Natural FFA4 receptor agonist and application thereof

Abstract

The invention discloses a natural-source FFA4 receptor agonist and application thereof, relates to the technical field of medicines, and in particular relates to discovery of a natural compound action target spot which is a G protein coupled receptor FFA4. The FFA4 receptor agonist is medolamic acid, oleanolic acid and hederagenin and / or corresponding pharmaceutically acceptable salts, solvates or polymorphs and pharmaceutical compositions thereof, and the FFA4 receptor agonist is one of medolamic acid, oleanolic acid and hederagenin and / or pharmaceutically acceptable salts, solvates or polymorphs and pharmaceutical compositions thereof. An in-vitro cell target experiment shows that the pachymic acid, the oleanolic acid and the hederagenin are FFA4 agonists. Researches show that the FFA4 receptor is related to non-alcoholic fatty liver disease, stomach injury induced by non-steroidal anti-inflammatory drugs, osteoporosis, inflammatory bowel disease and other diseases, so that the agonist has potential medicinal application.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a natural FFA4 receptor agonist and application thereof. Background Art

[0002] FFA4 (Free Fatty Acid receptor 4) is a G protein-coupled receptor that has been deorphaned (a member of the fatty acid receptor family), also known as GPR120 receptor, and its endogenous ligands are long-chain fatty acids, such as ALA (α-linolenic acid), EPA (eicosapentaenoic acid), DHA (docosahexaenoic acid), etc. FFA4 receptors are expressed in fat, pancreas, lung and colon cells. Studies have shown that FFA4 receptors are associated with non-alcoholic fatty liver disease (G-Protein-Coupled Receptors120Agonist III Improves Hepatic Inflammation and ER Stress in Steatohepatitis.), inflammatory bowel disease (GPR120 Inhibits Colitis Through Regulation of CD4+T Cell Interleukin 10Production. Gastroenterology. 2022; 162(1): 150-165), and non-steroidal anti-inflammatory drug-induced gastric injury (Omega-3 Polyunsaturated Fatty Acids Protect NSAID-induced Gastric Injury through GPR120 Dependent Lipid Raft Preservation [C] / / Journal of Gastroenterology and Hepatology. 111 RIVER ST, HOBOKEN 07030-5774, NJ USA: WILEY-BLACKWELL, 2012, 27: 164-165.), osteoporosis (Ahn SH, Park SY, Baek JE, et al. Free fatty acid receptor 4 (GPR120) stimulates bone formation and suppresses bone resorption in the presence of elevated n-3 fatty acid levels [J]. Endocrinology, 2016, 157 (7): 2621-2635.). At present, the structural diversity of FFA4 agonists reported in the literature is relatively low. There are two main types of synthetic agonists, namely, the core structure containing carboxyl and the core structure containing sulfonyl imide. In addition, there are few reported FFA4 agonists from natural sources.Therefore, the discovery of new FFA4 ligands not only has potential significance for the treatment of inflammatory bowel diseases, but also enriches the structural diversity of FFA4 ligands, providing a reference for future research on new and efficient FFA4 ligands.

[0003] It has not been reported whether oleanolic acid, oleanolic acid and hederagenin have FFA4 activation effects. Summary of the invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a natural source FFA4 receptor agonist and its application.

[0005] The technical solution of the present invention is as follows:

[0006] A natural FFA4 receptor agonist comprises at least one of selenoic acid, oleanolic acid, hederagenin and pharmaceutically acceptable solvates thereof.

[0007] The invention also discloses a pharmaceutical composition, comprising the above-mentioned natural source FFA4 receptor agonist.

[0008] Furthermore, it also includes a pharmaceutically acceptable carrier or excipient.

[0009] Furthermore, the dosage form includes one of tablets, capsules, granules, suspensions, pellets, injections and aerosols.

[0010] The present invention also discloses a use of oleanolic acid in the above-mentioned natural source FFA4 receptor agonist or the above-mentioned pharmaceutical composition in the preparation of a drug for treating inflammatory bowel disease.

[0011] The present invention also discloses a use of the above-mentioned natural source FFA4 receptor agonist, oleanolic acid, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating osteoporosis.

[0012] The present invention also discloses a use of the above-mentioned natural source FFA4 receptor agonist or the above-mentioned pharmaceutical composition in preparing a drug for treating gastric damage induced by non-steroidal anti-inflammatory drugs.

[0013] The present invention also discloses a use of the above-mentioned natural source FFA4 receptor agonist, selenoic acid, or the above-mentioned pharmaceutical composition in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0014] The beneficial effects of the present invention are:

[0015] In vitro cell target test experiments show that the present invention's selenoic acid, oleanolic acid and hederagenin act on FFA4 receptors, which are related to diseases such as non-alcoholic fatty liver disease, gastric damage induced by non-steroidal anti-inflammatory drugs, osteoporosis and inflammatory bowel disease, so the clinical application range of such substances in related diseases can be expanded. Moreover, according to research, the agonist structure is different from the parent nucleus structure of the FFA4 agonist currently published in the literature, so it can also provide a new framework for the design of FFA4 agonists and enrich the structural diversity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The agonistic effect of selenoic acid on FFA4 receptor; A: agonistic experiment, concentration-response curve of selenoic acid on CHO-K1-FFA4 cells; B: desensitization experiment, concentration-response curve of selenoic acid on CHO-K1-FFA4 cells after pretreatment with TUG891 for 1 hour; C: antagonistic experiment, effect of FFA4 antagonist AH7614 on selenoic acid agonistic signal in CHO-K1-FFA4 cells.

[0017] Figure 2 The agonistic effect of oleanolic acid on FFA4 receptor; A: agonistic experiment, concentration-response curve of oleanolic acid on CHO-K1-FFA4 cells; B: desensitization experiment, oleanolic acid was pretreated on CHO-K1-FFA4 cells for 1 hour, and then TUG891 was added to treat the cells for 1 hour, and then the concentration-response curve was plotted; C: antagonistic experiment, the effect of FFA4 antagonist AH7614 on the agonistic signal of oleanolic acid on CHO-K1-FFA4 cells.

[0018] Figure 3 The agonist effect of hederagenin on FFA4 receptor; A: agonist experiment, concentration-response curve of hederagenin on CHO-K1-FFA4 cells; B: desensitization experiment, concentration-response curve of hederagenin on CHO-K1-FFA4 cells after pretreatment for 1 hour and then treatment with TUG891 for 1 hour. DETAILED DESCRIPTION

[0019] The present invention relates to the discovery of the action targets of natural compounds, specifically the discovery of the action targets of selenoic acid, oleanolic acid and hederagenine. The compounds are selenoic acid, oleanolic acid and hederagenine and / or their corresponding pharmaceutically acceptable salts, solvates or polymorphs and drug combinations; the target is the FFA4 receptor; and the application is specifically the application in the preparation of drugs for treating non-alcoholic fatty liver disease, gastric damage induced by non-steroidal anti-inflammatory drugs, osteoporosis and inflammatory bowel disease.

[0020] Use of selenoic acid, oleanolic acid and hederagenin in the preparation of FFA4 agonists and drugs for receptor-related diseases.

[0021] The FFA4-related disease is selected from non-alcoholic fatty liver disease, non-steroidal anti-inflammatory drug-induced gastric damage, osteoporosis and inflammatory bowel disease.

[0022] The chemical structures of the present invention's selenoic acid (CAS No. 13849-91-7), oleanolic acid (CAS No. 17990-42-0) and hederagenin (CAS No. 465-99-6) are as follows, wherein the chemical structure of selenoic acid is as shown in Formula 1, the chemical structure of oleanolic acid is as shown in Formula 2, and the chemical structure of hederagenin is as shown in Formula 3:

[0023]

[0024]

[0025] The drug contains one or more of selenoic acid, oleanolic acid and hederagenin or pharmaceutically acceptable solvates thereof as active ingredients.

[0026] A pharmaceutical composition comprises selenoic acid, oleanolic acid and hederagenin as active ingredients and a pharmaceutical carrier.

[0027] A pharmaceutical composition, the dosage form of which is one of tablets, capsules, granules, suspensions, dripping pills, injections and aerosols.

[0028] The present invention is further described with reference to examples, which are intended to illustrate the present invention but not to limit the present invention.

[0029] The present invention is described below by means of specific embodiments, but the present invention is not limited thereto.

[0030] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0031] Selmol acid, oleanolic acid and hederagenin were purchased from Chengdu Pusi Co., Ltd., Chinese hamster ovary cells (CHO-K1) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and CHO-K1-FFA4 cells were derived from transfection (the transfection process was consistent with the reference [RSC Adv. 2019; 9(26): 15073-15083.]); probe molecules TUG891 and AH7614 were purchased from TOCRIS, USA. F12 culture medium was purchased from Thermo Fisher Scientific, USA (Cat. No.: C11765500BT); fetal bovine serum (FBS) was PAN brand (Cat. No.: ST30-3302); buffer salt HBSS was Gibco brand (Cat. No.: 14065056); buffer salt HEPES was Gibco brand (Cat. No.: 15630080). Each compound was dissolved in DMSO to prepare a 100 mM stock solution. The test buffer was prepared in the following volume ratio: HEPES: HBSS: ultrapure water = 1:10:89, which was used for compound dilution during the Epic experiment. The detection platform was Corning's third-generation Epic imager, and the detected signal was the wavelength shift caused by the cell dynamic mass resetting (DMR).

[0032] Example 1 Selmol acid is an agonist of FFA4

[0033] CHO-K1-FFA4 cells in the logarithmic growth phase were inoculated into In the 384-well biosensor microplate, the volume of cell suspension (F12 culture medium containing cells) inoculated in each well is 40 μL, and the number of cells inoculated in each well is 15,000. Then the 384-well plate is placed in a cell culture incubator (air with a volume concentration of 5% CO2, 37°C) and cultured for 24 hours. The Epic DMR signal detection experiment can be started when the cell confluence reaches 90%. Before the Epic detection, the culture medium in the 384-well plate is discarded, and then 30 μL of test buffer (volume ratio: HEPES: HBSS: ultrapure water = 1:10:89) is replaced in each well with cells.

[0034] The process of the stimulation experiment and desensitization experiment on CHO-K1-FFA4 cells is as follows: DMSO is used to prepare a 100 mM stock solution, which is diluted into a working solution using the test buffer. First, 10 μL of different concentrations of selenoic acid working solution (320 μM, 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM) were directly added to a 384-well cell plate containing 30 μL of test buffer, so that the final concentration of selenoic acid in each well of the 384-well cell plate was 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively, and the signal was monitored on the Epic instrument for 1 hour; then 10 μL of TUG891 working solution (TUG891 was prepared into a 100 mM stock solution in DMSO and then diluted to a 5 μM working solution with test buffer) was added to the above 384-well cell plate containing selenoic acid, and the final concentration of TUG891 in the 384-well cell plate was 1 μM, and the signal was monitored on the Epic instrument for another 1 hour. The results are as follows Figure 1 As shown in A, selenoic acid can induce DMR response signals in CHO-K1-FFA4 cells in a concentration-dependent manner, and can also reduce the DMR signal of the FFA4 receptor agonist TUG891 ( Figure 1 B), and showed concentration dependence, with EC50 values ​​of stimulation and desensitization being 7.925±0.402μM and 8.374±0.882μM, respectively, indicating that penicillin can stimulate FFA4 receptors.

[0035] The antagonism experiment on CHO-K1-FFA4 cells was performed as follows: first, 10 μL of AH7614 working solution of different concentrations was directly added to a 384-well cell plate containing 30 μL of test buffer (the AH7614 working solution was prepared by diluting 100 mM AH7614 stock solution using test buffer), so that the final concentrations of AH7614 in the 384-well cell plate were 2000 nM, 666.67 nM, 222.2 nM, 74.1nM, 24.7nM, 8.2nM, 2.7nM, and the signal was monitored on the Epic instrument for 1 hour; 10μL of the working solution of selenoic acid was added to the 384-well cell plate containing AH7614 (100mM selenoic acid stock solution was diluted to 50μM working solution with test buffer, and 10μL of the working solution was added to the 384-well cell plate, and the final concentration in the well was 10μM), and the signal was monitored on the Epic instrument for 1 hour. The results are as follows Figure 1 C, indicating that selenoic acid has receptor specificity for FFA4.

[0036] The above results show that selenoic acid has FFA4 receptor agonist activity and is a FFA4 receptor agonist.

[0037] Example 2 Oleanolic acid is an agonist of FFA4

[0038] CHO-K1-FFA4 cells in the logarithmic growth phase were inoculated into In the 384-well biosensor microplate, the volume of cell suspension (F12 culture medium containing cells) inoculated in each well is 40 μL, and the number of cells inoculated in each well is 15,000. Then the 384-well plate is placed in a cell culture incubator (air with a volume concentration of 5% CO2, 37°C) and cultured for 24 hours. The Epic DMR signal detection experiment can be started when the cell confluence reaches 90%. Before the Epic detection, the culture medium in the 384-well plate is discarded, and then 30 μL of test buffer (volume ratio: HEPES: HBSS: ultrapure water = 1:10:89) is replaced in each well with cells.

[0039] The process of the stimulation experiment and desensitization experiment on CHO-K1-FFA4 cells is as follows: DMSO is used to prepare a 100 mM stock solution, which is diluted into a working solution using the test buffer. First, 10 μL of oleanolic acid working solution of different concentrations (320 μM, 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM) was directly added to a 384-well cell plate containing 30 μL of test buffer, so that the final concentration of oleanolic acid in each well of the 384-well cell plate was 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively, and the signal was monitored on the Epic instrument for 1 hour; then 10 μL of TUG891 working solution (TUG891 was prepared into a 100 mM stock solution in DMSO and then diluted to a 5 μM working solution with test buffer) was added to the above 384-well cell plate containing oleanolic acid, and the final concentration of TUG891 in the 384-well cell plate was 1 μM, and the signal was monitored on the Epic instrument for another 1 hour. The results are as follows Figure 2 As shown in A, oleanolic acid can induce DMR response signals in CHO-K1-FFA4 cells in a concentration-dependent manner, and can reduce the DMR signal of FFA4 receptor agonist TUG891 ( Figure 2 B), and showed concentration dependence, with EC50 values ​​of stimulation and desensitization being 18.70±3.635μM and 15.48±4.557μM, respectively, indicating that oleanolic acid can stimulate FFA4 receptor.

[0040] The antagonism experiment process on CHO-K1-FFA4 cells is as follows: first, 10 μL of AH7614 working solution of different concentrations (AH7614 working solution is prepared by diluting 100 mM AH7614 stock solution with test buffer) is 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 are 2000 nM, 666.67 nM, 222.2 nM, respectively. , 74.1nM, 24.7nM, 8.2nM, 2.7nM, and monitor the signal on the Epic instrument for 1h; continue to add 10μL of oleanolic acid working solution to the 384-well cell plate containing AH7614 (dilute the 100mM oleanolic acid stock solution with test buffer to 50μM working solution, take 10μL of working solution and add it to the 384-well cell plate, the final concentration in the well is 10μM), monitor the signal on the Epic instrument for 1h, the results are as follows Figure 2 C, indicating that oleanolic acid has receptor specificity for FFA4.

[0041] The above results show that oleanolic acid has FFA4 receptor agonist activity and is a FFA4 receptor agonist.

[0042] Example 3 Hederagenin is an agonist of FFA4

[0043] CHO-K1-FFA4 cells in the logarithmic growth phase were inoculated into In the 384-well biosensor microplate, the volume of cell suspension (F12 culture medium containing cells) inoculated in each well is 40 μL, and the number of cells inoculated in each well is 15,000. Then the 384-well plate is placed in a cell culture incubator (air with a volume concentration of 5% CO2, 37°C) and cultured for 24 hours. The Epic DMR signal detection experiment can be started when the cell confluence reaches 90%. Before the Epic detection, the culture medium in the 384-well plate is discarded, and then 30 μL of test buffer (volume ratio: HEPES: HBSS: ultrapure water = 1:10:89) is replaced in each well with cells.

[0044] The process of the stimulation experiment and desensitization experiment on CHO-K1-FFA4 cells is as follows: DMSO is used to prepare a 100 mM stock solution, which is diluted into a working solution using the test buffer. First, 10 μL of different concentrations of hederagenin working solution (320 μM, 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM) were directly added to a 384-well cell plate containing 30 μL of test buffer, so that the final concentration of hederagenin in each well of the 384-well cell plate was 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM, respectively, and the signal was monitored on the Epic instrument for 1 hour; then 10 μL of TUG891 working solution (TUG891 was prepared into a 100 mM stock solution in DMSO and then diluted to a 10 μM working solution with test buffer) was added to the above 384-well cell plate containing hederagenin, and the final concentration of TUG891 in the 384-well cell plate was 2 μM, and the signal was monitored on the Epic instrument for another 1 hour. The results are as follows: Figure 3 As shown in A, hederagenin can induce DMR response signals in CHO-K1-FFA4 cells in a concentration-dependent manner, and can reduce the DMR signal of FFA4 receptor agonist TUG891 ( Figure 3 B), and showed concentration dependence, with EC50 values ​​of stimulation and desensitization of ~40μM and 40.03±0.945μM, respectively, indicating that hederagenin can stimulate FFA4 receptors.

[0045] The above results show that hederagenin has FFA4 receptor agonist activity and is a FFA4 receptor agonist.

[0046] In addition, FFA4 receptors are associated with diseases such as non-alcoholic fatty liver disease, non-steroidal anti-inflammatory drug-induced gastric damage, osteoporosis and inflammatory bowel disease, and therefore, the agonist has potential medicinal uses.

[0047] The above-mentioned embodiments only express the preferred implementation modes of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all these changes and deformations should fall within the scope of protection of the claims of the present invention.

Claims

1. A natural source FFA4 receptor agonist, characterized in that: The invention comprises at least one of selenoic acid, oleanolic acid, hederagenin and pharmaceutically acceptable solvates thereof.

2. A pharmaceutical composition, characterized in that The invention comprises the naturally derived FFA4 receptor agonist as claimed in claim 1.

3. A pharmaceutical composition according to claim 2, characterized in that: Also included are pharmaceutically acceptable carriers or excipients.

4. A pharmaceutical composition according to claim 2, characterized in that: The dosage form includes tablets, capsules, granules, suspensions, pellets, injections and aerosols.

5. Use of oleanolic acid in the natural FFA4 receptor agonist according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a drug for treating inflammatory bowel disease.

6. Use of oleanolic acid in the natural FFA4 receptor agonist according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a drug for treating osteoporosis.

7. Use of the natural FFA4 receptor agonist according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a drug for treating gastric damage induced by nonsteroidal anti-inflammatory drugs.

8. Use of selenoic acid in the natural FFA4 receptor agonist according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a drug for treating non-alcoholic fatty liver disease.

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