Pharmaceutical composition and pharmaceutical use of jnk inhibitor and parg inhibitor in combination

Abstract

The application discloses a medicine composition and pharmaceutical use of a combination of a JNK inhibitor and a PARG inhibitor. The application discloses for the first time that the JNK inhibitor (such as BIRB 796) and the PARG inhibitor (such as PDD00017273) can improve the synergistic effect of treating gastric cancer, the two inhibitors have a synergistic effect when used in combination, have a synergistic effect, realize a significant improvement of an anti-tumor effect, and do not increase the toxicity to various organs by the combination, but rather reduce the dosage of various drugs, thereby reducing the side effects, and have a significant significance for the application of the combination of the PARG inhibitor and the JNK inhibitor to the treatment of gastric cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for the combined use of JNK inhibitors and PARG inhibitors and its pharmaceutical applications. Background Technology

[0002] Gastric cancer (GC) ranks fifth in both incidence and mortality worldwide. Treatment options include surgery, chemotherapy, immunotherapy, and targeted therapy. In recent years, targeted therapy has not only transformed the treatment landscape for gastric cancer but has also become a crucial research direction in the treatment of solid tumors. Its core lies in achieving efficient recognition and elimination of tumor cells through targeted molecules. However, similar to many other cancers, existing single-target therapies are prone to developing resistance in gastric cancer treatment. Furthermore, due to the high heterogeneity of tumors, the beneficiary population for a single targeted drug is relatively limited, leading to poor efficacy or eventual disease progression in some patients. Therefore, exploring multi-target combination therapies to overcome drug resistance, enhance anti-tumor targeting effects, and expand the beneficiary population has gradually become a vital research direction in the treatment of gastric cancer and even pan-cancer therapies.

[0003] Synthetic lethality is the phenomenon where the simultaneous inactivation of two genes (A and B) leads to cell death, while the cell can survive if either gene is inactivated alone. When tumor cells carry BRCA1 / 2 mutations (homologous recombination repair deficiency, HRD), they rely on poly(ADP-ribose) polymerase (PARP)-mediated base excision repair (BER) to maintain DNA stability. Using PARP inhibitors can specifically kill BRCA1 / 2 mutant tumor cells.

[0004] Poly(ADP-ribose) glycohydrolase (PARG) and PARP dynamically control the synthesis and degradation of ADP-ribose polymers (PAR chains), maintaining genome stability. PARG inactivation leads to PAR chain deposition, retention of repair proteins on DNA, impaired DNA replication, and even DNA double-strand breaks. Currently, research on PARG inhibitors has entered the clinical translation stage, with several PARG inhibitors already in clinical trials, such as IDE-161, ETX-19477, and DAT-2645. Although PARP inhibitors are already used in clinical treatment, drug resistance is common. The tumor cell killing induced by PARG inhibitors not only broadens the therapeutic window of PARP inhibitors but also provides insights into combination therapies with other treatments.

[0005] JNK (c-Jun N-terminal kinase) is a member of the mitogen-activated protein kinase (MAPK) family, playing a crucial regulatory role in cellular stress responses, apoptosis, inflammation, and proliferation. In tumors, JNK signaling is often abnormally activated, participating in maintaining tumor cell survival and drug resistance. JNK inhibitors, as a class of compounds targeting c-Jun N-terminal kinases, have seen significant progress in the development of drugs for oncology, inflammation, and neurodegenerative diseases. In cancer treatment, the JNK pathway is an important target due to its central role in stress response, apoptosis regulation, and chemotherapy resistance. Although no JNK inhibitors have yet been approved for use alone in gastric cancer, numerous preclinical studies have shown that they can significantly enhance the anti-tumor effects of various chemotherapeutic drugs (such as platinum-based and benzalkonium chloride) and targeted therapies, particularly in inducing apoptosis and overcoming drug resistance. In terms of clinical research, several JNK inhibitors have entered the clinical trial stage, such as AS602801 and CC-930. Although these studies are not focused on gastric cancer, they have confirmed that JNK inhibitors are tolerable and have preliminary activity in humans, providing important clinical evidence and safety data for their expansion into combination therapy for gastric cancer. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a pharmaceutical composition for the combined use of JNK inhibitors and PARG inhibitors and their pharmaceutical applications.

[0007] CRISPR screening based on synthetic lethal effects (using the GECKO V2 whole-genome knockout library) revealed that among the significant targets for combined lethality with PARG inhibitors, MEK4, MEK7, and MAPK8, which are key components of the MEK4 / 7-JNK1 signaling pathway, are among the top targets. This finding not only suggests mechanistically that the JNK pathway is a key breakthrough in enhancing the efficacy of PARG inhibitors, but also directly points to JNK inhibitors as a highly promising clinical combination therapy option.

[0008] The present invention first provides a pharmaceutical composition for the combined use of JNK inhibitors and PARG inhibitors, the pharmaceutical composition being used for anti-tumor treatment, wherein the tumor type is gastric cancer, lung cancer, colon cancer, liver cancer, pancreatic cancer, breast cancer, or esophageal cancer.

[0009] Preferably, the JNK inhibitor is at least one of BSJ-04-122, DB07268, and benzomarine.

[0010] Preferably, the PARG inhibitor is at least one of IDE-161, ETX-19477, DAT-2645, and PDD00017273.

[0011] PARG inhibitor PDD00017273, chemical formula C 23 H 26 N6O4S2, CAS Registry Number 1945950-21-9, structural formula is shown in Equation 1:

[0012] Formula 1.

[0013] The JNK inhibitor bentamapimod (AS 602801), with the chemical formula C 25 H 23 N5O2S, CAS Registry Number 848344-36-5, has the structure shown in Equation 2:

[0014] Equation 2.

[0015] MKK4 / 7 dual inhibitor BSJ-04-122, chemical formula C 15 H 12 ClN5O, CAS Registry Number 2513289-74-0, structural formula is shown in Equation 3:

[0016] Formula 3.

[0017] JNK1 inhibitor DB07268, chemical formula C 17 H 15 N5O2, CAS Registry Number 929007-72-7, structural formula is shown in Equation 4:

[0018] Formula 4.

[0019] Preferably, the concentration of BSJ-04-122 is not less than 2 μM, the concentration of DB07268 is not less than 0.2 μM, and the concentration of benzodiazepines is not less than 10 μM. The concentration of PARG inhibitors is not less than 10 μM.

[0020] The present invention further provides the pharmaceutical use of a combination of JNK inhibitors and PARG inhibitors, wherein the pharmaceutical composition is used to prepare an antitumor drug, wherein the tumor type is gastric cancer, lung cancer, colon cancer, liver cancer, pancreatic cancer, breast cancer, or esophageal cancer.

[0021] Preferably, the JNK inhibitor is at least one of BSJ-04-122, DB07268, and benzomarine.

[0022] Preferably, the PARG inhibitor is at least one of IDE-161, ETX-19477, DAT-2645, and PDD00017273.

[0023] Preferably, the administration route of the combined drug composition is at least one of intratumoral injection, peritumoral injection, intravenous injection, and oral administration.

[0024] Preferably, the concentration of BSJ-04-122 is not less than 2 μM, the concentration of DB07268 is not less than 0.2 μM, and the concentration of benzodiazepines is not less than 10 μM. The concentration of PARG inhibitors is not less than 10 μM.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time the discovery of the synergistic effect of JNK inhibitors (such as benzomarmo) and PARG inhibitors (such as PDD00017273) in improving the efficacy of gastric cancer treatment. The two inhibitors have a synergistic effect when used in combination, resulting in a significant improvement in anti-tumor efficacy. Moreover, the combination of drugs does not increase the toxicity to various organs, but rather reduces the dosage of various drugs, thereby reducing toxic side effects. This has significant implications for the application of PARG inhibitors and JNK inhibitors in the treatment of gastric cancer. Attached Figure Description

[0026] Figure 1 Screening for synthetic lethal drugs with PARG deficiency for JNK inhibitors.

[0027] Figure 2 To verify the synthetic lethality of PARG inhibitors and JNK inhibitors in gastric cancer cell lines through in vitro morphological experiments. Figure 2 In the figure, A represents the effect of PARG inhibitor combined with different concentrations of DB07268 on the cell confluence of AGS gastric cancer cells. The scale bar length in the figure is 50µm. Figure 2 In the figure, B represents the result of the lethality test of the synthesis of PARG inhibitor and BSJ-04-122; Figure 2 In this context, C represents the result of the synthetic lethality test of PARG inhibitors combined with DB07268. Figure 2 D in the figure represents the lethality test result of the synthesis of PARG inhibitors and benzalkonium chloride.

[0028] Figure 3 The combination of PARG inhibitors and JNK inhibitor bentamamo significantly inhibited the in vivo growth of HGC27 cell line-derived xenograft tumors. Figure 3 In the image, A represents a tumor photograph; Figure 3 In this context, B represents the tumor weight statistics. Figure 3 In the figure, C represents the tumor volume statistics.

[0029] Figure 4 The combination of PARG inhibitors and JNK inhibitor bentamamo significantly inhibited tumor growth in patient-derived xenografts. Figure 4 In the image, A represents a tumor photograph; Figure 4 In this context, B represents the tumor weight statistics. Figure 4 In the figure, C represents the tumor volume statistics.

[0030] Figure 5 This is the result of immunohistochemical detection. Among them, Figure 5 In the image, A represents an immunohistochemical image. Figure 5 B in the figure represents the statistical result of the relative positivity rate of Ki67; Figure 5 C in the figure represents the statistical result of apoptosis rate.

[0031] Figure 6 H&E staining was performed to evaluate the toxic effects of the PARG inhibitor combined with the JNK inhibitor benzalkonium chloride on organs in nude mice. Figure 6 In this context, A represents the CDX model; Figure 6 In this context, B represents the PDX model.

[0032] Figure 7 To evaluate the efficacy of PARG inhibitors combined with bentammamo in different tumors by assessing cell survival rates after combination therapy. Figure 7 In this context, A represents A549 cells; Figure 7 B in the text represents HCT116 cells; Figure 7 C in the text represents HepG2 cells; Figure 7 D in the text represents KYSE150 cells; Figure 7 E in the text refers to MDA-MB-231 cells; Figure 7 F in the text represents PANC-1 cells. Detailed Implementation

[0033] Example 1: Screening of JNK Inhibitors

[0034] This invention aims to reveal key genes that confer sensitivity or resistance to PARG inhibitors by performing genome-wide CRISPR knockout screening under PARG inhibitor treatment conditions, and to establish predictive biomarkers for patient stratification.

[0035] Cell culture was performed first: human cell lines AGS (CRL-1739), HEK293T (CRL-3216), and HGC27 (TCHu22) were cultured in DMEM medium supplemented with 10% fetal bovine serum. The cells were then incubated in a humidified incubator at 37°C with 5% CO2.

[0036] Subsequently, a genome-wide CRISPR knockout screening based on synthetic lethality was conducted to identify key target genes that significantly reduced cell survival when treated with PARG inhibitors. The screening used a GECKO v2 genome-wide knockout library containing multiple sgRNA sequences covering the human genome. AGS cells in logarithmic growth phase were infected with lentivirus at an appropriate multiplicity of infection (MOI ≤ 0.3) to ensure that single-cell integration was predominant. After infection, puromycin was used for selection to obtain a stable Cas9 / sgRNA-expressing cell population. To ensure screening coverage, effective coverage of library complexity was maintained (preferably ≥ 300 × coverage), and the cell count was kept at least as high as the coverage requirement throughout the screening process.

[0037] After the cell library was constructed, the cells were divided into a control group and a treatment group. The control group was treated with an equal volume of solvent, while the treatment group was continuously cultured with a predetermined concentration of PARG inhibitor. Multiple passages were performed under conditions where the treatment pressure was sufficient to produce a selection effect without causing instantaneous population collapse. Cells were collected at the start of selection (T0) and the end of treatment (Tf), genomic DNA was extracted, sgRNA barcode regions were amplified, and high-throughput sequencing was performed. Statistical analysis of the abundance changes of each sgRNA in the treatment group relative to the control group (e.g., using MAGeCK or an equivalent enrichment / depletion algorithm) was conducted to obtain sgRNAs that were significantly depleted under PARG inhibitor pressure and their corresponding gene sets. These significantly depleted genes were defined as candidate targets that exhibit synthetic lethality or sensitization effects with PARG inhibitors.

[0038] The screening results showed that among the significant targets that could cause death when combined with PARG inhibitors, the top-ranking targets included MEK4 (also known as MAP2K4), MEK7 (also known as MAP2K7), and MAPK8 (also known as JNK1). Figure 1 The aforementioned genes are all key components of the MEK4 / 7-JNK1 signaling pathway. Based on this, this invention proposes that inhibiting the MEK4 / 7-JNK1 axis can produce synergistic cytotoxic effects in the context of PARG inhibitors, thereby constituting a druggable combination therapy strategy and providing a detectable combination of predictive biomarkers for patient stratification (e.g., expression / mutation / copy number alterations of MEK4, MEK7, and MAPK8, or readouts of their downstream signaling activities).

[0039] To validate the screening findings and achieve pharmacological translation, this invention further selects inhibitors targeting this pathway for in vitro synergistic validation: the preferred inhibitors are MEK4 / 7 inhibitor BSJ-04-122, JNK1 broad-spectrum inhibitor DB07268, and JNK inhibitor Bentamapimod.

[0040] In the further screening of candidate drugs, this invention comprehensively considers the strength of pharmacodynamic synergy, the reproducibility of cell models, and the translational potential reflected in publicly available clinical research data. Ultimately, Bentamapimod was selected as the preferred JNK pathway inhibitor for in vivo experiments, to be used for efficacy and safety evaluation in subsequent animal models when administered in combination with PARG inhibitors.

[0041] Example 2: Preparation of stock solutions of PDD00017273, BSJ-04-122, DB07268, and Bentamapimod

[0042] Accurately weigh appropriate amounts of PDD00017273 (PARG inhibitor, purchased from MCE, HY-108360), BSJ-04-122 (MEK4 / 7 inhibitor, purchased from MCE), DB07268 (JNK1 broad-spectrum inhibitor, purchased from MCE, HY-15737), and Bentamapimod (JNK inhibitor, purchased from MCE). Using DMSO as the solvent, prepare stock solutions with concentrations of 10 mM (5.15 mg / mL), 10 mM (3.14 mg / mL), 10 mM (3.21 mg / mL), and 10 mM (4.58 mg / mL), respectively. Aliquot into 1.5 mL EP tubes, seal with sealing film, and store at -20°C.

[0043] Example 3: Verification by in vivo morphological experiment

[0044] (1) Cell confluence analysis: To verify the effect of combined treatment with PARG inhibitor (PDD00017273) and JNK1 inhibitor (DB07268) on AGS gastric cancer cells, this experiment evaluated the inhibitory effect of the combined effect on cell proliferation through cell confluence analysis. First, AGS gastric cancer cells were seeded in 24-well cell culture plates and cultured at 37°C and 5% CO2 for 24 h until the cell confluence reached approximately 30%-40%. Then, different concentrations (0, 1, 5, 10, 25, 50 μM) of JNK1 inhibitor solutions were prepared, and 10 μM of PARG inhibitor was added simultaneously for combined treatment. After replacing the original culture medium with the corresponding drug, the cells were incubated for another 24 h. After treatment, the cells in each group were observed and photographed using an inverted microscope for subsequent analysis of cell confluence and proliferation inhibition.

[0045] The results showed that, compared with the DMSO control, the combined treatment with the PARG inhibitor (PDD00017273) and the JNK1 inhibitor (DB07268) significantly reduced cell confluence and increased inhibition rate. Figure 2 In the figure, A represents a scale bar with a length of 50µm.

[0046] (2) Clonal formation: AGS gastric cancer cells were divided into approximately 10 3 Cells were seeded per well in 6-well cell culture plates and cultured at 37°C and 5% CO2 for 12–24 h until cell attachment. Drug treatment was then initiated. Cells received the following combined treatments: 0.2 μM MEK4 / 7 inhibitor (BSJ-04-122) combined with 0.20 μM PARG inhibitor (PDD00017273); 0.2 μM JNK1 inhibitor (DB07268) combined with 0.10 μM PARG inhibitor (PDD00017273); and 0.10 μM JNK inhibitor (bentamamo) combined with 0.20 μM PARG inhibitor (PDD00017273). Cells were cultured for another 12 days after drug treatment, with the medium replaced with fresh medium containing the same concentration of drug as needed. After culture, colony formation, including colony number and morphological characteristics, was observed and recorded using an inverted microscope in each treatment group. The culture medium was then discarded, and the cells were washed twice with 1 mL of PBS buffer per well. Next, 1 mL of paraformaldehyde solution was added to each well for fixation at room temperature for 15 min. After fixation, the cells were washed twice more with PBS. Then, 1 mL of 0.1% crystal violet staining solution was added to each well, and staining was performed at room temperature for 30 min. After staining, the cells were washed twice with PBS to remove excess stain. Finally, the 6-well plate was placed in a well-ventilated and dry place to air dry naturally, and the cell clones were imaged and recorded using a scanner for subsequent quantitative analysis.

[0047] The results showed that the combination therapy group exhibited stronger inhibitory effects on proliferation in the AGS cell line compared to the single therapy group. Figure 2 Specifically, in the clonogenic assay, BSJ-04-122 monotherapy (2 μM) showed an inhibition rate of 27.04%, PARG inhibitor monotherapy (20 μM) showed an inhibition rate of 17.61%, and the combined therapy showed an inhibition rate of 61.0%; DB07268 monotherapy (0.2 μM) showed an inhibition rate of 34.48%, PARG inhibitor monotherapy (10 μM) showed an inhibition rate of 47.29%, and the combined therapy showed an inhibition rate of 85.71%; Bentamamo monotherapy (10 μM) showed an inhibition rate of 39.41%, PARG inhibitor monotherapy (20 μM) showed an inhibition rate of 42.94%, and the combined therapy showed an inhibition rate of 98.24%.

[0048] In summary, these results confirm that JNK inhibitors (bentamamamo, BSJ-04-122, or DB07268) and PARG inhibitors have a synergistic effect in inhibiting the proliferation of gastric cancer cells.

[0049] Example 4: Subcutaneous tumor formation in nude mice

[0050] For the CDX model, specific tumor cells in the logarithmic growth phase were collected by trypsin digestion, centrifuged, resuspended in sterile PBS, and counted. The cell density was adjusted to 1 × 10⁻⁶ cells per milliliter. 7 Each tumor was diluted 1:1 with matrix gel and then inoculated into four 4-week-old male BALB / c nude mice. 200 μL of the mixture was injected subcutaneously into the right axilla of each mouse. When the long diameter of the tumor exceeded 1 cm, the mice were euthanized by cervical dislocation. The tumor was harvested and aseptically cut into small pieces of approximately 1 cubic millimeter in volume, placed in PBS, and then inoculated subcutaneously into 24 experimental nude mice using a tumor grafting needle. For the PDX model, well-grown passaged tumors (derived from gastric cancer patients at Zhejiang Cancer Hospital, ethics approval number IIT-2025-247) were harvested, aseptically cut into small pieces of approximately 1 cubic millimeter in volume, placed in sterile PBS to maintain viability, and then transplanted subcutaneously into the ipsilateral back of each nude mouse using a tumor grafting needle. All nude mice were housed in an SPF-grade environment with constant temperature and humidity, and their general condition was observed daily. When the tumor volume reached approximately 80 cubic millimeters, the tumor-bearing mice were randomly divided into four groups (n=6 per group): a control group, a betamamapimod monotherapy group (Bentamapimod, 50 mg / kg, gavage, every 2 days), a PDD0017273 monotherapy group (PDD, 36 mg / kg, intraperitoneal injection, every 2 days), and a combination therapy group. The drug intervention lasted approximately 16 days, while the control group received an equal volume of saline. During the drug administration period, the long and short diameters of the tumor were measured every two days using electronic calipers, and the results were calculated according to the formula (length × width). 2 The approximate tumor volume was calculated by dividing the tumor size by 2, and the weight of the nude mice was recorded to assess the potential toxicity of the drug. At the end of the experiment, the nude mice were euthanized by cervical dislocation, the tumor tissue was completely dissected, weighed, and photographed. The tumor tissue was then divided into multiple portions. One portion was immediately flash-frozen in liquid nitrogen and then transferred to a -80°C freezer for storage for subsequent molecular biological analysis; another portion was fixed in 10% neutral formalin solution for paraffin embedding and histopathological analysis.

[0051] The results showed that after 16 days of treatment, compared with monotherapy, the combination therapy group had a more significant reduction in tumor growth rate, volume, and weight. Figure 3 , Figure 4 ).

[0052] In the CDX model, the mean tumor volume in the untreated group was 1314.04 mm. 3 The mean tumor volume in the benzamamo monotherapy group was 991.508 mm. 3 The mean tumor volume in the PDD0017273 monotherapy group was 760.875 mm. 3 The mean tumor volume in the PARG inhibitor combined with benzalkonium chloride treatment group was 491.166 mm.3 The mean tumor weight was 1.610 g in the untreated group, 0.9733 g in the benzamamo monotherapy group, 0.7233 g in the PDD0017273 monotherapy group, and 0.2025 g in the PARG inhibitor combined with benzamamo therapy group.

[0053] In the PDX model, the mean tumor volume in the untreated group was 1072.27 mm. 3 The mean tumor volume in the benzamamo monotherapy group was 854.49 mm. 3 The mean tumor volume in the PDD0017273 monotherapy group was 695.823 mm. 3 The mean tumor volume in the PARG inhibitor combined with benzalkonium chloride treatment group was 271.063 mm. 3 The mean tumor weight was 0.9475 g in the untreated group, 0.8188 g in the benzamamo monotherapy group, 0.6313 g in the PDD0017273 monotherapy group, and 0.3300 g in the PARG inhibitor combined with benzamamo therapy group.

[0054] These results indicate that the combination of PARG inhibitors and bentamamoxifen not only exhibits basic antitumor activity in CDX models, but also achieves consistent and significant inhibitory effects in PDX models that better mimic clinical tumor characteristics. This together suggests that the combined use of these two drugs has clear development prospects and clinical translational value.

[0055] Example 5: Immunohistochemistry

[0056] To further investigate the mechanism of action of the drug on tumor growth in vivo, we performed immunohistochemical analysis on transplanted tumor tissues. Tumor blocks (from PDX model nude mice in Example 4) were stained with Ki67, TUNEL, and H&E staining, and the expression of Ki67 and TUNEL staining was analyzed. Paraffin sections were dewaxed, hydrated, and subjected to heat-induced antigen retrieval using sodium citrate buffer (pH 6.0), followed by blocking endogenous peroxidase activity in 3% hydrogen peroxide. After blocking with 5% bovine serum albumin at room temperature for 30 minutes, the sections underwent specific incubation: for Ki67 staining, rabbit anti-Ki67 primary antibody working solution diluted 1:300 was added and incubated overnight at 4°C; for TUNEL staining, terminal deoxynucleotidyl transferase (TdT) was reacted with fluorescein-labeled dUTP at 37°C for 1 hour, followed by incubation at room temperature for 30 minutes with horseradish peroxidase-labeled antifluorescein antibody. After overnight incubation at 4°C, Ki67 sections were washed with PBS and incubated with horseradish peroxidase polymerase secondary antibody of the corresponding species for 1 hour at room temperature. All sections were developed with freshly prepared diaminobenzidine substrate solution for approximately 3 minutes, with the degree of development controlled under a microscope. Subsequently, the cell nuclei were counterstained with hematoxylin, dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and observed and images acquired under an optical microscope.

[0057] The results showed that Ki67 expression was significantly inhibited after combination therapy, while Tunel expression was significantly increased. Figure 5 Quantitative analysis showed that, compared with the control group (Ki67 positivity rate 92.40%), the cell proliferation activity (Ki67 positivity rate) in the benzodiazepine monotherapy group, the PARG inhibitor monotherapy group, and the combination therapy group decreased to 38.76%, 37.43%, and 10.86%, respectively, with corresponding proliferation inhibition rates of 58.06%, 59.49%, and 88.25%. Simultaneously, in terms of apoptosis induction, compared with the control group (Tunel positivity rate 0.10%), the proportion of apoptotic cells in the above three groups increased to 3.37%, 4.63%, and 16.20%, respectively, approximately 34.34 times, 47.20 times, and 165.14 times that of the control group. These results, from both inhibiting proliferation and promoting apoptosis, collectively demonstrate that the combination therapy regimen significantly inhibits tumor growth in vivo.

[0058] Example 6: Toxicity Impact Assessment

[0059] To evaluate the toxic effects of combining PARG inhibitors with benzodiazepines in nude mice, the changes in mouse weight after drug treatment were further investigated. During drug treatment, the weight of the mice was measured and recorded every two days. After data processing, a line graph of weight changes in the mice was plotted using Graphpad.

[0060] The results show that ( Figure 6In the CDX and PDX models, compared to the control group, there were no significant changes in body weight in the benzodiazepine (PARG) group, the PDD group, and the combination therapy group. Furthermore, no mice in any group died during the experiment. Therefore, all the above experimental results indicate that the combined use of PARG inhibitors and benzodiazepine has no significant toxic effects on nude mice.

[0061] Example 7: Evaluation of the efficacy of combination therapy in different tumors

[0062] In this embodiment, the following groups were set up: Ctrl group (control group, 1‰ dimethyl sulfoxide), PARGi group (PARG inhibitor group, plus PDD00017273), bentamapimod group, and Combination group (combined treatment group). A549 cells (lung cancer cell line), HCT116 cells (human colon cancer cells), HepG2 cells (human liver cancer cell line), PANC-1 cells (human pancreatic cancer cell line), MDA-MB-231 cells (human breast cancer cell line), and KYSE150 cells (human esophageal cancer cell line) were treated with the corresponding drugs, respectively.

[0063] After digesting the target cells into a cell suspension, cell counts were performed, and 3 × 10⁶ cells were collected. 3 A549, HCT116, HepG2, PANC-1, KYSE150, and MDA-MB-231 cells were seeded at 100 μL per well in 96-well plates. After 24 h of culture, the culture medium was discarded. Bentamapimod concentrations of 25, 20, 15, 10, 5, and 0 μM were added sequentially to rows 2–7 of the 96-well plates, and PARGi concentrations of 25, 12.5, 6.3, 3.1, 1.6, 0.8, and 0 μM were added sequentially to columns 2–8 of the 96-well plates. A control group (no drugs, only cells) and a blank group (no drugs, no cells) were also set up, with three replicates for each concentration. The plates were incubated at 37°C with 5% CO2 for 48 h. The culture medium in the 96-well plates was then discarded, and fresh medium containing 10% CCK-8 was added to each well, followed by incubation with cells for another 0.5–4 h. After incubation, the absorbance of each well at 450 nm was measured using a microplate reader. Cell viability was calculated using the following equation: Viability = (OD experimental group - OD blank group) / (OD control group - OD blank group) × 100%. GraphPadPrism 9 software was used to process the experimental data and calculate cell viability.

[0064] The results show that ( Figure 7The combined drug group exhibited the strongest growth inhibition effect in all tested cancer cell lines, with significantly lower cell survival rates than the single-drug groups and the control group. Further calculation of the inhibition rate (based on control group cell viability, inhibition rate = 100% - survival rate) showed that the combined drug group exhibited the strongest inhibitory effect in all tested cell lines. Specifically, the inhibition rates of the PARGi group, bentamimod group, and combination group were 23.50%, 19.04%, and 49.45% in A549 cells, respectively; 26.82%, 20.78%, and 61.29% in HCT116 cells; 16.66%, 14.75%, and 44.38% in HepG2 cells; 17.11%, 18.72%, and 54.03% in KYSE150 cells; 14.22%, 12.02%, and 39.22% in MDA-MB-231 cells; and 15.54%, 14.39%, and 50.77% in PANC-1 cells. In all six cell lines, the inhibition rate of the combination therapy group was significantly higher than that of any single-drug group. This indicates that the combination of PARG inhibitors and benzamamo has significant efficacy in various cancers, including lung cancer, colon cancer, liver cancer, pancreatic cancer, breast cancer, and esophageal cancer.

Claims

1. A pharmaceutical composition for the combined use of a JNK inhibitor and a PARG inhibitor, wherein the pharmaceutical composition is used for antitumor treatment, the tumor type being gastric cancer, and the PARG inhibitor is PDD00017273. When the JNK inhibitor is benzomarine, the concentration of benzomarine is 10 μM and the concentration of PDD00017273 is 20 μM. When the JNK inhibitor is BSJ-04-122, the concentration of BSJ-04-122 is 2 μM and the concentration of PDD00017273 is 20 μM; When the JNK inhibitor is DB07268, the concentration of DB07268 is 0.2 μM and the concentration of PDD00017273 is 10 μM.

2. Pharmaceutical use of a combination of a JNK inhibitor and a PARG inhibitor, wherein the pharmaceutical composition is used to prepare an antitumor drug, the tumor type being gastric cancer, and the PARG inhibitor being PDD00017273. When the JNK inhibitor is benzomarine, the concentration of benzomarine is 10 μM and the concentration of PDD00017273 is 20 μM. When the JNK inhibitor is BSJ-04-122, the concentration of BSJ-04-122 is 2 μM and the concentration of PDD00017273 is 20 μM; When the JNK inhibitor is DB07268, the concentration of DB07268 is 0.2 μM and the concentration of PDD00017273 is 10 μM.

3. The pharmaceutical use according to claim 2, characterized in that, The combined drug composition can be administered via at least one of the following methods: intratumoral injection, peritumoral injection, intravenous injection, and oral administration.

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