Application of loganin and / or hydroxygenkwanin in preparation of EGFR (epidermal growth factor receptor) inhibitor for treating inflammatory bowel disease

Abstract

The invention discloses application of loganin and / or hydroxygenkwanin in preparation of an EGFR (epidermal growth factor receptor) inhibitor for treating inflammatory bowel diseases. Molecular docking experiments find that loganin and hydroxygenkwanin can be specifically bound with EGFR protein, the binding energy is lower than-5kcal / mol, and the loganin and hydroxygenkwanin can inhibit EGFR protein expression, block downstream RAS / ERK signal channels, relieve intestinal inflammation and repair mucosal barriers. Animal experiments show that in a DSS-induced UC mouse model, the pathological manifestation of a mouse can be remarkably improved by administration of loganin, and the effect of a 100mg / kg dose group is remarkably superior to that of a positive drug control. The two natural small molecule compounds are used for preparing the EGFR inhibitor for treating the inflammatory bowel disease for the first time, a low-toxicity and high-efficiency brand-new scheme is provided for IBD treatment, the problems of drug resistance and toxic and side effects of existing drugs are hopefully solved, and wide clinical application prospects are achieved.

Description

Technical Field

[0001] This invention belongs to the field of natural small molecule compound application technology, specifically relating to the application of loganin and / or hydroxygenistein in the preparation of EGFR inhibitors for the treatment of inflammatory bowel disease. Background Technology

[0002] Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that influences cell proliferation, differentiation, migration, and survival by regulating multiple downstream signaling pathways. In inflammatory bowel disease (IBD), EGFR overactivation promotes abnormal proliferation of intestinal epithelial cells, release of inflammatory factors, and damage to the mucosal barrier, which is closely related to the pathological process of IBD. Existing EGFR inhibitors, such as gefitinib and osimertinib, generally suffer from drug resistance and significant toxic side effects, limiting their clinical application. IBD is a chronic inflammatory bowel disease, and existing treatments (such as glucocorticoids, immunosuppressants, and biologics) have significant side effects, unstable efficacy, or high costs, necessitating novel, highly effective, and low-toxicity treatment options. Loganin is a cyclohexene ether terpene natural compound, mainly derived from plants in the Cornaceae family. Current research focuses on its metabolic regulation and analgesic effects, but it remains unclear whether it can target EGFR to treat IBD. Chinese patent CN202211291695 proposes that loganin derivatives can be used to treat diabetic nephropathy by inhibiting α-glucosidase and alleviating renal oxidative stress. Hydroxygenin is a methoxyflavonoid compound isolated from *Daphne genkwa*. Recent studies have gradually revealed its anti-tumor potential, but mechanistic exploration is limited, and it is unclear whether it can target EGFR to treat IBD. Apigenin, also a flavonoid, has been shown to inhibit EGFR phosphorylation, suggesting that hydroxygenin may have similar potential. Patent CN201810558941 reports that hydroxygenin has a significant inhibitory effect on various tumor cells, but its application in the treatment of IBD has not yet been discussed. Currently, no published literature or patents reveal that loganin, hydroxygenin, or a combination thereof can target and inhibit EGFR to treat IBD. The purpose of this invention is to reveal the potential of loganin and hydroxygenin as EGFR inhibitors for the treatment of IBD, and to provide a novel, highly effective, and low-toxicity treatment regimen. Summary of the Invention

[0003] Addressing the shortcomings of existing technologies, the present invention aims to provide the application of loganin and / or hydroxygenistein in the preparation of EGFR inhibitors for the treatment of inflammatory bowel disease (IBD), offering a novel and highly effective treatment regimen using natural small molecule compounds to overcome the problems of drug resistance and toxic side effects associated with existing treatments. Through in-depth research and extensive experiments, this invention is the first to discover that loganin and hydroxygenistein can specifically target EGFR, inhibit its protein expression, and block the downstream RAS / ERK signaling pathway, thereby effectively reducing intestinal inflammation and repairing the mucosal barrier, providing a completely new approach and method for the treatment of IBD. The technical solution of this invention is innovative and practical, significantly improving the pathological state of IBD and possessing significant clinical application value.

[0004] The technical solution adopted in this invention is as follows: The application of loganin and / or hydroxygenistein in the preparation of EGFR inhibitors for the treatment of inflammatory bowel disease, wherein the EGFR inhibitors specifically inhibit EGFR protein expression and block the downstream RAS / ERK signaling pathway, thereby reducing intestinal inflammation and repairing the mucosal barrier, and are used for the treatment of inflammatory bowel disease.

[0005] To better realize the present invention, the EGFR inhibitor is further used to treat EGFR-overexpressing diseases, especially inflammatory bowel disease.

[0006] To better realize the present invention, the EGFR-overexpressing disease further includes inflammatory bowel disease, which includes ulcerative colitis.

[0007] A combination therapy comprising therapeutically effective amounts of loganin and hydroxygenistein, and a pharmaceutically acceptable carrier, characterized in that the weight ratio of said loganin and hydroxygenistein is 1:1 to 1:5, and said therapeutically effective amount is 50 to 200 mg of loganin and 10 to 50 mg of hydroxygenistein per kilogram of body weight.

[0008] Terminology Explanation: abbreviations Full English name Full Chinese name EGFR epidermal growth factor receptor Epidermal growth factor receptor RAS Rat sarcoma c-ras expression product of proto-oncogene ERK Extracellular regulated protein kinases Extracellular regulated protein kinases HGK Hydroxyenkwanin Hydroxygenazoline LOG Loganin Strychnoside DSS Dextran sulfate sodium salt Sodium dextran sulfate UC Ulcerative colitis Ulcerative colitis IBD Inflammatory bowel disease Inflammatory bowel disease CM Conditioned medium Conditional culture medium Beneficial effects

[0009] The beneficial effects of this invention are as follows: 1. This study reveals for the first time the application of loganin and / or hydroxygenistein as EGFR inhibitors in the treatment of inflammatory bowel disease (IBD), providing a novel treatment option using natural small molecule compounds and broadening the application prospects of EGFR inhibitors in the treatment of IBD.

[0010] 2. Loganin and hydroxygenistein of the present invention can specifically inhibit EGFR protein expression and block the downstream RAS / ERK signaling pathway, thereby effectively reducing intestinal inflammatory response, repairing the mucosal barrier, and significantly improving the pathological state of IBD, and have significant therapeutic effects.

[0011] 3. Compared with existing drugs for treating IBD, the pharmaceutical composition of the present invention has advantages such as low toxicity and side effects and high efficacy, which can overcome the problems of drug resistance and large toxic side effects of existing drugs, and provide patients with a safer and more effective treatment option.

[0012] 4. The combination therapy of the present invention further improves the therapeutic effect by optimizing the ratio and dosage of loganin and hydroxygenistein, providing a more promising drug combination scheme for the clinical treatment of IBD. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the docking of loganin and hydroxygenistein with the EGFR protein.

[0014] Figure 2 This study investigated the regulatory effects of loganin on EGFR, RAS, ERK, and p-ERK.

[0015] Figure 3 This describes the regulatory effects of hydroxygenistein on EGFR, RAS, ERK, and p-ERK.

[0016] Figure 4 Pathological characteristics of UC mice treated with loganin: (A) Changes in mouse body weight; (B) DAI score; (CD) Colon length; (E) Spleen index. (Note: *Compared with the DSS group.)

[0017] Figure 5 To illustrate the regulatory effects of loganin on EGFR, p-ERK, and ERK. Detailed Implementation

[0018] The following will clearly and completely describe the concept, specific structure, and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features, and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. The quantitative experiments in the following embodiments are all set up with three replicate experiments, and the data are the average of the three replicate experiments or the average ± standard deviation. Example

[0019] Molecular docking experiment To verify whether loganin and hydroxygenkwanin can specifically bind to EGFR protein and thus exert their therapeutic effect, we employed molecular docking technology. Loganin and hydroxygenkwanin were selected for molecular docking experiments with EGFR protein, respectively. Docking prediction was performed using AutoDockVina software, and the docking results were visualized using Pymol software. The docking results showed that both loganin and hydroxygenkwanin exhibited good binding interactions with EGFR protein, with binding energies both below -5 kcal / mol. Loganin binds to EGFR protein at an energy of -6.573 kcal / mol, forming covalent bonds with amino acid residues such as SER-720, LYS-745, THR-854, and ASN-842 on the EGFR protein. Hydroxygenoside binds to EGFR protein at an energy of -8.681 kcal / mol, forming covalent bonds with amino acid residues such as MET-793 and ASN-842 on the EGFR protein (see Table 1). This indicates that loganin and hydroxygenoside can specifically bind to EGFR protein, providing a molecular basis for their therapeutic effect on IBD by targeting EGFR.

[0020] Table 1. Results of the docking of LOG and MOR with EGFR Molecular Calculated binding energy (kcal / mol) Covalent bonds Loganin -6.573 SER-720, LYS-745, THR-854, ASN-842 Hydroxygenkwanin -8.681 MET-793, ASN-842 Example

[0021] Regulatory effects of loganin on the EGFR / RAS / ERK signaling pathway To further verify whether loganin regulates the EGFR / RAS / ERK signaling pathway, we used Western blotting (WB). Human colon cancer cells (Caco2) were cultured to 70-80% confluence and treated with different concentrations of loganin. After 24 hours of treatment, cellular proteins were lysed using RIPA lysis buffer, separated by SDS-PAGE gel electrophoresis, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 hours, then incubated overnight at 4°C with the corresponding primary antibody. After washing with TBST, HRP-labeled secondary antibody was added, and the membrane was incubated at room temperature for 2 hours. Finally, the membrane was developed using ECL in a darkroom, and X-ray images were taken. The grayscale values ​​of the images were analyzed using GAPDH as an internal control, and compared with a Ctrl control group. The experimental results showed that in the conditioned medium (CM) induced cell model, the expression levels of EGFR, RAS and p-ERK proteins were significantly increased. The treatment with loganin was able to inhibit the abnormal activation of this pathway by downregulating the expression of EGFR, indicating that loganin has a significant regulatory effect on the EGFR / RAS / ERK signaling pathway (see Figure 2). Example

[0022] Regulatory effects of hydroxygenistein on the EGFR / RAS / ERK signaling pathway Using the same method as in Example 2, the regulatory effect of hydroxygenistein on the EGFR / RAS / ERK signaling pathway was examined. The results showed that in the CM-induced cell model, hydroxygenistein could also inhibit the abnormal activation of the EGFR / RAS / ERK signaling pathway by downregulating EGFR expression, thereby exerting an inhibitory effect on intestinal inflammation (see Example 2). Figure 3 This result further confirms that hydroxygenistein, like loganin, has the potential to target EGFR for the treatment of IBD, providing more sufficient experimental evidence for the application of this invention. Example

[0023] The therapeutic effect of loganin in a DSS-induced UC mouse model To simulate the complex pathological and signal transduction environment in the body, we established a mouse model of ulcerative colitis (UC) induced by DSS (dextrose sulfate) to experimentally verify the therapeutic effect and mechanism of action of loganin. Wild-type UC model mice were randomly divided into several treatment groups, which were treated by gavage with 50 mg / kg, 100 mg / kg, and 200 mg / kg of loganin, respectively, with 5-aminosalicylic acid as a positive control. After a period of treatment, the pathological manifestations of the mice in each treatment group were observed. The experimental results showed that all treatment groups had a certain alleviating effect on the pathological manifestations of the mice. The treatment effect of the 100 mg / kg loganin medium-dose group (LOG-M) was particularly significant. The mice in this group showed significant recovery of body weight, a significant reduction in disease activity index (DAI), an increase in colon length, and a significant decrease in spleen coefficient (see Figure 4). This indicates that loganin has a good therapeutic effect in the DSS-induced UC mouse model, and within a certain dose range, its therapeutic effect is better than that of the positive drug control group, further demonstrating the effectiveness and feasibility of loganin as an EGFR inhibitor for the treatment of IBD. Example

[0024] Regulatory effects of loganin on EGFR-related pathways in UC mouse model Building upon Example 4, to further investigate the regulatory role of loganin on EGFR-related pathways in a UC mouse model, we used Western blotting to detect the expression levels of EGFR and p-ERK proteins in mouse tissues. The experimental methods were the same as before. The results showed that in DSS-induced IBD model mice, loganin significantly downregulated EGFR expression and effectively inhibited the activation of the EGFR / RAS / ERK signaling pathway. This indicates that loganin exerts its anti-inflammatory and mucosal barrier repair effects by regulating EGFR and its downstream related signaling pathways, thereby alleviating DSS-induced intestinal inflammation and barrier damage (see Figure 5). This result further supports the innovation and efficacy of loganin as an EGFR inhibitor in the treatment of IBD in this invention at the molecular mechanism level. Example

[0025] Comparative experiments with existing drugs To more intuitively demonstrate the advantages of the pharmaceutical composition of this invention, we conducted a comparative experiment with existing drugs for treating IBD. In a DSS-induced UC mouse model, mice were treated with the combination therapy of this invention (loganin 100 mg / kg + hydroxygenistein 30 mg / kg), 5-aminosalicylic acid (positive control), and placebo, respectively. The results showed that the combination therapy of this invention was superior to 5-aminosalicylic acid in improving pathological manifestations, reducing intestinal inflammation, and repairing the mucosal barrier in mice, and no obvious toxic side effects were observed during the treatment. This further demonstrates the significant advantages and clinical application potential of the pharmaceutical composition of this invention in the treatment of IBD. Example

[0026] Treatment efficacy for Crohn's disease and radiation enteropathy For Crohn's disease models Animal model establishment: An experimental Crohn's disease mouse model was selected, and intestinal inflammatory features similar to those of human Crohn's disease were induced through specific modeling methods.

[0027] Dosage regimen: Loganin and hydroxygenistein were administered to the model mice by gavage according to the ratio and dosage of the combined therapeutic drug described in this invention (loganin 100 mg / kg and hydroxygenistein 30 mg / kg), while a placebo control group was set up.

[0028] Observation indicators: Observe the clinical symptoms of mice, including changes in body weight and disease activity index (DAI) scores.

[0029] Dissect mice and observe the pathological morphological changes in intestinal tissue.

[0030] Detect the levels of inflammatory factors associated with Crohn's disease in intestinal tissue.

[0031] Experimental Results: The results showed that, compared with the control group, the treatment group did not show significant improvements in body weight change, DAI score, degree of intestinal pathological damage, or inflammatory factor levels. This indicates that the EGFR inhibitor described in this invention has no significant therapeutic effect on Crohn's disease model mice.

[0032] For radiation enteropathy model Animal model establishment: A radiation enteropathy mouse model was used. The pathological changes of radiation enteropathy were induced by irradiating specific parts of the mouse intestine with radiation.

[0033] Dosage regimen: The same combination of loganin and hydroxygenistein (100 mg / kg loganin and 30 mg / kg hydroxygenistein) was administered for intervention, with a placebo control group.

[0034] Observation indicators: Observe the mice's survival status, weight changes, and other general conditions.

[0035] Examine the extent of damage to intestinal tissues, including typical pathological features of radiation enteropathy such as mucosal damage and vascular lesions.

[0036] Detect the expression levels of biomarkers associated with radiation enteropathy.

[0037] Experimental results: The results showed that there were no significant differences between the treatment group and the control group in the above-mentioned observation indicators, indicating that the EGFR inhibitor described in this invention did not show a significant therapeutic effect on the radiation enteropathy model mice.

[0038] In summary, this invention, through multi-level studies including molecular docking experiments, cell experiments, animal experiments, and comparative experiments with existing drugs, fully validates the application value of loganin and hydroxygenistein as EGFR inhibitors in the treatment of inflammatory bowel disease (IBD). They can specifically target EGFR, inhibit its protein expression, and block downstream signaling pathways, thereby effectively reducing intestinal inflammation and repairing the mucosal barrier. This provides a novel and significantly advantageous treatment option of natural small molecule compounds for IBD, particularly ulcerative colitis, and is expected to overcome many shortcomings of existing therapeutic drugs, meeting the urgent clinical need for novel, highly effective, and low-toxicity IBD treatments.

[0039] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of loganin and / or hydroxygenistein in the preparation of EGFR inhibitor drugs for the treatment of inflammatory bowel disease, characterized in that, The application of loganin and / or hydroxygenistein in the preparation of EGFR inhibitors, which specifically inhibit EGFR protein expression and block the downstream RAS / ERK signaling pathway, thereby alleviating intestinal inflammation and repairing the mucosal barrier, and are used for the treatment of inflammatory bowel disease.

2. The use of loganin and / or hydroxygenistein according to claim 1 in the preparation of EGFR inhibitor drugs for the treatment of inflammatory bowel disease, characterized in that, The EGFR inhibitors are used to treat diseases with high EGFR expression, and are particularly suitable for treating inflammatory bowel disease.

3. The use of loganin and / or hydroxygenistein according to claim 2 in the preparation of EGFR inhibitor drugs for the treatment of inflammatory bowel disease, characterized in that, The EGFR-overexpressing diseases include inflammatory bowel disease, which includes ulcerative colitis.

4. A combination therapy comprising therapeutically effective amounts of loganin and hydroxygenistein, and a pharmaceutically acceptable carrier, characterized in that, The weight ratio of loganin and hydroxygenistein is 1:1 to 1:5, and the therapeutically effective dose is 50 to 200 mg of loganin and 10 to 50 mg of hydroxygenistein per kilogram of body weight.

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