Use of integrin signal pathway as target in preparation of medicine for preventing or treating pancreatitis

Abstract

The present application relates to a new strategy for preventing and treating acute pancreatitis, and particularly relates to the application of integrin signal pathway as a target in the preparation of a drug for preventing or treating pancreatitis. The present application creatively finds that pancreatitis can induce the activation of integrin signal pathway in the pancreas, so the integrin signal pathway is expected to become a drug target for preventing and treating pancreatitis, and it is verified through research that the integrin antagonist can effectively alleviate the development and deterioration of acute pancreatitis, which provides a new strategy for preventing and treating pancreatitis, especially acute pancreatitis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a new strategy for the prevention and treatment of acute pancreatitis. Specifically, it relates to the application of integrin signaling pathway as a target in the preparation of drugs for the prevention or treatment of pancreatitis, and particularly to the application of integrin α5 antagonists in the preparation of drugs for the prevention or treatment of pancreatitis. Background Technology

[0002] The pancreas is the second largest digestive gland in the human body. It consists of two parts: the endocrine part and the exocrine part. The endocrine part, namely the islets of Langerhans, is distributed throughout the glandular parenchyma and produces insulin, glucagon, and other peptides to maintain glucose metabolism. The exocrine part is composed of acinar cells and releases pancreatic juice, which includes various digestive enzymes such as amylase, trypsin, collagenase, and lipase to promote nutrient digestion. Therefore, the health of the pancreas is closely related to maintaining metabolism and balance in daily life.

[0003] Pancreatitis, or inflammation of the pancreas, is one of the most common gastrointestinal diseases requiring hospitalization. Mild cases of pancreatitis can resolve on their own, requiring only hospitalization for observation. Acute pancreatitis, however, is usually more severe; severe acute pancreatitis often progresses to sepsis or multiple organ failure. Pancreatitis can also induce acinar duct metaplasia, which, combined with mutations in genes such as KRAS in pancreatic cells, can lead to more malignant pancreatic cancer. Therefore, the development of effective treatments for acute pancreatitis is urgently needed to prevent it from worsening into more serious pancreatic diseases.

[0004] However, effective treatments for relieving pancreatitis symptoms are extremely rare. Existing clinical treatments mainly include: (1) intravenous fluid infusion to replenish body fluids; (2) analgesia to relieve severe pain caused by pancreatitis; and (3) endoscopic removal of damaged pancreatic tissue cells from the pancreas. For pancreatitis caused by different reasons, the following treatments may also be included: (1) pancreatitis caused by bile duct stenosis or obstruction may require surgical unblocking of the bile duct; (2) pancreatitis caused by gallstones may require surgical removal of the gallstones; and (3) pancreatitis caused by alcohol addiction may require treatment for alcohol addiction.

[0005] Therefore, current clinical treatments for pancreatitis are mostly symptomatic, and there is an urgent need for new targets, mechanisms, drugs, and strategies for the treatment of pancreatitis.

[0006] CN115590843A discloses a composition for treating acute pancreatitis and its application, wherein the composition comprises resveratrol, emodin, and rhein. Experimental results show that the composition is comparable in efficacy to pancreatolytic agents for treating acute pancreatitis and can be used to prepare a drug for treating acute pancreatitis with controllable quality and stable efficacy.

[0007] CN112972447A discloses the application of CaMK II inhibitors in the preparation of drugs for the prevention and / or treatment of acute pancreatitis. Two clinically relevant acute pancreatitis disease models were simulated: ① intraperitoneal injection of crotonin to induce acute pancreatitis in C57BL / 6 mice; ② bile duct ligation to induce acute pancreatitis in C57BL / 6 mice. Experiments demonstrated that CaMK II participates in regulating crotonin-induced acute pancreatitis in mice and is positively correlated with disease severity. The application of the CaMK II inhibitor KN93 significantly improved the disease progression of acute pancreatitis induced by the above two models. Furthermore, KN93 significantly prolonged the survival rate of mice with bile duct ligation-induced acute pancreatitis, thus showing promise for drug development research in the treatment of acute pancreatitis.

[0008] Currently, there is still a lack of specific drugs for acute pancreatitis; and existing technologies have very limited strategies for the prevention or treatment of acute pancreatitis. Therefore, it is very meaningful to develop more strategies for the prevention or treatment of acute pancreatitis. Summary of the Invention

[0009] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new strategy for the prevention and treatment of acute pancreatitis, specifically involving the application of integrin signaling pathway as a target in the preparation of drugs for the prevention or treatment of pancreatitis, and particularly providing the application of integrin α5 antagonists in the preparation of drugs for the prevention or treatment of pancreatitis.

[0010] To achieve this objective, the present invention adopts the following technical solution:

[0011] This invention provides the application of integrin signaling pathway as a target in the preparation of drugs for the prevention or treatment of pancreatitis.

[0012] This invention creatively discovers that pancreatitis induces the activation of the integrin signaling pathway in the pancreas. Therefore, the integrin signaling pathway is expected to become a drug target for the prevention and treatment of pancreatitis. Research has verified that integrin signaling pathway antagonists, especially integrin α5 antagonists, can effectively alleviate the development and exacerbation of acute pancreatitis. Specifically, this manifests as: (1) restoring the overall morphology of the pancreas to normal; (2) significantly alleviating acinar duct metaplasia and significantly reducing the number of CK19-positive ductal metaplastic cells; (3) alleviating the abnormal angiogenesis caused by pancreatitis; and (4) alleviating the increased release of Spp1 (also known as OPN, osteopontin) induced by pancreatitis. This provides a new strategy for the prevention or treatment of pancreatitis, especially acute pancreatitis.

[0013] This invention also provides the use of integrin α5 antagonists in the preparation of medicaments for the prevention or treatment of pancreatitis.

[0014] Preferably, integrin α5 antagonists include ATN-161 (CAS No. 262438-43-7, chemical structure shown below), or a pharmaceutically acceptable salt thereof (e.g., trifluoroacetate), or a pharmaceutically acceptable derivative thereof.

[0015]

[0016] This invention repositions the pharmacological function of ATN-161, further expanding its new applications in the treatment of pancreatitis and providing potential treatment ideas and references for clinical practice.

[0017] In this invention, the drug alleviates acinar ductalization.

[0018] In this invention, the drug alleviates abnormal angiogenesis.

[0019] In this invention, the drug inhibits osteopontin expression.

[0020] In this invention, the drug inhibits the expression of intercellular adhesion molecule-1 (Icam-1) and platelet-endothelial cell adhesion molecule (Pecam1 or CD31).

[0021] Preferably, the pancreatitis is acute pancreatitis.

[0022] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0023] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

[0024] In this invention, the dosage form of the drug is any pharmaceutically acceptable dosage form.

[0025] This invention also provides the use of integrin α5 antagonists in the preparation of drugs to alleviate acinar ductalization and abnormal angiogenesis induced by acute pancreatitis.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention creatively discovers that pancreatitis induces the activation of the integrin signaling pathway in the pancreas. Therefore, the integrin signaling pathway is expected to become a drug target for the prevention and treatment of pancreatitis. Research has verified that integrin signaling pathway antagonists, especially integrin α5 antagonists, can effectively alleviate the development and deterioration of acute pancreatitis. Specifically, this manifests in the following ways: (1) restoring the overall morphology of the pancreas to normal; (2) significantly alleviating acinar duct metaplasia and significantly reducing the number of CK19-positive ductal metaplastic cells; (3) alleviating the abnormal angiogenesis caused by pancreatitis; and (4) alleviating the increased release of Spp1 (also known as OPN, osteopontin) induced by pancreatitis. This provides a new strategy for the prevention and treatment of pancreatitis, especially acute pancreatitis. Attached Figure Description

[0028] Figure 1 This is a Sirius red staining result of pancreatic tissue sections from mice in the normal control group and the model group;

[0029] Figure 2 This image shows the immunohistochemical staining results (CK19 and CD31) of pancreatic tissue sections from normal control group and model group mice.

[0030] Figure 3 This is a graph showing the quantitative expression results of CK19 in the pancreatic tissue of mice in the normal control group and the model group;

[0031] Figure 4 This is a graph showing the statistical results of blood vessel length in the pancreatic tissue of mice in the normal control group and the model group;

[0032] Figure 5 The images show the immunohistochemical staining results (Icam1, CK19, CD31) of pancreatic tissue sections from normal control group and model group mice.

[0033] Figure 6 This is a graph showing the quantitative expression results of Icam1 in the pancreatic tissue of mice in the normal control group and the model group;

[0034] Figure 7It consists of single-cell transcriptome maps at different time points, including acinar, ductal, endothelial, and islet cells (where A is the cell transcriptome grouping results of the main pancreatic cells (acini, ducts, blood vessels, islets) in the control group and pancreatitis treatment group, B is the time-labeled results of different cell groups, and C is the grouping and time-labeled results of vascular endothelial cells in the control group and pancreatitis treatment group).

[0035] Figure 8 This is a diagram showing the results of cell-cell interaction analysis based on single-cell transcriptomics.

[0036] Figure 9 This is a diagram showing the expression results of the integrin α5 receptor (encoded by the Itga5 gene) in capillaries and veins.

[0037] Figure 10 These are immunohistochemical staining results (EMCN, CK19, OPN) of pancreatic tissue sections from normal control group and model group mice;

[0038] Figure 11 This is a graph showing the quantitative expression results of Spp1 in the pancreatic tissue of mice in the normal control group and the model group;

[0039] Figure 12 The images show the Sirius red staining results of pancreatic tissue sections from mice in the control and intervention groups.

[0040] Figure 13 The images show the immunohistochemical staining results (Icam1, CK19, CD31) of pancreatic tissue sections from mice in the control and intervention groups.

[0041] Figure 14 This is a graph showing the quantitative expression results of blood vessel density, blood vessel length, Icam1, and CK19 in the pancreatic tissue of mice in the control and intervention groups.

[0042] Figure 15 Immunohistochemical staining results of pancreatic tissue sections from mice in the control and intervention groups (EMCN, CK19, OPN staining performed);

[0043] Figure 16 This is a graph showing the quantitative expression results of Spp1 in the pancreatic tissue of mice in the control and intervention groups. Detailed Implementation

[0044] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0045] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods. The reagents and materials involved in the embodiments are all commercially available. All quantitative experiments involved in the embodiments are performed in at least three replicates, and the results are averaged.

[0046] The experimental animals used in the examples were C57BL / 6 mice, purchased from Guangdong Yaokang Biotechnology Co., Ltd., aged 6-8 weeks, weighing 21-26g, and male.

[0047] The rain frog extract involved in the embodiments was purchased from MCE, model number HY-A0190; the ATN-161 involved was purchased from MCE, model number HY-13535A.

[0048] Example 1

[0049] Construction of a mouse model of acute pancreatitis induced by taurine:

[0050] (1) Construction method: The experimental animals were divided into a normal control group and a model group. Mice in the model group were injected intraperitoneally with baicalin (75 μg / kg each time, 8 times a day, 1 hour apart, for two consecutive days) to establish an acute pancreatitis model. Mice in the control group were given a strict PBS control intraperitoneal injection.

[0051] (2) Model validation:

[0052] (2.1) Staining of pancreatic tissue sections:

[0053] Methods: Mice were euthanized by cervical dislocation, and pancreatic tissue was rapidly extracted, fixed and dehydrated, and frozen sections were prepared for Sirius red staining.

[0054] The results are as follows Figure 1 As shown in the figure, compared with the control group, the amount of red collagen deposition in pancreatic tissue was significantly increased in mice with acute pancreatitis induced by taurine, and the overall pancreatic tissue connections were relatively loose.

[0055] (2.2) Immunohistochemical staining:

[0056] Methods: Mice were euthanized by cervical dislocation, and pancreatic tissue was rapidly extracted, fixed and dehydrated, and frozen sections were prepared for immunohistochemical staining of CK19 and CD31.

[0057] The results are as follows Figure 2 As shown in the figure, compared with the control group, in mice with taurine-induced acute pancreatitis, CK19+ cells were significantly increased, early acinar duct metaplasia appeared, and the density and length of CD31+ (platelet-endothelial cell adhesion molecule) vessels were also significantly increased, surrounding the newly emerging CK19+ cells. The quantitative expression results of CK19 are shown in the figure. Figure 3 As shown, the length of the blood vessel was quantitatively calculated as follows: Figure 4As shown.

[0058] (2.3) Immunohistochemical staining:

[0059] Methods: Mice were euthanized by cervical dislocation, and pancreatic tissue was rapidly extracted, fixed and dehydrated, and frozen sections were prepared for immunohistochemical staining of Icam1, CK19, and CD31.

[0060] The results are as follows Figure 5 As shown in the figure, in the control group, Icam1 (intercellular adhesion molecule-1) was expressed only in normal periductal vessels. In mice with hymenoplasmin-induced acute pancreatitis, the expression of Icam1 (intercellular adhesion molecule-1) was significantly increased. The quantitative expression results of Icam1 are shown in the figure below. Figure 6 As shown.

[0061] The above experimental results show that the mouse model of acute pancreatitis has been successfully established.

[0062] (3) Investigating the integrin signaling pathway as a pancreatitis-specific enriched signaling pathway:

[0063] (3.1) Construction of single-cell transcriptome atlas of pancreatic tissue:

[0064] Methods: Mice were euthanized by cervical dislocation, and pancreatic tissue was rapidly extracted for single-cell transcriptome analysis, including single-cell capture, library construction, sequencing, and bioinformatics analysis.

[0065] The results are as follows Figure 7 As shown, single-cell transcriptome maps of acinar, ductal, endothelial, and islet cells were successfully obtained at different time points after pancreatitis. A shows the results of cell transcriptome grouping of major pancreatic cells (acini, ducts, blood vessels, and islets) in the control and pancreatitis treatment groups. B shows the time-labeled results of different cell groups. C shows the grouping and time-labeled results of vascular endothelial cells in the control and pancreatitis treatment groups (WT represents the control group, CAE represents violarin, and D1, D7, and D28 represent days 1, 7, and 28 after violarin treatment).

[0066] (3.2) Cell-to-cell interaction analysis based on single-cell transcriptome:

[0067] Methods: Based on the single-cell transcriptome data mentioned above, Cell Chat and iTalk bioinformatics analysis software were used to identify interactions at different time points after pancreatitis, in order to obtain pancreatitis-specific interaction pathways.

[0068] The results are as follows Figure 8As shown, pancreatic ductal cells were found to release Spp1 (also known as Osteopontin, OPN) and act on integrin receptors in vascular endothelial cells (indicated by red arrows). This signaling pathway is absent or very weak in normal pancreas, but is significantly enhanced after pancreatitis and persists until 28 days after taurine injection. Expression of the integrin receptor Integrinα5 (encoded by the gene Itga5) in capillaries and veins was significantly enhanced after pancreatitis (e.g., ...). Figure 9 (As shown in the figure) (CAE represents basilin, and D1, D7, and D28 represent the 1st, 7th, and 28th days after basilin treatment).

[0069] (3.3) Immunohistochemical staining:

[0070] Methods: Mice were euthanized by cervical dislocation, and pancreatic tissue was rapidly extracted, fixed and dehydrated, and frozen sections were prepared for immunohistochemical staining of EMCN, CK19, and OPN.

[0071] The results are as follows Figure 10 As shown in the figure, compared with the control group, the release of Spp1 (also known as Osteopontin, OPN, osteopontin), a ligand of the integrin signaling pathway, was significantly increased in mice with acute pancreatitis induced by taurine. CK19-positive ductal cells showed significant co-localization with Spp1. The quantitative expression results of Spp1 are shown in the figure. Figure 11 As shown, Spp1 expression is significantly increased in the pancreas.

[0072] The above experimental results show that the integrin signaling pathway is significantly enhanced in acute pancreatitis.

[0073] Example 2

[0074] Application of integrin signaling pathway antagonists in the prevention / treatment of acute pancreatitis:

[0075] (1) Construction of a mouse model of acute pancreatitis induced by taurine: Same as in Example 1, the first day of taurine injection was recorded as the first day of the experiment.

[0076] (2) Administration method: The model mice were randomly divided into two groups, namely the control group and the intervention group, with 6 mice in each group. The control group was injected with PBS buffer (pH=7.2, once a day for two consecutive days, 100ul / mouse each time) via the tail vein on the first and second days of the experiment. The intervention group was injected with ATN-161 (once a day for two consecutive days, 200μg / 100ul / mouse each time) via the tail vein on the first and second days of the experiment.

[0077] (3) Results Analysis:

[0078] (3.1) Staining of pancreatic tissue sections:

[0079] Methods: Mice were euthanized by cervical dislocation, and pancreatic tissue was rapidly extracted, fixed and dehydrated, and frozen sections were prepared for Sirius red staining.

[0080] The results are as follows Figure 12 As shown in the figure, compared with the control group, the pancreatic tissue morphology of the intervention group mice was significantly restored and collagen deposition was reduced.

[0081] (3.2) Immunohistochemical staining:

[0082] Methods: Mice were euthanized by cervical dislocation, and pancreatic tissue was rapidly extracted, fixed and dehydrated, and frozen sections were prepared for immunohistochemical staining of Icam1, CK19, and CD31.

[0083] The results are as follows Figure 13 and Figure 14 As shown in the figure, the control group mice still had a large number of CK19-positive cells generated by acinar duct metaplasia in their pancreas. The overall expression of Icam1 (intercellular adhesion molecule-1) and CD31 (platelet-endothelial cell adhesion molecule) in the pancreas was significantly increased, and larger blood vessels appeared. In contrast, the number of CK19-positive cells in the pancreas of the intervention group mice was significantly reduced, and the overall expression of Icam1 (intercellular adhesion molecule-1) and CD31 (platelet-endothelial cell adhesion molecule) in the pancreas was significantly decreased. Quantitative calculations of vascular density, vascular length, and the expression results of CK19 and Icam1 showed that, compared with the control group, the intervention group had significantly reduced vascular density and vascular length, and significantly reduced expression levels of CK19 and Icam1.

[0084] (3.3) Immunohistochemical staining:

[0085] Methods: Mice were euthanized by cervical dislocation, and pancreatic tissue was rapidly extracted, fixed and dehydrated, and frozen sections were prepared for immunohistochemical staining of EMCN, CK19, and OPN.

[0086] The results are as follows Figure 15 As shown in the figure, compared with the control group, the number of CK19 positive cells in the pancreas of the intervention group mice was significantly reduced, and the expression of EMCN (endomucin) and Spp1 (also known as OPN, osteopontin) in the pancreas was significantly decreased. The quantitative expression results of OPN are shown in the figure. Figure 16 As shown.

[0087] The above experimental results show that integrin signaling pathway antagonists have excellent efficacy in preventing / treating acute pancreatitis.

[0088] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0089] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0090] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of integrin α5 antagonist ATN-161 in the preparation of drugs for treating acute pancreatitis.

2. The application according to claim 1, characterized in that, The drug alleviates acinar ductalization.

3. The application according to claim 1, characterized in that, The drug alleviates abnormal vascular proliferation.

4. The application according to claim 1, characterized in that, The drug inhibits osteopontin expression.

5. The application according to claim 1, characterized in that, The drug inhibits the expression of intercellular adhesion molecule-1 and platelet-endothelial cell adhesion molecule.

6. The application according to claim 1, characterized in that, The drug also contains pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, coating materials, colorants, antioxidants, antibacterial agents, or buffers. The dosage form of the drug can be any pharmaceutically acceptable dosage form.

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