5-ht2a / drds dual-target agonist and use thereof in preparation of antidepressant

Abstract

The application provides a 5-HT2A / DRD1 dual-target agonist and application thereof in preparation of an antidepressant drug, wherein the dual-target agonist comprises saikosaponin A and saikosaponin D. The application proves for the first time that saikosaponin A and saikosaponin D are dual-target agonists of 5-HT2A receptors and DRD1 receptors. Compared with existing drugs that only act on a single neurotransmitter system, the multi-component regulation mode is more in line with the pathological characteristics of multi-neurotransmitter disorders of depression.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, and in particular relates to a 5-HT2A / DRD1 dual-target agonist and its application in the preparation of antidepressant drugs. Background Technology

[0002] Depression is a type of affective disorder characterized by low mood, slowed thinking, and loss of interest. Currently, Western medicine mainly treats depression by addressing symptoms, and there are no specific drugs available. Furthermore, existing clinical antidepressants have problems such as slow onset of action, significant side effects, and poor efficacy in some patients. Therefore, finding safe, effective, and low-side-effect antidepressants has become a research hotspot and urgent need in the current medical field.

[0003] Bupleurum, a commonly used traditional Chinese medicine, was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). It possesses the effects of dispersing heat, soothing the liver and relieving depression, and raising yang qi. Its liver-soothing and depression-relieving effects make it a core herb in traditional Chinese medicine for treating depression, and it is widely used clinically. Related formulas such as Chaihu Shugan San (Bupleurum Liver-Soothing Powder), Xiaoyao San (Free and Easy Wanderer Powder), and Xiao Chaihu Tang (Minor Bupleurum Decoction) all use Bupleurum as the principal ingredient. Numerous clinical and experimental studies have confirmed that Bupleurum and its related formulas have a definite therapeutic effect on depression, effectively improving patients' depressive state and accompanying symptoms such as sleep disorders and autonomic nervous system dysfunction. However, although the antidepressant effect of Bupleurum is widely recognized, the specific material basis (i.e., the core active monomer component) of its antidepressant effect, as well as its target and pathway of action, are not yet fully understood. This has become a key bottleneck restricting the modern development and application of Bupleurum-based antidepressant drugs. Summary of the Invention

[0004] In view of this, the present invention aims to overcome the deficiencies in the prior art and proposes a 5-HT2A / DRD1 dual-target agonist and its application in the preparation of antidepressant drugs.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a 5-HT2A / DRD1 dual-target agonist, the dual-target agonist comprising saikosaponin A and saikosaponin D.

[0006] Secondly, the present invention also provides the application of a 5-HT2A / DRD1 dual-target agonist in the preparation of antidepressant drugs.

[0007] Thirdly, the present invention also provides a method for screening antidepressant monomeric compounds, comprising the following steps: S1. Separate the cell membranes of cells overexpressing the target receptor separately, and then prepare the target receptor cell membrane chromatographic column by separating the cell membrane and modified silica gel separately; S2. The solutions of the Bupleurum monomer compounds to be tested were placed into the target receptor cell membrane chromatography column, and the active ingredients were screened by HPLC-CMC method. S3. Bioactivity was verified using a calcium flux fluorescence detection system, and saikosaponin A and saikosaponin D were obtained through screening.

[0008] Preferably, the cells in step S1 are HEK293 human embryonic kidney cells.

[0009] Preferably, the cells overexpressing the target receptor in step S1 are HEK293 human embryonic kidney cells overexpressing the 5-HT2A receptor and the DRD1 receptor.

[0010] Preferably, the method for preparing the modified silica gel in step S1 includes the following steps: Aminosilicone was added to tetrahydrofuran to obtain a mixture. The mixture was cooled to 0°C in an ice bath. Cyanuryl chloride and ethyl diisopropylamine were added under stirring at a temperature not exceeding 5°C for 2 hours. After filtration, the mixture was washed with ethanol to obtain cyanuryl chloride-modified silica. The cyanuryl chloride-modified silica was then dissolved in N,N-dimethylformamide. O6-[4-(aminomethyl)benzyl]guanine was added, and the mixture was refluxed at 95°C for 8 hours. After reflux, the product was filtered and washed with N,N-dimethylformamide to obtain modified silica with bonded groups.

[0011] Preferably, the ratio of aminosilicone, cyanuric chloride, and ethyl diisopropylamine is 1.4g:0.36g:1.4mL.

[0012] Preferably, the mass ratio of the cyanuric chloride-modified silica gel to O6-[4-(aminomethyl)benzyl]guanine is 1.4:0.05.

[0013] Preferably, the preparation method of the target receptor cell membrane chromatography column in step S1 includes the following steps: S11. Cell membranes of cells overexpressing the target receptor were separated by Tris-HCl hypotonic buffer and ultrasonic disruption. S12. Using modified silica gel as a carrier, the cell membrane is uniformly coated on the carrier surface through cell membrane phospholipid self-fusion to prepare a cell membrane stationary phase suspension. S13. Pack the cell membrane stationary phase suspension into the cell membrane chromatography column core, pressurize at a flow rate of 1 mL / min until the column inlet pressure stabilizes, repeat the process of adding cell membrane stationary phase suspension and pressurizing until the column is full, and seal the column for later use.

[0014] Preferably, the specific steps of step S2 are as follows: S21. Set the chromatographic parameters: mobile phase: pure water; column temperature: 37℃; flow rate: 0.2 mL / min; analysis time: 120 min; injection volume: 2 μL; S22. Perform a full-wavelength scan on the solution of the Bupleurum monomer compound to be tested, using distilled water as a blank control, to determine the maximum absorption wavelength of each Bupleurum monomer compound to be tested and screen and analyze it.

[0015] Compared with the prior art, the present invention has the following advantages: (1) This invention achieves precise dual-target identification and a clear mechanism of action. This invention demonstrates for the first time through experiments that saikosaponin A and saikosaponin D are dual-target agonists of the 5-HT2A receptor and the DRD1 receptor. Compared with existing drugs that only act on a single neurotransmitter system, this multi-component regulatory approach is more consistent with the pathological characteristics of multi-neurotransmitter imbalance in depression; (2) The screening results of this invention exhibit extremely high bioactivity (high binding affinity, high activating rate, and low effective concentration). In the HPLC-CMC screening, saikosaponins A and D showed extremely strong receptor binding ability. Saikosaponin A had a retention time of 49.426 min on the 5-HT2A receptor column and 20.194 min on the DRD1 receptor column, which was significantly better than other tested monomers. Calcium flow detection results showed that saikosaponins A and D had extremely strong activation effects on the receptors. The maximum activating rate of saikosaponin A on DRD1 was 239.5%, while the activating rate of saikosaponin D on DRD1 was as high as 314.5%. The screened components had extremely low half-maximum effective concentrations (EC50). For example, the EC50 of saikosaponin D on DRD1 was only 2.511 µM, and the EC50 of ferulic acid on 5-HT2A was only 0.536 µM, proving that these monomers can exert significant biological effects at extremely low doses. (3) This invention provides a quantitative evaluation standard, improving R&D efficiency. This invention not only identifies the active ingredient, but also establishes a precise drug price evaluation system through dose-response curves. Compared with traditional apparent efficacy observation, the EC50 data provided by this invention provides a scientific and objective quantitative indicator for subsequent development of antidepressant lead drugs and quality control; (4) This invention solves the problem of unclear basis of active substances in traditional Chinese medicine. Through the use of modern chromatography and molecular biology techniques, this invention clarifies the core monomeric components that enable Bupleurum to exert its antidepressant effect, providing a solid theoretical basis and material foundation for the modernization of Bupleurum-based prescriptions and the development of new monomeric drugs. Attached Figure Description

[0016] Figure 1 The chromatogram of the blank control on a 5-HT2A receptor cell membrane chromatography column; Figure 2The chromatogram of the positive control drug mirtazapine on a 5-HT2A receptor cell membrane chromatography column; Figure 3 The chromatogram of saikosaponin A on a 5-HT2A receptor cell membrane chromatography column; Figure 4 The chromatogram of saikosaponin C on a 5-HT2A receptor cell membrane chromatography column; Figure 5 The chromatogram of ferulic acid on a 5-HT2A receptor cell membrane chromatography column; Figure 6 The chromatogram of scopolamine lactone on a 5-HT2A receptor cell membrane chromatography column is shown. Figure 7 The chromatogram of saikosaponin B3 on a 5-HT2A receptor cell membrane chromatography column; Figure 8 The chromatogram of rutin on a 5-HT2A receptor cell membrane chromatography column; Figure 9 The chromatogram of chlorogenic acid on a 5-HT2A receptor cell membrane chromatography column; Figure 10 The chromatogram of kaempferol on a 5-HT2A receptor cell membrane chromatography column; Figure 11 The chromatogram of isorhamnetin on a 5-HT2A receptor cell membrane chromatography column; Figure 12 The chromatogram of the blank control on the DRD1 receptor cell membrane column; Figure 13 The chromatogram of the positive control drug dopamine on a DRD1 receptor cell membrane chromatography column; Figure 14 The chromatogram of saikosaponin A on a DRD1 receptor cell membrane chromatography column; Figure 15 The chromatogram of saikosaponin C on the DRD1 receptor cell membrane column; Figure 16 The chromatogram of ferulic acid on a DRD1 receptor cell membrane chromatography column; Figure 17 The chromatogram of scopolamine lactone on a DRD1 receptor cell membrane chromatography column; Figure 18 The chromatogram of saikosaponin B3 on a DRD1 receptor cell membrane chromatography column; Figure 19 The chromatogram of rutin on a DRD1 receptor cell membrane chromatography column; Figure 20 The chromatogram of chlorogenic acid on a DRD1 receptor cell membrane chromatography column; Figure 21 The chromatogram of kaempferol on a DRD1 receptor cell membrane chromatography column; Figure 22 The chromatogram of saikosaponin D on the DRD1 receptor cell membrane column; Figure 23 The dose-response curve of the agonistic effect of saikosaponin A on DRD1; Figure 24 The dose-response curve of the agonistic effect of saikosaponin D on DRD1; Figure 25 The dose-response curve shows the agonistic effect of chlorogenic acid on DRD1. Figure 26 The dose-response curve of the agonistic effect of saikosaponin A on 5-HT2A; Figure 27 The dose-response curve of the agonistic effect of saikosaponin D on 5-HT2A; Figure 28 The dose-response curve of the agonist effect of isorhamnetin on 5-HT2A; Figure 29 The dose-response curve of the agonistic effect of kaempferol on 5-HT2A; Figure 30 This is the dose-response curve of the agonistic effect of ferulic acid on 5-HT2A. Detailed Implementation

[0017] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0018] In this document, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0019] In this document, when values ​​are described as ranges, it should be understood that such disclosure includes disclosure of all possible subranges within that range, as well as the specific numerical values ​​falling within that range, regardless of whether the specific numerical value or specific subrange is explicitly specified.

[0020] In this article, the terms "multiple" or "more than" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0021] In this document, the terms "preferred" and "more preferred" are used only to describe implementation methods or embodiments with better effects, and should be understood as not constituting a limitation on the scope of protection of this invention.

[0022] In this document, terms such as "further" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0023] In this article, the term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0024] In this document, the term "about" means a specified value of + / - 10%, preferably + / - 5%, and more preferably + / - 1%.

[0025] In this article, the terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are not limited to.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0027] This invention provides a group of Bupleurum monomeric compounds with antidepressant activity and their application in regulating 5-HT2A and DRD1 receptors. This invention confirms the efficacy of specific monomeric compounds through a combination of physical binding screening and bioactivity verification. Specifically: (1) Constructing a receptor-specific screening platform Cell model establishment: HEK293 human embryonic kidney cells overexpressing 5-HT2A receptor and HEK293 human embryonic kidney cells overexpressing DRD1 receptor were selected as target cells.

[0028] Cell membrane chromatography column preparation: Cell membranes were separated using hypotonic buffer and ultrasonic disruption. Modified silica gel was used as a carrier, and receptor cell membranes were coated onto the silica gel surface using phospholipid self-fusion technology to prepare a receptor-specific chromatographic stationary phase. This step utilizes the drug-receptor interaction properties to translate the in vivo mechanism of action into dynamic retention behavior within the chromatographic column.

[0029] (2) Screening and identification of active ingredients The monomeric compounds of Bupleurum chinense to be tested were screened by HPLC-CMC technology, and the retention time (Rt) was used as the evaluation index.

[0030] (3) Confirmation of the biological effects of the core components (calcium flow detection) To confirm that the selected binding components possess biological agonistic activity, this invention introduces calcium flux fluorescence detection technology. HEK239 cells containing the receptor were plated (5-HT2A: 30,000 cells / well; DRD1: 50,000 cells / well), labeled with fluorescent probes, and intracellular calcium signal changes triggered by receptor activation were captured in real time.

[0031] The present invention will be described in detail below with reference to embodiments.

[0032] Example 1: Preparation of Cell Membrane Chromatography Column (1) Isolation and extraction of cell membrane HEK293 human embryonic kidney cells overexpressing 5-HT2A and DRD1 receptors were collected separately, centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. 5 mL of pre-cooled Tris-HCl hypotonic buffer was added, vortexed, and then sonicated on ice for 30 min (until no intact cells were found). Stepwise centrifugation: the supernatant was collected at 5000 r / min (10 min), and the precipitate was collected at 12000 r / min (20 min). The precipitate was resuspended in physiological saline and centrifuged again at 12000 r / min for 20 min. The final cell membrane precipitate was homogenized with 1 mL of physiological saline by sonication to obtain a cell membrane suspension.

[0033] (2) Preparation of modified silica gel First, 1.4 g of aminosilica gel was added to 14 mL of tetrahydrofuran, and the mixture was cooled to 0 °C in an ice bath. Then, 0.36 g of cyanuric chloride and 1.4 mL of ethyl diisopropylamine were slowly added with stirring at a temperature not exceeding 5 °C for 2 hours. After filtration, the cyanuric chloride-modified silica gel (SiO2-TCT) was thoroughly washed with ethanol. Then, 1.4 g of SiO2-TCT was dissolved in N,N-dimethylformamide (50 mL). Then, 50 mg of O6-[4-(aminomethyl)benzyl]guanine was added, and the mixture was refluxed at 95 °C for 8 hours. After reflux, the product was filtered and completely washed with N,N-dimethylformamide to obtain BG (bonding group)-modified silica gel (SiO2-BG).

[0034] (3) Preparation of cell membrane stationary phase Weigh 0.05 g of modified silica gel as a carrier and add it to 5 mL of cell membrane suspension. After shaking for 5 min, transfer it to a beaker. Stir at 4℃ for 12 h to promote the self-fusion of cell membrane phospholipids and uniformly coat the carrier. Then centrifuge (1000 r / min, 5 min), discard the supernatant, wash the precipitate twice with pre-cooled Tris-HCl buffer to remove free cell membranes. Resuspend the precipitate in an appropriate amount of Tris-HCl buffer to prepare cell membrane stationary phase (CMSP) suspension, and store at 4℃ for later use.

[0035] (4) Packing of cell membrane chromatography column After cleaning the empty column core (10 mm × 2 mm ID) with distilled water, install the column sleeve, gasket, and sieve plate according to the orientation; inject CMSP suspension into the column core and pressurize at a low flow rate until the pressure at the column inlet is stable; repeat the operation of adding CMSP suspension and pressurizing until the column is filled to obtain 5-HT2A receptor cell membrane chromatography column and DRD1 receptor cell membrane chromatography column respectively. After filling, seal the column to ensure uniform distribution of packing material.

[0036] (5) Establishment of chromatographic model 1) Chromatographic parameters: Column: 5-HT2A / DRD1 receptor cell membrane chromatographic column (HPLC system); Mobile phase: pure water; Column temperature: 37℃; Flow rate: 0.2 mL / min; Analysis time: 120 min; Injection volume: 2 μL. The system needs to be equilibrated with the mobile phase until the baseline is stable before operation.

[0037] 2) Perform a full wavelength scan (190-800 nm) on the monomer solution to be tested, using distilled water as a blank control, to determine the maximum absorption wavelength of each compound.

[0038] The results are as follows Figure 1-22 As shown, among the 17 monomers, saikosaponin A, saikosaponin D, saikosaponin B3, saikosaponin C, ferulic acid, kaempferol, rutin, and chlorogenic acid exhibit significant retention behavior on the 5-HT2A receptor column. Simultaneously, saikosaponin A, saikosaponin D, saikosaponin B3, ferulic acid, and kaempferol also demonstrate strong binding affinity on the DRD1 receptor cell membrane chromatography column. Notably, saikosaponin A and D show significantly longer retention times on both receptor cell membrane chromatography columns compared to the other monomers.

[0039] Example 2: Screening of receptor agonists (1) Determination of cell plating density The calcium flow assay requires a cell count between 20,000 and 80,000 cells per well in a 96-well plate. Therefore, eight cell coagulation densities were set: 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, and 80,000 cells per well, with six replicates per density. After incubation at 37°C for 24 hours, the culture medium was removed. Under light-protected conditions, 100 μL of 10% CCK-8 solution was added to each well of the cell culture plate, and incubation continued for 1 hour. The absorbance was measured at 450 nm using a microplate reader to determine the optimal cell coagulation density.

[0040] (2) Detection method for receptor agonists Recombinant cell lines HEK239 / 5HT2A and HEK239 / DRD1, which showed good cell condition after microscopic observation, were digested and counted. The cells were then arranged at different densities (3 × 10⁻⁶). 4 5×10 4(50 analytes / well) were seeded into Matrigel pre-coated 96-well plates and incubated overnight at 37°C with 5% CO2. The next day, 100 μL of loading buffer was added to each well, and the plates were incubated in the dark for 2 h before detection. The analytes were added to the plates at concentration gradients of 50 μmol / L, 10 μmol / L, 2 μmol / L, 0.4 μmol / L, 0.08 μmol / L, and 0.0016 μmol / L (the volume of the compound in the plate is generally at least 70 μL). 50 μL of the compound was automatically added to each well using a FlexStation instrument, and the calcium flux fluorescence signal value was acquired and recorded in real time.

[0041] (3) Monomer compounds to be screened Saikosaponin A, Saikosaponin D, 6-O-acetylsalicosaponin A, Saikosaponin E, Saikosaponin F, Saikosaponin C, Saikosaponin B1, Saikosaponin B3, isorhamnetin, rutin, ferulic acid, scopolamine lactone, quercetin, chlorogenic acid, isochlorogenic acid A, kaempferol, sucrose.

[0042] (4) Data Analysis Calcium flow data detected by the calcium flow model were converted into relative fluorescence units (RFU), with the highest values ​​represented for all wells. Preliminary data acquisition and processing were performed using ScreenWorks software. EC50 values ​​were determined through nonlinear regression fitting analysis of the dose-response curves. EC50 was calculated using GraphPad Prism fitting.

[0043] The results are as follows Figure 23-30 As shown, the efficacy verification results can be seen: 5-HT2A receptor activity: Saikosaponin A, Saikosaponin D and ferulic acid were confirmed as agonists of the 5-HT2A receptor, with EC50 values ​​of 9.144 µM, 5.671 µM and 0.536 µM, respectively.

[0044] DRD1 receptor activity: Saikosaponin A and Saikosaponin D were confirmed to have a strong agonistic effect on the DRD1 receptor, with maximum agonistic rates of 239.5% and 314.5%, respectively, and EC50 values ​​of 5.501 µM and 2.511 µM, respectively.

[0045] In summary, this invention, through initial screening using HPLC-CMC physical adsorption and secondary screening using calcium flux detection technology, identified saikosaponin A and saikosaponin D as the core antidepressant active components. These two, acting as "dual-target" agonists, exert their antidepressant effects by synergistically regulating the 5-HT and dopamine systems, and are key active ingredients in the preparation of antidepressant drugs.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A 5-HT2A / DRD1 dual-target agonist, characterized in that: The dual-target agonists include saikosaponin A and saikosaponin D.

2. The application of a 5-HT2A / DRD1 dual-target agonist in the preparation of antidepressant drugs.

3. A method for screening antidepressant monomeric compounds, characterized in that: Includes the following steps: S1. Separate the cell membranes of cells overexpressing the target receptor separately, and then prepare the target receptor cell membrane chromatographic column by separating the cell membrane and modified silica gel separately; S2. The solutions of the Bupleurum monomer compounds to be tested were placed into the target receptor cell membrane chromatography column, and the active ingredients were screened by HPLC-CMC method. S3. Bioactivity was verified using a calcium flux fluorescence detection system, and saikosaponin A and saikosaponin D were obtained through screening.

4. The screening method according to claim 3, characterized in that: The cells in step S1 are HEK293 human embryonic kidney cells.

5. The screening method according to claim 3, characterized in that: The cells overexpressing the target receptor in step S1 are HEK293 human embryonic kidney cells overexpressing the 5-HT2A receptor and the DRD1 receptor.

6. The screening method according to claim 3, characterized in that: The method for preparing modified silicone in step S1 includes the following steps: Aminosilicone was added to tetrahydrofuran to obtain a mixture. The mixture was cooled to 0°C in an ice bath. Cyanuryl chloride and ethyl diisopropylamine were added under stirring at a temperature not exceeding 5°C for 2 hours. After filtration, the mixture was washed with ethanol to obtain cyanuryl chloride-modified silica. The cyanuryl chloride-modified silica was then dissolved in N,N-dimethylformamide. O6-[4-(aminomethyl)benzyl]guanine was added, and the mixture was refluxed at 95°C for 8 hours. After reflux, the product was filtered and washed with N,N-dimethylformamide to obtain modified silica with bonded groups.

7. The screening method according to claim 6, characterized in that: The ratio of aminosilicone, cyanuric chloride, and ethyl diisopropylamine is 1.4g:0.36g:1.4mL.

8. The screening method according to claim 6, characterized in that: The mass ratio of the cyanuric chloride-modified silica gel to O6-[4-(aminomethyl)benzyl]guanine is 1.4:0.

05.

9. The screening method according to claim 3, characterized in that: The preparation method of the target receptor cell membrane chromatography column in step S1 includes the following steps: S11. Cell membranes of cells overexpressing the target receptor were separated by Tris-HCl hypotonic buffer and ultrasonic disruption. S12. Using modified silica gel as a carrier, the cell membrane is uniformly coated on the carrier surface through cell membrane phospholipid self-fusion to prepare a cell membrane stationary phase suspension. S13. Pack the cell membrane stationary phase suspension into the cell membrane chromatography column core, pressurize at a flow rate of 1 mL / min until the column inlet pressure stabilizes, repeat the process of adding cell membrane stationary phase suspension and pressurizing until the column is full, and seal the column for later use.

10. The screening method according to claim 3, characterized in that: The specific steps of step S2 are as follows: S21. Set the chromatographic parameters: mobile phase: pure water; column temperature: 37℃; flow rate: 0.2 mL / min; analysis time: 120 min; injection volume: 2 μL; S22. Perform a full-wavelength scan on the solution of the Bupleurum monomer compound to be tested, using distilled water as a blank control, to determine the maximum absorption wavelength of each Bupleurum monomer compound to be tested and screen and analyze it.

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