Application of cordycepin metabolites in the preparation of drugs for treating acute pancreatitis

Abstract

This invention provides the application of cordycepin metabolites in the preparation of medicaments for treating acute pancreatitis, wherein the cordycepin metabolite is 2,3-dihydroxybenzoic acid. The application of cordycepin metabolites in the preparation of medicaments for treating acute pancreatitis provided by this invention offers a new approach to the treatment of pancreatitis, opening up new avenues for the treatment of acute pancreatitis globally, and is expected to fundamentally improve patients' quality of life and survival rates. Furthermore, 2,3-dihydroxybenzoic acid, as a metabolite of cordycepin, is derived from a natural substance, possesses high safety and tolerability, and may have fewer side effects compared to traditional treatment methods, making it more attractive for clinical application.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the use of cordycepin metabolites in the preparation of drugs for treating acute pancreatitis. Background Technology

[0002] The annual incidence of acute pancreatitis (AP) is (13–45) per 100,000 people, and approximately 20% may progress to moderately severe acute pancreatitis (MSAP) or severe acute pancreatitis (SAP). The global obesity epidemic may also contribute to the increased global incidence of acute pancreatitis.

[0003] The key cellular changes in the pathogenesis of acute pancreatitis include pathological calcium signaling, mitochondrial dysfunction, premature activation of trypsinogen in acinar and macrophage cells, endoplasmic reticulum stress, impaired unfolded protein response, and impaired autophagy. These are triggered by common acinar cytotoxic agents such as alcohol, nicotine, and bile acids. Intraductal events, such as increased pressure due to pancreatic duct obstruction, luminal acidification, and exposure of pancreatic duct cells to bile acids, can also indirectly trigger these events. The interaction between acinar cells and the immune system perpetuates the inflammatory response. At the local level, the regulatory role of intrapancreatic and peripancreatic fat saponification and mesenteric lymph under ischemic conditions in the severity of acute pancreatitis is well-established. Despite the heavy global disease burden, there are currently no effective drugs to treat or prevent acute pancreatitis. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the purpose of this invention is to provide the application of cordycepin metabolites in the preparation of drugs for treating acute pancreatitis, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0005] The first aspect of the present invention provides the use of cordycepin metabolites in the preparation of medicaments for treating acute pancreatitis, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0006] In one embodiment, the drug has at least one of the effects of (1) to (3):

[0007] (1) Inhibits serum pancreatic amylase activity;

[0008] (2) Inhibits lipase activity;

[0009] (3) Inhibit the expression of anti-inflammatory factors.

[0010] A second aspect of the present invention provides the use of cordycepin metabolites in the preparation of products that inhibit serum pancreatic amylase activity, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0011] A third aspect of the present invention provides the use of cordycepin metabolites in the preparation of products that inhibit lipase activity, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0012] The fourth aspect of the present invention provides the use of cordycepin metabolites in the preparation of products that inhibit the expression of anti-inflammatory factors, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0013] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: The application of the cordycepin metabolites provided by the present invention in the preparation of drugs for treating acute pancreatitis provides a new idea for the treatment of pancreatitis, opens up new avenues for the treatment of acute pancreatitis worldwide, and is expected to fundamentally improve the quality of life and survival rate of patients; In addition, 2,3-dihydroxybenzoic acid, as a metabolite of cordycepin, is derived from natural substances, has high safety and tolerability, and may have fewer side effects compared with traditional treatment methods, making it more attractive in clinical applications. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. In the drawings:

[0015] Figure 1 Figure showing the results of amylase level detection in mice with acute pancreatitis induced by crotonin treatment;

[0016] Figure 2 Figure showing the results of lipase level detection in mice with acute pancreatitis induced by crotonin treatment;

[0017] Figure 3 Figure showing the results of detecting tumor necrosis factor α levels in mice with acute pancreatitis induced by crotonin treatment;

[0018] Figure 4 Figure showing the results of interleukin-6 level detection in mice with acute pancreatitis induced by cordycepin treatment;

[0019] Figure 5 Figure showing the results of interleukin 1β level detection in mice with acute pancreatitis induced by crotonin treatment;

[0020] Figure 6Figure showing the results of interleukin-18 level detection in mice with acute pancreatitis induced by crotonin treatment;

[0021] Figure 7 Figure 1 shows the HE staining results of pancreatic tissue in a mouse model of acute pancreatitis induced by taurine after different treatments.

[0022] Figure 8 Figure showing the results of transforming growth factor β level detection in acinar cell supernatant after different treatments;

[0023] Figure 9 Figure showing the results of α-interferon levels in acinar cell supernatant after different treatments;

[0024] Figure 10 Figure showing the results of gamma interferon levels in acinar cell supernatant after different treatments;

[0025] Figure 11 Figure 1 shows the results of interleukin-6 level detection in acinar cell supernatant after different treatments;

[0026] Figure 12 Figure showing the results of interleukin-1β level detection in acinar cell supernatant after different treatments;

[0027] Figure 13 Figure showing the results of interleukin-10 levels in acinar cell supernatant after different treatments;

[0028] Figure 14 Figure showing the results of amylase level detection in mice with acute pancreatitis induced by cordycepin and sodium taurine.

[0029] Figure 15 The image shows the results of lipase level detection in mice with acute pancreatitis induced by cordycepin and sodium taurine.

[0030] Figure 16 Figure showing the results of detecting tumor necrosis factor α levels in mice with acute pancreatitis induced by cordycepin and sodium taurine.

[0031] Figure 17 Figure showing the results of interleukin-6 level detection in mice with acute pancreatitis induced by cordycepin and sodium taurine.

[0032] Figure 18 Figure showing the results of interleukin 1β level detection in mice with acute pancreatitis induced by cordycepin and sodium taurine.

[0033] Figure 19 Figure showing the results of interleukin-18 level detection in mice with acute pancreatitis induced by cordycepin treatment with sodium taurocholate;

[0034] Figure 20 The figure shows the results of metabolomics analysis of a mouse model of acute pancreatitis induced by taurine.

[0035] Figure 21 Figure showing the results of differential gene expression detection of cordycepin and its metabolites;

[0036] Figure 22 The image shows the results of differential gene expression detection for taurine, 2,3-dihydroxybenzoic acid and 2,6-dihydroxybenzoic acid.

[0037] Figure 23 Figure 1. Results of interleukin-6 level detection in acinar cells after 24 hours of treatment with sodium taurocholate for cordycepin and its metabolites.

[0038] Figure 24 Figure 1. Results of tumor necrosis factor α level detection 24 hours after treatment of acinar cells with sodium taurocholate for cordycepin and its metabolites.

[0039] Figure 25 Figure showing the interleukin-1β level detection results 24 hours after treatment of acinar cells with cordycepin and its metabolites with sodium taurine. Detailed Implementation

[0040] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several changes and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0041] Acute pancreatitis (AP) is a common acute abdominal condition characterized by acinar necrosis and localized or systemic inflammatory responses. AP is closely associated with multi-organ damage and increased mortality; therefore, preventing disease progression is paramount in its treatment. Despite the development of numerous treatments for AP over the past few decades, no specific drug or effective target has yet emerged. Therefore, developing treatments for AP is a priority.

[0042] In 1950, Cunningham et al. first isolated cordycepin, the main active ingredient, from Cordyceps militaris. Cordycepin is the first discovered natural derivative of adenosine, appearing as needle-like or plate-like white crystals. Its structural formula is similar to that of adenosine, and its molecular formula is C1. 10 H 13 N5O3 has a molecular weight of 251.24. Unlike adenosine, cordycepin lacks a 3'-hydroxyl group in its ribose moiety, which is replaced by hydrogen, forming a deoxy analogue of adenosine.

[0043] Cordycepin has multiple intracellular targets and can act on multiple signaling pathways. Cordycepin exhibits broad therapeutic potential in anti-tumor, anti-inflammatory, anti-oxidative stress, and hypoglycemic and hypolipidemic effects. Current research on cordycepin mainly focuses on its anti-cancer, anti-inflammatory, antioxidant, and immunomodulatory effects. A 2019 study by Fu Xiaobing et al. showed that in terms of antioxidant and anti-inflammatory effects, on the one hand, cordycepin upregulates the expression of cellular antioxidant proteases and inhibits the generation of radiation-induced reactive oxygen species (ROS), reducing oxidative stress-induced cell damage; on the other hand, cordycepin can effectively prevent and treat radiation-induced ulcer damage and inhibit inflammation and aging through the NRF2 and AMPK dual pathways, demonstrating significant clinical development value. Cordycepin also has a significant effect in treating metabolic inflammation. By targeting intestinal barrier integrity and gut microbiota, cordycepin alleviates metabolic inflammation in mice fed a Western diet, inhibiting intestinal inflammation, oxidative stress damage, reducing intestinal epithelial cell apoptosis and pyroptosis, and significantly improving intestinal barrier damage caused by a Western diet. Meanwhile, cordycepin exerts a significant protective effect against hepatic steatosis, inflammation, liver damage and fibrosis in metabolically stressed mice by activating the AMP-activated protein kinase signaling pathway, and improves non-alcoholic steatohepatitis.

[0044] Cordycepin is clearly a valuable natural medicinal ingredient. However, it still has certain limitations in its application. Because its structure is similar to adenosine, cordycepin is rapidly metabolized into 3'-deoxyinosine by adenosine deaminase (ADA) after entering the body. 3'-Deoxyinosine is an inactive metabolite of cordycepin. Studies have shown that when cordycepin is administered to rats orally and intravenously, the pharmacokinetic results show a rapid decrease in blood cordycepin levels, indicating that cordycepin has a short elimination half-life and high clearance rate. The unfavorable pharmacokinetic characteristics of cordycepin severely limit its further application. Therefore, researching methods to slow down its deamination reaction to improve its bioavailability and efficacy, and expand its application scope, is of great significance.

[0045] This invention utilizes taurine to construct cell and animal models of acute pancreatitis, validating the anti-inflammatory effect of cordycepin on acute pancreatitis. Simultaneously, we also verified the inhibitory effect of cordycepin on pancreatic necrosis in sodium taurine-induced severe acute pancreatitis. Based on the above, we further utilized metabolomics to analyze the mechanism of action and differential metabolite changes in acute pancreatitis after cordycepin treatment. GSEA gene set enrichment analysis showed that taurine, 2,3-dihydroxybenzoic acid, and 2,6-dihydroxybenzoic acid were differentially expressed endogenous metabolites of cordycepin. Compared with the control group, the levels of these three metabolites increased after cordycepin treatment. These data indicate that the metabolites in mice with acute pancreatitis changed before and after cordycepin treatment.

[0046] Furthermore, this invention validated the effects of cordycepin and its metabolites in a cell model of acute pancreatitis induced by crotonin. By detecting typical biomarkers of acute pancreatitis, the results demonstrated that 2,3-dihydroxybenzoic acid exhibited superior therapeutic efficacy.

[0047] The first aspect of the present invention provides the use of cordycepin metabolites in the preparation of medicaments for treating acute pancreatitis, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0048] In one embodiment, the drug has at least one of the effects of (1) to (2):

[0049] (1) Inhibits serum pancreatic amylase activity;

[0050] (2) Inhibits lipase activity;

[0051] (3) Inhibit the expression of anti-inflammatory factors.

[0052] A second aspect of the present invention provides the use of cordycepin metabolites in the preparation of products that inhibit serum pancreatic amylase activity, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0053] A third aspect of the present invention provides the use of cordycepin metabolites in the preparation of products that inhibit lipase activity, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0054] The fourth aspect of the present invention provides the use of cordycepin metabolites in the preparation of products that inhibit the expression of anti-inflammatory factors, wherein the cordycepin metabolites are 2,3-dihydroxybenzoic acid.

[0055] The present invention will be further illustrated by the following examples.

[0056] Example

[0057] An acute pancreatitis mouse model induced by crotonin was established. Crotonin (20-100 μg / kg, commonly 50 μg / kg) was administered intraperitoneally seven times at one-hour intervals. Different drug treatments were given after the final model establishment. The cordycepin treatment group received an intraperitoneal injection of cordycepin (200 mg / kg) after the final model establishment, while the modeling group received an equal volume of saline after the final model establishment. The sham-operated group received no treatment, and the cordycepin control group received an intraperitoneal injection of cordycepin (200 mg / kg).

[0058] Twelve hours later, serum was collected for ELISA testing. In this example, the ELISA test was performed according to the ELISA kit from Daktronics Corporation and the instructions from Shanghai Enzyme-Linked Biotechnology Co., Ltd. The levels of amylase ( ) in mouse serum were measured. Figure 1 ), lipase level ( Figure 2 ), levels of the inflammatory cytokine tumor necrosis factor α ( ) Figure 3 ), interleukin-6 level ( Figure 4 ), interleukin-1β level ( Figure 5 ) and interleukin-18 levels ( Figure 6 Specific operating steps: Before use, equilibrate the kit at room temperature for 30 minutes; Blank wells: Do not add sample, only add colorimetric reagents A and B and stop solution for zeroing; Standard wells: Add 100 μL of diluted standard to each well, then add 50 μL of biotin antigen working solution; Zero wells: Add 100 μL of standard / sample diluent, then add 50 μL of biotin antigen working solution; Sample wells: Add 100 μL of sample, then add 50 μL of biotin antigen working solution; Gently shake, cover with sealing film, and incubate at 37°C for 90 minutes; Dilute the 50-fold concentrated wash buffer 50 times with distilled water for later use; First wash: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with wash buffer, let stand for 60 seconds and then discard, repeat this 4 times, pat dry; Add 100 μL Add TMB to the zero, standard, and sample wells, gently shake, cover with sealing film, and incubate at 37°C for 30 min; Second wash: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with washing solution, let stand for 60 seconds, then discard, repeat this 4 times, and pat dry; Color development: Add 100 μL of colorimetric reagent to each well, gently shake to mix, and develop at 37°C for 10 min; Termination: Add 100 μL of stop solution to each well to terminate the reaction; Measurement: Zero the sample with a blank well and measure the absorbance of each well at 450 nm. In the acute edematous pancreatitis group, the increase in serum pancreatic amylase was positively correlated with the severity of pancreatitis. Intraperitoneal injection of crotonin significantly induced increases in amylase, lipase, and pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, interleukin, and interleukin-18 in mice, while cordycepin treatment significantly inhibited the increase in amylase, lipase, and pro-inflammatory factor levels. Figures 1-6 ).

[0059] The results showed that compared with mice in the group with acute pancreatitis induced by crotonin, cordycepin significantly reduced serum amylase and lipase levels, inhibited the increase of inflammatory factors, and alleviated pancreatic edema. Cordycepin treatment had a significant protective effect against crotonin-induced acute pancreatitis. Hematoxylin-eosin staining of pancreatic tissue also demonstrated that cordycepin could reduce pancreatic damage. Figure 7 ).

[0060] In this embodiment, the therapeutic effect of cordycepin was also verified at the cellular level. Figures 8-13 An acute pancreatitis model was established in MPC-83 mice by inducing pancreatic acinar cells with cordycepin. The cordycepin control group and cordycepin treatment group were pretreated with 50 μM and 100 μM cordycepin, respectively, for 30 minutes. The blank control group and the model group were treated with the same volume of PBS solution. After 30 minutes of pretreatment, the cordycepin treatment group and the model group were treated with 10 μM cordycepin. -7Cells were treated with 1 Mol / L cordycepin for 24 hours. Equal volumes of PBS were added to the blank control group and the cordycepin control group. After 24 hours, the cell supernatant from each group was collected, centrifuged at 3000 rpm for 15 minutes at 4°C to remove particles and polymers, and the supernatant was collected for ELISA detection. In this example, the ELISA detection was performed according to the instructions of the Dako Biotechnology Co., Ltd. Results showed that cordycepin significantly reduced growth factor β (…). Figure 8 ), alpha interferon ( Figure 9 ), Gamma interferon ( Figure 10 Interleukin-6 (IL-6) Figure 11 ), interleukin-1β level ( Figure 12 It also increased the level of the anti-inflammatory factor interleukin-10 ( ), and improved the level of interleukin-10. Figure 13 Consistent with results in animals, cordycepin reduced acinar cell damage induced by taeniacin.

[0061] This embodiment also tested the therapeutic effect of cordycepin in a mouse model of severe acute pancreatitis induced by sodium taurocholate. Both the modeling group and the cordycepin treatment group received retrograde infusion of 50 μL of 3 mM sodium taurocholate (TLCS) into the pancreatic duct using a microinfusion pump. After modeling, the cordycepin treatment group received intraperitoneal injection of cordycepin (200 mg / kg), while the modeling group received an equal volume of saline. The sham-operated group underwent no surgery, and the cordycepin control group received intraperitoneal injection of cordycepin (200 mg / kg). Twelve hours later, mouse serum was collected for ELISA testing. In this embodiment, the ELISA test was performed according to the ELISA kit from Daco Biotechnology Co., Ltd. and the instructions from Shanghai Enzyme-Linked Biotechnology Co., Ltd., with the specific procedures as described above. The results also showed that cordycepin could improve pancreatic tissue necrosis induced by sodium taurocholate in severe acute pancreatitis and reduce serum amylase (…). Figure 14 ) and lipase ( Figure 15 ) levels, significantly reduced pro-inflammatory factor levels ( Figures 16-19 ).

[0062] In this study, metabolomics analysis was performed on the composition of endogenous metabolites in mice with severe acute pancreatitis induced by cordycepin treatment with sodium taurocholate. Both the modeling group and the cordycepin treatment group underwent retrograde infusion of 50 μL of 3 mM sodium taurocholate (TLCS) into the pancreatic duct using a microinfusion pump. After modeling, the cordycepin treatment group received intraperitoneal injection of cordycepin (200 mg / kg), while the modeling group received an equal volume of saline. The sham-operated group was not operated on, while the cordycepin control group received intraperitoneal injection of cordycepin (200 mg / kg). Analysis revealed that compared to the sham-operated group, the levels of metabolites such as taurine and 2,3-dihydroxybenzoic acid were increased after cordycepin treatment. Figure 20), and through differential gene expression of metabolites, enrichment was achieved in three metabolites with significant differences: taurine, 2,3-dihydroxybenzoic acid, and 2,6-dihydroxybenzoic acid. Figure 21 , Figure 22 ).

[0063] Metabolomics analysis revealed that, in a model of acute pancreatitis induced by sodium taurocholate in acinar cells, the therapeutic effects of equal concentrations of cordycepin, taurine, 2,3-dihydroxybenzoic acid, and 2,6-dihydroxybenzoic acid were detected by ELISA. In this example, the ELISA assay was performed according to the ELISA kit from Daco Biotechnology Co., Ltd. and the instructions from Shanghai Enzyme-Linked Biotechnology Co., Ltd., with the specific procedures described above. Compared with cordycepin, taurine, and 2,6-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid significantly reduced the 24-hour inflammatory factor interleukin-6 (IL-6). Figure 23 ), tumor necrosis factor α ( Figure 24 ) and interleukin 1β levels ( Figure 25 The metabolite 2,3-dihydroxybenzoic acid showed superior efficacy in the treatment of acute pancreatitis.

[0064] Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. Unless otherwise specified, the embodiments and features described in this application can be arbitrarily combined with each other.

Claims

1. The use of cordycepin metabolites in the preparation of drugs for treating acute pancreatitis, characterized in that, The cordycepin metabolite is 2,3-dihydroxybenzoic acid.

2. The use of the cordycepin metabolite according to claim 1 in the preparation of a medicament for treating acute pancreatitis, characterized in that, The drug has at least one of the effects of (1) to (3): (1) Inhibits serum pancreatic amylase activity; (2) Inhibits lipase activity; (3) Inhibit the expression of anti-inflammatory factors.

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