Pharmaceutical composition
By developing a pharmaceutical composition containing AXL kinase inhibitor R428 and MMP inhibitor, the problem of pancreatic cancer resistance to existing therapeutic methods has been solved, significantly inhibited the growth of pancreatic cancer tumors and improved the therapeutic effect.
Patent Information
- Application Number
- CN202411598759.4
- 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 detect early, and is less sensitive to existing chemotherapy and radiotherapy, resulting in poor prognosis in patients.
A pharmaceutical composition, including AXL kinase inhibitor R428 and MMP inhibitor, is developed to synergistically inhibit the growth of pancreatic cancer tumors by inhibiting the activity of AXL kinase and MMP protein.
This pharmaceutical composition significantly inhibits the growth of pancreatic cancer tumors and improves the effect of treating pancreatic cancer, especially when combined with AXL kinase inhibitors, which enhances the therapeutic effect on pancreatic cancer.
Smart Images
Figure CN119587700A_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 that there is a significant correlation between the shedding of receptor tyrosine kinase AXL and pancreatic cancer metastasis, and its shedding is mediated by the matrix metalloproteinase (MMP) family. Therefore, the applicant creatively discovered a new target and its composition method.
[0005] In view of this, in a first aspect, the present invention provides a pharmaceutical composition comprising:
[0006] AXL kinase inhibitor R428 or a pharmaceutically acceptable salt thereof, and
[0007] A MMP inhibitor or a pharmaceutically acceptable salt thereof.
[0008] In some preferred embodiments, the present invention provides a pharmaceutical composition comprising:
[0009] (a) a first active ingredient: a therapeutically effective amount of an AXL kinase inhibitor R428 or a pharmaceutically acceptable salt thereof; and
[0010] (b) Second active ingredient: a therapeutically effective amount of a MMP inhibitor.
[0011] 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%.
[0012] 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%.
[0013] In some preferred embodiments, the molar ratio of the first active ingredient to the second active ingredient in the composition is 0.1-9.9:9.9-0.1, more preferably, 1-5:5-1, and most preferably, 1-3:3-1.
[0014] The MMP inhibitors include interfering molecules (shRNA, siRNA, etc.) that can inhibit MMP protein expression or gene transcription, or any compound that can reduce the activity of MMP protein (such as any antibody, ligand, protein inhibitor, protein binding molecule that can inhibit the activity of MMP protein).
[0015] In some specific embodiments, the MMP inhibitor can be shRNA, or siRNA.
[0016] Specifically, the siRNA may be as shown in the following SEQ ID NOs. 1 to 4.
[0017]
[0018] Specifically, the shRNA may be as shown in the following SEQ ID NOs. 5 to 12.
[0019]
[0021] In some specific embodiments, the MMP inhibitor may be a compound that reduces the activity of MMP protein, such as anti-MMP antibodies, MMP inhibitor Marimastat, MMP inhibitor MMI-270, MMP inhibitor MDI-301.
[0022] In some preferred embodiments, the MMP protein is MMP1, MMP9, MMP11 and MMP15. Preferably, the MMP protein is MMP1 and MMP11.
[0023] In some specific embodiments, the MMP inhibitor can be a broad-spectrum inhibitor, or a non-broad-spectrum inhibitor.
[0024] In a specific embodiment, the MMP inhibitor is an MMP1 inhibitor, an MMP9 inhibitor, an MMP11 inhibitor, and an MMP15 inhibitor. In a preferred embodiment, the MMP inhibitor is an MMP1 inhibitor and an MMP11 inhibitor.
[0025] The MMP1 inhibitor, MMP9 inhibitor, MMP11 inhibitor and MMP15 inhibitor are specific inhibitors of MM1, MMP9, MMP11 and MMP15, respectively, but not broad-spectrum inhibitors.
[0026] In some preferred embodiments, the pharmaceutically acceptable salts include but are not limited to hydrochlorides, sulfates, and the like.
[0027] In some specific embodiments, the present invention provides a pharmaceutical composition, comprising the pharmaceutical composition as described above, and a pharmaceutically acceptable carrier.
[0028] 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.
[0029] Taking the MMP 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 MMP 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 dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 500 mg / kg body weight per day.
[0030] A second aspect of the present invention provides a medicine kit, comprising:
[0031] (a) a first formulation comprising a therapeutically effective amount of an AXL kinase inhibitor R428 or a pharmaceutically acceptable salt thereof;
[0032] (b) a second formulation comprising a therapeutically effective amount of a MMP inhibitor or a pharmaceutically acceptable salt thereof; and
[0033] Optionally, (c) instructions for use.
[0034] In some preferred embodiments, the instructions for use indicate that the first preparation and the second preparation are used in combination to prevent and / or treat cancer.
[0035] In some preferred embodiments, the first agent and the second agent are administered simultaneously, separately or sequentially in the prevention and / or treatment of cancer.
[0036] In some preferred embodiments, the first formulation and the second formulation are independent of each other.
[0037] In some preferred embodiments, the first formulation and the second formulation are combined.
[0038] The third aspect of the present invention provides a use of a MMP inhibitor for preparing a drug for treating and / or preventing cancer.
[0039] In a preferred embodiment, the cancer is pancreatic cancer. More preferably, the cancer is pancreatic ductal adenocarcinoma.
[0040] 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.
[0041] In a preferred embodiment, the cancer is pancreatic cancer. More preferably, the cancer is pancreatic ductal adenocarcinoma.
[0042] 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.
[0043] In a preferred embodiment, the cancer is pancreatic ductal adenocarcinoma.
[0044] Further, the administration dosage is 10 mg / Kg-300 mg / Kg, preferably, 10 mg / Kg-100 mg / Kg, and more preferably, 10 mg / Kg-50 mg / Kg.
[0045] The present invention creatively finds that when the activity of MMP protein is inhibited, the therapeutic effect of AXL kinase inhibitor R428 is better, that is, AXL kinase inhibitor R428 and MMP inhibitor have a synergistic effect in treating pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0046] Figure 1 The results of 118 proteases that were significantly changed between tumor and normal samples;
[0047] Figure 2 The result is that MMP family proteases are upregulated in tumors;
[0048] Figure 3 The ES result diagram is a Western blot (WB) to verify that MMP1 and MMP11 are involved in regulating AXL;
[0049] Figure 4 is the relationship between the mRNA expression level of MMP1 in organoids and the synergistic effect. The larger the ΔAUC value, the better the synergistic effect.
[0050] Figure 5 is the relationship between the mRNA expression level of MMP11 in organoids and the synergistic effect. The larger the ΔAUC value, the better the synergistic effect.
[0051] Figure 6 The sensitivity of tumor cells with stable knockdown of MMP1 or 11 to R428 in the clonogenic assay;
[0052] Figure 7 for Figure 6 The statistical analysis results of
[0053] Figure 8 MMP inhibitors inhibit the shedding of the extracellular domain of AXL;
[0054] Figures 9-10 To verify that sAXL binds to and terminates GAS6 signaling by immunoblotting and PRM;
[0055] Fig.11 Schematic diagram of the validation of the synergistic effect between AXL tyrosine kinase inhibitor R428 and MMP inhibitors for organoids;
[0056] Fig.12 The drug action curves of 66 organoids;
[0057] Fig.13 Validate the synergistic effect of R428 and MMP inhibitors for xenograft tumor models;
[0058] Fig.14 The synergistic effect of R428 and MMP inhibitors was verified in an orthotopic tumor model. DETAILED DESCRIPTION
[0059] 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.
[0060] Example 1: Samples used in the present invention
[0061] Cell lines
[0062] The origin of human pancreatic cancer cell lines PANC1 (CRL-1469) and MIA PaCa2 has 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.).
[0063] Mouse
[0064] KPC (Kras) for isolating primary tumor cells to establish orthotopic implant models 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.). NSG mice were used to construct an orthotopic pancreatic ductal adenocarcinoma (PDAC) model. SCID mice were used to construct a transplant tumor model. 5-week-old NSG mice were purchased from Shanghai Southern Model Organisms Technology Development Co., Ltd. 6-week-old SCID mice were purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. 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.
[0065] Organoids
[0066] The 66 PDAC patient-derived organoids used in this study have been described in previous studies (Shi, X., Li Y., Yuan Q., Tang S., Guo S., Zhang Y., He J., Zhang X., Han M., Liu Z., et al. (2022). Integrated profiling of human pancreatic cancer organoids reveals chromatin accessibility features associated with drug sensitivity. Nature communications 13, 2169.)
[0067] Clinical samples
[0068] 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.
[0069] Example 2: MMP knockout cells are more sensitive to R428
[0070] In the tissue S-PM proteomic dataset, 118 proteases were found to be significantly altered between tumor and normal samples ( Figure 1 Of these, 23 were from the classic ADAM and matrix metalloproteinase (MMP) families, with MMP family proteases being more significantly upregulated in tumors than ADAM family proteases, suggesting that MMP family members may be more important than ADAM family members in regulating extracellular domain shedding (ES) in PDAC ( Figure 2 To explore potential proteases responsible for regulating the ES of 22 membrane proteins in tumors, we evaluated four MMPs because they were significantly upregulated in tumor tissues, especially compared with the known AXL shedding enzymes ADAM10 and ADAM17.
[0071] RNA interference sequences targeting the mRNA of four genes (MMP1, MMP9, MMP11, and MMP15) were designed and chemically synthesized (RiboBio), and the target cell lines were transfected using the Lipofectamine RNAiMax kit according to the supplier's instructions. Secretome analysis revealed that knockdown of MMP1 and MMP11 in PCC significantly reduced the ES of most of the 22 membrane proteins. We verified the involvement of MMP1 and MMP11 in regulating the ES of AXL by immunoblotting (WB). Figure 3). In summary, MMP-mediated membrane receptor shedding can generally serve as another dimension of intercellular signaling regulation. The higher the mRNA expression levels of MMP1 and MMP11, the more significant the synergistic effect of the two drugs in organoids, which is consistent with the expected regulatory effect of AXL shedding. Moreover, tumor cells with stable knockdown of MMP1 or 11 were more sensitive to R428 in the clonogenic assay, as shown in the following results. Figures 4-5 shown. Figure 6 and 7 Sensitivity of tumor cells with stable knockdown of MMP1 or 11 to R428 in clonogenic assay. Figure 6 For experimental photos, Figure 7 For statistical analysis results.
[0072] Example 3: Combination of R428 and MMP inhibitors to inhibit pancreatic cancer
[0073] The inventors treated cancer cells with the broad-spectrum MMP inhibitor Marimastat. WB analysis showed that Marimastat almost abolished AXL shedding into conditioned medium (CM) compared with whole cell lysate (WCL) ( Figure 8 Given the extremely high binding affinity between AXL and GAS6 (K d 33pM), AXL can act as a high-affinity decoy receptor to neutralize GAS6 and attenuate AXL signaling. WB and parallel reaction monitoring (PRM) analysis confirmed that when GAS6 was first incubated with PANC1 CM containing sAXL, GAS6-stimulated activation of three pTyr sites on AXL and its key signaling node AKT was almost abolished, and when CM was collected with Marimastat, activation was complemented ( Fig. 9 , 10 ). Phenotypically, the AXL kinase inhibitor R428 and MMP inhibitor, which have entered Phase II clinical trials (ClinicalTrials.gov identification code NCT03649321), showed significant synergistic effects in a 66-organoid administration experiment ( Fig.11 , 12 ) and was validated in an orthotopic tumor model in mice ( Fig.13 Similarly, co-administration of R428 and Marimastat reduced tumor growth in an orthotopic mouse model of pancreatic cancer ( Fig.14 ).
Claims
1. A pharmaceutical composition comprising: AXL kinase inhibitor R428 or a pharmaceutically acceptable salt thereof, and A MMP 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 AXL kinase inhibitor R428 or a pharmaceutically acceptable salt thereof ranges from 1% to 99%, and the content of the MMP inhibitor or a pharmaceutically acceptable salt thereof ranges from 1% to 99%.
3. The composition according to claim 2, characterized in that Based on the total weight of the composition, the content of the AXL kinase inhibitor R428 or a pharmaceutically acceptable salt thereof is 50%, and the content of the MMP inhibitor or a pharmaceutically acceptable salt thereof is 50%.
4. The composition according to any one of claims 1 to 3, characterized in that The MMP inhibitors include interfering molecules capable of inhibiting MMP protein expression or gene transcription, or any compounds capable of reducing the activity of MMP proteins.
5. The composition according to claim 4, characterized in that The MMP inhibitor is an MMP1 inhibitor or an MMP11 inhibitor.
6. The composition according to claim 4, wherein the nucleotide sequence of the interfering molecule is one or more of SEQ ID NOs. 1 to 12.
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 an AXL kinase inhibitor R428 or a pharmaceutically acceptable salt thereof; and (b) a second formulation comprising a therapeutically effective amount of a MMP inhibitor or a pharmaceutically acceptable salt thereof.
10. The medicine box according to claim 9, characterized in that: The MMP inhibitors include interfering molecules that can inhibit the expression of MMP proteins or gene transcription, or any compounds that can reduce the activity of MMP proteins.