSO as a control. The single-drug groups of Fenofibrate, MBX8025, and Rosiglitazone were given at concentrations of 80, 40, 20, 10, 5, and 2.5 μM, respectively. The single-drug groups of GW501516 were given at concentrations of 90, 30, 10, 3.3, 1.1, and 0.6 μM, respectively. The combination groups of Fenofibrate, MBX8025, Rosiglitazone, and USP7 inhibitors were given at a fixed concentration of 2.5 μM LX04-104, which was combined with Fenofibrate, MBX8025, and Rosiglitazone at the above concentration gradients, respectively. The combination groups of GW501516 and USP7 inhibitors were given at concentrations of 40, 13.3, 4.4, 1.5, 0.5, and 0.2 μM LX04-104, which were combined with GW501516 at the above concentration gradients, respectively. 72 h after drug administration, an equal volume of CellCounting-Lite 2.0 Luminescent CellViability Assay was added, and the mixture was shaken on a shaker for 15 min before signal values ​​were detected. Within a certain cell number range, the absorbance value was directly proportional to the number of viable cells. Figure 1 As shown, when USP7 inhibitors are used in combination with multiple candidate drugs, a specific combination (USP7 inhibitor + fenofibrate) exhibits a significant synergistic anti-tumor effect in C666-1 cells. However, USP7 inhibitors and GW501516 only produce a certain synergistic effect at specific concentrations. Since this concentration is far beyond the conventional pharmacological activity range, this combination is not used as the target combination for subsequent experiments. Most other combinations do not have obvious synergistic effects.

[0044] ② Preliminary verification of the combined effects and mechanism of Fenofibrate and USP7 inhibitor in C666-1 cells. C666-1 cells in logarithmic growth phase were collected, seeded in 96-well plates at 7000 cells / well, and 100 μL of cell culture was added. After 24 hours of incubation, the drugs were added. For efficacy verification of the combination therapy, DMSO was used as a control in the blank group; the USP7 inhibitor single-drug groups were administered LX04-104 at concentrations of 10, 5, 2.5, and 1.25 μM, and LML-17-133 at concentrations of 40, 20, 10, and 5 μM. The combination therapy groups used 20 μM of fenofibrate as a fixed concentration, combined with LX04-104 and LML-17-133 at the above concentration gradients, respectively. Figure 2 A and Figure 2As shown in Figure B, Fenofibrate enhanced the inhibitory effect of USP7 inhibitors on C666-1 nasopharyngeal carcinoma cells in a dose-dependent manner, with a combination index (CI) <1, confirming a synergistic effect. In the mechanism validation section, the blank group received DMSO as a control, the GW6471 monotherapy groups received 4, 2, 1, and 0.5 μM, and the combination therapy groups received 2.5 μM LX04-104 and 20 μM Fenofibrate combined with GW6471 at the above concentration gradients. 72 h after drug administration, an equal volume of CellCounting-Lite 2.0 Luminescent Cell Viability Assay was added, and the mixture was shaken on a shaker for 15 min before signal values ​​were detected. Within a certain cell number range, the absorbance value was directly proportional to the number of viable cells. Figure 2 As shown in C, the PPARα antagonist GW6471 cannot block this synergistic effect, demonstrating that the synergistic effect of fenofibrate does not depend on PPARα pathway activation.

[0045] ③ Validation of the combined effect of Fenofibrate and USP7 inhibitor on SW1990 cells SW1990 cells in logarithmic growth phase were collected, seeded in 96-well plates at 3000 cells / well, and 100 μL of cell culture was added. After 24 hours of incubation, drugs were added. The control group received DMSO. The USP7 inhibitor monotherapy groups received LX04-104 at concentrations of 5, 2.5, 1.25, and 0.6 μM, and LML-17-133 at concentrations of 50, 25, 12.5, and 6.2 μM. The combination groups received 20 μM Fenofibrate at a fixed concentration, combined with LX04-104 and LML-17-133 at the aforementioned concentration gradients. 72 hours after drug administration, an equal volume of CellCounting-Lite 2.0 Luminescent Cell Viability Assay was added, and the cells were shaken for 15 minutes before signal values ​​were detected. Within a certain cell number range, the absorbance value was directly proportional to the number of viable cells. Figure 4 As shown, the combination of fenofibrate and the USP7 inhibitor produced a significant synergistic inhibitory effect on the human pancreatic cancer cell line SW1990 (CI<1).

[0046] ④ Validation of the combined effect of Fenofibrate and USP7 inhibitor on KPIC cells KPIC cells in logarithmic growth phase were collected, seeded in 96-well plates at 3000 cells / well, and 100 μL of cell culture was added. After 24 hours of incubation, the drugs were added. The control group received DMSO; the Fenofibrat monotherapy groups were administered at concentrations of 80, 40, 20, 10, 5, and 2.5 μM, while the combination groups were administered at fixed concentrations of 2 and 2.5 μM LX04-104 and 30 and 40 μM LML-17-133, respectively, in combination with Fenofibrate monotherapy at the aforementioned concentration gradients. 72 hours after drug administration, an equal volume of CellCounting-Lite 2.0 Luminescent Cell Viability Assay was added, and the cells were shaken on a shaker for 15 minutes before signal values ​​were detected. Within a certain cell number range, the absorbance value was directly proportional to the number of viable cells. Figure 5 As shown.

[0047] (2) Results: Through drug screening, fenofibrate was found to significantly enhance the antitumor effect of USP7 inhibitors (see Figure 1). In the initial screening stage, we first evaluated the difference between the combination therapy group and the optimal single-drug group using statistical methods. Only when the effect of the combination therapy group was significantly better than that of the optimal single-drug group (p<0.05) were we determined that the combination had a synergistic effect, and further used CompuSyn software to perform combination index (CI) analysis to quantify its synergistic effect. For combinations that did not reach statistical significance, they were directly determined to have no significant synergistic effect, and no CI value was calculated. Meanwhile, if... Figure 2 , Figure 4 and Figure 5 As shown, this combination therapy exhibits a synergistic anti-tumor effect in various types of tumor cells; Figure 2 The results further indicate that the enhancing effect of fenofibrate on the antitumor efficacy of USP7 inhibitors is independent of the PPARα pathway. Furthermore, experimental results show that the synergistic effect varies in different tumor cell types at the same molar ratio, for example, in… Figure 2 A, Figure 4 In A, the synergistic effect of fenofibrate and LX04-104 at a concentration ratio of 8:1 was significantly stronger in C666-1 cells than in SW1990 cells and KPIC cells.

[0048] 4.2 Cell morphology images taken with an inverted microscope (1) Experimental procedure: C666-1 cells in logarithmic growth phase were collected, seeded in 6-well plates at 7000 cells / well, and 100 μL of cell culture solution was added. After incubation for 24 hours, the drugs were added. The blank group was given DMSO as a control; the single-drug groups were given 20 μM Fenofibrate and 2.5 μM LX04-104, respectively, and the combination group was given 2.5 μM LX04-104 added to the medium containing 20 μM Fenofibrate. The cell morphology was photographed under an inverted microscope 48 h after drug administration.

[0049] (2) The results showed that C666-1 cells treated with fenofibrate in combination with a USP7 inhibitor exhibited typical apoptosis and cell death characteristics, manifested as shrunken cell morphology, impaired cell membrane integrity, and a large number of dead cells. See Figure 3 .

[0050] 4.3 CFSE Cell Division Tracker Kit for Detecting Cell Proliferation (1) Experimental procedure: Prepare a 5 μM working solution of CSFE dye, collect C666-1 cells in the logarithmic growth phase, centrifuge and count them, and then dilute them in CFSE working solution at a concentration of 2 x 10⁻⁶. 7 Resuspend the cells in working medium at a density of 10 cells / mL. Incubate the cells at 37°C in the dark for 20 minutes. Add 5 times the volume of the original chromosomes in cell culture medium containing 10% FBS to quench staining, then centrifuge. After centrifugation, seed the cells into 6-well plates (40 x 10⁻⁶). 4 / well, 1ml cell culture. After 24 hours of incubation, the drugs were added. The blank group was given DMSO as a control; the single-drug groups were given 20μM Fenofibrate and 2.5μM LX04-104, respectively, and the combination group was given 2.5μM LX04-104 added to the medium containing 20μM Fenofibrate. After 48 hours, the cells were gently digested with trypsin, centrifuged at 500×G for 5min to remove the medium, resuspended in PBS, and the fluorescence intensity was detected by flow cytometry.

[0051] (2) The results showed that both the combination therapy group and the USP7 inhibitor monotherapy group inhibited the proliferation of C666-1 cells. See Figure 6 This indicates that the combination therapy group retained the proliferation-inhibiting effect of the single-drug group.

[0052] 4.4 Apoptosis Detection (1) Experimental procedure: C666-1 cells in logarithmic growth phase were collected, seeded in 6-well plates at 400,000 cells / well, and 100 μL of cell culture solution was added. After incubation for 24 hours, drugs were added. The blank group was given DMSO as a control; the single-drug groups were given 20 μM Fenofibrate and 2.5 μM LX04-104, respectively, and the combination group was given 2.5 μM LX04-104 added to the medium containing 20 μM Fenofibrate. 72 h after drug administration, the cells were gently digested with trypsin to prepare a single-cell suspension, centrifuged at 300×g for 5 min, the supernatant was discarded, the cells were collected, washed once with PBS, gently resuspended and counted. 1×10 5 Resuspend the cells and centrifuge at 300×G for 5 min, discarding the supernatant. Wash the cells once with PBS, centrifuge again, discard the supernatant, and resuspend the cells in 100 μL of diluted 1×AnnexinV Binding Buffer. Then add 2.5 μL of AnnexinV-FITC Reagent and 2.5 μL of PI Reagent. Gently vortex to mix, and incubate at room temperature in the dark for 15–20 min. After incubation, add 400 μL of diluted 1×AnnexinV Binding Buffer and mix the sample thoroughly. Analyze immediately.

[0053] (2) The results showed that the combination of fenofibrate and the USP7 inhibitor significantly increased the apoptosis rate of C666-1 cells, indicating that the combination of drugs could significantly promote apoptosis in C666-1 cells. See Figure 6 .

[0054] 4.5 Mouse tumor suppression experiment (1) Construction of pancreatic cancer orthotopic xenograft model KPIC-luc cells in logarithmic growth phase with a cell density of approximately 80%-90% were prepared by trypsin digestion to prepare a single-cell suspension. After centrifugation, the cells were counted and 1×10⁶ cells were collected. 7 After centrifugation, the supernatant was discarded, and the cells were resuspended in 200 μL of serum-free culture medium. The cell suspension and ABW Matrigengel high-concentration matrix gel were diluted at a ratio of 1:1 at 4°C.

[0055] The mice were prepared one day before surgery, and the hair on the upper left side of their abdomen was clipped. The mice were injected intraperitoneally with an appropriate amount of anesthetic (10 μl / g). After the mice were anesthetized, the skin on the left side of the mouse's abdomen was wiped with alcohol and iodine, respectively.

[0056] Make a 1cm incision in the left abdomen of the mouse (the general area of ​​the spleen). Similarly, grasp the smooth muscle at the top of the spleen and make an incision to access the peritoneal cavity. Gently grasp the tail end of the spleen and pull it out. The pancreas will be attached to the spleen. Use a moist, sterile cotton swab to unfold the pancreas and locate its tail. Inject 20 µl of injection solution into the pancreatic tail, leave the needle inside for 10 seconds, and then slowly rotate the needle away from the pancreas. Successful implantation will appear as a surface bubble without any leakage. Afterward, return the pancreas and spleen to the peritoneal cavity. Suture the muscles first, then suture the skin separately. Close the incision using 6-0 sutures or staples.

[0057] Wrap the animal in toilet paper and place it on a heating pad until it recovers from anesthesia, then return it to its cage. Monitor for pain at the same time.

[0058] (2) Drug administration to mice Twenty-one days after modeling, mice were randomly divided into a model group, an LX04-104 group, a fenofibrate group, and a combination group. The model group was administered corn oil by gavage at 100 mg / kg / day and saline by intraperitoneal injection daily. The LX04-104 monotherapy group was administered LX04-104 dissolved in saline and intraperitoneally at 3 mg / kg / day. The fenofibrate monotherapy group was administered fenofibrate dissolved in corn oil by gavage at 100 mg / kg / day. The LX04-104 combined with fenofibrate group was administered fenofibrate dissolved in corn oil by gavage at 100 mg / kg / day daily and LX04-104 dissolved in saline and intraperitoneally at 3 mg / kg / day. The administration was carried out for a total of 15 days.

[0059] (3) The results showed that the combination therapy group significantly inhibited the growth rate of in situ pancreatic tumors in mice, effectively reduced tumor volume and weight, and the tumor-suppressing effect was significantly better than that of each single-drug treatment group. See Table 1 and... Figure 7 As shown.

[0060] Table 1 Grouping of tumor-bearing mice and tumor weight in mice

[0061] 4.6 Calculation of the Combination Drug Use Index (CI value) The effects of drug combination therapy were quantitatively analyzed using CompuSyn software and the Chou-Talalay combination index method. Based on the cell proliferation inhibition rates of each drug individually and in combination, corresponding dose and effect data were input, and CI values ​​were calculated using CompuSyn software. Where: CI < 1 indicates synergistic effect between drugs, CI = 1 indicates additive effect, and CI > 1 indicates antagonistic effect between drugs. 4.7 Statistical Analysis All experimental data are expressed as mean ± standard error (mean ± SEM), and in vitro experimental data are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 9 software. Analysis of differences among multiple groups was performed using unpaired Student's t-test or one-way ANOVA combined with Dunnett's multiple comparison test. P ≤ 0.05 was considered statistically significant.

Claims

1. A pharmaceutical composition, characterized in that, The active ingredient contains a USP7 inhibitor and fenofibrate, wherein the USP7 inhibitor is selected from at least one of LX04-104 and LML-17-133, and the molar ratio of the USP7 inhibitor to fenofibrate is 0.015625 to 0.625:

1.

2. The pharmaceutical composition according to claim 1, characterized in that, The USP7 inhibitor is LX04-104, and the molar ratio of LX04-104 to fenofibrate is 0.0625 to 0.25:

1.

3. The pharmaceutical composition according to claim 2, characterized in that, The molar ratio of LX04-104 to fenofibrate is 0.125:

1.

4. The pharmaceutical composition according to claim 1, characterized in that, The USP7 inhibitor is LML-17-133, and the molar ratio of LML-17-133 to fenofibrate is 0.31 to 0.625:

1.

5. The pharmaceutical composition according to claim 4, characterized in that, The molar ratio of LML-17-133 to fenofibrate is 0.31 to 0.5:

1.

6. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The pharmaceutical composition is an oral or injectable formulation.

7. Use of the pharmaceutical composition according to any one of claims 1-5 in the preparation of a medicament for treating tumors.

8. The application according to claim 7, characterized in that, The tumor is either nasopharyngeal carcinoma or pancreatic carcinoma.

9. The application according to claim 8, characterized in that, The nasopharyngeal carcinoma mentioned is EBV-related nasopharyngeal carcinoma.

10. The application according to claim 8, characterized in that, The pancreatic cancers mentioned include pancreatic cancers derived from KPIC cells and pancreatic cancers derived from SW1990 cells.

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