An FFA4 receptor agonist or drug and its application

Abstract

This invention discloses the application of psoralen in the preparation of FFA4 receptor agonists and drugs for FFA4 receptor-related diseases. Cellular experiments show that this compound is a concentration-dependent FFA4 agonist; animal experiments show that this compound has significant efficacy in a DSS-induced colitis model, reducing DSS-induced colonic shortening, improving weight loss, and alleviating splenic swelling. This compound can be used in the preparation of FFA4 agonists and in the prevention and treatment of FFA4-related diseases such as inflammatory bowel disease.

Description

Technical Field

[0001] This invention relates to a novel use of psoralen as an FFA4 (Free Fatty Acid receptor 4) receptor agonist for the treatment of inflammatory bowel disease, and to its use in the pharmaceutical field. Background Technology

[0002] The FFA4 (Free Fatty Acid receptor 4) is a G protein-coupled receptor that has now been freed from orphanage (belonging to the fatty acid receptor family), also known as the GPR120 receptor. Its endogenous ligands are long-chain fatty acids, such as ALA (alpha-linolenic acid), EPA (eicosapentaenoic acid), and DHA (docosahexaenoic acid). The FFA4 receptor is expressed in adipose, pancreatic, lung, and colonic cells. Studies have shown that the FFA4 receptor is associated with diseases such as inflammatory bowel disease (GPR120 Inhibits Colitis Through Regulation of CD4+ T Cell Interleukin 10Production. Gastroenterology. 2022; 162(1):150-165). Currently, the reported structural diversity of FFA4 agonists is low. The synthesized agonists mainly fall into two categories: those with a carboxyl group and those with a sulfonamide group. Moreover, there are very few reported naturally occurring FFA4 agonists. Therefore, the discovery of novel FFA4 ligands not only has potential significance for the treatment of diseases such as inflammatory bowel, but also enriches the structural diversity of FFA4 ligands, providing a reference for future research on novel and efficient FFA4 ligands.

[0003] There are currently no reports on whether psoralen has FFA4 agonist activity or its application in the prevention and treatment of inflammatory bowel disease. Summary of the Invention

[0004] The purpose of this invention is to provide the application of psoralen in the preparation of FFA4 agonists and drugs for receptor-related diseases. Specifically, one objective of this invention is to provide that the target of psoralen is the FFA4 receptor; another objective is to provide the application of psoralen in the treatment of inflammatory bowel disease. Cellular experiments have shown that this compound is an FFA4 agonist, and animal experiments have shown that this compound can reduce colonic shortening, improve weight loss, and reduce splenic swelling in a DSS-induced inflammatory bowel disease model, providing a foundation for the development of anti-inflammatory bowel disease drugs. The specific details are as follows:

[0005] Application of psoralen in the preparation of FFA4 agonists or drugs for FFA4 receptor-related diseases.

[0006] The aforementioned FFA4-related diseases are characterized in that the diseases or symptoms are selected from inflammatory bowel disease.

[0007] The drug comprises one or more of psoralen or pharmaceutically acceptable solvates thereof as active ingredients.

[0008] The application is characterized in that the drug is a pharmaceutical composition made of psoralen as an active ingredient and a pharmaceutical carrier.

[0009] The pharmaceutical composition is characterized in that it is a tablet, capsule, granule, suspension, pellet, injection, or aerosol.

[0010] Beneficial effects of the present invention

[0011] This invention discloses the application of psoralen in the preparation of FFA4 receptor agonists and drugs for receptor-related diseases. This invention confirms that psoralen has concentration-dependent FFA4 receptor agonist activity and is an FFA4 agonist. Animal experimental models have demonstrated that psoralen has a therapeutic effect on inflammatory bowel disease and has good prospects for drug development.

[0012] Animal studies have shown that this compound has significant efficacy in a DSS-induced colitis model, reducing DSS-induced colonic shortening, improving weight loss, reducing splenic swelling, and alleviating inflammatory responses. This compound can be used in the preparation of FFA4 agonists and in the prevention and treatment of FFA4-related diseases such as inflammatory bowel disease. Attached Figure Description

[0013] Figure 1 Psoralen alpha-4 agonist effect on FFA4 receptors. Figure 1 A: Stimulation experiment, concentration-response curve of psoralen in CHO-K1-FFA4 cells; Figure 1 B: Desensitization experiment: psoralen was pretreated on CHO-K1-FFA4 cells for 1 h, and then treated with TUG891 for 1 h. Concentration-response curve. Figure 1 C: Antagonism experiment, the effect of the FFA4 antagonist AH7614 on psoralen-44 agonist signaling in CHO-K1-FFA4 cells.

[0014] Figure 2 The alleviating effect of psoralen on DSS-induced enteritis. Figure 2 A: Schematic diagram of the effect of psoralen on colon length in mice with DSS-induced ulcerative colitis; Figure 2 B: Trend graph of mouse body weight over time in each group; Figure 2 C: Trend graph of DAI score of each group of mice over time; Figure 2D: Statistical graph of colon length in each group of mice; Figure 2 E: Statistical chart of spleen weight to body weight ratio in each group of mice. Detailed Implementation

[0015] The present invention will now be further illustrated with examples. These examples are merely illustrative and not intended to limit the scope of the invention.

[0016] The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

[0017] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and biological materials described are commercially available unless otherwise specified.

[0018] Example 1: Psoralen as an agonist of FFA4

[0019] Psoralen was purchased from Chengdu Pusi Technology Co., Ltd., and Chinese hamster ovary cells (CHO-K1) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. CHO-K1-FFA4 cells were derived from transfection (the transfection process was consistent with the literature [RSC Adv. 2019; 9(26):15073-15083.]). Probe molecules TUG891 and AH7614 were purchased from TOCRIS, USA. F12 medium was purchased from Thermo Fisher Scientific, USA (catalog number: C11765500BT); fetal bovine serum (FBS) was from PAN (catalog number: ST30-3302); buffered salt HBSS was from Gibco (catalog number: 14065056); buffered salt HEPES was from Gibco (catalog number: 15630080). All compounds were dissolved in DMSO to prepare 100 mM stock solutions. The test buffer was prepared in a volume ratio of HEPES:HBSS:ultrapure water = 1:10:89, used for compound dilution during Epic experiments. The detection platform was a Corning Epic 3rd generation imager, and the detected signal was the wavelength shift caused by dynamic mass resetting (DMR).

[0020] CHO-K1-FFA4 cells in the logarithmic growth phase were seeded into... In a 384-well biosensor microplate, each well was seeded with 40 μL of cell suspension (F12 culture medium containing cells), with 15,000 cells seeded per well. The 384-well plate was then placed in a cell culture incubator (5% CO2 air, 37°C) and cultured for 24 h. Once the cell confluence reached 90%, the Epic DMR signal detection experiment could begin. Before performing the Epic detection, the culture medium in the 384-well plate was discarded, and 30 μL of test buffer (volume ratio: HEPES:HBSS:ultrapure water = 1:10:89) was added to each well containing cells.

[0021] The activation and desensitization experiments on CHO-K1-FFA4 cells were performed as follows: Psoralen was prepared into a 100mM stock solution using DMSO and diluted into a working solution using test buffer. First, 10 μL of different concentrations of psoralen working solution (320 μM, 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM) were directly added to 384-well cell culture plates containing 30 μL of test buffer, resulting in final concentrations of psoralen in each well of the 384-well cell culture plate of 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively. The signal was monitored on an Epic instrument for 1 hour. Then, 10 μL of TUG891 working solution (TUG891 was prepared as a 100 mM stock solution in DMSO and then diluted to 5 μM working solution with test buffer) was added to the above 384-well cell culture plates containing psoralen, resulting in a final concentration of 1 μM of TUG891 in the 384-well cell culture plate. The signal was monitored on an Epic instrument for another 1 hour. The results are as follows. Figure 1 As shown in Figure A, psoralen methyl ether elicits a concentration-dependent DMR response in CHO-K1-FFA4 cells and reduces the DMR signal of the FFA4 receptor agonist TUG891. Figure 1 B), and showed a concentration-dependent effect, with EC50 values ​​of 14.95±1.52μM for activation and 14.62±0.92μM for desensitization, indicating that psoralen can activate FFA4 receptors.

[0022] The antagonistic experiment on CHO-K1-FFA4 cells was conducted as follows: First, 10 μL of AH7614 working solution at different concentrations (AH7614 working solution was prepared by diluting 100 mM AH7614 stock solution with test buffer) was directly added to a 384-well cell plate containing 30 μL of test buffer, so that the final concentrations of AH7614 in the 384-well cell plate were 2000 nM, 666.67 nM, and 222.2 nM, respectively. The concentrations were 74.1 nM, 24.7 nM, 8.2 nM, and 2.7 nM. The signal was monitored for 1 hour using an Epic instrument. Then, 10 μL of psoralen working solution (100 mM psoralen stock solution was diluted to 75 μM with test buffer, and 10 μL of the working solution was added to each well of the 384-well cell plate, resulting in a final concentration of 15 μM) was added. The signal was monitored for 1 hour using an Epic instrument. The results are as follows: Figure 1 C indicates that psoralen has receptor specificity for FFA4.

[0023] The results in summary indicate that psoralen has FFA4 receptor agonist activity and is an FFA4 receptor agonist.

[0024] Example 2: The ameliorative effect of psoralen on DSS-induced ulcerative colitis

[0025] Eighteen 6-week-old male C57BL / 6J mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd. The animals were housed separately and acclimatized for one week, with free access to water and food during the experiment. The ambient temperature was 22±2℃, and the relative humidity was 55±5%, with a 12-hour light-dark cycle daily. Sodium dextran sulfate (DSS) was purchased from MP Biomedicals (catalog number: 216011080). The solvent was DMSO:castor oil polyoxyethylene ether:physiological saline = 5:5:90 (volume ratio).

[0026] Experimental grouping and establishment of animal models: Mice were randomly divided into three groups: control group, DSS modeling group, and psoralen group, with six mice in each group. At the beginning of the experiment, the control group had free access to distilled water daily; the DSS modeling group and the psoralen group had free access to distilled water containing 2% DSS daily. The psoralen group was administered 0.1 mL / 10 g (mice's body weight, in a DMSO:castor oil polyoxyethylene ether: physiological saline = 5:5:90 (volume ratio) solution via intraperitoneal injection once daily. The psoralen dosage was 10 mg / kg (mice's body weight). The control group and DSS model group were administered the same volume of solution (DMSO:castor oil polyoxyethylene ether: physiological saline = 5:5:90) via intraperitoneal injection daily as the psoralen group. Mouse body weight, fecal condition, and fecal hemorrhage were recorded daily during the experiment. At the end of the 8-day experiment, mice were sacrificed, and the colon and spleen were harvested. Colon length was measured, and spleen weight was measured. Three representative (mid-length) colons from each group were photographed. Figure 2 A).

[0027] Each mouse was weighed and its weight recorded daily. The daily weight change rate was calculated using the following formula: [(body weight - initial body weight) / initial body weight] × 100. The results showed that the control group mice experienced a slight increase in body weight, while the DSS model group mice experienced a severe decrease in body weight. The psoralen group showed a lower degree of weight loss than the DSS group. Figure 2 B) indicates that psoralen can alleviate DSS-induced weight loss. Mice in the DSS model group had loose, unformed feces, severe bloody stools, and their Disease Activity Index (DAI) scores (scoring criteria consistent with Acta Pharm Sin B. 2020; 10(3):447-461) gradually increased over time. Mice in the psoralen-treated group showed a lower DAI score compared to the DSS group. Figure 2 C) indicates that psoralen can reduce DAI scores. Colon length is an important indicator of DSS-induced enteritis. We compared the effects of different groups on colon length and found that the colon length in the psoralen-treated group was slightly restored compared to the DSS group. Figure 2 A, 2D), indicating that psoralen can alleviate DSS-induced colonic shortening. Subsequently, the spleen-to-body weight ratio was compared among the groups, showing that the psoralen group reduced the DSS-induced increase in the spleen / body weight ratio (…). Figure 2 E). The above data indicates that psoralen has the efficacy in preventing and treating enteritis and has potential for development.

Claims

1. Application of psoralen in the preparation of FFA4 agonists or drugs for FFA4 receptor-related diseases.

2. The application according to claim 1, characterized in that, FFA4 receptor-related diseases refer to inflammatory bowel disease.

3. An FFA4 agonist or drug for treating inflammatory bowel disease, characterized in that, The agonist or drug comprises one or more of psoralen or pharmaceutically acceptable solvates thereof as active ingredients.

4. The FFA4 agonist or medicament for treating inflammatory bowel disease according to claim 3, characterized in that, The FFA4 agonist or inflammatory bowel disease treatment drug is a pharmaceutical composition made of one or more of psoralen or its pharmaceutically acceptable solvates as active ingredients and a pharmaceutically acceptable carrier.

5. The drug according to claim 4, characterized in that: The pharmaceutical composition is any one of tablets, capsules, granules, suspensions, pellets, injections, or aerosols.

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