A cannabinoid receptor 2 agonist and its application in atrial fibrillation and heart failure

Abstract

The present invention relates to an agonist of cannabinoid receptor 2 and its application in atrial fibrillation and heart failure. The agonist is yellow lupin ketone or its salt or its pharmaceutically acceptable drug carrier. The present study shows that yellow lupin ketone has better effects of inhibiting cAMP formation and reducing intracellular calcium ion signals; yellow lupin ketone has a strong binding ability with CB2R. At the animal level, multiple experiments have confirmed that yellow lupin ketone can better improve the structural remodeling and electrical remodeling of the atrium of diabetic mice. The yellow lupin ketone proposed by the present invention as an agonist of cannabinoid receptor 2 can be prepared into a drug for treating atrial fibrillation and improving atrial fibrillation susceptibility, and has good application prospects in the biomedical field.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a cannabinoid receptor 2 agonist, lupiwighteone, and application thereof in atrial fibrillation and heart failure. Background Art

[0002] Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, which is closely associated with severe disease burden and mortality. By 2020, the number of AF patients worldwide has reached nearly 50 million, with a prevalence of 2.9%. With the aging of the population and changes in lifestyle, the incidence of AF is on the rise. AF can lead to serious consequences, including stroke and heart failure. AF can increase the risk of stroke by 5 times and the risk of heart failure by 2-3 times. Current studies have shown that structural remodeling and electrical remodeling of the atrium are important foundations for the onset of AF, but the upstream mechanisms that promote atrial structural remodeling and electrical remodeling are still unclear, which hinders the progress of drug research. Therefore, finding new treatment strategies for atrial fibrillation is the current research focus.

[0003] Cannabinoid receptor 2 (CB2R) belongs to the rhodopsin subfamily of G protein-coupled receptors and is involved in multiple molecular pathways that regulate cell function. Studies in recent years have found that CB2R plays an important role in the cardiovascular system. Activating CB2R can reduce the inflammatory response of endothelial cells and reduce myocardial fibrosis. A study published in Circulation in 2023 showed that under high glucose conditions, decreased expression of CB2R can lead to diabetic myocardial necrosis, and re-expression of CB2R in the heart rescued diabetes-induced myocardial cell necrosis and cardiac dysfunction. Previous studies by the research team found that compared with patients in the SR group, the expression of CB2R in the atrial tissue of patients in the AF group was low. At the same time, the expression changes of CB2R in the atria of mice with atrial fibrillation induced by Ang II were detected, and the results showed that the expression of CB2R in the Ang II group was significantly decreased compared with the Control group. These results suggest that CB2R excitation is related to the pathological progression of atrial fibrillation.

[0004] Lupiwighteone is an isoflavone found in licorice roots. It can induce cytotoxicity, apoptosis and anti-angiogenesis in DU-145 prostate cancer cells, cell cycle arrest, apoptosis and activation of the Nrf2 / ARE pathway in SH-SY5Y neuroblastoma cells, and can also inhibit the PI3K / Akt / mTOR pathway to induce apoptosis in human breast cancer cells in a caspase-dependent and -independent manner. However, the application of Lupiwighteone in cardiovascular diseases is still in a blank stage. Summary of the invention

[0005] The present invention aims to provide a cannabinoid receptor 2 agonist Lupiwighteone and its application in atrial fibrillation and heart failure.

[0006] Technical solution:

[0007] The first aspect of the present invention provides an agonist of cannabinoid receptor 2, wherein the agonist is yellow lupin whit ketone or its salt or a pharmaceutically acceptable drug carrier thereof, and the SMILES of yellow lupin whit ketone is:

[0008] CC(=CCC1=C2C(=C(C=C1O)O)C(=O)C(=CO2)C3=CC=C(C=C3)O)C.

[0009] The second aspect of the present invention proposes the use of yellow lupin weite ketone as an agonist of cannabinoid receptor 2 in the preparation of drugs for treating or improving atrial fibrillation and heart failure.

[0010] The third aspect of the present invention proposes the use of yellow lupin weterone as an agonist of cannabinoid receptor 2 in the preparation of a drug for treating or improving atrial fibrillation and heart failure in diabetic patients.

[0011] The fourth aspect of the present invention proposes the use of yellow lupin weterone as an agonist of cannabinoid receptor 2 in the preparation of drugs for treating or improving atrial fibrillation and improving susceptibility to atrial fibrillation.

[0012] Preferably, the atrial fibrillation and improved atrial fibrillation susceptibility include: diabetes-induced atrial fibrillation susceptibility, Ang II-induced atrial fibrillation susceptibility, TAC-induced atrial fibrillation susceptibility, obesity-induced atrial fibrillation susceptibility and circadian rhythm-induced atrial fibrillation susceptibility.

[0013] The fifth aspect of the present invention provides a composition comprising the yellow lupin whitnerone, or a salt thereof or a pharmaceutically acceptable drug carrier of the yellow lupin whitnerone.

[0014] Preferably, the composition is in the form of a pill, capsule, tablet, injection, sustained-release formulation or nanoformulation.

[0015] The present invention has the following advantages and effects:

[0016] The present invention proposes an agonist of cannabinoid receptor 2 and its application in atrial fibrillation and heart failure, and proposes that yellow lupin weite ketone (Lupiwighteone) can treat atrial fibrillation and improve atrial fibrillation susceptibility by stimulating cannabinoid receptor 2 (CB2R). Activation of CB2R usually inhibits the formation of intracellular cyclic adenosine monophosphate (cAMP) by inhibiting the activity of adenylate cyclase; at the same time, stimulating CB2R can reduce the increased calcium signal. The present invention first verifies the stimulatory effect of 9 traditional Chinese medicine monomers screened by AI virtual screening on CB2R through cell experiments, and confirms that Lupiwighteone has better inhibitory effects on cAMP formation and reducing intracellular calcium ion signals than CB2R agonist Hu308. At the same time, CETSA experiments also verify that Lupiwighteone has a strong binding ability with CB2R. At the animal level, multiple experiments have confirmed that Lupiwighteone can better improve the structural remodeling and electrical remodeling of the atrium of diabetic mice compared with the currently recognized CB2R agonist AM1241. Therefore, the present invention may form a drug for treating atrial fibrillation and improving susceptibility to atrial fibrillation, and has a good application prospect in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the HTRF-cAMP results of the 9 Chinese medicine monomers and the CB2R agonist Hu308 and antagonist AM630 in Example 1; A is the inhibition results of the 9 Chinese medicine monomers and the CB2R agonist Hu308 and antagonist AM630 on cAMP; B is the IC of Lupiwighteone to inhibit cAMP 50 C is the IC value of CB2R agonist Hu308 inhibiting cAMP 50 value;

[0018] Figure 2 The results of the Fluo-4AM calcium ion fluorescence experiment of the nine Chinese medicine monomers and the CB2R agonist Hu308 and antagonist AM630 in Example 2;

[0019] Figure 3 is the result of the CETSA experiment on the binding ability of Lupiwighteone to CB2R in Example 3;

[0020] Figure 4is the transthoracic echocardiographic test result of the diabetic induced atrial fibrillation mouse in Example 4; A is the echocardiogram in M-mode; B is the left atrial systolic area (LA systolic area); C is the left atrial diastolic area (LA diastolic area); D is the left atrial diameter (LAD); E is the ejection fraction (EF); F is the left ventricular fraction shortening (FS);

[0021] Figure 5 The results of electrocardiogram detection and transjugular atrial electrical stimulation of mice with diabetes-induced atrial fibrillation in Example 5; A is the electrocardiogram record of mice in each group after transjugular stimulation; B is the susceptibility of atrial fibrillation (n=8); C is the duration of atrial fibrillation;

[0022] Figure 6 These are the results of the Langendorff perfused mouse isolated hearts in Example 6. A is a representative electrophysiological spectrum of the left atrium during sinus rhythm; B is the average conduction velocity of each group of mice; C is the absolute heterogeneity of each group of mice; and D is the heterogeneity index of each group of mice. DETAILED DESCRIPTION

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0024] Studies have shown that CB2R activation usually inhibits the formation of intracellular cyclic adenosine monophosphate (cAMP) by inhibiting the activity of adenylate cyclase; at the same time, stimulating CB2R can reduce the increased calcium signal. Therefore, the present invention first verified the stimulatory effect of 9 traditional Chinese medicine monomers screened by AI virtual screening on CB2R through cell experiments.

[0025] Example 1: HTRF-cAMP results of 9 traditional Chinese medicine monomers and CB2R agonist Hu308 and antagonist AM630.

[0026] Experimental methods:

[0027] HTRF-cAMP assay was performed using the Revvity HTRF-cAMP-GI KIT (Part#: 62AM9PEB (1,000 tests)) kit. HEK293-CB2 cells were resuspended in HBSS buffer containing 10 mM HEPES and 500 μM 3-isobutyl-1-methylxanthine according to the manufacturer's instructions. The final cell density was 1 × 10 6 Cells / mL. The cell suspension (5 μL, 5000 cells / well) was loaded onto a 96-well plate and incubated with 5 μM concentrations of compounds (Mulberrin; 2,3-Dehydrokievitone; Morusinol; Isoangustone A; Morusin; Lupiwighteone; Limonianin; Xanthohumol D; Xanthohumol; Hu308; AM630) at room temperature for 15 min. The adenylate cyclase activator forskolin (FK) (3 μM) was then treated for 30 min, and then the reaction was terminated by adding the detection buffer consisting of cAMP-d2 (5 μL) and anti-cAMP-cryptate (5 μL). After incubation for 2 hours in the dark, the reaction was performed using High frequency signals were recorded by a F500 imaging reader (Tecan, Switzerland).

[0028] Experimental results:

[0029] Studies have shown that the activation of CB2R usually inhibits the formation of intracellular cyclic adenosine monophosphate (cAMP) by inhibiting the activity of adenylate cyclase. Therefore, in order to screen the agonist effects of 9 traditional Chinese medicine monomers (Mulberrin; 2,3-Dehydrokievitone; Morusinol; Isoangustone A; Morusin; Lupiwighteone; Limonianin; Xanthohumol D; Xanthohumol;) on CB2R, we performed HTRF-cAMP experiments. Figure 1 As shown in A, compared with the other eight Chinese medicine monomers, lupiwighteone and the CB2R agonist Hu308 significantly inhibited the forskolin-stimulated cAMP production in CB2-transfected HEK293 cells. Figure 1 As shown in B, Lupiwighteone's IC 50 is 1.402nM, such as Figure 1 As shown in C, the IC of Hu308 50The result showed that Lupiwighteone has better inhibitory effect on cAMP than Hu308 after CB2R stimulation.

[0030] Example 2: Fluo-4AM calcium ion fluorescence experiments of 9 traditional Chinese medicine monomers and CB2R agonist Hu308 and antagonist AM630

[0031] Experimental methods:

[0032] The calcium mobilization assay was used to evaluate the calcium mobilization activities of nine traditional Chinese medicine monomers and the CB2R agonist Hu308 and antagonist AM630. Fluo-4 AM fluorescent indicator was used to detect intracellular calcium (Ca 2+ HEK293-CB2 cells (5×10 4 Cells / well) were seeded in 96-well microplates and incubated for 24 hours, followed by serum starvation for 24 hours to eliminate the effects of growth factors in serum on cells. Then the cells were treated with 2μM Fluo-4 AM for 45min. After washing three times with phosphate-buffered saline (PBS), 100μL of Hank's Balanced Salt Solution (HBSS) was added to each well, and the intracellular Ca2+ fluorescence intensity was detected using a microplate reader (FLUOstar Omega, Omega). Then 100ul of 5μM concentration of compounds (Mulberrin; 2,3-Dehydrokievitone; Morusinol; Isoangustone A; Morusin; Lupiwighteone; Limonianin; Xanthohumol D; Xanthohumol; Hu308; AM630) was added, and the changes in fluorescence intensity were immediately detected.

[0033] Experimental results:

[0034] like Figure 2 The results showed that compared with the control group, Lupiwighteone and CB2R agonist Hu308 significantly inhibited the influx of calcium ions, and Lupiwighteone had the strongest activity in inhibiting the influx of calcium ions.

[0035] Example 3: CETSA experiment on the binding ability of Lupiwighteone to CB2R

[0036] Experimental methods:

[0037] HEK293-CB2 cells were exposed to 5 μM Lupiwighteone or dimethyl sulfoxide (DMSO) solution for 24 hours. The cells were lysed in a lysis buffer containing protease inhibitors and phosphatase inhibitors. Centrifuged at 12,000 rpm for 10 minutes at 4°C. Subsequently, the supernatant was divided into 6 equal parts, heated at different temperatures (47°C, 52°C, 57°C, 62°C, 67°C and 72°C), and then another round of centrifugation was performed under the same conditions, and the supernatant was retained and then analyzed using immunoblotting technology.

[0038] Experimental results:

[0039] We used the CETSA method to detect the direct interaction between lupiwighteone and CB2R. Figure 3 As shown, lupiwighteone increased the thermostability of CB2R compared to the DMSO control.

[0040] Example 4: Transthoracic echocardiography experiment in diabetic mice with atrial fibrillation

[0041] Experimental methods:

[0042] Open the oxygen tank of the gas anesthesia machine and maintain the oxygen concentration at 5%. Pour isoflurane into the gas anesthesia machine and maintain the concentration at 1.5%. Place the mouse in the anesthesia tank and wait for 3 to 5 minutes until the mouse is anesthetized successfully. Put on the anesthesia mask, lie on the fixed board in supine position, and fix the limbs with medical tape; apply depilatory cream on the left chest and wipe it clean with a sterile cotton ball after 2 minutes. Cover the left chest of the mouse with ultrasound coupling agent, the long axis of the ultrasound probe is parallel to the sternum, the probe logo is located on the upper left, and gently place it on the coupling agent to perform cardiac long axis ultrasound detection. Obtain ventricular and atrial ultrasound images in M ​​mode, record video and image data; each recording is maintained for at least three cardiac cycles and repeated three times. Data analysis and recording: left atrial diameter (LAD), left atrial systolic area (LA systolic area), left atrial diastolic area (LA diastolic area), ejection fraction (EF) and left ventricular short axis shortening (FS).

[0043] Experimental groups:

[0044] To evaluate the effect of Lupiwighteone on improving cardiac remodeling in diabetic mice, 8-week-old db / m mice (n=8) were used as the control group; 8-week-old db / db mice were randomly divided into the model group (n=8): intraperitoneal injection of control solvent (2% DMSO + 40% PEG300 + 5% Tween-80 + 53% Saline); low-dose group (n=8): intraperitoneal injection of Lupiwighteone (5 mg / kg / d); medium-dose group (n=8): intraperitoneal injection of Lupiwighteone (10 mg / kg / d); high-dose group (n=8): intraperitoneal injection of Lupiwighteone (20 mg / kg / d); positive drug group (n=8): intraperitoneal injection of CB2R agonist AM1241 (10 mg / kg / d).

[0045] Experimental results:

[0046] Atrial dilatation and fibrosis are important manifestations of structural remodeling in atrial fibrillation. Therefore, this study first used transthoracic echocardiography to evaluate the atrial cavity area and length. Figure 4 The results in A showed that the systolic left atrial area (LA systolic area), diastolic left atrial area (LA diastolic area) and mean left atrial diameter (LAD) of mice in the high-dose drug group (db / db+Lup(H)) were significantly lower than those in the model group (db / db), and the atrial remodeling of diabetic mice was better improved compared with the positive drug group (db / db+AM1241). Figure 4 BD). For the ejection fraction (EF) and the fraction shortening (FS) of the left ventricular short axis, the high-dose administration group (db / db+Lup(H)) mice had significantly higher ejection fraction (EF) and the fraction shortening (FS) of the left ventricular short axis than the model group (db / db), and the high-dose administration group (db / db+AM1241) had better improved the ventricular ejection capacity of diabetic mice ( Figure 4 EF). In conclusion, administration of medium and high doses of Lupiwighteone to diabetic mice can significantly improve atrial and ventricular remodeling in mice.

[0047] Example 5: Electrocardiogram detection and transjugular atrial electrical stimulation experiment in diabetic mice with atrial fibrillation

[0048] Experimental methods:

[0049] The mice were weighed and injected with 4% chloral hydrate solution intraperitoneally according to the standard of 1ml / 100g. After successful anesthesia, they were fixed in the supine position on the operating table. The skin of the right neck was wiped with alcohol cotton balls, and the hair was shaved with a blade to prepare the skin; the three-lead ECG detection wire was fixed to the distal ends of the right upper, left upper and right lower limbs of the mouse, the recorder was turned on, the interference was adjusted, and a continuous ECG was obtained; the skin of the right neck was cut open with ophthalmic scissors, the muscle tissue was bluntly separated layer by layer, the right external jugular vein was fully exposed, and the distal end was ligated; a 1ml syringe needle was used to perform venous puncture in the proximal segment of the ligation, the bleeding was cleaned, the electrode catheter was inserted from the puncture port, and the top electrode sheet was run along the external jugular vein to the right atrium, and the catheter was fixed; the physiological signal acquisition and stimulation system was used for intra-atrial pulse pacing, with an amplitude of 6V, a cycle length of 40ms, a pulse duration of 6ms, and a stimulation time of 5s. The stimulation was repeated 3 times, and the incidence and duration of atrial fibrillation were recorded. The duration of AF was defined as the time from the onset of AF to the return to sinus rhythm.

[0050] Experimental groups:

[0051] To evaluate the effect of Lupiwighteone on improving the susceptibility of diabetic mice to induced atrial fibrillation, the grouping was consistent with that in Example 4.

[0052] Experimental results:

[0053] To evaluate the susceptibility of diabetic mice to induced AF, transjugular atrial pacing was performed to induce AF in each group of mice. Figure 5 As shown in Figure B, 100% of the mice in the db / db group were induced into atrial fibrillation, 75% of the mice in the db / db+Lup(L) group were induced into atrial fibrillation, 50% of the mice in the db / db+Lup(M) group were induced into atrial fibrillation, 25% of the mice in the db / db+Lup(H) group were induced into atrial fibrillation, and 62.5% of the mice in the db / db+AM1241 group were induced into atrial fibrillation. Figure 5 A and 5C show that the duration of atrial fibrillation in db / db+Lup(H) group mice was significantly lower than that in other groups. These data suggest that administration of high doses of Lupiwighteone to db / db mice can significantly reduce the susceptibility and persistence of atrial fibrillation.

[0054] Example 6: Langendorff perfusion mouse isolated heart experiment

[0055] Experimental methods:

[0056] The isolated mouse heart was fixed on the Langendorff perfusion system. After stable perfusion, the 64-channel multi-electrode array probe was attached to the surface of the atrium to record the electrical activity synchronously. The field point signals were recorded and the data were saved using the matching Emapercard 5.0 software. The obtained data were analyzed using EMapScope 5 software to calculate the average conduction velocity, absolute inhomogeneity and inhomogeneity index.

[0057] Experimental groups:

[0058] To evaluate the effects of lupiwighteone on atrial electrophysiological parameters in diabetic mice, the grouping was consistent with that in Example 4.

[0059] Experimental results:

[0060] Slowing of atrial conduction velocity and increasing heterogeneity of refractory period are considered to be the main electrophysiological causes of intra-atrial reentry, which facilitates the initiation and maintenance of atrial fibrillation. Figure 6 As shown in A, in this experiment, the left atrium of each group of mice was electrophysiologically mapped in vivo. The test found that the average conduction velocity of the high-dose drug group (db / db+Lup(H)) was significantly higher than that of the model group (db / db) ( Figure 6 B), the absolute heterogeneity and heterogeneity index were significantly lower than those in the model group (db / db) ( Figure 6 C, 6D). The average conduction velocity of the medium-dose group (db / db+Lup(H)) was also higher than that of the positive drug group (db / db+AM1241) ( Figure 6 B), the absolute heterogeneity and heterogeneity are also lower than those of the positive drug group (db / db+AM1241) ( Figure 6 C, 6D). The above results suggest that Lupiwighteone has a protective effect on atrial electrical activity in db / db mice, and its efficacy is better than that of positive drugs.

[0061] In summary, the cannabinoid receptor 2 agonist yellow lupin weite ketone proposed in the present invention can be used to treat or improve atrial fibrillation and heart failure. It is suitable for treating or improving diabetes-induced atrial fibrillation susceptibility, Ang II-induced atrial fibrillation susceptibility, TAC-induced atrial fibrillation susceptibility, obesity-induced atrial fibrillation susceptibility and circadian rhythm-induced atrial fibrillation susceptibility, etc.

[0062] The invention discloses a composition comprising yellow lupin whit ketone, or a salt thereof or a pharmaceutical carrier of a pharmaceutically acceptable yellow lupin whit ketone, and the dosage form thereof is a pharmaceutically acceptable dosage form such as a pill, a capsule, a tablet, an injection, a sustained-release agent or a nano-preparation.

[0063] Any feature disclosed in this specification, unless otherwise stated, can be replaced by other equivalent or alternative features with similar purposes. That is, unless otherwise stated, each feature is only an example of a series of equivalent or similar features.

[0064] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A cannabinoid receptor 2 agonist, characterized in that The agonist is yellow lupin ketone or its salt or a pharmaceutically acceptable drug carrier thereof, and the SMILES of yellow lupin ketone is: CC(=CCC1=C2C(=C(C=C1O)O)C(=O)C(=CO2)C3=CC=C(C=C3)O)C.

2. Use of yellow lupin weterone as an agonist of cannabinoid receptor 2 in the preparation of drugs for treating or improving atrial fibrillation and heart failure.

3. The use of yellow lupin weterone as an agonist of cannabinoid receptor 2 in the preparation of drugs for treating or improving atrial fibrillation and heart failure in diabetic patients.

4. Use of yellow lupin weterone as an agonist of cannabinoid receptor 2 in the preparation of drugs for treating or improving atrial fibrillation and improving susceptibility to atrial fibrillation.

5. The use according to claim 3, characterized in that: The atrial fibrillation and improvement of atrial fibrillation susceptibility include: diabetes-induced atrial fibrillation susceptibility, Ang II-induced atrial fibrillation susceptibility, TAC-induced atrial fibrillation susceptibility, obesity-induced atrial fibrillation susceptibility and circadian rhythm-induced atrial fibrillation susceptibility.

6. A composition, characterized in that The invention comprises the yellow lupin whit ketone as claimed in claim 1, or a salt thereof or a pharmaceutical carrier of the yellow lupin whit ketone which is pharmaceutically acceptable.

7. The composition according to claim 6, characterized in that The dosage form of the composition is pills, capsules, tablets, injections, sustained-release preparations or nano preparations.

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