Use of htra1 inhibitors in the manufacture of a medicament for treating pancreatitis-carcinoma transition

Abstract

The application discloses application of an HTRA1 inhibitor in preparation of a drug for treating pancreatitis-cancer transformation, and belongs to the technical field of medicines.The HTRA1 inhibitor is screened through a drug database, the HTRA1 inhibitor is used to treat a pancreatic cancer cell line and a mouse pancreatitis-cancer transformation in-vivo model, it is proved that the HTRA1 inhibitor Carfilzomib can inhibit the malignant phenotype of Panc1 cells in vitro, and can inhibit the transformation of a KC mouse stimulated by cerulean into a pancreatic ductal adenocarcinoma in vivo, and the application provides a basis for using the HTRA1 inhibitor as the drug for treating pancreatitis-cancer transformation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of HTRA1 inhibitors in the preparation of drugs for treating pancreatitis-cancer transformation. Background Technology

[0002] Pancreatic cancer (PC) is one of the deadliest gastrointestinal malignancies and is projected to become the second leading cause of cancer-related death by 2030. Pancreatic ductal adenocarcinoma (PDAC) primarily originates from pancreatic duct epithelium and acinar cells, accounting for over 90% of all PC cases. Most PDAC patients are diagnosed at an advanced stage due to early asymptomatic or atypical symptoms, missing the optimal window for surgical treatment. While radiotherapy and chemotherapy techniques for PDAC have been continuously updated in recent years, current clinical treatments only extend patient survival by a few months, and the 5-year survival rate remains below 10%. Therefore, clarifying the pathogenesis of PDAC is crucial for early intervention. The KRAS mutation frequency is close to 100% in PDAC patients, and KRAS mutations are present in over 95% of precancerous lesions. Although KRAS mutation is a significant driver of PDAC, it is insufficient to promote tumorigenesis in the absence of sustained environmental stress. Pancreatitis is an independent risk factor for PDAC. Acinar cells undergo acinar-ductal metaplasia (ADM) during pancreatitis. As a major form of pancreatic dysplasia, ADM exhibits oncogenic potential in the presence of KRAS mutations. Therefore, targeted blocking of persistent inflammatory stimulation is crucial for the effective prevention of KRAS mutation-induced pancreatic dysplasia-associated diabetic angina (PDAC).

[0003] High-temperature demand factor A1 (HTRA1) is the first member of the human HTRA serine protease family to be discovered. As a secreted protein, HTRA1 is involved in the degradation of the extracellular matrix. In addition to serving as a tumor marker and / or prognostic factor, HTRA1 is also closely related to tumorigenesis and development by regulating tumor cell proliferation, migration, apoptosis, and differentiation. Our previous studies found that HTRA1 is highly expressed in pancreatitis and acute dementia (ADM). Furthermore, HTRA1 protein and mRNA levels are significantly upregulated in pancreatic cancer tissues and cells, and knockdown of HTRA1 can significantly inhibit the malignant phenotype of pancreatic cancer cells in vitro and inhibit pancreatitis-cancer transformation in vivo. Therefore, HTRA1 is a key target for inhibiting pancreatitis-induced PDAC progression. Summary of the Invention

[0004] To address the problems existing in the background art, the purpose of this invention is to provide the application of HTRA1 inhibitors in the preparation of drugs for treating pancreatitis-cancer transformation. This invention screens HTRA1 inhibitors based on a drug bank, and after in vitro and in vivo toxicity and pharmacodynamic evaluation, finally screens out one effective drug for inhibiting pancreatitis-cancer transformation.

[0005] The objective of this invention is achieved through the following means:

[0006] This invention provides the use of HTRA1 inhibitors in the preparation of medicaments for treating pancreatitis-cancer transformation.

[0007] Based on the above technical solution, the HTRA1 inhibitor further includes Carfilzomib.

[0008] Based on the above technical solution, the drug further includes an effective amount of an HTRA1 inhibitor and a pharmaceutically acceptable salt / carrier.

[0009] Based on the above technical solution, the pharmaceutically acceptable salts further include sodium salts and potassium salts.

[0010] Based on the above technical solution, the pharmaceutically acceptable carriers further include fillers, diluents, binders, disintegrants, emulsifiers, and drug-carrying carriers without toxic side effects.

[0011] Based on the above technical solution, the drug is further prepared into a pharmaceutically permissible dosage form, which includes tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

[0012] Based on the above technical solution, the drug is further prepared in the form of a single-dose drug.

[0013] Based on the above technical solution, the single-dose drug further comprises 1-1000mg of HTRA1 inhibitor.

[0014] Based on the above technical solution, the drug can further reduce the number and size of pancreatic tumors, reduce high-grade PanIN lesions in the pancreas, and improve collagen deposition in pancreatic tissue.

[0015] The advantages of this invention over the prior art are as follows:

[0016] This invention uses HTRA1 inhibitors to treat pancreatic cancer cell lines and a mouse pancreatitis-cancer transformation in vivo model, demonstrating that the HTRA1 inhibitor Carfilzomib can inhibit the malignant phenotype of Panc1 cells in vitro; in vivo, it can inhibit the transformation of KC mice into pancreatic ductal adenocarcinoma stimulated by taurine. This invention provides a basis for using HTRA1 inhibitors as drugs to treat pancreatitis-cancer transformation. Attached Figure Description

[0017] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0018] Figure 1The three-dimensional structure of HTRA1 is shown in (a), and four compounds obtained through screening are shown in (b).

[0019] Figure 2 The results of cytotoxicity experiments for Pipecuronium bromide (a), Carfilzomib (b), Succinycholine chloride (c), and Choline chloride (d) are presented.

[0020] Figure 3 The results of in vitro pharmacodynamic experiments of the HTRA1 inhibitor Carfilzomib are shown, where (a): cell cycle, (b): apoptosis, (c): tumor adhesion, (d): tumor migration, and (e): tumor invasion.

[0021] Figure 4 The results of in vivo pharmacodynamic experiments of the HTRA1 inhibitor Carfilzomib are shown in the following figures: (a) pancreatic tissue appearance, (b) HE staining, (c) Alcian blue staining, and (d) Sirius red staining. Detailed Implementation

[0022] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0023] Example 1

[0024] Screening for HTRA1 inhibitors:

[0025] The structure of HTRA1 was downloaded from the RCSB PDB database, PDB ID 3NZI2. The Dock module in MOE v2015.1001 was used for receptor-based virtual screening (SBVS). HTRA1 protein was defined as a receptor. Approximately 2800 approved drug molecules were selected from Drugbank as a virtual screening library. Receptor binding sites were selected near HTRA1 residues. Finally, four compounds (Pipecuronium bromide, Carfilzomib, Succinycholine chloride, and Choline chloride) were selected based on their top-scoring scores and suitability for injection. The three-dimensional structure of HTRA1 and the structures of the four selected compounds are shown below. Figure 1 As shown.

[0026] Example 2

[0027] Toxicity test:

[0028] Panc1 cells in logarithmic growth phase were used at a density of 1 × 10⁻⁶ cells per well. 4 Cells were seeded in 96-well cell culture plates. After cell attachment, a blank control group, a control group, and a drug group were set up, with 6 replicates in each group. The blank control group was incubated with DMEM medium, the control group was incubated with DMEM medium, and the drug group was incubated with DMEM containing compounds 1-4 at final concentrations of 10, 1, 0.1, 0.01, and 0.001 μM, respectively. After 24 h of cell incubation, 10 μL of CCK8 solution was added to each well, and the cells were cultured for another 2 h. The absorbance of each well was measured at 450 nm to screen for HTRA1 inhibitors that were toxic to Panc1 cells.

[0029] Cell viability = (Drug group - Blank control group) / (Control group - Blank control group) × 100%

[0030] Cytotoxicity test results as follows Figure 2 As shown, among the four inhibitors (Pipecuronium bromide, Carfilzomib, succinycholine chloride, and choline chloride), only Carfilzomib was found to be toxic to Panc1 cells at ≥0.01 μM.

[0031] Example 3

[0032] Drug efficacy experiment:

[0033] 1. In vitro efficacy verification

[0034] 1.1 Apoptosis

[0035] Cells were treated with the minimum toxic dose for 24 h, followed by digestion with 0.25% trypsin and washing twice with PBS. Cells were then stained with 5 μL Annexin V-FITC + 5 μL PI and incubated at room temperature in the dark for 15 min. Apoptosis results were analyzed by flow cytometry.

[0036] 1.2 Cell Adhesion

[0037] Panc1 cells were treated with the minimum toxic dose for 24 h, and then pretreated with serum-free medium containing 0.5 mg / mL Matrigel matrix in 96-well plates for 2 h. The cell concentrations of the control and drug groups were adjusted to 5 × 10⁻⁶ cells / well. 5 / mL, seed 100μL / well into a 96-well plate, incubate for 3h, and then use CCK8 to detect the number of adherent cells.

[0038] 1.3 Cell Cycle

[0039] Panc1 cells were treated with the minimum toxic dose for 24 h, and then collected. The cells were washed once with PBS, fixed in 75% ethanol, and incubated overnight at 4°C. Before analysis, the cells were washed again with PBS, and then 10 μL of DNase A and 25 μL of PI were added. The cells were incubated at 37°C in the dark for 30 min. Flow cytometry was used for analysis.

[0040] 1.4 Cell migration

[0041] Panc1 cells were cultured to achieve a density of 95%–100% in 6-well plates. Cells were treated with the maximum non-toxic dose, and a scratch was made in each well using a 200 μL pipette tip. Cell migration was imaged using a phase-contrast microscope at 0, 24, and 48 h after scratching. To create a reference point for repeated imaging of cell migration, a mark was made on the bottom of the plate outside the well before the cell scratch. This method allows for imaging of the same area at all sampling times.

[0042] 1.5 Cell Invasion

[0043] Cell invasion was detected using 8 μm pore size Transwell plates. After treating Panc1 cells with the maximum non-toxic dose for 24 h, cells (5 × 10⁻⁶) were... 4 (Number of cells) were resuspended in 100 μL of serum-free medium and added to the upper chamber of the transwell. 800 μL of medium containing 10% fetal bovine serum was added to the lower chamber of the transwell. After 24 h, the infiltrating cells were fixed with 10% methanol and stained with 0.1% crystal violet. Images were taken under a microscope, and the infiltrating cells were counted. 400 μL of 33% acetic acid was added to each upper chamber of the transwell to elute the crystal violet. The eluent was transferred to a 96-well plate, and the absorbance was read at 590 nm using a microplate reader.

[0044] 2. In vivo efficacy verification

[0045] Eight KC mice were randomly divided into two groups of four each using a random number table: the KC group and the KC+drug group. Mice were injected intraperitoneally with lecithin (50 μg / kg) four times a day on Mondays, Wednesdays, and Fridays. The KC+drug group was injected intravenously with the HTRA1 inhibitor Carfilzomib (5 mg / kg / week) every Tuesday. The KC group was injected with saline as a control.

[0046] Five weeks after the start of modeling, mice were anesthetized with isoflurane to collect samples. The mice were placed in a supine position with their heads and limbs fixed to a mouse board, and their backs were elevated to fully expose the abdominal aorta. The surgical area was routinely disinfected, and the abdomen was opened along the midline. The intact pancreas was harvested, photographed, weighed, and fixed in 4% paraformaldehyde solution for histopathological observation.

[0047] The tissue testing methods are as follows:

[0048] 2.1 Hematoxylin & Eosin (HE) staining

[0049] Pancreatic tissue fixed in 4% paraformaldehyde was extracted, embedded in paraffin, cut into thin sections of approximately 5 μm, and fixed onto poly-L-lysine-treated slides. The paraffin sections were then sequentially immersed in xylene, 95%, 85%, and 75% ethanol. After hematoxylin and eosin (HE) staining, the sections were examined histopathologically under a light microscope.

[0050] 2.2 Alcian blue staining

[0051] Pancreatic tissue paraffin sections were routinely dewaxed and hydrated. The paraffin sections were stained with alexandrite blue for 15 minutes and nucleoside red for 3 minutes. They were then immersed in anhydrous ethanol and cleared with xylene. After being mounted with neutral resin, they were observed under a light microscope.

[0052] 2.3 Sirius Red Staining

[0053] Paraffin sections of pancreatic tissue were routinely dewaxed and hydrated, stained with Sirius red for 30 minutes, then immersed in anhydrous ethanol, cleared with xylene, and finally mounted with neutral resin. The sections were then observed under a light microscope.

[0054] The results of in vitro pharmacodynamic experiments are as follows Figure 3 As shown, compared with the control group, the HTRA1 inhibitor Carfilzomib significantly reduced the percentage of Panc1 cells in the G0 / G1 phase and increased the percentage of cells in the G2 / M phase. These results indicate that the HTRA1 inhibitor Carfilzomib can arrest the cell cycle of Panc1 cells in the G2 / M phase. Figure 3 a). Simultaneously, the HTRA1 inhibitor Carfilzomib can promote Panc1 cell apoptosis and inhibit its adhesion (a). Figure 3 bc), the migration and invasion abilities of Panc1 cells treated with the HTRA1 inhibitor Carfilzomib were significantly lower than those of the control group. Figure 3 de).

[0055] Results of in vivo pharmacodynamic experiments as follows Figure 4 As shown, observation of the pancreas in KC mice revealed that treatment with the HTRA1 inhibitor Carfilzomib significantly reduced tumor size. Figure 4 a) HE staining and Alcian blue staining showed that, compared with untreated KC mice, intervention with the HTRA1 inhibitor Carfilzomib significantly reduced high-grade PanIN lesions in the pancreas of KC mice. Figure 4bc), in addition, Sirius red staining revealed that intervention with the HTRA1 inhibitor Carfilzomib significantly reduced collagen deposition in the pancreatic tissue of KC mice. Figure 4 d).

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of HTRA1 inhibitors in the preparation of drugs for intervening in pancreatitis-cancer transformation, characterized in that, The HTRA1 inhibitor is Carfilzomib; The drug can reduce the number and size of pancreatic tumors, reduce high-grade PanIN lesions in the pancreas, and improve collagen deposition in pancreatic tissue.

2. The application according to claim 1, characterized in that, The drug comprises an effective amount of an HTRA1 inhibitor and a pharmaceutically acceptable salt / carrier.

3. The application according to claim 2, characterized in that, The pharmaceutically acceptable salts include sodium and potassium salts.

4. The application according to claim 2, characterized in that, The pharmaceutically acceptable carriers include fillers, binders, disintegrants, and emulsifiers.

5. The application according to claim 1, characterized in that, The drug is prepared into a pharmaceutically permissible dosage form, including tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

6. The application according to claim 1, characterized in that, The drug is prepared in single-dose form.

7. The application according to claim 6, characterized in that, The single-dose medication contains 1-1000 mg of HTRA1 inhibitor.

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