Inhibitor composition for pancreatic ductal adenocarcinoma

Abstract

The inhibitor composition for pancreatic ductal adenocarcinoma comprises an AhR inhibitor and a PRMT5 inhibitor. The AhR inhibitor can be CH223191, and the PRMT5 inhibitor can be GSK3326595. The invention further discloses a preparation method of the compound. According to the application disclosed by the invention, the development of pancreatic tumors of mice is remarkably inhibited by using combined small-molecule inhibitors, namely the AhR inhibitor and the PRMT5 inhibitor for the first time.

Description

Technical Field

[0001] The present invention generally relates to small molecule inhibitor compositions for pancreatic ductal adenocarcinoma. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is highly malignant and has a very poor prognosis, with an average five-year survival rate of less than 10%, and its pathogenesis is still unclear. Surgery and adjuvant chemotherapy can help some patients prolong survival, but they are only suitable for a small number of patients with resectable tumors. Existing chemotherapy such as the FORFIRINOX regimen or the gemcitabine combined with albumin-paclitaxel regimen has very limited survival improvement for most patients with unresectable tumors, and is significantly associated with toxic and side effects. Except for the less than 1% of patients with microsatellite instability, most PDAC patients find it difficult to benefit from existing immunotherapy regimens. The reason is related to the histological characteristics of PDAC: typical PDAC manifests as an immune "cold tumor" - that is, it presents a suppressive immune microenvironment, and there is a lack of CD8 with killing function around the tumor. + There are also obstacles in recruiting innate immune cells such as T cells and other adaptive immune response helper cells, antigen presenting cells, macrophages and natural killer cells. Therefore, PDAC presents the characteristics of immunotherapy resistance, and the tumor cannot be effectively recognized and killed by the adaptive immune response.

[0003] Improving the PDAC immune microenvironment can help improve the response to immunotherapy, thereby effectively inhibiting tumor progression. Currently, the academic community focuses on enhancing endogenous T cell function, initiating anti-tumor specific T cell immune responses, and improving interstitial components to facilitate immune cell recruitment to achieve regulation of the tumor microenvironment (TME). Tumor cells are the source of anti-tumor immune responses. Recognizing endogenous tumor antigens plays a vital role in initiating local immune responses and recruiting tumor-specific immune cells. This process is one of the key ways to enhance the efficacy of immune checkpoint inhibitors.

[0004] Immune surveillance not only depends on the expression of antigens by tumor cells, but also on the effective presentation of tumor antigens to immune cells by tumor cells through the major histocompatibility complex (MHC). The loss of expression or dysfunction of MHC molecules is one of the important mechanisms for tumors to escape immune surveillance. The main causes of MHC molecule abnormalities include heterozygous deletions and somatic mutations. Abnormal histone function has also been proven to be an important factor leading to the dysfunction of MHC class I tumor cell antigen presentation. For example, abnormal trimethylation of histone H3K27 inhibits the expression of MHC class I molecules in leukemia and head and neck squamous cell carcinoma. Abnormal gene methylation in the promoter or enhancer region of human leukocyte-associated antigen (HLA) genes, B2M genes, and Tap1 genes can lead to abnormal transcription of MHC-related genes, thereby weakening the tumor antigen presentation function associated with MHC class I molecules. Disorders in the promoter methylation regulation of genes such as type II transactivator protein genes and CD74 can lead to the loss of MHC class II molecule expression in tumor cells.

[0005] Aryl hydrocarbon receptor (AhR) is an important nuclear transcription factor that participates in cell proliferation and differentiation, stress and metabolism, inflammatory response and other processes, regulates the expression of multiple genes, and has been proven to be closely related to the occurrence and development of various tumors. As a type of ligand-activated nuclear transcription factor, in the classically activated AhR signaling pathway, AhR protein can bind to tryptophan metabolites, arachidonic acid metabolites, indole and other substances, recognize specific gene binding sequences, and thus initiate the downstream signal transduction process. AhR is closely related to the function of immune cells. On the one hand, it can affect the function of innate immune cells such as macrophages and dendritic cells. On the other hand, it can also affect the immune regulation by affecting the differentiation of T helper cells such as Th17 and Th22 cells, and can also directly change CD4 + and CD8 + The effectiveness of T cells in adaptive immune responses. Previous research results have found that there are local high levels of AhR ligands in the tumor microenvironment: tryptophan metabolite kynurenine (KYN), and AhR in dendritic cells and regulatory T cells is activated, inducing immune tolerance, increasing the content of inhibitory cytokines such as interleukin-10 in the immune microenvironment, and T cell function is also inhibited by AhR signal activation.

[0006] It has been found that the use of indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors can inhibit AhR activity in PDAC tissues and cells and improve the immunosuppressive state of PDAC mouse models. It has also been reported that high AhR activity exists in PDAC tissues, and AhR knockout in bone marrow cells leads to an increase in dendritic cells around tumors and enhanced function, CD8 + INF-γ +The above studies reveal that AhR has an immunosuppressive effect on PDAC. However, its mechanism research is still mainly based on the immune cell components in the TME, and it is unable to explain the effect of high expression of AhR in tumor cells themselves on the formation of immunosuppressive TME, and the role of AhR in tumors in immune escape is still unclear, especially for tumor-killing specific CD8 + The mechanism of action of T cells needs to be further elucidated and utilized. Summary of the invention

[0007] The purpose of the present invention is to provide a pharmaceutical composition capable of effectively inhibiting pancreatic ductal adenocarcinoma.

[0008] According to a first aspect of the present invention, a pharmaceutical composition is provided, comprising an aryl hydrocarbon receptor (AhR) inhibitor and an arginine methyltransferase 5 (PRMT5) inhibitor.

[0009] According to the present invention, the AhR inhibitor may be CH223191, and the PRMT5 inhibitor may be GSK3326595.

[0010] The inhibitor of the present invention can be in any suitable dosage form. For example, CH223191 can be in an injection form, and GSK3326595 can be in an oral form.

[0011] According to another aspect of the present invention, provided is the use of an AhR inhibitor and a PRMT5 inhibitor in preparing a medicament for treating pancreatic ductal adenocarcinoma.

[0012] Through cell experiments and animal models based on AhR knockout tumor cells, this application found that AhR does not affect the proliferation of tumor cells themselves, but reduces the immunogenicity of PDAC by affecting tumor antigen presentation, thereby reducing the infiltration of immune cells in the tumor microenvironment and escaping the specific killing of immune cells. Specifically, AhR inhibits tumor antigen presentation of MHC class I molecules and regulates the expression of key proteins, which ultimately leads to the immune escape of PDAC. Molecular mechanism studies have found that AhR inhibits the chromatin opening of MHC class I molecules and key molecular genes by interacting with the arginine methyltransferase PRMT5, thereby inhibiting gene expression.

[0013] Therefore, this application discovered the AhR-PRMT5-MHCⅠ molecular expression pathway for the first time, thereby revealing a new molecular mechanism of pancreatic cancer tumor immune escape: AhR interacts with PRMT5 to inhibit chromatin opening, thereby inhibiting the expression of MHCⅠ molecules in tumor cells.

[0014] The present application further discovered that combined targeting of AhR and PRMT5 proteins can significantly inhibit the growth of pancreatic tumors in mice.

[0015] Pancreatic cancer is insensitive to existing immune checkpoint inhibitors (such as PD-1 antibodies) due to its "cold tumor" characteristics. The present invention significantly inhibited the progression of pancreatic tumors in mice by using a combination of small molecule inhibitors (AhR inhibitors and PRMT5 inhibitors) for the first time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A This is a comparison of AhR expression levels between human PDAC tissue and normal pancreatic tissue in the TCGA and GTEx databases; Figure 1B This is a comparison chart of AhR expression levels between human PDAC tissue and normal pancreatic tissue in the GEO database; Figure 1C This is a survival analysis chart of PDAC patients in the TGCA database based on AhR expression.

[0017] Figure 2A AhR immunohistochemical staining of normal pancreatic tissue adjacent to the cancer; Figure 2B AhR immunohistochemical staining of PDAC tumor tissue; Figure 2C This is a scoring chart for immunohistochemical staining.

[0018] Figure 3A and Figure 3B They are Ahr - / - and wild-type Pan02 were subcutaneously inoculated into immune-competent mice. Ahr Pictures of tumors formed by genetic tumor cells and curves of mouse survival time; Figure 3C , Figure 3D and Figure 3E Respectively Ahr - / - and wild-type PANO2 cells were inoculated into the pancreatic tissue of immune-normal mice, and knockout Ahr Tumor formation pictures before and after gene expression, H&E staining pictures of tumors formed by wild-type cells, Ahr H&E staining images of pancreatic tissue after knockout cell inoculation; Figure 3F and Figure 3G Respectively Ahr - / - And pictures of tumors formed after subcutaneous inoculation of wild-type Pan02 cells into nude mice and a curve of nude mouse survival time.

[0019] Figure 4A-4D These are the histochemical staining images of different immune cells in tumor tissues of subcutaneously inoculated mice; Figure 4E and Figure 4F They are statistical graphs of immune cell staining counts; Figure 4G and Figure 4H These are the flow cytometry results of activated CD4 and CD8 T cells in mouse tumor tissues; Fig. 4I and Figure 4J Then Figure 4G and Figure 4H Statistical chart of flow cytometry detection results.

[0020] Figure 5A and Figure 5B The expression levels of cell membrane MHC I subunits B2M and H2-Kb are shown respectively; Figure 5C and Figure 5D The relative fluorescence intensities of B2M and H2-Kb expression are shown respectively.

[0021] Fig. 6A Post-IP proteins are shown; Figure 6B This is the typical peptide peak of PRMT5 obtained by mass spectrometry; Figure 6C It shows that AhR and PRMT5 were co-expressed in 293T, and the interaction between the two proteins was detected by CoIP; Fig.6D For interference Prmt5 After RNA, QPCR was used to detect the mRNA expression of MHC I and its key regulatory molecules; Fig. 6E and Fig. 6F These are the comparison charts of PRMT5 expression between human PDAC tissue and normal pancreatic tissue in TCGA, GTEx, and GEO databases; Figure 6G This is a survival analysis chart of PDAC patients in the TGCA database based on PRMT5 expression; Figure 6H and Fig.6I These are the PRMT5 immunohistochemical staining images of adjacent normal pancreatic tissue and PDAC tissue; Figure 6J This is a scoring chart for immunohistochemical staining.

[0022] Fig. 7A The images show PDAC subcutaneous tumors in mice treated with two inhibitors alone and in combination. Figure 7B The corresponding statistical results of tumor weight; Figure 7C Shown is the survival analysis of PDAC subcutaneous tumors in mice treated with the two inhibitors alone and in combination; Fig.7D , Fig. 7E , Figure 7F and Figure 7G The CD4+ / -4+ / -1 expression in PDAC subcutaneously inoculated tumor tissues of mice in the control group, two inhibitors alone, and combination treatment groups were detected by ISH. + T lymphocytes; Figure 7H for Figure 7D-Figure 7E CD4 + T lymphocyte count statistical results; Fig.7I , Figure 7J , Figure 7K and Figure 7LThe CD8 in the subcutaneously inoculated tumor tissues of PDAC mice were detected by ISH in the control group, two inhibitors alone and combination treatment groups. + T lymphocytes; Figure 7M for Figure 7I-7L CD8 + T lymphocyte count statistical results. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with the accompanying drawings. It should be understood by those skilled in the art that the embodiments described below are only exemplary descriptions of the present invention and are not intended to limit the present invention in any way.

[0024] Experimental process and analysis 1. High expression of AhR in pancreatic cancer cells regulates tumor growth through the immune system Figure 1A This is a comparison of AhR expression levels between human PDAC tissue and normal pancreatic tissue in the TCGA and GTEx databases; Figure 1B This is a comparison chart of AhR expression levels between human PDAC tissue and normal pancreatic tissue in the GEO database; Figure 1C This is a survival analysis chart of PDAC patients in the TGCA database based on AhR expression. Figure 1A and Figure 1B It can be seen that PDAC tumor tissue is more sensitive than normal pancreatic tissue. Ahr The mRNA expression level increased significantly. Figure 1C It can be seen that in PDAC patients, high expression Ahr PDAC patients with high mRNA expression have worse survival.

[0025] Further testing of paraffin sections of 34 PDAC patients: Figure 2A AhR immunohistochemical staining of normal pancreatic tissue adjacent to the cancer; Figure 2B AhR immunohistochemical staining of tumor tissue from a PDAC patient; Figure 2C This is an immunohistochemical staining score chart. It can be seen that immunohistochemistry shows that AhR expression in tumor cells in PDAC tissue is significantly increased, and it is positively colored in the nucleus of tumor cells. The expression in the tumor with immunohistochemical score is higher than that in the normal pancreatic duct of adjacent tissue. The above results reveal that AhR is highly expressed in tumor cells in human PDAC tissue, and patients with high expression have a poor prognosis. Therefore, it is speculated that the highly expressed AhR in pancreatic ductal cell carcinoma plays a specific function in the development of tumors.

[0026] To verify the role of AhR in PDAC, CRISPR / CAS9 technology was used to knock out AhR in the mouse pancreatic cancer cell line PANO2. Ahr Gene.

[0027] C57 / BL6 mice were inoculated subcutaneously Ahr + / + and Ahr - / - Pan02 cells, 1x10 6 Cells / piece; Figure 3A Shows knockout Ahr The corresponding tumor pictures after gene Figure 3B The corresponding survival curves of mice are shown. Figure 3C Figure 2 shows the in situ inoculation of pancreatic tissue in C57 / BL6 mice. Ahr + / + and Ahr - / - Pan02 cells, 1x10 6 The corresponding tumor pictures formed by cells / Figure 3D and Figure 3E The H&E staining images of wild-type tumors inoculated in situ in C57 / BL6 mice are shown respectively. Ahr H&E staining of knockout tumors. Ahr + / + and Ahr - / - Pan02 cells, 1x10 6 Cells / Figure 3F Corresponding pictures of subcutaneous tumors are shown. Figure 3G Shown is the subcutaneous inoculation of nude mice Ahr + / + and Ahr - / - Corresponding survival curves of Pan02 mice.

[0028] Will Ahr - / - and wild-type Pan02 were subcutaneously inoculated into immune-competent C57 / BL6 mice. Ahr After gene therapy, tumor size decreased, see Figure 3A ; In addition, the survival time of AhR gene knockout mice was significantly prolonged, see Figure 3B In order to better simulate the pancreatic in situ tumor microenvironment and in vivo anti-tumor immune process, the constructed mouse PDAC in situ tumor model found that Ahr The tumorigenicity of the knockout cells was significantly inhibited, with only 1 / 5 of the mice developing visible tumor masses, and no tumor masses were found in the remaining mice. Figure 3C ; H&E staining showed that the tumor formed by wild-type cells had unclear boundaries with the pancreatic tissue, and the pancreatic tissue had obvious acinar ductal metaplasia, see Figure 3D ; However, there is a clear boundary between the AhR knockout cell tumor and the pancreatic tissue, which is surrounded by a large number of interstitial cells infiltration, and no obvious acinar ductal metaplasia is observed, see Figure 3EAhR knockout did not affect the growth of subcutaneous tumors in immunodeficient mice, that is, AhR knockout had no significant effect on tumor appearance, volume growth rate, and tumor weight, and AhR knockout could not prolong the survival time of tumor-bearing nude mice, see Figure 3F and Figure 3G .

[0029] 2. Knockout of tumor cells Ahr Increased immune cell infiltration in the tumor microenvironment Figure 4A Shown is the subcutaneous inoculation of C57 / BL6 mice with Pan02 cells Ahr + / + Pan02 tumor cells, CD8 + IHC staining of T lymphocytes. Figure 4B Shown is the subcutaneous inoculation of C57 / BL6 mice with Pan02 cells Ahr - / - Pan02 tumor cells, CD8 + IHC staining of T lymphocytes. Figure 4C Shown is the subcutaneous inoculation of C57 / BL6 mice with Pan02 cells Ahr + / + Pan02 tumor cells, CD4 + IHC staining of T lymphocytes. Figure 4D Shown is the subcutaneous inoculation of C57 / BL6 mice with Pan02 cells Ahr - / - Pan02 tumor cells, CD4 + IHC staining of T lymphocytes. Figure 4E C57 / BL6 mice are shown to be inoculated subcutaneously Ahr + / + and Ahr - / - Pan02 cells, tumor tissue after IHC staining, CD8 + T lymphocyte count statistics. Figure 4F C57 / BL6 mice are shown to be inoculated subcutaneously Ahr + / + and Ahr - / - Pan02 cells, tumor tissue after IHC staining, CD4 + T lymphocyte count statistics. Figure 4G C57 / BL6 mice are shown to be inoculated subcutaneously Ahr + / + and Ahr - / - Pan02 cells, flow cytometry detection of CD8 + IFγ+ T lymphocyte ratio. Figure 4H C57 / BL6 mice are shown to be inoculated subcutaneously Ahr + / + and Ahr - / - Pan02 cells, flow cytometry detection of CD4 + IFγ + T lymphocyte ratio. Fig. 4I Shows Figure 4G Statistical results of streaming detection; Figure 4J Shows Figure 4H Flow cytometry statistical results.

[0030] To specifically knock out tumor cells Ahr The immunological mechanism of inhibiting tumor growth is shown in the above histochemical staining of immune cells in mouse tumor tissue. As can be seen, Ahr - / - In the tumors formed by subcutaneous inoculation of cells into C57 / BL6 mice, CD4 + and CD8 + The infiltration of T cells was significantly higher than that of wild-type cells, see Figure 4A-4F In addition, in order to gain a more comprehensive understanding of the immune microenvironment of tumor tissue, the tumor tissue was digested and dissociated into single cells, and then flow cytometry analysis was performed; Ahr - / - A higher proportion of CD4 + IFN-γ + and CD8 + IFN-γ + Double-positive lymphocytes, see Figure 4G-4J The above results suggest that AhR may promote PDAC by constructing an inhibitory immune microenvironment and reducing the infiltration and activation of anti-tumor immune cells.

[0031] 3. Tumor cells Ahr Knockout leads to increased expression of MHC I genes in tumor cells Flow cytometry was used to analyze the expression levels of MHC class I heavy chains and β2-microglobulin (mainly including B2M and H2-Kb) on the membrane surface of wild-type cells and AhR knockout cells.

[0032] Figure 5A Flow cytometry analysis of wild type and Ahr - / - Expression level of MHC I subunit B2M on Pan02 cell membrane; Figure 5B Flow cytometry analysis of wild type and Ahr - / -Expression level of MHC I subunit H2-Kb on Pan02 cell membrane; Figure 5C The relative fluorescence intensity of B2M expression detected by flow cytometry is shown as a statistical analysis; Figure 5D Shown is the statistical analysis of the relative fluorescence intensity of H2-Kb expression detected by flow cytometry.

[0033] Figure 5A-5D The results showed that AhR knockout led to a significant increase in the amount of B2M and H2-Kb protein molecules on the surface of tumor cells. This confirmed that AhR has the effect of inhibiting the expression of MHC class I molecules on the surface of tumor cell membranes, thereby inhibiting tumor antigen presentation.

[0034] 4. AhR regulates the expression of MHC I molecules through PRMT5 interaction Fig. 6A The protein electrophoresis gel Coomassie blue staining after the tag antibody IP is shown, and the red box indicates the protein band corresponding to the molecular weight of AhR; Figure 6B For mass spectrometry detection Fig. 6A After IP, the protein was analyzed to obtain the typical peptide peak of PRMT5; Figure 6C The figure shows that AhR and PRMT5 were co-expressed in 293T cells, and the interaction between the two proteins was detected by CoIP. Fig.6D After interfering with Prmt5 gene expression, QPCR was used to detect the mRNA expression of MHC I and key regulatory molecules. Fig. 6E and Fig. 6F The comparison of PRMT5 expression between human PDAC tissue and normal pancreatic tissue in TCGA, GTEx, and GEO databases; Figure 6G This is a survival analysis chart of PDAC patients in the TGCA database based on PRMT5 expression. Figure 6H and Fig.6I These are the immunohistochemical staining images of PRMT5 in normal pancreatic tissue adjacent to PDAC cancer and tumor tissue; Figure 6J This is a scoring chart for immunohistochemical staining.

[0035] In order to explore the molecular mechanism of AhR regulating the expression of MHC class I molecules, an AhR recombinant plasmid containing two protein purification and detection tags (Streptavidin and FlagM2) was constructed. After the plasmid was transfected into 293T cells, the stably transfected mixed cells were selected with puromycin. After amplification, the total protein was collected and the tagged AhR was purified by tandem affinity. After SDS-PAGE electrophoresis and Coomassie blue staining, the purified protein was clearly visualized with blue dye concentration in the molecular weight region corresponding to AhR, proving that the protein purification achieved good results, see Fig. 6A. Then the gel was cut and protein spectrum was detected. Analysis of mass spectrometry results showed that the protein with the highest abundance related to chromatin activity was Protein arginine N-methyltransferase 5 (PRMT5), with a total of 9 peptides captured, and a Sum PEP Score of 31.165, see Figure 6B To verify the interaction between AhR and PRMT5, IP was performed on 293T stably transfected cells. The results showed that AhR could pull down PRMT5, proving that the two interact with each other. Figure 6C .

[0036] To confirm that AhR is achieved through interaction with PRMT5, PDAC cell lines were treated with its small interfering RNA (siRNA). The results showed that after PRMT5 was inhibited, the expression levels of mRNA of MHC class I molecules and its key regulatory molecules detected by qPCR also increased significantly, see Fig.6D This suggests that AhR regulates the expression of MHC I molecules through PRMT5.

[0037] Analysis of data from the public databases TCGA, GTEx, and GEO revealed that PDAC tumor tissues were more sensitive to inflammatory cytokines than normal pancreatic tissues. Prmt5 mRNA expression was significantly increased, see Fig. 6E and Fig. 6F ; In PDAC patients, high expression Prmt5 PDAC patients with mRNA have worse survival, see Figure 6G .

[0038] Further testing of paraffin sections of 34 PDAC patients revealed that immunohistochemistry showed that PRMT5 expression in tumor cells in PDAC tissues was significantly increased, and most of them were positive in the nuclei of tumor cells. The immunohistochemistry score was higher than that of normal pancreatic ducts in adjacent tissues. Figure 6H-6J The above results revealed that PRMT5 is highly expressed in tumor cells in human PDAC tissues, and patients with high expression have a poor prognosis. Therefore, it is speculated that the highly expressed AhR and PRMT5 in pancreatic ductal cell carcinoma play a specific function in the development of tumors.

[0039] 4. Combining AhR inhibitors and PRMT5 inhibitors can significantly inhibit the growth of pancreatic cancer tumors in mice The AhR inhibitor CH223191 (MCE, USA, #HY-12684) was used, and the dissolution method was to sequentially add 10% DMSO + 40% PEG300 + 5% Tween80 + 45% saline, with a working concentration of 1 mg / ml. The dosage for mice was 0.2 mg per mouse (about 20 mg / kg), and it was intraperitoneally injected three times a week for a total of 12 times. The PRMT5 inhibitor GSK3326595 (HY-101563; MedChemExpress) was orally administered at 50 mg / kg per day for a total of 15 days. Treatment began after the subcutaneous tumor grew to 0.3 cm. Four groups of experimental mice were set up: a blank control group (only injected with drug solvent), an AhR inhibitor CH223191 treatment group, a PRMT5 inhibitor GSK3326595 treatment group, and a combination treatment group of the two inhibitors; there were 8 mice in each group. Two cohorts were set up for each study, one for survival curve observation and the other for tumor tissue and biological observation. The survival curve observation group was observed until 60 days after subcutaneous tumor formation, and the tumor tissue biology observation group was killed on the 30th day after tumor formation.

[0040] Fig. 7A and Figure 7B The results of the treatment of PDAC subcutaneous tumors in C57 / BL6 mice with AhR inhibitor CH223191 and PRMT5 inhibitor GSK3326595 alone or in combination with the two inhibitors, as well as the statistical results of tumor weight. Figure 7C To analyze the survival of C57 / BL6 mice with PDAC subcutaneous tumors treated with AhR inhibitor CH223191 and PRMT5 inhibitor GSK3326595 alone or in combination. Fig.7D , Fig. 7E , Figure 7F and Figure 7G The CD4+ T cells in the tumor tissues of C57 / BL6 mice inoculated with PDAC tumors were detected by ISH in the control group, AhR inhibitor CH223191, PRMT5 inhibitor GSK3326595 alone, and the combination of the two inhibitors. + T lymphocytes; Figure 7H for Figure 7D-Figure 7E CD4 + T lymphocyte count statistical results; Fig.7I , Figure 7J , Figure 7K , Figure 7L CD8 in tumor tissues of C57 / BL6 mice inoculated with PDAC subcutaneously were detected by ISH in the control group, AhR inhibitor CH223191, PRMT5 inhibitor GSK3326595 alone, and the combination of the two inhibitors. +T lymphocytes; Figure 7M for Figure 7I-7L CD8 + T lymphocyte count statistical results.

[0041] In the mouse subcutaneous tumor model, the tumor growth rate was significantly reduced after treatment with AhR inhibitor CH223191 and PRMT5 inhibitor GSK3326595, and the therapeutic effect of the combination of GSK3326595 and CH223191 was significantly better than that of the two drugs alone. The tumor volume was smaller, the weight was lower, and the survival time of the mice was longer. Figure 7A-7C Histological examination also revealed that there were more CD4 in the tumor tissue of the double-drug combination treatment group (see Figure 7D-7H ) and CD8 T cell infiltration (see Figure 7I-7M ). Therefore, in the mouse subcutaneous tumor formation experiment, it was confirmed that the combination of AhR inhibitor and PRMT5 inhibitor can significantly inhibit the growth of pancreatic tumors inoculated in mice.

Claims

1. A pharmaceutical composition comprising an AhR inhibitor and a PRMT5 inhibitor.

2. The pharmaceutical composition according to claim 1, wherein the AhR inhibitor is CH223191 and the PRMT5 inhibitor is GSK3326595.

3. The pharmaceutical composition according to claim 1, wherein CH223191 is in an injection form and GSK3326595 is in an oral form.

4. Application of AhR inhibitors and PRMT5 inhibitors in the preparation of drugs for the treatment of pancreatic ductal adenocarcinoma.

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