Use of cenicriviroc in the treatment of cancer

Abstract

The application provides a drug use of a TGS1 inhibitor alone or in combination with a PARPi for treating pancreatic cancer, and particularly relates to the field of biological medicine technology. The application first proposes a new medical use of the TGS1 inhibitor, sinaphenidine, and proves through in-vitro and in-vivo experiments that sinaphenidine alone, or in combination with a PARPi or a chemotherapeutic drug, can be used for treating pancreatic cancer and can be used as a radiotherapy drug sensitizer. The application applies sinaphenidine to the preparation of a drug for preventing and treating pancreatic cancer, develops a new clinical use of sinaphenidine, and has the characteristics of biological pleiotropism, high safety, small toxic and side effects and the like, and is expected to become a new effective means for treating pancreatic cancer and has important conversion significance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of sinefenoxan in the treatment of cancer. Background Technology

[0002] Pancreatic adenocarcinoma (PAAD) is a highly lethal and aggressive solid malignant tumor. Most patients are diagnosed at an advanced stage with systemic metastases, making surgical treatment inadvisable. Standard treatment for pancreatic cancer includes radiotherapy and chemotherapy, which can also be used as adjuvant therapy after surgery. However, pancreatic cancer often exhibits resistance to these therapies, severely impacting treatment efficacy and patient prognosis. Therefore, overcoming drug resistance and adverse reactions to treatment regimens is crucial for enhancing treatment response. Based on genomic characteristics determined by whole-exome sequencing, pancreatic cancer tumors are classified into four subtypes: stable, locally rearranged, dispersed, and unstable. The unstable subtype, characterized by BRCA1 / 2, PALB2, RAD51C, RAD51D, and ATM, accounts for 4%–7% of cases. Due to synthetic lethal mechanisms, these genetic alterations make the tumors particularly sensitive to PARP inhibitors. Results from the Phase III POLO trial of olaparib (POLO) showed that patients with BRCA-mutated pancreatic cancer treated with olaparib had significantly longer progression-free survival than those receiving placebo. Currently, olaparib maintenance therapy for patients with germline BRCA-mutated metastatic pancreatic cancer has been approved in several countries and is recommended in national comprehensive cancer network clinical guidelines. Despite the success of olaparib and other treatments in clinical trials for BRCA-mutated pancreatic cancer, a significant proportion of patients without these mutations still do not respond well to conventional therapies. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the purpose of this invention is to provide new applications for cinnefenadine. Specifically, it proposes the use of cinnefenadine, alone or in combination with PARPi, for the treatment of pancreatic cancer. Based on in vitro and in vivo experiments, this invention is the first to demonstrate that cinnefenadine, alone or in combination with PARPi, can be used to treat pancreatic cancer. This overcomes the gene mutation limitations in pancreatic cancer treatment, expands the indications for PARPi in pancreatic cancer patients, enhances the therapeutic effect of combination therapy, prolongs the survival of pancreatic cancer patients, and allows patients to benefit more from it.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0005] This invention discloses the application of TGS1 inhibitors as the sole active ingredient in the preparation of drugs for treating tumors.

[0006] This invention also discloses the application of TGS1 inhibitor as the sole active ingredient in the preparation of drugs that inhibit the proliferation and invasion of tumor cells.

[0007] Furthermore, the above-described application is characterized in that the TGS1 inhibitor comprises sinefenidone or shRNA; the shRNA nucleotide sequence is shown in SEQ ID No. 1~3.

[0008] The present invention also discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a TGS1 inhibitor and a PARP inhibitor.

[0009] The present invention also discloses a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a TGS1 inhibitor and a chemotherapy drug.

[0010] Furthermore, the pharmaceutical composition described above is characterized in that the TGS1 inhibitor comprises cinnefenidone or shRNA; the shRNA nucleotide sequence is shown in SEQ ID No. 1-3.

[0011] The present invention also discloses a pharmaceutical preparation, characterized in that the pharmaceutical preparation comprises a TGS1 inhibitor or a combination of a TGS1 inhibitor and a PARP inhibitor or a combination of a TGS1 inhibitor and a chemotherapy drug.

[0012] The present invention also discloses a radiosensitizer / chemosensitizer, characterized in that it contains a TGS1 inhibitor.

[0013] Furthermore, the pharmaceutical preparation or the radio / chemosensitizer described above is characterized in that the TGS1 inhibitor comprises cinnefenidone or shRNA; the shRNA nucleotide sequence is shown in SEQ ID No. 1-3.

[0014] The present invention also discloses the use of the pharmaceutical composition, pharmaceutical preparation, or radio / chemosensitizer described above in the preparation of a medicament for treating tumors.

[0015] Trimethylguanosine synthase 1 (TGS1) is a conserved hypermethylase involved in the capping of mRNA and small nuclear RNA (snRNA) and in key biological processes, including glucose metabolism and embryonic development. In this study, our results indicate that TGS1 is a key player in DNA repair. The study showed that TGS1 is significantly overexpressed in pancreatic cancer and is associated with poor prognosis and increased chemotherapy resistance. Mechanistically, TGS1 is crucial for recruiting BRCA1 to DNA damage sites and promoting heart rate repair (HR) repair. Inhibiting TGS1 through gene knockdown or pharmacological intervention with sinefenidine (cinefenidine) disrupts HR repair and enhances the sensitivity of pancreatic cancer cells to genotoxic therapies. This study highlights the therapeutic potential of TGS1 inhibition as part of a combination strategy with existing treatments such as PARP inhibitors to improve the prognosis of pancreatic cancer patients.

[0016] In pancreatic cancer, elevated TGS1 expression is associated with defective homologous recombination repair. However, when TGS1 is suppressed, it leads to decreased BRCA1 levels, resulting in homologous recombination repair deficiency and significantly increasing the sensitivity of pancreatic cancer cells to PARPi.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] 1. Combining sinefenoxan with sinefenoxan results in a smaller effective dose, faster metabolism, and potentially fewer toxic side effects.

[0019] 2. Sinefungin targets TGS1, has a narrow spectrum of action and a low off-target rate, and is less likely to cause other toxic side effects.

[0020] 3. Sinaifeneol can significantly enhance the lethality of PARPi. Attached Figure Description

[0021] Figure 1The effect of TGS1 shRNA transfection on the sensitivity of PAAD cell lines to radiotherapy and chemotherapy was investigated. (A) Western blot analysis showed TGS1 expression in PAAD cell lines PANC-1, Mia-2, CFPAC-1, ASPC-1, BXPC-3, and SW1990; (B) Western blot analysis showed the protein level of TGS1 in PANC-1 cells transfected with TGS1 shRNA; and (C) Western blot analysis showed the protein level of TGS1 in Mia-2 cells transfected with TGS1 shRNA. (D) Western blot analysis showed the protein level of TGS1 in CFPAC-1 cells transfected with TGS1 shRNA; (EH) colony formation assays showed the cell viability of Mia-2 cells transfected with TGS1 shRNA after irradiation (E), olaparib (F), cisplatin (G), and etoposide (H); (IL) colony formation assays showed the cell viability of CFPAC-1 cells transfected with TGS1 shRNA after irradiation (I), olaparib (J), cisplatin (K), and etoposide (L). Data points with error bars represent mean ± standard deviation. Unpaired two-tailed ANOVA was used for statistical analysis. *P<0.05; **P<0.01.

[0022] Figure 2 Sensitivity of PANC-1 cells to olaparib was assessed after transfection with the TGS1 shRNA plasmid. The results of the colony formation assay (AD) showed cell viability in PANC-1 cells transfected with TGS1 shRNA after exposure to irradiation (A), olaparib (B), cisplatin (C), and etoposide (D). The flowchart of olaparib sensitivity in a PANC-1 cell-derived xenograft model (CDX) transfected with TGS1 shRNA (n=5 mice per group) was also presented. Tumor growth curves (F), representative tumor images (G), and tumor weight (H) in CDX model mice treated with PBS, olaparib, TGS1 shRNA, and olaparib + TGS1 shRNA; (I) Western blot analysis of protein levels of cleaved caspase3, γ-H2AX, pCHK2, and cleaved PARP in tumor tissues of CDX model mice treated with shTGS1 and olaparib; (J) Immunohistochemical analysis showing the expression of Ki-67, γ-H2AX, and cleaved caspase3 after olaparib treatment and TGS1 knockdown; (K) Immunohistochemical statistics showing the proportion of Ki-67, γ-H2AX, and cleaved caspase3 positive cells after olaparib treatment and TGS1 knockdown. Scale bar, 50 μm. Data points with error bars represent mean ± standard deviation. Unpaired t-tests and unpaired two-tailed ANOVA were used for statistical analysis. *P<0.05;**P<0.01.

[0023] Figure 3 This study investigated the sensitivity of pancreatic cancer cell lines Mia-2 and CFPAC-1 to olaparib induced by sinefenib inhibition of TGS1. Immunoblot analysis (AC) showed the TGS1 protein levels in PANC-1 (A), Mia-2 (B), and CFPAC-1 (C) cells treated with sinefenib (10 μM, 20 μM); colony formation assay (DG) showed the cell survival rate of Mia-2 cells treated with sinefenib exposed to radiation (D), olaparib (E), cisplatin (F), or etoposide (G); and colony formation assay (HK) showed the cell survival rate of CFPAC-1 cells treated with sinefenib exposed to radiation (H), olaparib (I), cisplatin (J), or etoposide (K). Data points with error bars represent the mean including standard deviation. Statistical analysis was performed using unpaired two-tailed ANOVA. *p<0.05; **p<0.01. NS indicates no difference.

[0024] Figure 4 This study demonstrates how cinnefenib inhibits TGS1 to induce sensitivity of the pancreatic cancer cell line PANC-1 to olaparib. (AD) shows the clonogenic assay, illustrating the effect of cinnefenib on the survival of PANC-1 cells exposed to irradiation (A), olaparib (B), cisplatin (C), and etoposide (D); (E) is a flowchart of the PANC-1 cell-derived xenograft (CDX) model; (FH) are representative images (F), tumor growth curves (G), and tumor weight (H) of CDX models treated with PBS, olaparib, cinnefenib, or a combination of olaparib and cinnefenib; (I) shows the expression levels of caspase3, γ-H2AX, pCHK2, and PARP in the CDX model using immunohistochemistry; (KN) shows the expression levels of Ki-67, γ-H2AX, and cleaved caspase3 in the CDX model using immunohistochemistry. Data points with error bars represent the mean including standard deviation. Statistical analysis was performed using unpaired t-tests and unpaired two-tailed ANOVA. *p<0.05; **p<0.01.

[0025] Figure 5To investigate the olaparib sensitivity of TGS1-induced pancreatic cancer patient-derived xenograft PDX 1301 induced by sinefenoxan. The results show: (A) Flowchart of olaparib and sinefenoxan treatment in the PDX model; (BD) Differences in serum AST (B), serum ALT (C), and mouse body weight (D) among PBS, olaparib, sinefenoxan, and olaparib + sinefenoxan in the PDX 1301 model; (EG) Representative images (E), tumor growth curves (F), and tumor weight (G) of PDX 1301 tumors treated with PBS, olaparib, or sinefenoxan; (HK) Representative images and quantifications of HE staining, Ki-67, γ-H2AX, and caspase 3 in PDX 1301 treated with PBS, olaparib, or sinefenoxan; (L) Protein expression of cleaved caspase 3, γ-H2AX, pCHK2, and PARP in the PDX 1301 model tumors. Data points with error bars represent the mean including the standard deviation. Statistical analysis was performed using unpaired t-tests and unpaired two-tailed ANOVA. *p<0.05; **p<0.01. NS indicates no difference.

[0026] Figure 6 This study aimed to investigate the effect of cinnefenoxan on inhibiting olaparib sensitivity in TGS1-induced Xenograft PDX 748 from pancreatic cancer patients. The data are presented as follows: (AC) Differences in serum AST (A), serum ALT (B), and mouse body weight (C) among PBS, olaparib, cinnefenoxan, and olaparib + cinnefenoxan in the PDX 748 model; (DF) Representative images (D), tumor growth curves (E), and tumor weight (F) of PDX 748 tumors treated with PBS, olaparib, or cinnefenoxan; (GJ) Representative images and quantifications of HE staining, Ki-67, γ-H2AX, and caspase 3 in PDX 748 treated with PBS, olaparib, or cinnefenoxan; (K) Protein expression of cleaved caspase 3, γ-H2AX, pCHK2, and PARP in the PDX 748 model tumors. Data points with error bars represent the mean including standard deviation. Statistical analysis was performed using unpaired t-tests and unpaired two-tailed ANOVA. *p<0.05; **p<0.01. NS indicates no difference. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0028] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0029] Example 1.

[0030] I. Materials and Methods.

[0031] 1. Cell culture.

[0032] PAAD cell lines were purchased from the Cell Resource Center of Peking Union Medical College (NSTI-BMCR, a national science and technology infrastructure, NSTI-BMCR, http: / / cellresource.cn). ASPC-1, BXPC-3, and PANC-1 cells were grown in Dulbecco-modified Eagle medium (DMEM) containing 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin. Mia-2 cells were cultured in DMEM containing 5% (v / v) horse serum. CFPAC-1 cells were maintained in IMDM medium containing 10% (v / v) FBS.

[0033] 2. Western blot and antibody.

[0034] Cells were harvested and lysed using NETN buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 with 10 mM NaF trypsin inhibitor and 20 mM β-glycerophosphate × saline solution). The immunoprecipitate was washed with NETN and then centrifuged at 500 × g for 1 min three times. 50 μL of the immunoprecipitate was reserved and added to 50 μL of 1 × Laemmli buffer, then boiled at 95°C for 10 min. Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane (Millibo, Germany). After blocking with 5% skim milk and 0.1% Tween-20 (A100777, Sangon Biotech, Shanghai, China) in TBST (Solepro, China), the membranes were incubated overnight at 4°C with TGS1 antibody (1:1000, Bethyl, A304-922A, USA) or GAPDH (1:10000, Proteintech, 10494-1-AP, USA), followed by incubation with secondary antibody (Jackson, USA) for 1 hour. The membranes were then washed three times with TBST, and protein bands were detected using Super ECL plus (NCM, P10300, China) on an Amersham ImageQuant 800 (Cytiva, US) instrument. Tumor tissue was collected, and total protein was extracted from each group. The protein cleavage products were incubated overnight with cleaved PARP (1:1000, 5625S, CST, USA), cleaved caspase3 (1:1000, 9661S, CST, USA), γ-H2AX (1:1000, 05-636, Millipore, USA) and pCHK2 (1:1000, 82263S, Proteintech, USA), as shown in the Western blot.

[0035] 3. Cell viability assay.

[0036] Cells were seeded at a concentration of 500–1000 cells per well in 6-well plates. The next day, PANC-1 / Mia-2 / CFPAC-1 cells were treated with different doses of cilenefentine (0, 10, 20 μM), olaparib (0, 0.5, 1, 2 μM), or X-rays (1 / 2 / 4 / 8 Gy). Cells were cultured for 10–14 days, and then the colonies were stained with 0.1% crystal violet solution. The number of colonies in each well was quantified, and the survival rate was calculated by comparing the number of colonies in treated and untreated wells. Viable cells were measured using a cell counting kit-8 (CCK-8, PSAITONG). Cells were then seeded into 96-well plates. On the second day, PANC-1 / Mia-2 / CFPAC-1 cells were treated with different doses of cisplatin (0, 6.25, 12.5, 25 μM) and etoposide (0, 3.125, 6.25, 12.5, 25 μM). Next, the cells were exposed to 8 μL of CCK-8 reagent (100 μL of culture medium per well) and incubated at 37°C and 5% CO2 for 1 hour. Absorbance was measured at 450 nm using a BioTek microplate reader (800TS).

[0037] 4. Validation of xenograft models derived from pancreatic cancer cells and patients.

[0038] A cell-derived xenograft (CDX) model was established using 5-6 week old female thymic nude mice (BALB / c-nu). Cells containing 2×10⁻⁶ cells were used to establish the CDX model. 6 A suspension of pancreatic cancer cells (including PANC-1 and TGS1 shRNA-transfected PANC-1 cell lines) was mixed with 50% Matrigel and subcutaneously injected into the anterior region of each mouse. Approximately 20 days after inoculation, mice were divided into different treatment groups, including a TGS1 inhibition group (cilefentine, 20 mg / kg, intraperitoneal injection, every other day), an olaparib group (20 mg / kg, intraperitoneal injection, every other day for two weeks), and a combination therapy group of TGS1 inhibitor and olaparib. The different groupings are illustrated in the attached figure.

[0039] Patient-derived xenograft (PDX) models of pancreatic cancer were developed by Nanjing Chang Le Biotechnology Co., Ltd. TGS1-high expression models (PDX-1301 and 748) were selected for explantation. Biopsies of PDX tumors were subcutaneously implanted into female BALB / c-nu mice. Once the average tumor volume of the PDX model reached 100 mm², the transplantation was completed. 3 Mice were randomly assigned to treatment cohorts, including those receiving PBS alone, TGS1 inhibition (sinefentanyl, 20 mg / kg, intraperitoneal injection, every other day), olaparib (20 mg / kg, intraperitoneal injection, every other day), and sinefentanyl plus olaparib combination therapy.

[0040] In all mouse studies, the length and width of each tumor were measured every other day using electronic calipers. Tumor volume was calculated using the formula: Volume = 0.5 (Length × Width). 2 At the end of the experiment (the endpoint being a tumor diameter not exceeding 1.5 cm), the animals were euthanized, and a portion of the tumor tissue was rapidly fixed in 4% paraformaldehyde for immunohistochemical (IHC) analysis. The remaining tumor tissue was stored at -80°C. Tumor tissue from CDX and PDX was collected for immunohistochemical staining. Fresh tumor tissue was fixed overnight in 4% paraformaldehyde fixative, dehydrated, and embedded in paraffin. Samples underwent immunohistochemical staining. Tissues were stained with H&E, Ki-67 (1:1000, 28074-1-AP, Proteintech, USA), cleaved caspase 3 (1:1000, 9661S, CST, USA), and γ-H2AX (1:1000, 05-636, Millipore, USA) antibodies. Images of 10 randomly stained microscopic areas in each slice were recorded using ImageJ software for quantitative analysis.

[0041] II. Experimental Results.

[0042] 1. TGS1 shRNA inhibits TGS1-induced sensitivity of pancreatic cancer cell lines to olaparib.

[0043] Experimental results are as follows Figure 1 As shown in Figure 2. Colony formation and cell proliferation assays demonstrated that the combined action of TGS1 shRNA with radiotherapy, olaparib, cisplatin, and etoposide significantly inhibited cell viability in PANC-1, Mia-2, and CFPAC-1 cells. PANC-1 cells and a Xenograft model (CDX) of PANC-1 cells transfected with TGS1 shRNA were treated with PBS and olaparib. Based on representative images, growth curves, and tumor weight results, the combination of TGS1 shRNA and olaparib showed optimal efficacy. Immunohistochemistry confirmed that the TGS1 shRNA combined with olaparib treatment group showed increased expression of caspase3, γ-H2AX, pCHK2, and PARP; decreased Ki-67 expression; and increased expression of γ-H2AX and cleaved caspase3.

[0044] 2. Experimental study on the sensitivity of sinefenoxan to TGS1-induced pancreatic cancer cell lines to olaparib.

[0045] Experimental results are as follows Figure 3As shown in Figure 4. Colony formation and cell proliferation assays demonstrated that different doses of cilexetil, combined with radiotherapy, olaparib, cisplatin, and etoposide, significantly inhibited the survival rate of PANC-1 cells. In a PANC-1 cell-derived xenograft model (CDX), treatment with PBS, olaparib, cilexetil, or a combination of olaparib and cilexetil, based on representative images, growth curves, and tumor weight results, showed that the combination of olaparib and cilexetil was optimal. Immunohistochemistry confirmed that the olaparib and cilexetil combination treatment group showed increased expression of caspase3, γ-H2AX, pCHK2, and PARP; decreased Ki-67 expression; and increased expression of γ-H2AX and cleaved caspase3.

[0046] 3. Sensitivity assay of olaparib in a TGS1-inhibited pancreatic cancer patient tissue-derived xenograft (PDX) model.

[0047] Experimental results are as follows Figure 5 As shown in Figure 6, for pancreatic cancer patients with tissue-derived xenograft (PDX) models, treatment with PBS, olaparib, cinnefenoxan, or a combination of olaparib and cinnefenoxan was conducted. Based on representative images, growth curves, and tumor weight results, the combination of olaparib and cinnefenoxan was optimal. Immunohistochemistry confirmed that the olaparib and cinnefenoxan combination treatment group showed increased expression of caspase3, γ-H2AX, pCHK2, and PARP; decreased Ki-67 expression; and increased expression of γ-H2AX and cleaved caspase3.

[0048] In summary, the combined use of sinefenoxan and a PARP inhibitor significantly inhibited tumor growth in TGS1-overexpressing cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models. This invention not only highlights the potential of sinefenoxan as a therapeutic agent to improve the effectiveness of existing treatments for pancreatic cancer, but also provides a new avenue for addressing the significant clinical challenges associated with pancreatic cancer.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a TGS1 inhibitor as the sole active ingredient in the preparation of a drug for treating pancreatic cancer, wherein the TGS1 inhibitor is sinefenidine.

2. The application as described in claim 1, characterized in that, The application of TGS1 inhibitor as the sole active ingredient in the preparation of drugs that inhibit the proliferation and invasion of pancreatic cancer cells, wherein the TGS1 inhibitor is sinefenidine.

3. The application of TGS1 inhibitors and PARP inhibitors in the preparation of drugs for treating pancreatic cancer, wherein the TGS1 inhibitor is sinefenoxan; and the PARP inhibitor is olaparib.

Images