Application of VNN1 inhibitors in the preparation of drugs for chronic pancreatitis

Abstract

The present invention discloses the use of a VNN1 inhibitor in the preparation of a drug for treating chronic pancreatitis, and belongs to the field of medical technology. The present invention screens 10 VNN1 inhibitors through a drug database, uses VNN1 inhibitors to treat an in vitro pancreatic acinar cell injury model, and proves that the VNN1 inhibitor Capmatinib can significantly inhibit the release of ROS in vitro, and can reduce pancreatic damage and fibrosis induced by chronic pancreatitis in vivo, and can be used in the preparation of drugs for treating chronic pancreatitis. The present invention provides a basis for using VNN1 inhibitors as drugs for treating chronic pancreatitis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the application of a VNN1 inhibitor in the preparation of a medicine for treating chronic pancreatitis. Background Art

[0002] Chronic pancreatitis (CP) is a complex, chronic, progressive digestive disease characterized by pancreatic acinar cell damage, persistent inflammation, and irreversible pancreatic fibrosis. It is estimated that the incidence of CP is 4-14 cases per 100,000 people per year, and the prevalence is 120-143 cases per 100,000 people. At the same time, CP is considered a risk factor for pancreatic cancer (PC). Within five years after the diagnosis of CP, the risk of patients suffering from PC increases by as much as eight times. At present, the main clinical treatments for CP are surgery or symptomatic supportive treatment. Active treatment can relieve symptoms, but these methods cannot restore pancreatic function or reverse the process of pancreatic fibrosis. It is difficult to cure it, and patients may die of complications in the late stage. As a proinflammatory mediator, reactive oxygen species (ROS) are one of the important reasons for the persistence of CP. It can cause damage to pancreatic acinar cells and activation of stellate cells, leading to fibrosis. Therefore, inhibiting the production of ROS is of great significance in slowing down the occurrence and development of CP.

[0003] Vascular non-inflammatory protein 1 (VNN1) is a pantothenate hydrolase that can hydrolyze pantothenate to generate pantothenic acid and cysteamine through the dissimilatory pathway of CoA. In addition, VNN1 limits the synthesis of reduced glutathione (GSH) by inhibiting glutamylcysteine ​​synthetase (γ-GCS), which can lead to untimely clearance of ROS and their accumulation, resulting in oxidative stress. Inhibition of VNN1 can reduce the degree of damage caused by oxidative stress. Therefore, VNN1 may be a key target in the treatment of CP. Summary of the invention

[0004] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide the use of VNN1 inhibitors in the preparation of drugs for the treatment of chronic pancreatitis. The present invention screens VNN1 inhibitors based on the drug database (FDA), and finally screens out a drug that effectively inhibits ROS release by targeted inhibition of VNN1, thereby treating chronic pancreatitis through in vitro and in vivo toxicity and pharmacodynamics evaluation.

[0005] The object of the present invention is to achieve the following:

[0006] The present invention provides use of a VNN1 inhibitor in preparing a medicine for treating chronic pancreatitis.

[0007] Based on the above technical solution, further, the VNN1 inhibitor includes Capmatinib.

[0008] Based on the above technical solution, further, the drug comprises an effective amount of a VNN1 inhibitor and a pharmaceutically acceptable salt / carrier.

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

[0010] Based on the above technical solution, further, the pharmaceutically acceptable carrier includes a filler, a diluent, a binder, a disintegrant, an emulsifier and a drug-carrying carrier without toxic side effects.

[0011] Based on the above technical solution, further, the drug is prepared into pharmaceutically acceptable dosage forms, including tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

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

[0013] Based on the above technical solution, further, the single-dose drug contains 1-1000 mg VNN1 inhibitor.

[0014] Based on the above technical solution, further, the drug can accelerate the removal of reactive oxygen species (ROS) and reduce ROS in pancreatic acinar cells by promoting the synthesis of γ-GCS and GSH in acinar cells.

[0015] The beneficial effects of the present invention compared with the prior art are as follows:

[0016] The present invention uses a VNN1 inhibitor to treat an in vitro mouse acinar cell injury model and an in vivo mouse CP model, and proves that the VNN1 inhibitor Capmatinib can promote the synthesis of γ-GCS and GSH of acinar cells and inhibit the generation of ROS in vitro, and reduce CP-induced acinar cell damage and pancreatic fibrosis in vivo. The present invention provides a basis for using the VNN1 inhibitor as a drug for treating chronic pancreatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0018] Figure 1 The three-dimensional structure of VNN1 (a) and the structural formulas of the top 20 screened 10 compounds suitable for injection (b).

[0019] Figure 2 The cytotoxicity test results of 10 compounds suitable for injection for screening the Top 20, compared with the control group (Ctrl), *P<0.05, **P<0.01.

[0020] Figure 3 Effects of VNN1 inhibitors Indacaterol, Capmatinib, Ezetimibe, Nafitifine, Bictegravir, and Haloperidol on intracellular GSH (A) and γ-GCS (B). Compared with the model group (STC), **P<0.01.

[0021] Figure 4 The flow cytometry graphs show the effects of VNN1 inhibitors Capmatinib, Ezetimibe, and Haloperidol on intracellular ROS production. Compared with the model group (STC), *P<0.05, **P<0.01.

[0022] Figure 5 The effects of VNN1 inhibitors Capmatinib, Ezetimibe and Haloperidol on serum ALT, AST and BUN in CP mice.

[0023] Figure 6 The pathological effects of VNN1 inhibitors Capmatinib, Ezetimibe and Haloperidol on the heart, spleen, lung, kidney, brain and intestinal tissues of CP mice.

[0024] Figure 7 To investigate the effects of VNN1 inhibitors Capmatinib, Ezetimibe and Haloperidol on VNN1 protein expression and pancreatic damage in CP mice. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0026] Example 1

[0027] Screening of VNN1 inhibitors: The crystal structure of human VNN1 protein (the structure of VNN1 was downloaded from the RCSB PDB database, PDB code: 4CYG) was used as the receptor, and the inhibitor ligand in the crystal structure 4CYG was used as the positive control molecule. A three-dimensional similarity search was performed on about 2,600 molecules in the FDA-listed drug library to obtain similar molecules, and the similar molecules were docked with the receptor protein. The top 100 molecules with docking scores were taken as hit compounds. Finally, 10 compounds with the top 20 scores and suitable for injection were selected (DB05039: Indacaterol, DB11791: Capmatinib, DB04794: Bifonazole, DB06589: Pazopanib, DB01012: Cinacalcet, DB00973: Ezetimibe, DB00735: Naftifine, DB11799: Bictegravir, DB00481: Raloxifene, DB00502: Haloperidol).

[0028] The three-dimensional structure of VNN1 and the structures of the 10 compounds obtained by screening are shown in Figure 2. Figure 1 shown.

[0029] Example 2

[0030] Toxicity experiment: 266-6 mouse pancreatic acinar cell line growing in logarithmic phase was cultured at 1×10 4 The cells were inoculated in a 96-well cell culture plate. After the cells adhered to the wall, a blank control group, a control group and a drug group were set up, with 10 replicate wells in each group. The blank control group was incubated with DMEM medium, the control group was incubated with DMEM medium, the drug group was incubated with DMEM medium prepared with a final concentration gradient of 10 μM, 1 μM, 100 nM, 10 nM, and 1 nM for Indacaterol, Pazopanib, Cinacalcet, Ezetimibe, Naftifine, Raloxifene, and Haloperidol, and the cells were incubated with DMEM medium prepared with a final concentration gradient of 5 μM, 0.5 μM, 50 nM, 5 nM, and 0.5 nM for Capmatinib, Bifonazole, and Bictegravir. After 24 hours, 10 μL of CCK8 solution was added to each well, and the culture was continued for 1 hour. The absorbance of each well was measured at 450 nm to evaluate the in vitro cytotoxicity of the 10 VVN1 inhibitors.

[0031] Cell survival rate = (drug group - blank control group) / (control group - blank control group) × 100%.

[0032] The results of cytotoxicity experiments were Figure 2As shown, the maximum non-toxic concentrations of the 10 inhibitors are: Indacaterol (1μM), Capmatinib (5μM), Bifonazole (1nM), Pazopanib (100nM), Cinacalcet (100nM), Ezetimibe (1μM), Naftifine (10μM), Bictegravir (500nM), Haloperidol (10μM), and Raloxifene (10nM).

[0033] Example 3

[0034] In this example, six inhibitors and concentrations with relatively low cytotoxicity were selected for pharmacodynamic experiments: Indacaterol (1 μM), Capmatinib (5 μM), Ezetimibe (1 μM), Naftifine (10 μM), Bictegravir (500 nM), and Haloperidol (10 μM).

[0035] 1. In vitro efficacy verification

[0036] 1.1 γ-GCS determination

[0037] Sodium taurocholate (STC) 80 μM was used to induce cell injury in the mouse pancreatic acinar cell line 266-6 in vitro, and the above six small molecule inhibitors were given at the maximum non-toxic dose for 24 hours, then the 266-6 cells were digested with 0.25% trypsin, washed twice with PBS solution, and centrifuged to collect the cell precipitate. The γ-GCS kit (purchased from Nanjing Jiancheng Bioengineering Institute) was used according to the process, and finally detected by microplate reader.

[0038] The results are as follows Figure 3 As shown in (B), compared with the model group (STC), VNN1 inhibitors Capmatinib, Ezetimibe and Haloperidol can significantly increase the content of intracellular γ-GCS, thereby effectively clearing ROS and reducing the degree of damage caused by oxidative stress.

[0039] 1.2GSH

[0040] STC 80 μM was used to induce cell injury in the mouse pancreatic acinar cell line 266-6 in vitro, and the above six small molecule inhibitors were given at the maximum non-toxic dose for 24 hours. Then, 266-6 cells were digested with 0.25% trypsin, washed twice with PBS solution, and the cell precipitate was collected by centrifugation. GSH kit (Beijing Solebold Technology Co., Ltd.) was used according to the process and detected by microplate reader.

[0041] The results are as follows Figure 3As shown in (A), compared with the model group (STC), VNN1 inhibitors Capmatinib, EzetimibeNaftifine and Haloperidol can significantly increase the intracellular GSH content, thereby effectively clearing ROS and reducing the degree of oxidative stress damage.

[0042] 1.3ROS

[0043] STC 80 μM was used to induce cell injury in the mouse pancreatic acinar cell line 266-6 in vitro, and the above six small molecule inhibitors were given at the maximum non-toxic dose for 24 hours, then the 266-6 cells were digested with 0.25% trypsin, washed twice with PBS solution, and centrifuged to collect the cell precipitate. Finally, a ROS probe (purchased from Nanjing Jiancheng Bioengineering Institute) was used according to the process, and the ROS level was detected by flow cytometry.

[0044] The results are as follows Figure 4 As shown in the results, compared with the model group (STC), VNN1 inhibitors Capmatinib, Ezetimibe and Haloperidol could significantly reduce the level of ROS in cells and alleviate the degree of damage caused by oxidative stress.

[0045] comprehensive Figure 3-4 Based on the experimental results, Capmatinib, Ezetimibe and Haloperidol are preferred VNN1 inhibitors, and the CP mouse model is used to evaluate the in vivo toxicity and efficacy of these three VNN1 inhibitors.

[0046] Example 4

[0047] In this example, three VNN1 inhibitors, Capmatinib, Ezetimibe and Haloperidol, screened out in vitro, were selected for in vivo toxicity and pharmacodynamics experiments.

[0048] After one week of adaptation, 24 C57BL / 6 mice were randomly divided into 4 groups: model group (CP), CP+Capmatinib group, CP+Ezetimibe group, and CP+Haloperidol group (n=6) to evaluate the in vivo toxicity and efficacy of the three VNN1 inhibitors. All C57BL / 6 mice were constructed into CP model by intraperitoneal injection of caerulein, and caerulein (50μg / kg) was injected intraperitoneally on Mondays, Wednesdays, and Fridays every week, with an interval of 1h / time, 6 times a day, for 8 weeks; the three VNN1 inhibitors were administered by gavage 4 weeks after the start of CP modeling (Capmatinib, 100mg / kg / day; Ezetimibe, 1mg / kg / day; Haloperidol, 0.5mg / kg / day; all for 4 weeks); the three drugs were prepared into suspensions with 0.5% CMC-Na, and the mice in the CP group were gavaged with 0.5% CMC-Na at the same administration frequency. After 8 weeks of the experiment, C57BL / 6 mice were anesthetized with isoflurane, and blood was collected from the inner canthus and the serum was frozen in a -80°C refrigerator. Biochemical kits were then used to detect liver and kidney function indicators such as ALT, AST, and BUN in the mouse serum. Tissues such as the pancreas, heart, spleen, lung, kidney, brain, and intestine were fixed in 10% formaldehyde and stained with HE, Masson, and IHC to detect the in vivo toxicity and efficacy of the three VNN1 inhibitors.

[0049] 1. In vivo toxicity evaluation

[0050] 1.1 ALT, AST and BUN

[0051] The test results of the kit are as follows Figure 5 As shown, after 4 weeks of administration of three VNN1 inhibitors, Capmatinib (100 mg / kg / day), Ezetimibe (1 mg / kg / day), and Haloperidol (0.5 mg / kg / day), there was no significant change in the ALT, AST, and BUN levels in the mouse serum compared with the CP group.

[0052] 1.2 HE staining

[0053] HE staining results Figure 6 As shown, the heart, spleen, lung, kidney, brain and small intestine of mice in the three VNN1 inhibitor-treated groups were normal in morphology, and there was no obvious pathological damage compared with the mice in the CP group.

[0054] comprehensive Figure 5-6 The experimental results showed that oral administration of three VNN1 inhibitors, Capmatinib (100 mg / kg / day), Ezetimibe (1 mg / kg / day) and Haloperidol (0.5 mg / kg / day), had no obvious toxic side effects on C57BL / 6 mice.

[0055] 2. In vivo efficacy evaluation

[0056] 2.1IHC staining

[0057] IHC staining results Figure 7 As shown, the VNN1-positive area (brown area) in the pancreatic tissue of mice in the three VNN1 inhibitor-treated groups was smaller than that in the CP group, indicating that the expression of VNN1 protein was inhibited after the administration of VNN1 inhibitors, especially in the Capmatinib-treated group.

[0058] 2.2 HE staining and Masson staining

[0059] HE and Masson staining results are as follows Figure 7 As shown in the data, the pancreatic tissue atrophy, acinar-ductal metaplasia and acinar loss were obvious in the CP group mice. The degree of pancreatic tissue damage in the mice treated with the three VNN1 inhibitors was improved to varying degrees, and the improvement was particularly obvious with Capmatinib. This suggests that the VNN1 inhibitor Capmatinib can significantly reduce pancreatic damage and fibrosis caused by CP.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. Application of VNN1 inhibitors in the preparation of drugs for the treatment of chronic pancreatitis; The VNN1 inhibitor is Capmatinib.

2. The use according to claim 1, characterized in that: The drug comprises an effective amount of a VNN1 inhibitor and a pharmaceutically acceptable salt / carrier.

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

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

5. The use according to claim 1, characterized in that: The drug is prepared into pharmaceutically acceptable dosage forms, including tablets, granules, oral liquid preparations, drops, injection preparations and capsule preparations.

6. The use according to claim 1, characterized in that: The medicine is prepared in the form of a single-dose medicine.

7. The use according to claim 6, characterized in that: The single-dose medicine contains 1-1000 mg of VNN1 inhibitor.

8. The use according to claim 1, characterized in that: The drug can reduce the expression of VNN1 protein and the level of reactive oxygen species (ROS) in pancreatic acinar cells, thereby treating chronic pancreatitis.

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