Pharmaceutical composition containing USP2 inhibitor ML364 and sorafenib and application thereof

Abstract

The invention provides a pharmaceutical composition and application thereof in preparation of a medicine for treating liver cancer. It is found for the first time that when oral small-molecule multi-kinase inhibitor sorafenib and deubiquitinase USP2 inhibitor ML364 for treating liver cancer are combined for use, hepatoma cell ferroptosis can be obviously induced, sensitivity of hepatoma cells to sorafenib is improved, and therefore the progress of liver cancer is effectively inhibited. The pharmaceutical composition provided by the invention has a wide application prospect in preparation of medicines for treating liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals, and specifically relates to a pharmaceutical composition for improving the therapeutic effect of liver cancer, comprising an oral small molecule multi-kinase inhibitor sorafenib and a deubiquitinating enzyme USP2 inhibitor ML364, which can effectively inhibit the progression of liver cancer. Background Art

[0002] Liver cancer is a common malignancy worldwide, with consistently high morbidity and mortality rates. This not only severely impacts patients' health and quality of life, but also places a significant burden on global healthcare systems. Liver cancer has become a major public health challenge in my country, with its high morbidity and mortality rates highlighting the urgency and arduousness of liver cancer prevention and treatment in my country. Therefore, developing new targets and strategies for liver cancer prevention, diagnosis, and treatment has become a critical issue that needs to be addressed.

[0003] Sorafenib is a small molecule, multi-targeted tyrosine kinase inhibitor with dual anti-proliferative and anti-angiogenic effects. In 2008, the US FDA approved it as a first-line treatment for advanced liver cancer. Clinical research data show that sorafenib can prolong the median overall survival of patients with advanced liver cancer by approximately three months. However, only approximately 30% of liver cancer patients benefit from sorafenib treatment, and these patients typically develop sorafenib resistance within six months. Sorafenib resistance has become a major obstacle to the effectiveness of this clinical drug.

[0004] In summary, the problems and shortcomings of the existing technology are as follows: Sorafenib is a first-line drug for the clinical treatment of liver cancer. Most liver cancer patients develop resistance to sorafenib due to acquired factors. Therefore, increasing sorafenib sensitivity and enhancing its tumor-killing ability are key to improving sorafenib's efficacy and prognosis for liver cancer patients. Therefore, the development of pharmaceutical compositions that increase liver cancer cell sensitivity to sorafenib and thereby enhance its efficacy is of great significance.

[0005] USP2 is the second member of the ubiquitin-specific hydrolase (USP) family of deubiquitinating enzymes, which promotes substrate deubiquitination and stabilizes substrate proteins. Recent studies have found that the USP2 gene is highly expressed in liver cancer and promotes liver cancer progression, suggesting that USP2 may be a new target for liver cancer treatment. ML364 is a specific enzyme inhibitor of USP2 that exhibits antitumor activity in various tumors, but its effect on liver cancer progression and its combination with sorafenib for liver cancer treatment have not been reported to date, and the mechanism by which its combination with sorafenib inhibits liver cancer progression requires further investigation. Summary of the Invention

[0006] In view of the problems and defects in the prior art, an object of the present invention is to provide a pharmaceutical composition comprising a deubiquitinating enzyme USP2 inhibitor ML364 and sorafenib.

[0007] Furthermore, the concentration ratio of ML364 to sorafenib in the pharmaceutical composition can be 0.8:1 to 1.5:1.

[0008] Furthermore, the chemical name of ML364 is: 2-[[(4-methylphenyl)sulfonyl]amino]-N-(4-phenyl-2-thiazolyl)-4-(trifluoromethyl)benzamide, molecular formula: C 24 H 18 F3N3O3S2, molecular weight: 517.54, CAS No.: 1991986-30-1, its chemical structure is as follows:

[0009]

[0010] Another object of the present invention is to provide the use of the pharmaceutical composition in preparing a drug for treating liver cancer.

[0011] Another object of the present invention is to provide the use of the deubiquitinating enzyme USP2 inhibitor ML364 in the preparation of an anti-liver cancer drug sensitizer.

[0012] Furthermore, the anti-liver cancer drug is sorafenib.

[0013] Another object of the present invention is to provide the use of the deubiquitinating enzyme USP2 inhibitor ML364 as a ferroptosis inducer.

[0014] Another object of the present invention is to provide the use of the deubiquitinating enzyme USP2 inhibitor ML364 in the preparation of a drug for treating liver cancer.

[0015] The present invention discovered that USP2 protein levels are significantly elevated in tumor tissues of liver cancer patients. When the USP2 inhibitor ML364 inhibits USP2 enzymatic activity, it significantly induces ferroptosis in liver cancer cells and significantly inhibits their growth. Mechanistic studies have revealed that ML364 primarily affects ferroptosis and inhibits liver cancer cell growth by affecting the DCAF7-BMAL1-HIF1α-SLC7A11 signaling pathway. This mechanism is the first discovered by the present invention.

[0016] The DCAF7-BMAL1-HIF1α-SLC7A11 signaling pathway is a new signaling pathway discovered by the inventors of the present invention that regulates ferroptosis of liver cancer cells. The scaffolding protein DCAF7 interacts with the circadian clock protein BMAL1 by recruiting the deubiquitinating enzyme USP2, mediating its deubiquitination and inhibiting its degradation through the autophagy pathway, thereby affecting the HIF1α-SLC7A11 signaling pathway downstream of BMAL1 and affecting ferroptosis of liver cancer.

[0017] The present invention further discovered that when a combination of the USP2 inhibitor ML364 and sorafenib is used, the sensitivity of liver cancer cells to sorafenib can be increased by significantly inducing ferroptosis of liver cancer cells, thereby improving the efficacy of sorafenib and inhibiting the growth of liver cancer cells in vitro and in vivo.

[0018] In summary, the composition comprising the USP2 inhibitor ML364 and sorafenib provided by the present invention has broad application prospects in the preparation of drugs for treating liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 2 shows the experimental results of USP2 protein and transcription levels expressed in tumor tissues of clinical liver cancer patients.

[0020] Figure 2 This figure shows the experimental results showing that the USP2 inhibitor ML364 induces ferroptosis in liver cancer cells and inhibits the growth of liver cancer cells.

[0021] Figure 3 This figure shows the experimental results showing that the USP2 inhibitor ML364 induces ferroptosis at the cellular level and increases sorafenib sensitivity.

[0022] Figure 4 This figure shows the experimental results showing that the USP2 inhibitor ML364 induces ferroptosis and increases sorafenib sensitivity in mice. DETAILED DESCRIPTION

[0023] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0024] Unless otherwise specified, the reagents and equipment used in the present invention can be obtained by purchasing commercial products.

[0025] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1: USP2 protein and transcript levels are significantly elevated in tumor tissues of clinical liver cancer patients

[0027] To detect the expression of USP2 in liver cancer tissues, 27 pairs of clinical liver cancer tissues and their corresponding adjacent tissues were collected, and tissue whole proteins and whole mRNA were extracted. Immunoblotting and qPCR experiments were used to detect USP2 protein and transcription changes, respectively.

[0028] The results showed that the two subtypes of USP2 protein, USP2a and USP2b, were significantly increased in tumor tissues of patients with liver cancer ( Figure 1 AG), and its mRNA level is also elevated in most tumor tissues ( Figure 1 H). The results suggest that USP2 may be a potential target for liver cancer treatment.

[0029] Example 2: USP2 inhibitor ML364 induces ferroptosis in liver cancer cells and inhibits their growth

[0030] To test the effect of USP2 inhibitor ML364 on ferroptosis of liver cancer cells, 10 4 Huh7 cells ( Figure 2 A) or SNU-449 cells ( Figure 2 B), four replicate wells were set up for each group. After the cells adhered, the corresponding concentrations of ML364 (0, 2, 4 μM) and 2 μM (for Huh7) or 1 μM (for SNU-449) of RSL3 were added and treated for 24 hours. The effect of ML364 treatment on the sensitivity of liver cancer cells to RSL3 was detected using CCK-8. The results showed that treatment of liver cancer cells (Huh7, SNU-449) with the USP2 inhibitor ML364 significantly increased the sensitivity of liver cancer cells to the ferroptosis inducer RSL3 ( Figure 2 AB).

[0031] In addition, to test the effect of ferroptosis inhibitor Fer-1 on this process, 10 4 Huh7 cells ( Figure 2 C) or SNU-449 cells ( Figure 2 D), four replicate wells were set up in each group. After the cells adhered, ML364 (0, 4 μM) at the corresponding concentrations shown in the figure was added, and 2 μM (for Huh7) or 1 μM (for SNU-449) of RSL3 and 1 μM of the ferroptosis inhibitor Fer-1 were added for 24 hours. The effect of the ferroptosis inhibitor Fer-1 on the sensitivity of ML364 to RSL3 was detected by CCK-8. The results showed that the process by which the USP2 inhibitor ML364 increased the sensitivity of liver cancer cells to the ferroptosis inducer RSL3 could be significantly blocked by the ferroptosis inhibitor Fer-1 ( Figure 2 CD), the above results indicate that ML364 can induce ferroptosis in liver cancer cells.

[0032] At the same time, in order to further verify that ML364 can induce ferroptosis in liver cancer cells, Huh7 cells or SNU-449 cells were treated with ML364 (0, 5, 10 μM) at the corresponding concentrations shown in the figure for 12 hours, and then the cells were collected. The changes in the levels of glutathione (GSH), lipid peroxidation product malondialdehyde (MDA), and reactive oxygen species (ROS) in the cells after treatment with the corresponding concentrations of ML364 were detected using the corresponding kits ( Figure 2 EG). The results showed that ML364 could dose-dependently reduce the GSH content in liver cancer cells ( Figure 2 E), and increased MDA ( Figure 2 F) and ROS( Figure 2 G) content, further confirming that ML364 can induce ferroptosis in liver cancer cells.

[0033] In addition, to detect the effect of ML364 on the growth of liver cancer cells, 2 × 10 3 SNU-449 cells ( Figure 2 H) and Huh7 cells ( Figure 2 I), four replicate wells were set up in each group, and 2μM and 4μM ML364 were added after the cells adhered to the wall. The OD values at a wavelength of 450nm were detected using CCK-8 at 0, 24, 48, 72 and 96 hours. The results showed that ML364 can also inhibit the growth of liver cancer cells in a dose-dependent manner ( Figure 2 To further explore its regulatory mechanism, when the HepG2 cell density reached 80%, ML364 (0, 5, 10 μM) at the corresponding concentrations shown in the figure was added and treated for 12 hours. The cells were then collected and immunoblotting was used to detect changes in the protein levels of USP2 substrates DCAF7 and BMAL1, as well as HIF1α and SLC7A11 downstream of BMAL1. The results showed that ML364 affected liver cancer cell ferroptosis by downregulating the DCAF7-BMAL1-HIF1α-SLC7A11 pathway proteins ( Figure 2 J).

[0034] Example 3: USP2 inhibitor ML364 induces ferroptosis at the cellular level and increases sorafenib sensitivity

[0035] Inducing ferroptosis is an effective way to increase the sensitivity of liver cancer cells to sorafenib. The results of Example 2 show that ML364 can inhibit the growth of liver cancer cells by significantly inducing ferroptosis in liver cancer cells. Therefore, it is suggested that ML364 is a potential small molecule inhibitor that increases the sensitivity of liver cancer cells to sorafenib. In order to detect the effect of ML364 on the sensitivity of liver cancer cells to sorafenib, 10 4 Huh7 cells ( Figure 3 A) or SNU-449 cells ( Figure 3 B) Four replicate wells were set up in each group. After the cells adhered, ML364 (0, 2, 4 μM) and 20 μM (for Huh7) or 10 μM (for SNU-449) sorafenib were added at the corresponding concentrations shown in the figure and treated for 24 hours. The effect of ML364 treatment on the sensitivity of liver cancer cells to sorafenib was detected using CCK-8. Indeed, the inventors further found that ML364 can significantly increase the sensitivity of liver cancer cells to sorafenib ( Figure 3 AB). In addition, to explore whether ML364 affects the sensitivity of liver cancer cells to sorafenib by affecting ferroptosis, 10 4 Huh7 cells ( Figure 3 C) or SNU-449 cells ( Figure 3 D), four replicate wells were set up in each group. After the cells adhered, ML364 (0, 2, 4 μM) at the corresponding concentrations shown in the figure was added. At the same time, 20 μM (for Huh7) or 10 μM (for SNU-449) sorafenib and 1 μM Fer-1 were added for 24 hours. The effect of the ferroptosis inhibitor Fer-1 on the ML364-induced sorafenib sensitivity of liver cancer cells was detected using CCK-8. The results showed that the process by which the USP2 inhibitor ML364 increased the sensitivity of liver cancer cells to sorafenib could be significantly blocked by the ferroptosis inhibitor Fer-1 ( Figure 3 CD), indicating that ML364 increases the sensitivity of HCC cells to sorafenib mainly by inducing ferroptosis.

[0036] In addition, to examine the effect of the combination of ML364 and sorafenib on the growth of liver cancer cells, 2 × 10 3 Huh7 cells ( Figure 3 E) and SMMC-7721 cells ( Figure 3 F), four replicate wells were set up in each group, and after the cells adhered, 0, 2, and 4 μM ML364 and 0, 2, and 4 μM sorafenib were added for treatment alone or in combination, and the OD value at a wavelength of 450 nm was detected by CCK-8 at 0, 24, 48, 72, and 96 hours. The results showed that at the same active ingredient concentration (4 μM), the combination of ML364 and sorafenib (ML364-2 μM + sorafenib-2 μM) significantly increased the inhibitory effect of sorafenib on the growth of liver cancer cells (Huh-7, SMMC-7721) compared with the administration of sorafenib alone (4 μM) ( Figure 3 EF).

[0037] This example demonstrates that the USP2 inhibitor ML364 induces ferroptosis at the cellular level and increases sorafenib sensitivity. Furthermore, the combination of ML364 and sorafenib is more effective than sorafenib alone, better inhibiting the growth of liver cancer cells and exhibiting a synergistic effect. Furthermore, this example demonstrates that ML364 itself also has the effect of inhibiting the growth of liver cancer cells.

[0038] Example 4: USP2 inhibitor ML364 induces ferroptosis in mice and increases sorafenib sensitivity

[0039] 1×10 7 Huh7 cells were resuspended in 100 μL PBS and injected subcutaneously into the base of the right hind limb of nude mice. After successful tumor implantation, the length (L) and width (W) of the tumor were measured using a vernier caliper to calculate the tumor volume (V = L × W 2 / 2). When the tumor volume reaches about 100-200mm 3 Mice were randomly divided into 4 groups (n=6): control group (control, oral administration control group: 2% DMSO, 40% PEG300, 5% Tween-80, 53% Saline; intraperitoneal injection control group: 0.5% sodium carboxymethylcellulose solution); sorafenib alone group (Sora, 20 mg / kg); ML364 alone group (ML364, 30 mg / kg); and combination group (Sora (20 mg / kg) + ML364 (30 mg / kg)). Sorafenib was administered orally at a dose of 20 mg / kg, while the control group was administered orally with a corresponding volume of control solvent (2% DMSO, 40% PEG300, 5% Tween-80, 53% Saline); ML364 was administered orally at a dose of 30 mg / kg, while the control group was administered orally with a corresponding volume of 0.5% sodium carboxymethylcellulose solution. The drug was administered once every other day. The weight of the mice and the length and width of the tumor were weighed before each administration to calculate the tumor volume. After 7 doses, the nude mice were anesthetized with isoflurane and then killed by cervical dislocation (to ensure that the tumor volume did not exceed 2000 mm 3 ) and took pictures. Then the tumor tissue was peeled off, weighed and photographed ( Figure 4 A).

[0040] Depend on Figure 4 The results showed that ML364 and sorafenib alone could inhibit the volume and weight of tumors in mice and increase the MDA content in tumor tissues, while the combination of ML364 and sorafenib could significantly enhance this effect. Figure 4 BE). On the other hand, ML364 and sorafenib administered alone or in combination had no significant effect on the body weight of mice ( Figure 4F), blood routine and blood biochemistry results also showed that combined administration had no significant effect on related indicators ( Figure 4 G), indicating that the combined administration regimen has significant tumor inhibition effect and good tolerability.

[0041] This example demonstrates that the USP2 inhibitor ML364 induces ferroptosis in mice and increases sorafenib sensitivity. The combination of ML364 and sorafenib can have a more significant tumor inhibitory effect and good tolerability.

[0042] The above description shows that the combination of USP2 inhibitor ML364 and sorafenib has broad application prospects in the preparation of drugs for the treatment of liver cancer.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the deubiquitinating enzyme USP2 inhibitor ML364 and sorafenib.

2. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of ML364 to sorafenib in the pharmaceutical composition is 0.8:1 to 1.5:

1.

3. Use of the pharmaceutical composition according to claim 1 in preparing a medicament for treating liver cancer.

4. Application of deubiquitinating enzyme USP2 inhibitor ML364 in the preparation of ferroptosis inducers.

5. Application of deubiquitinase USP2 inhibitor ML364 in the preparation of anti-liver cancer drug sensitizers.

6. The use according to claim 5, characterized in that The anti-liver cancer drug is sorafenib.

7. Application of the deubiquitinating enzyme USP2 inhibitor ML364 in the preparation of drugs for the treatment of liver cancer.

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