Application of adenosine A2a / A2b receptor dual antagonist in preparation of antidepressant drug

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of an adenosine A2a / A2b receptor dual antagonist in preparation of an anti-depression medicine and a medicine for preventing or treating depression. The invention provides a new application of the adenosine A2aR / A2bR double antagonist, animal experiments prove that the adenosine A2aR / A2bR double antagonist AB928 can effectively treat depression of mice, does not have obvious toxic or side effects, increases indications of the AB928, and has a prospect of being applied as an anti-depression drug in practice, so that the medical application of the adenosine A2aR / A2bR double antagonist AB928 is further expanded.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of an adenosine A2a / A2b receptor dual antagonist in the preparation of antidepressant drugs. Background Technology

[0002] Adenosine, as a neurotransmitter and regulator, is widely distributed in the nervous system, cardiovascular system, kidneys, and gastrointestinal tract, regulating various important physiological functions. Adenosine participates in the physiological processes of multiple diseases by binding to adenosine receptors (A1R, A2aR, A2bR, and A3R). Adenosine receptors are typical G protein-coupled receptors, playing a crucial role in the regulation of normal human physiological functions and the development of major diseases. Drugs targeting all four members of the Adenosine receptor family have been approved by the FDA and are widely used in the treatment or clinical trials of cancer, heart disease, neurodegenerative diseases, and autoimmune diseases. However, there are very few reports on the application of these drugs in antidepressant research.

[0003] Adenosine A2a and A2b receptors (A2aR / A2bR) are important drug targets, playing crucial roles in various physiological and pathological processes, such as motor control, neurodegenerative diseases, tumor immunomodulation, antidepressant effects, cardiac diseases, nerve regeneration, and immune regulation. However, the development of their ligands has primarily focused on non-psychiatric diseases, particularly tumor immunotherapy. Currently, A2aR and / or A2bR antagonists in clinical trials are mainly indicated for Parkinson's disease and various cancers. Those skilled in the art generally believe that the main and clear clinical application prospects of A2aR / A2bR antagonists lie in motor disorders and tumor immunotherapy, rather than in antidepressant effects related to mental and emotional disorders. Although some basic research indicates that A2aR / A2bR may play an important role in the regulation of depression, and that depressive behavior induced by chronic stress is associated with the upregulation of A2aR / A2bR expression, current clinical research on their antidepressant effects remains lacking.

[0004] As neuromodulatory receptors, A2aR / A2bR have been supported by numerous epidemiological, genetic, and pharmacological studies on their regulatory role in depression, but their specific mechanism of action remains unclear.

[0005] Caffeine, as a widely used non-selective adenosine receptor antagonist, may reduce the risk of depression through its antagonistic function against A2aR. Through brain-region-specific administration, the adenosine A2aR antagonist itratheline (KW6002, an effective, selective, orally available A2aR antagonist with a Ki value of 2.2 nM in a Parkinson's disease model trial) offers the possibility of targeting A2aR signaling to treat depression. KW6002 was approved by the US FDA in 2019 for the treatment of Parkinson's disease.

[0006] A2bR is expressed at relatively low levels in the brain and has a low affinity for adenosine, typically being activated only in high concentrations of adenosine (e.g., under stress, ischemia, or inflammatory conditions). Therefore, it was once considered a "minor" receptor. However, recent studies have revealed its important role in neuroinflammation, glial cell function, and blood-brain barrier regulation. Evidence suggests significant neuroinflammation and systemic inflammation in patients with chronic stress and depression. Elevated levels of pro-inflammatory cytokines (such as IL-6, IL-1β, and TNF-α) can affect neurogenesis and synaptic plasticity. A2bR is highly expressed on immune cells (such as microglia and macrophages) and astrocytes. When activated, it promotes the release of pro-inflammatory cytokines, exacerbating neuroinflammation. Therefore, it may be possible to inhibit this inflammatory response by blocking A2bR, thereby producing an antidepressant effect (Biomed Pharmacother. 2021, 135: 111164).

[0007] Therefore, although some basic research suggests that A2aR or A2bR may be involved in emotion regulation, this field remains significantly uncertain and complex. First, there is the diversity of target functions: A2aR and A2bR are distributed and function in different tissues such as the immune, cardiovascular, and nervous systems. Specifically regulating a particular pathway while avoiding other side effects presents a significant challenge. For example, non-selective adenosine receptor antagonists have complex and even contradictory effects on mood, such as potentially inducing anxiety.

[0008] Secondly, the "minor" role and mechanism of A2bR are unclear: A2bR is expressed at low levels in the brain, and its function, especially its role in depression, is far from clear. Although some studies speculate that it may indirectly affect mood through pathways such as neuroinflammation, direct evidence is scarce, and the causal relationship is unclear.

[0009] Third, there are unknown differences between "single-target" and "dual-target" approaches: Existing research mostly focuses on the antidepressant potential of single receptors (such as the use of A2aR antagonists KW6002 or A2bR antagonists PSB-603). Whether simultaneously antagonizing A2aR and A2bR will produce synergistic effects, antagonistic effects, or introduce unpredictable side effects is currently completely unknown.

[0010] Currently, WO 2004 / 089942, WO 2005 / 000842, and WO 2006 / 008041 disclose benzothiazole derivatives, including Tozadenant, as A2aR inhibitors for the treatment of Parkinson's disease. WO 2004 / 092171 and WO 2005 / 028484 disclose similar thiazopyridine and pyrazolopyrimidine derivatives, also used as A2aR inhibitors for the treatment of Parkinson's disease. However, no mention is made of these compounds being used to treat depression.

[0011] The core symptoms of depression include depressed mood, slowed thinking, and reduced willpower, often accompanied by cognitive impairment and somatic symptoms (such as sleep disturbances and pain). Depression severely impacts patients' normal work and life, and increases their economic burden, making it a major global public health problem. Current antidepressants are mainly based on the monoamine neurotransmitter hypothesis, acting by regulating the serotonin (5-HT), norepinephrine, and dopamine systems. They can be divided into five main classes: tricyclic antidepressants, monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SSRIs), and atypical antidepressants (such as mirtazapine, bupropion, and ketamine). However, these drugs generally suffer from delayed onset of action (usually requiring 2-4 weeks), low response rates (only about 30%-50% of patients experience significant relief), and significant side effects. The side effects of existing antidepressants in treating depression remain a significant challenge in the field of psychiatry. Adverse reactions to existing drugs severely impact patients' treatment adherence and quality of life; therefore, there is an urgent need to develop new target antidepressants.

[0012] Therefore, it is essential to find an antidepressant based on the adenosine A2aR / A2bR dual antagonist pathway. Summary of the Invention

[0013] The purpose of this invention is to address the current situation where existing antidepressants have significant side effects and lack novel targets, by using AB928, a drug with dual antagonistic effects on adenosine A2aR / A2bR, to treat depression, as an effective measure for treating depression in practice.

[0014] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides the use of an adenosine A2aR / A2bR double antagonist in the preparation of a drug for the prevention and treatment of depression, wherein the A2aR / A2bR double antagonist is AB928 and its pharmaceutically acceptable salt; The molecular structure of AB928 is shown below:

[0015] The molecular formula is C 23 H 22 N8O has a molecular weight of 426.47.

[0016] Secondly, the present invention also provides a medicament for the prevention and treatment of depression, wherein the medicament is an adenosine A2aR / A2bR double antagonist as an active ingredient, wherein the A2aR / A2bR double antagonist is AB928 and its pharmaceutically acceptable salt.

[0017] Preferably, the drug is a pharmaceutical composition and further comprises one or more pharmaceutically acceptable carriers, diluents, and excipients.

[0018] Preferably, the pharmaceutically acceptable carrier, diluent, or excipient includes water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil, and polyalkylene glycol.

[0019] Preferably, the pharmaceutical composition is a tablet, capsule, solution, suspension, injection, or patch.

[0020] Preferably, the pharmaceutical composition is administered orally, by injection, or by spray.

[0021] Preferably, the dosage of AB928 in the drug is from 1 mg / kg to 20 mg / kg.

[0022] The clinical validation of compound AB928 in this invention for use as a drug is as follows: This invention discovered that compound AB928 (chemical name: 3-(2-amino-6-(1-((6-(2-hydroxypropyl)pyridin-2-yl)methyl)-1H-1,2,3-triazol-4-yl)pyrimidin-4-yl)-2-methylbenzonitrile; English name: Etrumadenant, AB928, CAS Registry Number: 2239273-34-6), which has good antidepressant effects, is an orally administered selective adenosine receptor (A2aR / A2bR) dual antagonist (Kd for A2aR / A2bR is 2 nM and 1.4 nM, respectively), which can alleviate adenosine-mediated immunosuppression and has immunomodulatory and antitumor activities. Currently, the existing use of the A2aR / A2bR dual antagonist Etrumadenant (AB928) as a drug is for immunomodulatory and antitumor activities, and it is in Phase II clinical trials. A Phase I clinical trial (Seitz L, et al. Safety, tolerability, and pharmacology of AB928, a novel dual adenosine receptor antagonist, in a randomized, phase I study in healthy volunteers. Invest NewDrugs. 2019 Aug; 37(4):711-721.) recruited 85 participants (randomized 3:1, AB928: placebo). The AB928 dose range for evaluation was 10 to 200 mg once daily and 100 mg twice daily. AB928 was well tolerable at the highest tested dose (200 mg once daily or 100 mg twice daily) and did not affect any physiological parameters that might be sensitive to adenosine inhibition; no safety issues were found. The PK curve of AB928 was linear and dose-proportional, showing a PK / PD correlation. Significant inhibition of adenosine receptor-mediated phosphorylated CREB was observed at peak plasma concentrations in all dose cohorts and at trough plasma concentrations in higher dose cohorts. Plasma AB928 levels ≥1 μM were associated with ≥90% adenosine receptor inhibition. The absorption rate of AB928 decreased after a meal, but the extent of absorption remained unchanged. These data support the prospect of further clinical development of oral AB928.

[0023] The beneficial effects of this invention are: This invention provides a novel application for adenosine A2aR / A2bR dual antagonists, particularly AB928, in the preparation of drugs for the prevention or treatment of depression, addressing the current lack of novel target antidepressants. This invention has demonstrated through various animal experiments that AB928 is effective in treating depression in mice without significant toxic side effects. AB928 is currently in Phase II clinical trials for antitumor effects, indicating good safety and efficacy in humans. This invention has also discovered that AB928 has therapeutic effects in various mouse models of depression [tail suspension test (TST), open field test (OFT), forced swimming test (FST), and fine behavior test (SAT)], expanding its indications and enabling its practical application as an antidepressant. Attached Figure Description

[0024] Figure 1 This represents the dose-response relationship of the antagonist AB928 against A2aR.

[0025] In the figure: (a) is the fluorescence signal curve change caused by AB928 with dose change; (b) is the IC50 value curve after logistic fitting.

[0026] Figure 2 This represents the dose-response relationship of the antagonist AB928 against A2bR.

[0027] In the figure: (a) is the fluorescence signal curve change caused by AB928 with dose change; (b) is the IC50 value curve after logistic fitting.

[0028] Figure 3 This diagram shows the molecular interaction between the antagonist AB928 and A2aR.

[0029] In the figure: (a) is the molecular binding signal diagram of AB928 and A2aR; (b) is the binding affinity KD value of AB928 and A2aR calculated by logistic fitting.

[0030] Figure 4 This graph shows the weight changes in a mouse depression model constructed with corticosterone.

[0031] In the figure: (a) is the animal's weight on the first day; (b) is the animal's weight on day 21 after modeling. Figure 5 This indicates the TST experiment's evaluation of the antidepressant effects of each drug administration group on mice.

[0032] Figure 6 This represents the behavioral trajectory diagram of mice in each drug administration group during the OFT experiment.

[0033] Figure 7 This indicates the OFT experiment evaluating the antidepressant effect of each drug administration group on mice.

[0034] Figure 8 This indicates the FST experiment's evaluation of the antidepressant effects of each drug administration group on mice.

[0035] Figure 9 This indicates the SAT experiment evaluation of the antidepressant effect of each drug administration group on mice. Detailed Implementation

[0036] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0037] The compounds described in this invention are not particularly limited, and those skilled in the art can prepare them based on common knowledge in the field, or obtain the compounds described in this invention through commercial sales.

[0038] The present invention will be described in detail below through embodiments.

[0039] In the following examples, unless otherwise specified, all reagents and materials used are commercially available.

[0040] In the following examples, the antagonistic effect of compound [AB928] on A2aR and A2bR was detected using a FLIPR instrument (Molecular Devices). The FLIPR detection method is a homogeneous, dynamic, cellular-level fluorescence detection method. When cell lines stably transfected with A2aR and A2bR are incubated with a calcium-sensitive fluorescent dye for a period of time, the dye enters the cells. When a compound with A2aR and A2bR antagonistic activity binds to A2aR and A2bR on the cell surface, it can cause a decrease in the elevated calcium ion signal induced by the intracellular agonist, thereby causing a change in the fluorescence signal of the calcium-sensitive fluorescent dye. Based on the changes in the fluorescence signal, the antagonistic activity of the compound can be detected in real time.

[0041] In the following examples, an animal depression model was established by feeding mice with corticosterone instead of drinking water for 21 days. The antidepressant activity of the compounds was evaluated using the tail suspension test (TST), open field test (OFT), forced swim test (FST), and spontaneous activity test (SAT).

[0042] In the following examples, the animals used were mice of the C57 BL / 6J strain, all purchased from Beijing Huafukang Biotechnology Co., Ltd. The animal use license number was SCXK (Beijing) 2019-0008. They were male, with a body weight of 20±2 g and a mouse age of 8 weeks. All animals were of SPF grade and were housed in an SPF-class experimental animal center. The rearing environment temperature was 22±2°C, the relative humidity was 50±10%, and a 12-hour light and dark cycle alternation (7:00~19:00) was maintained. The mice were randomly grouped, with 5 mice in each cage, and were allowed to freely eat and drink. After 1 week of adaptive feeding, the experiment was started to reduce the impact of environmental stress on the experimental results. All experiments were carried out between 8:00 and 18:00. One hour before each behavioral test, the test animals were transferred to the test room and adapted to the environment under the condition of no additional interference to exclude the potential impact of circadian rhythm and environmental changes on the behavioral data. This research protocol strictly follows international experimental animal welfare norms and related requirements to ensure that the experimental process meets ethical standards. Example 1 1: Antagonistic dose-effect relationship of antagonist AB928 on A2aR.

[0043] The antagonistic dose-effect relationship of A2aR was detected using A2aR-HEK293 cells stably transfected with A2aR. The reaction system was set to 31.25 μL, including 20 μL of FLIPR calcium flow detection fluorescent dye for incubating cells, 5 μL of antagonists at different concentrations, and 6.25 μL of agonist. The specific procedure was as follows: Cell plate preparation: A2aR-HEK293 cells stably transfected with A2aR were seeded into a 384-well black-bottom transparent culture plate at 25,000 cells / well. 20 μL of culture medium was added to each well and cultured at 37°C and 5% CO2 for 24 h to 36 h. When the cell confluence in the wells reached 80%, the culture medium in the wells was removed, and 20 μL of FLIPR calcium flow detection fluorescent dye was added to each well and incubated for 2 h under light protection at 37°C.

[0044] Antagonist plate preparation: A gradient concentration solution of antagonist AB928 (specification 10 mg, product of Shanghai Haoyuan Biomedical Technology Co., Ltd.) was prepared with PBS. The prepared concentrations were: 2.5, 0.5, 0.1, 0.2, 0.04 μmol / L (DMSO concentration was 1%). 30 μL of antagonists at different concentrations was added to each well of a 384-compound well plate, and 4 replicates were set for each concentration for standby.

[0045] Agonist plate preparation: A solution of agonist CGS21680 hydrochloride (specification 1 g, product of Shanghai Jiji Biochemical Co., Ltd.) was prepared with PBS at a concentration of 1.3 μmol / L. 30 μL of the agonist was added to each well of a 384-compound well plate for standby.

[0046] Antagonist detection: Instrument parameters were set as follows: excitation wavelength 470–495 nm; emission wavelength 515–575 nm; LED intensity 60%–80%; gain 6.5–8; exposure time 0.045–0.05 ms; detection interval 1 s; antagonist sample volume 5 μL; sample height 35 μL; aspiration rate 10 μL / s; dispensing rate 10 μL / s. Cell plates, antagonist plates, and agonist plates were placed in their respective positions on the FLIPR instrument (MD product). Using the instrument's automatic sample dispensing system, the antagonist AB928 test solution was transferred to the corresponding wells of the cell plate at a volume of 5 μL / well. After addition, the plates were incubated at room temperature in the dark for 20 min. Subsequently, 6.3 μL of the agonist CGS21680 hydrochloride solution was added again using an automated sample loading system. Detection was performed immediately after addition, with the following parameters: detection time 200 s, 1 scan per second; baseline detection (obtaining fluorescence signal B0) was performed in the first 10 s, and compound detection (obtaining fluorescence signal B1) was performed from the 11th to the 210th second. The changes in fluorescence signal over time caused by different concentrations of AB928 were detected using real-time sample loading and recording.

[0047] Statistical method: The fluorescence signals measured from 1 to 210 seconds were normalized for the first 10 seconds. Then, the minimum value was subtracted from the maximum value to obtain (ΔF: the changing fluorescence signal value), where ΔF = (Max - Min), and the relative fluorescence unit (RFU) was calculated as RFU = ΔF / F0 [(Max - Min) / B0]. A logistic curve was fitted using Origin 2019 software, with the common logarithmic value of the compound concentration on the x-axis and the fluorescence signal RFU on the y-axis.

[0048] Experimental results: Different concentrations of AB928 caused changes in fluorescence signal; as the concentration increased, the fluorescence value decreased. (See attached figure.) Figure 1 (a) The fluorescence signal decreases with increasing antagonist dosage, exhibiting a clear dose-dependent effect. Using the common logarithm of compound concentration as the x-axis and RFU as the y-axis, an inverted "S"-shaped curve was obtained by fitting a logistic curve using Origin 2019 software. The results are shown in [Figure number missing]. Figure 1 (b) Obtain the IC50 antagonistic effect of antagonist AB928 on A2aR. 50 The value was 7.76 ± 0.75 nmol / L, indicating that AB928 has a significant antagonistic effect on A2aR. 2: The dose-response relationship of antagonist AB928 to A2bR.

[0049] The dose-response relationship of A2bR antagonism was detected using A2bR-HEK293 cells stably transfected with A2bR. The reaction system was set to 31.25 μL, including 20 μL of FLIPR calcium flux assay dye for cell incubation, 5 μL of different concentrations of antagonist, and 6.25 μL of agonist. The specific procedure is as follows: Cell plate preparation: Stable A2bR-HEK293 cells transfected with A2bR were seeded at 25,000 cells / well into 384-well black-bottomed permeable culture plates. 20 μL of culture medium was added to each well, and the cells were cultured at 37°C and 5% CO2 for 24-36 h. When the cell confluence in the wells reached 80%, the culture medium was removed, and 20 μL of FLIPR calcium flux detection fluorescent dye was added to each well. The cells were then incubated at 37°C in the dark for 2 h.

[0050] Antagonist plate preparation: AB928 (10 mg, product of Shanghai Haoyuan Biomedical Technology Co., Ltd.) solutions of gradient concentrations were prepared with PBS, with concentrations of 2.5, 0.5, 0.1, 0.2, and 0.04 μmol / L (DMSO concentration of 1%). 30 μL of the antagonist solution of different concentrations was added to each well of a 384-well compound plate, with 4 replicates for each concentration.

[0051] Agonist plate preparation: Prepare a 1.3 μmol / L solution of agonist CGS21680 hydrochloride (1 g specification, product of Shanghai Jizhi Biochemical Co., Ltd.) using PBS. Add 30 μL of the agonist to each well of a 384-well compound plate for later use.

[0052] Antagonist detection: Instrument parameters were set as follows: excitation wavelength 470–495 nm; emission wavelength 515–575 nm; LED intensity 60%–80%; gain 6.5–8; exposure time 0.045–0.05 ms; detection interval 1 s; antagonist sample volume 5 μL; sample height 35 μL; aspiration rate 10 μL / s; dispensing rate 10 μL / s. Cell plates, antagonist plates, and agonist plates were placed in their respective positions on the FLIPR instrument (MD product). Using the instrument's automatic sample dispensing system, the antagonist AB928 test solution was transferred to the corresponding wells of the cell plate at a volume of 5 μL / well. After addition, the plates were incubated at room temperature in the dark for 20 min. Subsequently, 6.3 μL of the agonist CGS21680 hydrochloride solution was added again using an automated sample loading system. Detection was performed immediately after addition, with the following parameters: detection time 200 s, 1 scan per second; baseline detection (obtaining fluorescence signal B0) was performed in the first 10 s, and compound detection (obtaining fluorescence signal B1) was performed from the 11th to the 210th second. The changes in fluorescence signal over time caused by different concentrations of AB928 were detected using real-time sample loading and recording.

[0053] Statistical method: The fluorescence signals measured from 1 to 210 seconds were normalized for the first 10 seconds. Then, the minimum value was subtracted from the maximum value to obtain (ΔF: the changing fluorescence signal value), where ΔF = (Max - Min), and the relative fluorescence unit (RFU) was calculated as RFU = ΔF / F0 [(Max - Min) / B0]. A logistic curve was fitted using Origin 2019 software, with the common logarithmic value of the compound concentration on the x-axis and the fluorescence signal RFU on the y-axis.

[0054] Experimental results: Different concentrations of AB928 caused changes in fluorescence signal; as the concentration increased, the fluorescence value decreased. (See attached figure.) Figure 2 (a) The fluorescence signal decreases with increasing antagonist dosage, exhibiting a clear dose-dependent effect. Using the common logarithm of compound concentration as the x-axis and RFU as the y-axis, an inverted "S"-shaped curve was obtained by fitting a logistic curve using Origin 2019 software. The results are shown in [Figure number missing]. Figure 2 (b) Obtain the IC50 antagonistic effect of antagonist AB928 on A2bR. 50 The value was 28.34 ± 2.49 nmol / L, indicating that AB928 has a significant antagonistic effect on A2bR. 3: Molecular interaction between antagonist AB928 and A2aR.

[0055] The molecular interaction between the antagonist AB928 and A2aR was detected using the Bio-Layer Interferometry (BLI) method to obtain the binding constant (ka) and dissociation constant (kd), and the affinity (KD) was obtained through fitting calculation analysis.

[0056] Experimental materials: Ni-NTA sensor (96 sensors / plate, with His tag, Sartorius GmbH, Germany); black 96-well plate (655209, Greiner GmbH, Germany); human A2aR protein (50 μg, Ag35458, Wuhan Sanying Biotechnology Co., Ltd.); AB928 (Etrumadenant, 10 mg, Shanghai Haoyuan Biomedical Technology Co., Ltd.).

[0057] Experimental instrument: Octet® R8 biomolecular interaction analysis system (Sartorius GmbH, Germany).

[0058] Experimental methods: (1) Plate loading: Prepare a black 96-well plate as the sample plate (Plate 1). The first row A1-A12 are the experimental group, and the second row B1-B12 are the control group. The amount of solution added to each well is 200 μL. Among them, PBS buffer is added to wells A1, A3, B1, and B3-B8; A2aR solution (concentration of 0.5 μmol) is added to wells A2 and B2; the small molecule compound solution to be tested with concentrations of 0.001, 0.01, 0.1, 1, 10, and 100 μmol is added to wells A4-A9 respectively (the final concentration of DMSO is controlled to be 1%); glycine regeneration solution is added to wells A10 and B10; and neutralization solution (PBS in this experiment) is added to wells A11 and B11. (2) Sensor preparation: Place the Ni-NTA sensor on the sensor plate, add 200 μL of PBS solution to wells A1 and B1 of another 96-well plate, and immerse the sensor surface in the buffer to activate it for a period of time. (3) Instrument parameter settings: Open the Plate Definition interface of the instrument, set the wells at the corresponding positions on Plate 1, select the names of all added wells, and enter the molar concentration of the small molecule sample in the Conc column respectively; ① Open the Assay Definition interface, select A1, add it as Baseline, time is 120 s, this segment is the baseline measurement; ② Select A2, add it as Loading, time is 600 s, this segment is the chip loading; ③ Select A3, add it as Baseline2, time is 30 s, this segment is the chip equilibration in the neutralization solution, as the second baseline measurement; ④ Select A4, add it as Association, time is 300 s, protein binds to small molecule; ⑤ Select A3, add it as Dissociation, time is 300 s, protein dissociates from small molecule; ⑥ Select A9 and add it as a Regeneration device with a time of 30 seconds to regenerate the sensor.

[0059] Instrument Operation: Set the settings to "A3-A4-A3-A9-A10" five times, meaning that after the sensor is loaded with protein solution, it will undergo four steps of "baseline-binding-dissociation-regeneration" in wells A4-A9 containing small molecule samples of different concentrations. Open the SensorAssignment interface, select A1, B1, C1, and D1 in Sensor Tray to set the initial position of the sensor. Open the Review Experiment interface to check whether the settings of each well in Plate 1 and the running process are correct. Open the RunExperiment interface, set the data save location and naming file (detection time + protein and small molecule name). In the running settings, set Start after(s) to 600 s, keep other parameters unchanged, and run the experiment.

[0060] Data processing: (1) Open the instrument’s Data Analysis 12.0 software and select the named detection file in the Data Selection interface; (2) Open the Processing interface, select Sensor Selection in Step 1, select the sensor bound to the buffer solution, right-click and set it as a reference sensor; select Subtraction in Step 2, click Customize, set A1-B1, i.e., the experimental group minus the control group; in Step 3, set the Time Range to From 20 to 29.80, and the other options are the default settings, click Process Data, and the software will process the data; (3) After the data processing is completed, go to the Analyze interface, click Fit Curves, and you can output various kinetic parameters in the output table, mainly including Ka, Kd, ​​and KD values.

[0061] Experimental results: The membrane interference signal pattern caused by the combination of AB928 and A2aR is shown in the figure. Figure 3 (a) The concentration-signal fitting plot is shown in [the figure]. Figure 3 (b) As the concentration increases, the binding signal strengthens, and the binding constant ka between AB928 and A2aR is 1.98 × 10⁻⁶. 12 μM, dissociation constant kd = 3.13 × 10 6 μM, affinity KD=1.58 μM. Example 2 The aforementioned operations in this embodiment are the same as steps 1 to 3 of embodiment 1, to verify the antidepressant effect of AB928 in animals. 1: Weight changes in a mouse depression model constructed with corticosterone.

[0062] Body weight change is an important indicator for evaluating the effectiveness of a depression model. During the establishment of the mouse depression model, mouse weight was measured regularly to observe changes in body weight. 100 mg of corticosterone (CORT, 4 g, Hunan Yunbang Pharmaceutical Co., Ltd.) powder was dissolved in 10 mL of anhydrous ethanol (500 mL, Beijing Chemical Reagent Research Institute Co., Ltd.), sonicated for 5-10 minutes until completely dissolved, and then diluted with 990 mL of drinking water to 0.1 mg / mL. This solution was prepared fresh daily for 21 consecutive days, with the CORT solution being changed once daily. The experiment included a control group (controlled, given drinking water) of 8 mice and a model group (model, given CORT solution) of 8 mice. Body weight changes were recorded regularly. Figure 4 As shown. Compared with the control group, there was no significant difference in body weight between the two groups on day 1 of modeling [see...]. Figure 4 (a) On day 21 of modeling, the body weight of mice in the Model group decreased by 15.28% [see Figure 4 (b) indicates that replacing drinking water with 0.1 mg / mL CORT slows down the weight gain in mice. The difference in weight gain is consistent with the weight loss characteristics of a mouse depression model. 2: The TST experiment evaluated the antidepressant effects of each drug administration group on mice.

[0063] The TST experiment simulates an inescapable stressful situation. During the experiment, the mice are immobilized by their tails, placing them in a suspended position from which they cannot escape. The mice will instinctively exhibit struggling movements such as swinging their bodies and kicking their legs in an attempt to escape the adverse situation. Over time, the animals' struggling movements gradually decrease, and the duration of stillness significantly increases. This periodic state of stillness is considered a marker of "behavioral despair," and this behavioral change is a key indicator for screening potential antidepressants.

[0064] In the experiment, the mouse's tail tip was secured to the top support of the tail-suspending box 1-2 cm above the body using medical-grade tape, placing it in an inverted position with its head 5 cm above the bottom of the box. The total experiment duration was 6 minutes, with the first 2 minutes being an adaptation period. After excluding struggling behavior during this phase, the cumulative immobility time of the mouse in the following 4 minutes was recorded (defined as a state where the limbs did not move actively and only respiration was maintained). During the operation, gentle handling should be maintained to minimize the time spent grasping and restraining the animal, avoiding causing it anxiety and fear. Mice that successfully developed the model were randomly divided into four groups of eight mice each: a negative control group (Control, given drinking water), a model group (solvent composed of 5% DMSO: 40% PEG300: 3% Tween 80: 52% saline; PEG300, 500 mL, Beijing Innocare Technology Co., Ltd.; Tween 80, 500 mL, Shanghai Aladdin Biochemical Technology Co., Ltd.; saline, 500 mL, Shijiazhuang No. 4 Pharmaceutical Co., Ltd.), a positive control group (KW6002, 50 mg, Shanghai Jizhi Biochemical Co., Ltd., dose 1 mg / kg), and AB928 drug groups (1, 3, and 10 mg / kg). All groups received a single intraperitoneal injection of 20 μL / 100 g body weight. The antidepressant effect of the compound was evaluated using the TST test 2 h after administration.

[0065] A single mouse was secured to the top support of the tail suspension test system (model PHM-300, Med Associates, USA) approximately 1-2 cm from the tail tip using medical tape, placing the mouse in an inverted position with its head about 5 cm above the bottom of the test chamber. Immobility time was recorded continuously for 6 minutes, and the immobility time at the last 4 minutes was calculated. The immobility times of mice in each compound group and the Model group were statistically compared. Experimental data are expressed as mean ± standard deviation (Mean ± SD). Each group had n=8 mice. One-way ANOVA in GraphPad Prism 9.5 was used to analyze differences between groups. Compared with the Control group, significance was indicated by #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001; compared with the Model group, significance was indicated by... p<0.05, p<0.01, p<0.001 p<0.0001 indicates a significant difference.

[0066] The effect of compound AB928 on immobility time in mice was evaluated using the TST experiment. The experimental results are as follows: Figure 5 As shown, compared with the control group, the immobility time of mice in the Model group was significantly increased in the TST experiment (####p<0.0001), indicating that the depression model was successfully established; compared with the Model group, the dose of the antagonist positive control drug KW6002 was 1 mg / kg, and the immobility time of mice in the KW6002 group was significantly reduced ( p<0.01, indicating that KW6002 has an antidepressant effect; compared with the Model group, the AB928 drug group showed significant antidepressant effects at doses of 3 mg / kg and 10 mg / kg, and the effect was dose-related, with immobility time reduced by 5.16% compared with the Model group. p<0.05), 5.75% p<0.05). Evaluation by the TST experiment showed that the AB928 drug group had significant antidepressant effects at doses of 3 mg / kg and 10 mg / kg. 3: OFT experiment to evaluate the antidepressant effect of each drug administration group on mice.

[0067] In antidepressant drug research, the OFT experiment is commonly used to evaluate the antidepressant effect of drugs. Depressed mouse models typically exhibit reduced activity, fewer entries into the central region, and shorter dwell time. If a candidate compound can increase the total distance moved by mice, increase the frequency of entry into the central region, and increase the dwell time, it suggests that the compound may have an effect on improving depressive-like behavior. Mice that successfully developed the model were randomly divided into four groups of eight mice each: a negative control group (Control, given drinking water), a model group (solvent composed of 5% DMSO: 40% PEG300: 3% Tween 80: 52% saline), a positive control group (KW6002, 1 mg / kg), and an AB928 drug group (10 mg / kg). All groups received a single intraperitoneal injection of 20 μL / 100 g body weight. The OFT experiment was used to evaluate the antidepressant effect of the compound in each group 4 h after administration.

[0068] During the experiment, mice were gently placed in the center of the open field analysis system (model XR602, product of Shanghai Xinsoft Co., Ltd., China) to avoid causing additional stress to the animals. The test chamber was thoroughly cleaned after each animal's experiment, and the next animal was tested only after the odor had dissipated. Mice were placed against the wall in the open field chamber, and their activity was observed over 5 minutes using an automated observation and analysis imaging system. The movement distance, speed, immobility time, number of times entering the central area, movement distance, and movement time of mice in each drug group and the Model group were compared. Experimental data are expressed as mean ± standard deviation (Mean ± SD). Each group had n=8 mice. One-way ANOVA in GraphPad Prism 9.5 was used to analyze differences between groups. Compared with the Control group, significance was indicated by #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001; compared with the Model group, significance was indicated by... p<0.05, p<0.01, p<0.001 p<0.0001 indicates a significant difference.

[0069] The effects of KW6002 and AB928 on mouse behavioral trajectories were evaluated using OFT experiments. Figure 6 The trajectory index analysis results for each group are shown below. Figure 7 As shown. Figure 7 (a) indicates that depressed mice exhibited a significant reduction in movement distance across the total area. ## p<0.01), while administration of KW6002 and AB928 at 10 mg / kg significantly increased ( p<0.01, p<0.05); Figure 7 (b) indicates that the movement speed of depressed mice decreased in the total area. # p<0.05), while administration of KW60025 and AB928 at 10 mg / kg both showed significant increases (p<0.05). p<0.01, p<0.05). OFT test results showed that AB928 had a significant antidepressant effect at 10 mg / kg. 4: The FST experiment was used to evaluate the antidepressant effect of each drug administration group on mice.

[0070] Freezing test (FST) is a classic experimental method for evaluating the efficacy of antidepressants and studying depressive-like behaviors in animals. This experiment involves placing animals in an inescapable aquatic environment and observing their behavioral responses. Initially, the animals attempt to escape, but subsequently gradually enter a state of immobility, considered a manifestation of "behavioral despair," which shares some similarities with human depressive mood. Antidepressants can significantly shorten the immobility time in animals, thus allowing for the evaluation of their antidepressant effects. Mice that successfully developed the model were randomly assigned to groups of eight mice each: a negative control group (controlled, given drinking water), a model group (using a solution composed of 5% DMSO, 40% PEG300, 3% Tween 80, and 52% saline), a positive control group (KW6002, 1 mg / kg), and an AB928 drug group (10 mg / kg). All groups received a single intraperitoneal injection (20 μL / 100 g body weight). The FST test was performed 6 hours after administration to evaluate the antidepressant effect of the compound in each group.

[0071] In the experiment, mice were placed in the forced swimming test module Track 3D transparent glass tank (15 cm in diameter, 22 cm in height) of an animal movement tracking system (model EthoVision XT 15, Noldus, Netherlands). The water temperature was 24℃ and the water depth was 16 cm, ensuring that the mice could not touch the bottom or climb. The entire experiment was recorded for 6 minutes, with the first 2 minutes as the adaptation period and the last 4 minutes for recording immobility time. After the experiment, the mice were removed from the water, dried with a towel, and placed under a heat lamp to dry before being returned to their original cages. The antidepressant effect of the candidate compound was evaluated by comparing the differences in movement speed, movement time, and immobility time between the drug group and the model group. Each drug group (KW6002 dosage was 1 mg / kg, AB928 dosage was 10 mg / kg) was compared with the Model. Experimental data are expressed as mean ± standard deviation (Mean ± SD). Each group had n=8. One-way ANOVA in GraphPad Prism 9.5 was used to analyze differences between groups. Compared with the Control group, significance was indicated by #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001; compared with the Model group, significance was indicated by... p<0.05, p<0.01, p<0.001 p<0.0001 indicates a significant difference.

[0072] The antidepressant effects of KW6002 and AB928 were evaluated using the FST experiment, and the results were as follows: Figure 8 As shown. Compared with the Con group, the mice in the Model group showed significantly reduced FST movement speed and movement time (#p<0.05, ##p<0.01), and significantly increased immobility time (##p<0.01), indicating that the depressed model mice exhibited typical "behavioral hopelessness" behavior, indicating that the depression model was successfully established; compared with the Model group, the mice in the KW6002 group showed significantly increased FST movement speed and movement time ( p<0.05, p<0.01, and the immobility time was significantly reduced ( p<0.01), indicating that KW6002 has a significant antidepressant effect; compared with the Model group, the FST movement speed of the 10 mg / kg AB928 drug group was significantly lower. Figure 8 (a) , Exercise time Figure 8 (b) Significantly increased ( p<0.001, p<0.001), stationary time [ Figure 8 (c) Significantly reduced ( (p<0.0001), demonstrating a significant antidepressant effect. FST experimental results showed that 10 mg / kg of AB928 had a significant antidepressant effect. 5: The SAT experiment was used to evaluate the antidepressant effect of each drug administration group on mice.

[0073] The fine behavioral assessment (SAT) system for rats and mice primarily uses carbon fiber plates to identify and record stress changes resulting from different behaviors and their durations, such as crawling, standing, grooming, licking, drinking, and eating. The antidepressant effect of the drug was assessed by monitoring the animals' movement time and immobility time; the antidepressant drug significantly shortened the animals' immobility time. Mice that successfully developed the model were randomly divided into four groups of eight: a negative control group (Control, given drinking water), a model group (solvent composed of 5% DMSO: 40% PEG300: 3% Tween 80: 52% saline), a positive control group (KW6002, 1 mg / kg), and an AB928 drug group (10 mg / kg). All groups received a single intraperitoneal injection of 20 μL / 100 g body weight. The antidepressant effect of the compound was evaluated using the SAT test 8 h after administration.

[0074] In the experiment, mice were placed in cages using a fine behavior monitoring system (Laboras, Metris, Netherlands) with free access to water. Spontaneous behavioral activity of the mice was monitored and recorded over 10 minutes using the SAT (Simultaneous Attention Scale) system. After the experiment, the antidepressant effect of the candidate compounds was evaluated by comparing the differences in movement time and immobility time between the drug groups and the model group. Each drug group (KW6002 at 1 mg / kg, AB928 at 10 mg / kg) was compared with the model group. Experimental data are expressed as mean ± standard deviation (Mean ± SD). Each group had n=8 participants. One-way ANOVA in GraphPad Prism 9.5 was used to analyze differences between groups. Compared with the control group, significance was indicated by #p<0.05, ##p<0.01, ###p<0.001, and ####p<0.0001; compared with the model group, significance was indicated by... p<0.05, p<0.01, p<0.001 p<0.0001 indicates a significant difference.

[0075] The antidepressant effects of KW6002 and AB928 were evaluated using the SAT test, and the results were as follows: Figure 9As shown. Compared with the Control group, the mice in the Model group had significantly reduced SAT movement distance and movement time (###p<0.001, ###p<0.001), indicating that the depressed model mice exhibited typical "depressive" behavior, indicating that the depression model was successfully established; compared with the Model group, the mice in the KW6002 group had significantly increased movement distance and movement time ( p<0.001, (p<0.0001), indicating that KW6002 has a significant antidepressant effect; compared with the Model group, the mice in the SAT group in the 10 mg / kg group had a greater range of motion. Figure 9 (a) , Exercise time Figure 9 (b) Significantly increased ( p<0.05, (p<0.05). The SAT test results showed that 10 mg / kg of AB928 had a significant antidepressant effect.

[0076] As described above, the AB928 drug of the present invention can effectively treat depression in various animal models by regulating the activity of A2aR / A2bR adenosine receptors.

[0077] The foregoing description is based on embodiments of the present invention, but these are merely examples and not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various modifications and applications, not illustrated above, can be made without departing from the essential characteristics of the embodiments of the invention. For example, the constituent elements specifically shown in the embodiments of the invention can be modified and implemented. Furthermore, differences related to these modifications and applications should be interpreted as being included within the scope of the invention as defined in the appended claims.

Claims

1. The use of an adenosine A2aR / A2bR dual antagonist in the preparation of drugs for the prevention and treatment of depression, characterized in that, The A2aR / A2bR double antagonist is AB928 and its pharmaceutically acceptable salt; The molecular structure of AB928 is shown below: 。 2. A drug for the prevention and treatment of depression, characterized in that, The drug has an adenosine A2aR / A2bR double antagonist as its active ingredient, wherein the A2aR / A2bR double antagonist is AB928 and its pharmaceutically acceptable salt.

3. The drug for preventing and treating depression according to claim 2, characterized in that, The drug is a pharmaceutical composition, and also contains one or more pharmaceutically acceptable carriers, diluents, and excipients.

4. The drug for preventing and treating depression according to claim 3, characterized in that, Pharmaceutically acceptable carriers, diluents, or excipients include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oil, and polyalkylene glycols.

5. The drug for preventing and treating depression according to claim 2, characterized in that, The pharmaceutical composition can be administered orally, by injection, or by spray.

6. The drug for preventing and treating depression according to claim 2, characterized in that, The pharmaceutical composition is in the form of tablets, capsules, solutions, suspensions, injections, or patches.

7. The drug for preventing and treating depression according to claim 2, characterized in that, The dosage of AB928 is 1 mg / kg to 20 mg / kg.

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