CN116763899A - Application of HTRA1 inhibitor in preparation of medicine for treating pancreatitis-cancer transformation - Google Patents

Abstract

The invention discloses an application of an HTRA1 inhibitor in preparing a medicine for treating pancreatitis-cancer transformation, and belongs to the technical field of medicines. According to the invention, the HTRA1 inhibitor is screened through a drug database, and the HTRA1 inhibitor is adopted to treat pancreatic cancer cell lines and mouse pancreatitis-cancer transformant in vivo models, so that the HTRA1 inhibitor Carfilzomib can inhibit the malignant phenotype of Panc1 cells in vitro; the invention can inhibit the transformation of KC mice stimulated by ranpirin to pancreatic duct adenocarcinoma in vivo, and provides a basis for using HTRA1 inhibitor as a medicament for treating pancreatitis-cancer transformation.

Description

Application of HTRA1 inhibitors in the preparation of drugs for the treatment of pancreatitis-carcinoma transformation

Technical field

The invention belongs to the field of medical technology, and specifically relates to the application of HTRA1 inhibitors in the preparation of drugs for treating pancreatitis-cancer transformation.

Background technique

Pancreatic cancer (PC) is one of the most lethal gastrointestinal malignancies and will become the second leading cause of cancer-related mortality by 2030. Pancreatic ductal adenocarcinoma (PDAC) mainly originates from pancreatic duct epithelium and acinar cells, accounting for more than 90% of all PC cases. Most PDAC patients are diagnosed at an advanced stage due to early asymptomatic or atypical symptoms and miss the best opportunity for surgical treatment. In recent years, the technology of radiotherapy and chemotherapy for PDAC has been constantly updated, but the existing clinical treatment options can only prolong the survival of patients for a few months, and the 5-year survival rate of the disease is still less than 10%. Therefore, clarifying the pathogenesis of PDAC is of great significance for early intervention. The frequency of KRAS mutations in PDAC patients is close to 100%, and KRAS mutations are present in more than 95% of precancerous lesions. Although KRAS mutations are important drivers of PDAC, KRAS mutations alone are not sufficient to promote tumorigenesis in the absence of sustained environmental stress. Pancreatitis is one of the independent risk factors for PDAC. Acinar cells undergo acinar-ductal metaplasia (ADM) during pancreatitis, and as the main form of pancreatic atypical hyperplasia, ADM exhibits oncogenic potential in the presence of KRAS mutations. Therefore, targeted blockade of ongoing inflammatory stimuli is crucial to effectively prevent KRAS mutation-induced PDAC.

High temperature requirement factor A1 (HTRA1) is the first discovered member of the human HTRA serine protease family. 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 the occurrence and development of tumors by regulating the proliferation, migration, apoptosis and differentiation of tumor cells. Our previous studies found that HTRA1 is highly expressed during pancreatitis and ADM. In addition, HTRA1 protein and mRNA levels are significantly up-regulated in pancreatic cancer tissues and cells. 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 to inhibit the progression of pancreatitis-induced PDAC.

Contents of the invention

In order to solve the problems existing in the background technology, the purpose of the present invention is to provide the application of HTRA1 inhibitors in the preparation of drugs for the treatment of pancreatitis-cancer transformation. The present invention screens HTRA1 inhibitors based on the drug database (Drugbank). and pharmacodynamic evaluation, and finally screened out an effective drug that inhibits pancreatitis-cancer transformation.

The object of the present invention is achieved in the following ways:

The present invention provides the use of HTRA1 inhibitors in the preparation of drugs for treating pancreatitis-cancer transformation.

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

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

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

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

Based on the above technical solution, further, the medicine is prepared into pharmaceutically acceptable dosage forms, including tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

Based on the above technical solution, further, the medicine is prepared in the form of a single dose medicine.

Based on the above technical solution, further, the single dose medicine contains 1-1000 mg HTRA1 inhibitor.

Based on the above technical solution, further, 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.

The beneficial effects of the present invention compared with the prior art are as follows:

The present invention uses HTRA1 inhibitors to treat pancreatic cancer cell lines and mouse pancreatitis-cancer transformation in vivo models, proving that the HTRA1 inhibitor Carfilzomib can inhibit the malignant phenotype of Panc1 cells in vitro; in vivo, it can inhibit the KC cells stimulated by cerulein. The mice transform into pancreatic ductal adenocarcinoma, and the present invention provides a basis for using HTRA1 inhibitors as drugs for treating pancreatitis-carcinoma transformation.

Description of drawings

In order to explain the embodiments of the present invention more clearly, the drawings involved in the embodiments will be briefly introduced below.

Figure 1 shows the three-dimensional structure of HTRA1 (a) and the four compounds obtained through screening (b).

Figure 2 shows the cytotoxicity experimental results of Pipecuronium bromide (a), Carfilzomib (b), Succinycholine chloride (c), and Choline chloride (d).

Figure 3 shows the results of in vitro efficacy experiments of HTRA1 inhibitor Carfilzomib, where (a): cell cycle, (b): apoptosis, (c): tumor adhesion, (d): tumor migration, (e): Tumor invasion.

Figure 4 shows the results of the in vivo efficacy experiment of the HTRA1 inhibitor Carfilzomib, where (a): appearance of pancreatic tissue, (b): HE staining, (c): Alcian blue staining, (d): Sirius red staining.

Detailed ways

The present invention will be described in detail below with reference to the examples, but the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some of the embodiments of the present invention. For those skilled in the art, without paying any attention to Under the premise of creative labor, other similar embodiments can be obtained and all fall within the protection scope of the present invention.

Example 1

Screening of HTRA1 inhibitors:

The structure of HTRA1 was downloaded from the RCSB PDB database with the PDB ID 3NZI2. The Dock module in MOE v2015.1001 is used for receptor-based virtual screening (SBVS). The HTRA1 protein is defined as a receptor. About 2,800 approved drug molecules are selected from Drugbank as a virtual screening library. Receptor binding sites are selected near the residues of HTRA1. Finally, 4 of the top 10 scores are selected and suitable for injection administration. Compounds (Pipecuronium bromide, Carfilzomib, Succinycholine chloride, Choline chloride), the three-dimensional structure of HTRA1 and the structures of the four compounds obtained through screening are shown in Figure 1.

Example 2

Toxicity test:

Panc1 cells growing in the logarithmic phase were inoculated into a 96-well cell culture plate at 1×10 ⁴ cells per well. After the cells adhered, a blank control group, a control group and a drug group were set up, with 6 duplicate wells in each group. The blank control group was 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 incubating the cells for 24 hours, Add 10 μL of CCK8 solution to each well, continue to culture for 2 hours, measure the absorbance of each well at 450 nm, and screen out HTRA1 inhibitors that are toxic to Panc1 cells.

Cell survival rate = (drug group-blank control group)/(control group-blank control group)×100%

The results of the cytotoxicity experiment are shown in Figure 2. Among the four inhibitors (Pipecuronium bromide, Carfilzomib, succinycholine chloride, choline chloride), only Carfilzomib was toxic to Panc1 cells at ≥0.01 μM.

Example 3

Drug efficacy experiment:

1. In vitro drug efficacy verification

1.1 Apoptosis

The cells were treated with the minimum toxic dose for 24 hours, and then the Panc1 cells were digested with 0.25% trypsin and washed twice with PBS solution. Then stain with 5 μL Annexin V-FITC + 5 μL PI, and incubate at room temperature in the dark for 15 min. Apoptosis results were analyzed using flow cytometry.

1.2 Cell adhesion

Panc1 cells were treated with the minimum toxic dose for 24 h, and a 96-well plate was pretreated with serum-free medium containing 0.5 mg/mL Matrigel matrix for 2 h. The cell concentration of the control group and the drug group was adjusted to 5×10 ⁵ /mL, and 100 μL/well was inoculated into a 96-well plate. After incubation for 3 hours, CCK8 was used to detect the number of adherent cells.

1.3 Cell cycle

Panc1 cells were treated with the minimum toxic dose for 24 hours, collected, washed once with PBS, fixed in 75% ethanol, and kept overnight at 4°C. Before analysis, cells were washed again with PBS, then 10 μL DNase A and 25 μL PI were added, and incubated at 37°C in the dark for 30 min. Analysis was performed using flow cytometry.

1.4 Cell migration

Culture Panc1 cells to a density of 95%-100% in a 6-well plate. Use the maximum nontoxic dose to treat cells, and use a 200 μL pipette tip to leave a scratch on the cells in each well. At 0, 24 and 48 h after scratching, cell migration was photographed using a phase contrast microscope. 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 cell scratching. This way the same area can be photographed at all sampling times.

1.5 Cell invasion

Cell invasion was detected using 8 μm pore size transwell plates. After treating Panc1 cells with the maximum nontoxic dose for 24 hours, resuspend the cells (5 × 10 ⁴ cells) in 100 μL of serum-free medium and add it to the upper chamber of the transwell, and add 800 μL of medium containing 10% fetal bovine serum to the lower chamber of the transwell; After 24 h, the infiltrated cells were fixed with 10% methanol and stained with 0.1% crystal violet. Take images under a microscope and count the invading cells. Add 400 μL of 33% acetic acid to the upper chamber of each transwell to elute crystal violet. Transfer the eluate to a 96-well plate and read the absorbance value at 590 nm using a microplate reader.

2. In vivo drug efficacy verification

Eight KC mice were randomly divided into 2 groups according to the random number table method, with 4 mice in each group: KC group and KC+drug group. The mice were intraperitoneally injected with cerulein (50 μg/kg) four times a day every Monday, Wednesday, and Friday. The KC+ drug group was injected with the HTRA1 inhibitor Carfilzomib, 5 mg/kg/week, through the tail vein every Tuesday; the KC group was injected with normal saline as a control.

Experimental mice were anesthetized with isoflurane five weeks after the start of modeling to collect samples. The mice were placed in a supine position, with their heads and limbs fixed on mouse boards respectively, and their backs were elevated to fully expose the abdominal aorta. The surgical area was routinely disinfected, and a laparotomy was performed along the midline of the abdomen. The complete pancreas was photographed, weighed, and fixed in 4% paraformaldehyde solution for histopathological observation.

The tissue detection methods are as follows:

2.1 Hematoxylin-Eosin staining (Hematoxylin&Eosin, HE)

Pancreatic tissue fixed in 4% paraformaldehyde was removed, embedded in paraffin, cut into tissue slices of approximately 5 μm, and fixed on polylysine-treated glass slides. The paraffin sections were immersed in xylene, 95%, 85%, and 75% ethanol in sequence. The sections were stained with HE and then observed histopathologically under a light microscope.

2.2 Alcian blue staining (Alcian blue)

Paraffin sections of pancreatic tissue were routinely dewaxed and hydrated. The paraffin sections were stained with Alcian blue staining solution for 15 minutes and Nuclear Fast Red staining solution for 3 minutes, and then immersed in absolute ethanol in sequence. After xylene became transparent, neutral gum was added dropwise to seal the sections. Observe under a light microscope.

2.3 Sirius red staining (Sirius red)

Paraffin sections of pancreatic tissue were routinely dewaxed and hydrated, stained with Sirius red staining solution for 30 minutes, and then immersed in absolute ethanol. After xylene made them transparent, neutral gum was added dropwise to seal the sections. Observe under a light microscope.

The results of the in vitro efficacy experiment are shown in Figure 3. Compared with the control group, the HTRA1 inhibitor Carfilzomib can significantly reduce the percentage of Panc1 cells in the G0/G1 phase and increase the percentage of cells in the G2/M phase. These results indicate that HTRA1 inhibits The agent Carfilzomib can arrest the cell cycle of Panc1 cells in the G2/M phase (Figure 3a). At the same time, the HTRA1 inhibitor Carfilzomib can promote Panc1 cell apoptosis and inhibit their adhesion (Figure 3b-c). The migration and invasion abilities of Panc1 cells treated with the HTRA1 inhibitor Carfilzomib were significantly lower than those in the control group (Figure 3d-e).

The results of the in vivo drug efficacy experiment are shown in Figure 4. Observation of the appearance of the pancreas of KC mice showed that the tumor size was significantly reduced after treatment with the HTRA1 inhibitor Carfilzomib (Figure 4a). HE staining and Alcian blue staining showed that compared with untreated KC Compared with mice, the high-grade PanIN lesions in the pancreas of KC mice were significantly reduced after intervention with the HTRA1 inhibitor Carfilzomib (Figure 4b-c). In addition, Sirius red staining showed that the intervention with the HTRA1 inhibitor Carfilzomib could significantly reduce the number of PanIN lesions in the pancreas tissue of KC mice. Collagen deposition (Fig. 4d).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features can be equivalently replaced; and these modifications or substitutions do not deviate from the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention. scope.

Claims (9)

1. Application of HTRA1 inhibitors in the preparation of drugs for treating pancreatitis-cancer transformation. 2. The application according to claim 1, wherein the HTRA1 inhibitor includes Carfilzomib. 3. The application according to claim 1 or 2, characterized in that the drug includes an effective amount of HTRA1 inhibitor and a pharmaceutically acceptable salt/carrier. 4. Application according to claim 3, characterized in that the pharmaceutically acceptable salts include sodium salts and potassium salts. 5. Application according to claim 3, characterized in that the pharmaceutically acceptable carrier includes fillers, diluents, binders, disintegrants, emulsifiers and drug carriers without toxic side effects. 6. Application according to claim 1, characterized in that the medicine is prepared into pharmaceutically acceptable dosage forms, and the dosage forms include tablets, granules, oral liquid preparations, drops, injection preparations and capsules. preparation. 7. The use according to claim 1, characterized in that the medicament is prepared in the form of a single dose medicament. 8. Application according to claim 7, characterized in that the single dose medicine contains 1-1000 mg HTRA1 inhibitor. 9. The application according to claim 1, characterized in that 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.