Pharmaceutical composition
By discovering new paracrine signaling pathways in pancreatic cancer and developing pharmaceutical compositions containing PDGFR, PTPN11 and LIF inhibitors, the problem of poor sensitivity to existing therapies was solved, significantly inhibiting tumor growth and prolonging survival.
Patent Information
- Application Number
- CN202411600629.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-03-11
AI Technical Summary
Pancreatic cancer, especially pancreatic ductal adenocarcinoma, is difficult to diagnose early and has poor sensitivity to existing therapies, resulting in poor prognosis.
A pharmaceutical composition has been developed, including PDGFR inhibitors, PTPN11 inhibitors and LIF inhibitors, by discovering novel paracrine signaling pathways mediated by platelet-derived growth factor receptors (PDGFR), non-receptor-type protein tyrosine phosphatase 11 (PTPN11), and leukemia inhibitors.
The pharmaceutical composition significantly inhibits the growth of pancreatic cancer tumors and significantly prolongs survival than administration alone.
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Figure CN119587701A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and specifically, relates to a pharmaceutical composition and use thereof, and more specifically, relates to a pharmaceutical composition for treating pancreatic cancer and use thereof. Background Art
[0002] Pancreatic ductal adenocarcinoma (PDAC), also known as pancreatic cancer, is one of the most deadly cancers, with a 5-year survival rate of less than 10% and a median survival time of less than 6 months. The main reason is that most pancreatic cancers are difficult to detect early, and most are already in the middle and late stages when diagnosed, missing the best time for radical surgical resection. At the same time, poor sensitivity to comprehensive treatments such as chemotherapy and radiotherapy is also an important factor leading to the high mortality rate of pancreatic cancer patients. It is expected to become the second leading cause of cancer-related deaths by 2040. The lack of reliable early diagnostic biomarkers and tenacious resistance to almost all existing therapies are the main reasons for the poor prognosis.
[0003] Therefore, there is a need in the art for a pharmaceutical composition that can effectively treat pancreatic cancer and significantly inhibit the growth of pancreatic cancer tumors. Summary of the invention
[0004] The applicant creatively discovered a new paracrine signaling pathway between pancreatic cancer tumor cells (PCC) and pancreatic cancer stellate cells (PSC) mediated by platelet-derived growth factor receptor (PDGFR), non-receptor protein tyrosine phosphatase 11 (Protein Tyrosine Phosphatase Non-Receptor Type 11, PTPN11), and leukemia inhibitory factor (leukemia inhibitory factor, LIF). Therefore, the applicant discovered a new paracrine signaling pathway and a pharmaceutical composition involving a target of the signaling pathway.
[0005] In view of this, in a first aspect, the present invention provides a pharmaceutical composition comprising:
[0006] A PDGFR inhibitor or a pharmaceutically acceptable salt thereof;
[0007] A PTPN11 inhibitor or a pharmaceutically acceptable salt thereof; and
[0008] A LIF inhibitor or a pharmaceutically acceptable salt thereof.
[0009] In some preferred embodiments, the present invention provides a pharmaceutical composition comprising:
[0010] (a) a first active ingredient: a therapeutically effective amount of a PDGFR inhibitor or a pharmaceutically acceptable salt thereof;
[0011] (b) a second active ingredient: a therapeutically effective amount of a PTPN11 inhibitor or a pharmaceutically acceptable salt thereof; and
[0012] (c) a third active ingredient: a therapeutically effective amount of a LIF inhibitor or a pharmaceutically acceptable salt thereof.
[0013] In some preferred embodiments, the content of the first active ingredient ranges from 1% to 99%, based on the total weight of the active ingredients of the composition; more preferably, 10% to 90%; more preferably, 30% to 70%.
[0014] In some preferred embodiments, the content of the second active ingredient ranges from 1% to 99%, based on the total weight of the active ingredients of the composition; more preferably, 10% to 90%; more preferably, 30% to 70%.
[0015] In some preferred embodiments, the content of the third active ingredient ranges from 1% to 99%, based on the total weight of the active ingredients of the composition; more preferably, 10% to 90%; more preferably, 30% to 70%.
[0016] In the present invention, the term "XX inhibitor" includes interfering molecules (shRNA, siRNA, etc.) that can inhibit the expression of XX protein or the transcription of XX gene, or any compound that can reduce the activity of XX protein (such as any antibody, ligand, protein inhibitor, protein binding molecule that can inhibit the activity of XX protein).
[0017] In some specific embodiments, the inhibitor can be shRNA, or siRNA.
[0018] Specifically, the siRNA may be as shown in the following SEQ ID NOs. 1 to 4:
[0019]
[0020] In some specific embodiments, the PDGFR inhibitor may be a compound that reduces the activity of PDGFR protein, such as an anti-PDGFR antibody, a PDGFR inhibitor such as any one or more of crenolanib, Sorafenib, Sunitinib, Imatinib, Pazopanib, Regorafenib, Dasatinib, Nintedanib, Orantinib, Telatinib, Motesanib, CP-673451, Toceranib, and Tyrphostin AG 1296. Preferably, the PDGFR inhibitor may be crenolanib.
[0021] In some specific embodiments, the PTPN11 inhibitor may be a compound that reduces the activity of the PTPN11 protein, such as an anti-PTPN11 antibody, a PTPN11 inhibitor such as any one or more of SHP099, Azacitidine, Sotorasib, Phosphoric acid, NSC 87877, and NSC 87877. Preferably, the PTPN11 inhibitor may be SHP099.
[0022] In some specific embodiments, the LIF inhibitor can be a compound that reduces the activity of LIF protein, such as an anti-LIF antibody, a LIF inhibitor such as any one or more of Cisplatin, Estradiol, Bupivacaine, Capsaicin, Simvastatin, Thrombin, and Daidzein. Preferably, the LIF inhibitor can be an anti-LIF antibody.
[0023] In some specific embodiments, the inhibitor can be a broad-spectrum inhibitor, or a non-broad-spectrum inhibitor.
[0024] In some specific embodiments, the antibody can be a polyclonal antibody, or a monoclonal antibody.
[0025] In some preferred embodiments, the pharmaceutically acceptable salts include but are not limited to hydrochlorides, sulfates, and the like.
[0026] In some specific embodiments, the present invention provides a pharmaceutical composition, comprising the pharmaceutical composition as described above, and a pharmaceutically acceptable carrier.
[0027] The pharmaceutical compositions of the present invention, when applied (administered) therapeutically, can provide different effects. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.
[0028] Taking the PTPN11 inhibitor of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The PTPN11 inhibitor of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 500 mg / kg body weight per day.
[0029] A second aspect of the present invention provides a medicine kit, comprising:
[0030] (a) a first formulation comprising a therapeutically effective amount of a PDGFR inhibitor or a pharmaceutically acceptable salt thereof;
[0031] (b) a second formulation comprising a therapeutically effective amount of a PTPN11 inhibitor or a pharmaceutically acceptable salt thereof; and
[0032] (c) a third formulation comprising a therapeutically effective amount of a LIF inhibitor or a pharmaceutically acceptable salt thereof;
[0033] and
[0034] Optionally, (d) instructions for use.
[0035] Further, the kit comprises: (a) a first formulation comprising a therapeutically effective amount of a PDGFR inhibitor, Gemcitabine, or a pharmaceutically acceptable salt thereof;
[0036] (b) a second formulation comprising a therapeutically effective amount of the PTPN11 inhibitor SHP099 or a pharmaceutically acceptable salt thereof; and
[0037] (c) a third formulation comprising a therapeutically effective amount of a LIF inhibitor Anti-LIF or a pharmaceutically acceptable salt thereof
[0038] In some preferred embodiments, the instructions for use indicate that the first preparation, the second preparation, and the third preparation are used in combination to prevent and / or treat cancer.
[0039] In some preferred embodiments, the first agent, the second agent, and the third agent are administered simultaneously, separately, or sequentially in the prevention and / or treatment of cancer.
[0040] In some preferred embodiments, the first formulation, the second formulation, and the third formulation are independent of each other.
[0041] In some preferred embodiments, the first formulation, the second formulation, and the third formulation are combined.
[0042] The third aspect of the present invention provides a use of a PDGFR inhibitor, a PTPN11 inhibitor, or a LIF inhibitor for preparing a drug for treating and / or preventing cancer.
[0043] In a preferred embodiment, the cancer is pancreatic cancer. More preferably, the cancer is pancreatic ductal adenocarcinoma.
[0044] The fourth aspect of the present invention provides a use of the composition of the present invention for preparing a drug for treating and / or preventing cancer.
[0045] In a preferred embodiment, the cancer is pancreatic cancer. More preferably, the cancer is pancreatic ductal adenocarcinoma.
[0046] The fourth aspect of the present invention provides a method for treating pancreatic cancer, comprising administering the above composition of the present invention to an individual.
[0047] In a preferred embodiment, the cancer is pancreatic ductal adenocarcinoma.
[0048] Further, the administration dosage of the composition is 10 mg / Kg-300 mg / Kg, preferably, 10 mg / Kg-100 mg / Kg, and more preferably, 10 mg / Kg-50 mg / Kg.
[0049] The present invention creatively discovered that PDGFs secreted by PCCs regulate the expression and secretion of LIF by PSCs by activating the PDGFR-PTPN11-ERK-c-Fos signaling pathway of PSCs, and LIF regulates the proliferation of PCCs by binding to and activating the LIFR-GP130-STAT3 pathway on PCCs. The combined administration of PDGFR inhibitors, PTPN11 inhibitors, and LIF inhibitors to pancreatic cancer transgenic mice significantly inhibited tumor growth compared to single administration. BRIEF DESCRIPTION OF THE DRAWINGS
[0050] Figure 1 Verification of the paracrine signaling pathway between PCC and PSC cells mediated by PDGFR-PTPN11-ERK-c-Fos. (a) Expression levels of PDGFRA, PDGFRB and PDGFC in multidimensional proteomic data. (b) Tyrosine phosphorylation proteomic analysis of the main pathways of signal transduction from PCCs to PSCs. (c) Immunoblotting verified that PCCs conditioned medium activated the PDGFR signaling pathway in PSCs. (d) Immunoblotting verified the PDGFR-PTPN11-ERK signaling axis. (e) PSC secretome analysis. (f) Inhibitor treatment of PSCs verified that the PDGFR-PTPN11-ERK pathway regulates LIF expression. (g,h) Verification that c-Fos is a transcription factor that regulates LIF. (i) Schematic diagram of the PDGFR-PTPN11-ERK-c-Fos signaling pathway. (j) Results of the combined drug administration experiment in the KPC mouse model;
[0051] Figure 2 PDGFR knockdown attenuates pTyr activation of PTPN11 in PSCs;
[0052] Figure 3 The weakening of the PDGFR-PTPN11-ERK signaling axis will inhibit the expression and secretion of LIF protein. DETAILED DESCRIPTION
[0053] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.
[0054] Example 1: Samples used in the present invention
[0055] Cell lines
[0056] The sources of human pancreatic cancer cell lines PANC1 (CRL-1469), MIA PaCa2, and human pancreatic stellate cell line hPSC have been described in previous studies (Shi, Y., Gao, W., Lytle, NK, Huang, P., Yuan, X., Dann, AM, Ridinger-Saison, M., DelGiorno, KE, Antal, CE, Liang, G., et al. (2019). Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring. Nature 569, 131-135.). Human pancreatic stellate cell line HPaSteC was purchased from ScienCell (California, USA). Cell culture was carried out according to the instructions of the supplier.
[0057] Mouse
[0058] KPC (Kras LSL-G12D / + ;Trp53 flox / flox ; Pdx1-Cre) mouse model is described in the literature (Shi, Y., Gao, W., Lytle, NK, Huang, P., Yuan, X., Dann, AM, Ridinger-Saison, M., DelGiorno, KE, Antal, CE, Liang, G., et al. (2019). Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring. Nature 569, 131-135.). Mice were kept in a specific pathogen-free (SPF) animal room at a temperature of 28 ° C and a humidity of 50%. All animal experiments were approved by the Experimental Animal Ethics Committee of Southern University of Science and Technology and complied with relevant policies and regulations.
[0059] Clinical samples
[0060] Human pancreatic tissue samples were purchased from Tongji Hospital affiliated to Tongji Medical College of Huazhong University of Science and Technology, approved by the Medical Ethics Committee of Tongji Hospital. A total of 46 patients were included in this study for tissue sample collection, including 30 patients with PDAC and 16 patients diagnosed with cancer whose tumors were close to the pancreas (within 5 cm) and part of the normal pancreas needed to be removed.
[0061] Example 2: Paracrine signal transduction between cancer cells and stromal cells mediated by PDGFR-PTPN11-ERK-c-Fos
[0062] Based on multidimensional functional proteomics data, it was found that PDGFRA and PDGFRB were significantly more highly expressed and highly tyrosine phosphorylated in pancreatic cancer tumor tissues than in normal tissues. Figure 1 As shown in a. Spatial proteomic analysis showed that ligand proteins mainly originated from pancreatic cancer cells (PCC), while receptor proteins mainly originated from stromal cells (PSC). After stimulating PSC with PCC conditioned medium (CM), PDGFA and PTPN11 were the most significantly activated proteins. Figure 1 As shown in b.
[0063] Then, cells or lyophilized CM were lysed in RIPA buffer containing 1% (w / v) SDS, 1% (v / v) Triton X-100, 1% (w / v) sodium deoxycholate, 1M NaCl, 50mM Tris-HCl pH 7.4, and a cocktail of protease and phosphatase inhibitors, and sonicated to further disrupt the cells. The lysate was centrifuged and the protein concentration was measured by the BCA method. A loading buffer containing 10mM DTT was added to the protein extract and heated at 95°C for 5 minutes. The proteins were separated by 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was washed with 5% PBS containing 0.1% Tween The membranes were blocked with 5% (w / v) nonfat dry milk in 20% TBS (TBST) for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: β-actin (1:5000), PDGFRA (1:1000), p-PDGFRA-Y742 (1:1000), AXL-ectodomain (1:1000), PTPN11 (1:2000), p-PTPN11-Y542 (1:5000), AKT (1:2000), p-AKT-S473 (1:2000), ERK (1:2000), p-ERK-T202 / Y204 (1:2000), E-cadherin (1:1000), N-cadherin (1:1000), snail (1:1000), and ZEB1 (1:1000). After washing three times with TBST, the membrane was incubated with HRP-labeled goat anti-rabbit IgG (1:2000) or goat anti-mouse IgG (1:2000) at room temperature for 1 h. After washing three times with TBST, Clarity Western ECL substrate was added to the membrane, and the signal was detected using a Tanon 6100C gel imaging system (Shanghai, China). By immunoblotting, PTPN11 was verified as a downstream protein of PDGFR ( Figure 1c), and regulates the PDGFR-PTPN11-ERK signaling axis ( Figure 1 d).
[0064] Through PSC secretome analysis and inhibitor treatment of PSC, it was found that this signaling pathway can regulate the expression of LIF ( Figure 1 e,f). The specific experimental process of secretory proteome identification is as follows:
[0065] The collected conditioned media was collected and centrifuged at 12,000 × g, 4 ° C for 15 minutes to remove cell debris and concentrated using a 3 kDa centrifugal filter. Centrifugation from 15 mL to 200 μL at 5,000 × g took approximately 3 hours. Wash 200 μL of concentrated conditioned media with 15 mL of 1× PBS to remove media components and exchange the buffer with 1× PBS. Transfer 200 μL of concentrated conditioned media to a 1.5 mL tube and completely dry the sample using a Speed Vac.
[0066] The protein precipitate was dissolved in 115 μL 8M urea buffer, and the protein amount was re-measured by BCA to ensure good protein recovery. 2 μL 0.25M TCEP (286.65, 0.0716625g in 1mL water) was added to reduce the protein to a final concentration of 5mM, vortexed and incubated in the dark for 30 minutes. The sample was alkylated with 3.06 μL 0.5M IAA (184.96, 0.037g in 0.4mL water) to a final concentration of 15mM, vortexed and incubated in the dark for 30 minutes. An additional 2.1 μL 0.25M TCEP was added to a final concentration of 5mM, vortexed and incubated in the dark for 15 minutes to quench IAA. The urea concentration was diluted by adding 700 μL 100mM Tris (pH 8.0) and vortexing (7 times the initial volume to reduce the urea concentration in the lysis buffer from 8.0M to 1.0M). Add 8 μL of 100 mM CaCl2 (110.98, 0.011098 g in 1 mL of water) to a final concentration of 1 mM and mix. Digest the protein with trypsin at a ratio of 1:10 (trypsin to protein) at 37°C overnight (18 hours). After digestion is complete, acidify the peptide mixture with 10% FA (final 0.1% FA) in the digestion solution, acidify it to pH 2-3 and vortex immediately to check the pH. Centrifuge at 14,000 × g for 5 minutes at 4°C or room temperature and collect the supernatant into a new test tube. Finally, desalt with Stagetip C18 and lyophilize with SpeedVac to detect the peptide concentration for mass spectrometry.
[0067] Finally, c-Fos was also verified to be a transcription factor regulating LIF ( Figure 1g,h), the specific experiments mainly include chromatin immunoprecipitation (CHIP) and promoter activity detection. Therefore, a new paracrine signaling pathway between PCC and PSC was discovered, such as Figure 1 As shown in i.
[0068] Example 3: Using siRNA to influence the expression of other proteins
[0069] HPaSteCs were transfected with siRNA targeting PDGFRA or PDGFRB in normal medium for 24 h using siRNA as shown in Table 1 below as inhibitors, followed by transfection in FBS-free medium for 24 h, and finally stimulated with PANC1 CM for 5 min, followed by cell lysis for WB analysis. The results are shown in Figure 2 When PTPN11 is knocked down, the PDGFR-PTPN11-ERK signaling axis is inhibited, which has a strong weakening effect on the expression and secretion of LIF protein, as shown in Figure 3 This indicates that when these two proteins are inhibited, LIF expression is also weakened, which in turn affects the activity of tumor cells.
[0070] Table 1
[0071] siRNAs Sequence number Sequence (5'-3') PDGFRB#1 SEQ ID NO.1 GAAAGGAGACGUCAAAUAU PDGFRB#2 SEQ ID NO.2 GGAAUGAGGUGGUCAACUU PTPN11#1 SEQ ID NO.3 GAACAUCACGGGCAAUUAA PTPN11#2 SEQ ID NO.4 GAACACUGGUGAUUACUAU
[0072] Example 4: PDGFR inhibitors, PTPN11 inhibitors, and LIF inhibitors inhibit tumor growth more significantly than when administered alone
[0073] The specific dosing regimen is as follows: For the first 12 days, Anti-LIF monoclonal antibody (manufactured by Nanjing GenScript Biotech Co., Ltd., named SC2039-PF-antibody) was given every other day (Q2D), Gemcitabine was given every two days (Q3D), and PDGFR inhibitors were given twice a week, all by intraperitoneal injection. After 12 days, Anti-LIF was given three times a week (TIW), Gemcitabine was given twice a week (BIW), and PDGFR inhibitors were given twice a week, all until the animals were moribund. Figure 1 As can be seen from j, the combined administration of PDGFR inhibitors, chemotherapy drug Gemcitabine and LIF inhibitors significantly prolonged the survival period compared with other combined administration methods, indicating that the combined administration of the three inhibitors inhibited tumor growth and prolonged survival.
Claims
1. A pharmaceutical composition comprising: A PDGFR inhibitor or a pharmaceutically acceptable salt thereof; A PTPN11 inhibitor or a pharmaceutically acceptable salt thereof; as well as A LIF inhibitor or a pharmaceutically acceptable salt thereof.
2. The composition according to claim 1, characterized in that Based on the total weight of the composition, the content of the PDGFR inhibitor or a pharmaceutically acceptable salt thereof ranges from 1% to 99%, the content of the PTPN11 inhibitor or a pharmaceutically acceptable salt thereof ranges from 1% to 99%, and the content of the LIF inhibitor or a pharmaceutically acceptable salt thereof ranges from 1% to 99%.
3. The composition according to claim 2, characterized in that The PDGFR inhibitor is any one or more of anti-PDGFR antibodies, crenolanib, Sorafenib, Sunitinib, Imatinib, Pazopanib, Regorafenib, Dasatinib, Nintedanib, Orantinib, Telatinib, Motesanib, CP-673451, Toceranib, or Tyrphostin AG1296.
4. The composition according to claim 2, characterized in that The PDGFR inhibitor is any one or more of anti-PDGFR antibodies, crenolanib, Sorafenib, Sunitinib, Imatinib, Pazopanib, Regorafenib, Dasatinib, Nintedanib, Orantinib, Telatinib, Motesanib, CP-673451, Toceranib, or Tyrphostin AG1296.
5. The composition according to claim 2, characterized in that The LIF inhibitor is any one or more of anti-LIF antibody, Cisplatin, Estradiol, Bupivacaine, Capsaicin, Simvastatin, Thrombin, or Daidzein.
6. The composition according to claim 1, characterized in that The inhibitor may be an interfering molecule that inhibits gene transcription, and the nucleotide sequence of the interfering molecule may be one or more of SEQ ID NOs. 1 to 4.
7. Use of the composition according to any one of claims 1 to 6 for preparing a medicament for treating and / or preventing cancer.
8. The use according to claim 7, characterized in that The cancer is pancreatic cancer.
9. A medicine box, comprising: (a) a first formulation comprising a therapeutically effective amount of a PDGFR inhibitor or a pharmaceutically acceptable salt thereof; (b) a second formulation comprising a therapeutically effective amount of a PTPN11 inhibitor or a pharmaceutically acceptable salt thereof; and (c) a third formulation comprising a therapeutically effective amount of a LIF inhibitor or a pharmaceutically acceptable salt thereof.
10. The medicine box according to claim 9, characterized in that: include: (a) a first formulation comprising a therapeutically effective amount of a PDGFR inhibitor, Gemcitabine, or a pharmaceutically acceptable salt thereof; (b) a second formulation comprising a therapeutically effective amount of the PTPN11 inhibitor SHP099 or a pharmaceutically acceptable salt thereof; and (c) a third formulation comprising a therapeutically effective amount of a LIF inhibitor Anti-LIF or a pharmaceutically acceptable salt thereof.