Application of lingenamine in preparation of medicine for treating schizophrenia

Abstract

The invention discloses application of lingenamine in preparation of a medicine for treating schizophrenia, and belongs to the technical field of biological medicine. By antagonizing 5-HT2AR and D2R receptors, the lingenamine can improve positive symptoms and negative symptoms of schizophrenia patients at the same time, and the defect that the curative effect of an existing medicine on the negative symptoms is limited is overcome. Meanwhile, adverse reactions such as over-sedation cannot be caused in the treatment process, side effects such as extrapyramidal system symptoms are avoided, and the treatment safety is remarkably improved. The lingenamine and the salts thereof have a definite multi-target action mechanism, and a new choice is provided for the treatment of schizophrenia. In addition, the medicine can be prepared into various dosage forms such as tablets, sustained-release preparations and injections, individualized administration is facilitated clinically according to specific conditions of patients, the medication scheme is flexibly adjusted, and therefore the flexibility and effectiveness of treatment are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of linderine in the preparation of drugs for treating schizophrenia. Background Technology

[0002] Schizophrenia (SCZ), a severe mental illness with complex pathological mechanisms, is characterized by its rapid onset and diverse and severe symptoms. It has a high incidence rate worldwide, significantly impacting patients' quality of life, social functioning, and their families. Its clinical symptoms are mainly divided into positive symptoms, negative symptoms, and cognitive symptoms. Positive symptoms include hallucinations (such as auditory and visual hallucinations), delusions (such as persecutory delusions and delusions of reference), thought disorders, and behavioral disorders, which cause patients to become significantly detached from reality. Negative symptoms manifest as emotional blunting, diminished willpower, social withdrawal, and poverty of speech, severely affecting the recovery of patients' social functioning. Cognitive symptoms involve impairment in multiple cognitive domains, including attention, memory, and executive function, further hindering the patient's recovery process.

[0003] Typical antipsychotics, such as phenothiazines (chlorpromazine, perphenazine, etc.) and butyrophenones (haloperidol, etc.), primarily exert their therapeutic effects by blocking dopamine D2 receptors. These drugs are effective in controlling positive symptoms of schizophrenia, including hallucinations, delusions, and behavioral disturbances. However, they have significant side effects, especially extrapyramidal reactions (EPS), including acute dystonia, akathisia, Parkinson's syndrome, and tardive dyskinesia, which severely impact patients' quality of life and treatment adherence. Furthermore, typical antipsychotics are less effective at treating negative and cognitive symptoms, failing to comprehensively improve the patient's mental state. Atypical antipsychotics, such as olanzapine, risperidone, quetiapine, and aripiprazole, have become the mainstays of schizophrenia treatment in recent years. These drugs not only antagonize dopamine D2 receptors but also have a high affinity for serotonin 2A receptors (5-HT2AR), exerting their therapeutic effect by balancing dopaminergic and serotonergic neurotransmission. Compared to typical antipsychotics, atypical antipsychotics are effective for both positive and negative symptoms, and have relatively milder side effects such as extrapyramidal reactions. However, they also have some drawbacks. For example, some patients may experience weight gain, metabolic syndrome (such as elevated blood sugar and dyslipidemia), and cardiovascular adverse reactions. Long-term use may also increase the risk of developing diabetes, cardiovascular disease, and other physical illnesses. Furthermore, individual responses to atypical antipsychotics vary among patients, and some may experience poor efficacy or intolerable side effects. In addition to the aforementioned antipsychotics, there are also some adjunctive therapies and methods used in the treatment of schizophrenia. For example, antidepressants can be used to treat schizophrenia patients with depressive symptoms; mood stabilizers can be used to control patients' mood fluctuations; and electroconvulsive therapy (ECT) has some efficacy for patients with extreme excitement, agitation, severe suicidal tendencies, or those unresponsive to medication. However, these adjunctive treatments and methods also have their limitations. For instance, antidepressants may increase the risk of patients turning manic, and ECT may lead to cognitive impairment. Therefore, finding safer and more effective drugs to treat schizophrenia has become an important task in the medical and research fields. An ideal new antipsychotic drug should effectively improve the positive symptoms of schizophrenia, actively address negative symptoms and cognitive impairment, while minimizing or eliminating side effects associated with obesity, glucose and lipid metabolism syndrome. This requires not only precise neurotransmitter receptor regulation capabilities but also a gentler mechanism of action to avoid unnecessary burden on other systems in the body.

[0004] Linderine is an alkaloid extracted and isolated from traditional Chinese medicinal herbs such as Lindera root, lotus seeds, and jujube seeds. In recent years, with the deepening research on natural drugs, the pharmacological effects of linderine have gradually attracted attention. Studies have shown that linderine possesses various biological activities, including cardiovascular protection, anti-inflammatory effects, and antioxidant effects. In the cardiovascular system, linderine can dilate coronary arteries, increase coronary blood flow, and reduce myocardial oxygen consumption, thus providing some protection against myocardial ischemia-reperfusion injury. In terms of anti-inflammation, linderine can reduce inflammatory responses by inhibiting the release of inflammatory mediators and the activation of inflammatory signaling pathways. In terms of antioxidant effects, linderine can scavenge free radicals and improve the body's antioxidant capacity. However, research on the application of linderine in the field of mental illness is relatively limited. Although a few studies have shown that some natural alkaloids may have a certain ameliorative effect on mental symptoms, there are no systematic studies reported on the prevention and / or treatment of schizophrenia using linderine. Summary of the Invention

[0005] Given the current state of affairs in the treatment of schizophrenia, typical drugs have significant side effects and poor efficacy against negative or cognitive symptoms; atypical drugs carry risks such as weight gain and metabolic abnormalities, and exhibit significant individual variability; and adjunctive therapies also have limitations, this invention aims to provide the application of causticine in the preparation of drugs for the prevention and / or treatment of schizophrenia, solving the problem of finding safer and more effective drugs, enabling the drug to comprehensively improve schizophrenia symptoms, reduce the burden on other systems, and provide a new option for the prevention and / or treatment of schizophrenia.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides the application of causticine in the preparation of drugs for treating schizophrenia.

[0007] The lindera alkaloid is selected from lindera alkaloid itself or its pharmaceutically acceptable salt, ester, or prodrug form.

[0008] The acceptable salt is selected from any one of hydrochloride, phosphate, sulfate, maleate and oxalate.

[0009] The drug is used to improve both positive and negative symptoms of schizophrenia.

[0010] Furthermore, the drug is a drug that antagonizes 5-HT2AR and D2R receptors.

[0011] Furthermore, the drug exerts its effect through interaction with the non-positive phase point of 5-HT2AR via van der Waals forces and electrostatic forces.

[0012] Furthermore, the drug exerts its effect through interaction with the positive phase point of D2R via van der Waals forces and electrostatic forces.

[0013] The drug works by improving social preferences and social novelty behaviors.

[0014] The present invention provides a medicament for treating schizophrenia, the medicament comprising causticine and a pharmaceutically acceptable carrier or excipient.

[0015] The dosage form of the drug is any one of tablets, capsules, sustained-release formulations, and injections.

[0016] Compared with the prior art, the present invention achieves the following technical effects: The present invention relates to the application of lindermaine in the preparation of drugs for treating schizophrenia. Lindermaine and its salts, by antagonizing 5-HT2AR and D2R receptors, can simultaneously improve both positive symptoms (such as hallucinations and delusions) and negative symptoms (such as emotional blunting and anhedonia) in patients with schizophrenia, thereby overcoming the limitations of existing antipsychotic drugs in improving the negative symptoms of schizophrenia. Lindermaine and its salts, while improving schizophrenia symptoms, do not cause significant adverse reactions such as excessive sedation, thus avoiding the extrapyramidal symptoms and other side effects present in existing antipsychotic drugs and improving the safety of treatment. Lindermaine and its salts possess multi-target action characteristics of 5-HT2AR and D2R, and can exert therapeutic effects by regulating the activity of these two receptors. With a clear mechanism of action, it provides a new and effective option for the treatment of schizophrenia.

[0017] The drug for treating schizophrenia provided by this invention, causticin and its salts, can be formulated into various dosage forms (such as tablets, sustained-release preparations, injections, etc.), providing multiple options for individualized drug administration in clinical practice, facilitating adjustments to the medication regimen according to the patient's specific condition, and improving the flexibility and effectiveness of treatment. Attached Figure Description

[0018] Figure 1 This is the chemical structure of R-coclaurine.

[0019] Figure 2 The results show the interaction analysis between caudacin and the 5HT2A receptor. A represents the breakthrough curves of clozapine at different concentrations in the 5HT2AR / CMC ratio; B represents the regression analysis of the affinity between clozapine and 5HT2AR determined by frontier assay (1 / m). Lapp vs1 / [A]); C is the breakthrough curve of linderine at different concentrations in 5HT2AR / CMC; D is the regression analysis of the affinity between linderine and 5HT2AR determined by the frontier analysis method (1 / m Lappvs 1 / [A]; E is the chromatographic elution curve of clozapine competitively displacing clozapine on 5HT2AR / CMC; F is the regression analysis of clozapine competitively displacing clozapine (1 / k vs [A]); G is the chromatographic elution curve of clozapine competitively displacing linderine on 5HT2AR / CMC; H is the regression analysis of clozapine competitively displacing linderine (1 / k vs [A]); I is the molecular docking simulation of clozapine and linderine on 5HT2AR; J is the amino acid residue interaction between clozapine and the 5HT2A receptor binding site; K is the amino acid residue interaction between linderine and the 5HT2A receptor binding site; L is the RMSD result of the molecular dynamics simulation of clozapine and linderine; M is the RMSF result of the molecular dynamics simulation of clozapine and linderine; N is the SASA result of the molecular dynamics simulation of clozapine and linderine; O is the Rg result of the molecular dynamics simulation of clozapine and linderine. The number of hydrogen bonds formed at the 5HT2AR action site by P, clozapine, and cauliflower.

[0020] Figure 3 The results show the interaction between caudacin and dopamine D2 receptors. A represents the breakthrough curves of clozapine at different concentrations on the D2R / CMC; B represents the regression analysis of the affinity between clozapine and D2R determined by frontier analysis (1 / m). Lapp vs 1 / [A]); C is the breakthrough curve of linderine at different concentrations in D2R / CMC; D is the regression analysis of the affinity between linderine and D2R determined by the frontier analysis method (1 / m Lapp vs 1 / [A]; E is the chromatographic elution curve of clozapine competitively displacing clozapine on D2R / CMC; F is the regression analysis of clozapine competitively displacing clozapine (1 / k vs [A]); G is the chromatographic elution curve of clozapine competitively displacing linderine on D2R / CMC; H is the regression analysis of clozapine competitively displacing linderine (1 / k vs [A]); I is the molecular docking simulation of clozapine and linderine on D2R; J is the amino acid residue interaction between clozapine and the D2 receptor binding site; K is the amino acid residue interaction between linderine and the D2 receptor binding site; L is the RMSD result of the molecular dynamics simulation of clozapine and linderine; M is the RMSF result of the molecular dynamics simulation of clozapine and linderine; N is the SASA result of the molecular dynamics simulation of clozapine and linderine; O is the Rg result of the molecular dynamics simulation of clozapine and linderine; P is the number of hydrogen bonds formed at the D2R binding site of clozapine and linderine.

[0021] Figure 4Calcium imaging was performed to visualize calcium flux in SHSY-5Y cells induced by lincoclaurine antagonizing the 5HT2AR agonist TCB2. A shows the fluorescence images of calcium flux in SHSY-5Y cells under 10 nM TCB2 conditions, including the control group (vehicle), lincoclaurine (R-coclaurine), and the 5HT2AR antagonist M100907. B shows the IC50 curves of calcium flux fluorescence in SHSY-5Y cells antagonized by lincoclaurine and M100907 against TCB2. C shows the fluctuation curves of calcium flux fluorescence in SHSY-5Y cells antagonized by lincoclaurine and M100907 against TCB2. D shows the difference in intensity of calcium flux fluorescence changes in SHSY-5Y cells antagonized by lincoclaurine and M100907 against TCB2.

[0022] Figure 5 The effect of R-coclaurine blocking dopamine activation of the D2 receptor on cAMP, the second messenger in D2R-HEK293 cells, was determined by ELISA.

[0023] Figure 6 The effect of cauliflower alkaloid on the movement distance of MK801-induced schizophrenic mice.

[0024] Figure 7 To determine the effect of cauliflower alkaloid on the social behavior of MK801-induced schizophrenic mice in a three-box social experiment.

[0025] Figure 8 To determine the effect of cauliflower alkaloid on social novelty behavior induced by MK801 in schizophrenic mice in a three-box social experiment.

[0026] Figure 9 To determine the effect of cauliflower alkaloid on immobility time in MK801-induced schizophrenic mice in a forced swimming experiment. Detailed Implementation

[0027] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0028] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0029] The 5HT2AR-overexpressing and D2R-overexpressing human embryonic kidney 293 cells (HEK293 cells) used in this invention were constructed at the National-Local Joint Engineering Research Center for Screening and Analysis of Natural Vascular Drugs at Xi'an Jiaotong University. SHSY5Y cells were also obtained from the same research center. C57 / BL6 mice were purchased from the Experimental Animal Center of Xi'an Jiaotong University. Animal behavioral experimental results were processed using ANY-maze (version 7.33) animal behavioral analysis software.

[0030] The Innoval NH2 silica gel used in this invention (purchased from Agela Technologies Ltd.), 1 mm × 10 mm column sleeve and core (purchased from Dalian Ripuli Technology Co., Ltd.), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) purchased from Guangdong Guanghua Technology Co., Ltd., and clozapine and linderine hydrochloride (purchased from MedChemExpress). Chromatographic grade methanol, ethanol, and acetonitrile (purchased from Honeywell Trading (Shanghai) Co., Ltd.). Fetal bovine serum, PBS buffer for cell culture, penicillin and streptomycin antibiotics, and trypsin (EDTA-free) (purchased from Thermo Fisher Scientific (China) Co., Ltd.). Clozapine and linderine hydrochloride, MK801 maleate (purchased from MedChemExpress). Tween 80 (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.).

[0031] Direct-heated CO2 incubator (purchased from Thermo Fisher Scientific (China) Co., Ltd.), Cytation 5 live-cell imaging recorder (purchased from BioTek Instruments, Vermont, USA), TCB2 (purchased from MedChemExpress), FLIPR calcium 5 kit (purchased from Meigu Molecular Equipment Co., Ltd.), clozapine and lindane hydrochloride (purchased from MedChemExpress). Fetal bovine serum, PBS buffer for cells, penicillin and streptomycin antibiotics, and trypsin (EDTA-free) (purchased from Thermo Fisher Scientific (China) Co., Ltd.).

[0032] Example 1 1. Experimental Procedure (1) Preparation of cell membrane chromatography stationary phase The 5HT2AR-HEK293 cell line was cultured in DMEM medium containing 10% fetal bovine serum (FBS). The D2R-HEK293 cell line was cultured in DMEM medium containing 10% FBS and 3 μg / mL cyprodinil S. All cells were cultured at 37°C and 5% CO2.

[0033] Preparation of carboxylated silica gel: Aminosilica gel was suspended in 100 mL of 10% succinate DMF (dried in MgSO4), and carboxylation was carried out at room temperature for 3 h under nitrogen protection. The carboxylated silica gel was obtained by centrifugation. The silica gel was then suspended in 50 mL of deionized water, centrifuged three times, and dried in a 60 °C oven for storage. Preparation of activated silica gel: 100 mg of dried carboxylated silica gel was weighed into 20 mL of MES buffer (50 mM, pH 5.0), and 400 mg of EDC and 100 mg of NHS were added to form the reaction system. The mixture was stirred at room temperature for 4 h. After repeated centrifugation, the silica gel was washed three times with 50 mL of deionized water to obtain activated silica gel.

[0034] Preparation of cell membrane fragments: 5HT2AR, D2R, and H1R cell lines were cultured in two 75 cm² culture media. 2 In culture flasks; cells were digested with trypsin to bring them to a density of approximately 3 × 10⁻⁶. 7 The cells were cultured in a medium containing 10% FBS to terminate the culture and obtain a mixture. The mixture was then centrifuged at 5000 rpm and 4 ℃ for 10 minutes to collect the precipitate. The precipitate was resuspended in 5 mL of sterile physiological saline and then sonicated at 50 Hz on ice for 1 minute. The centrifugation was repeated (5000 rpm, 10 minutes, 4 ℃) to collect the precipitate. The precipitate was then resuspended in 5 mL of sterile physiological saline and sonicated at 120 Hz on ice for 1 minute to break up the cell membrane mixture and obtain cell membrane fragments.

[0035] Preparation of cell membrane self-assembly modified stationary phase: Add the prepared cell membrane physiological saline suspension to 50 mg activated silica gel and react overnight at 4 ℃ to obtain the cell membrane self-assembly modified stationary phase.

[0036] (2) Determination of equilibrium dissociation constant by chromatographic front analysis of cell membrane The binding affinity between the ligand and the receptor was determined by affinity chromatography, and frontier analysis was performed. Finally, the equilibrium dissociation constant (K0) was calculated by simulating the linear regression relationship between 1 / mLapp and the analyte concentration 1 / [A]. D At least five breakthrough curves were determined for the affinity of clozapine and causticine (R)-coclaurine for each receptor, repeated three times. Breakthrough curves were determined under the same conditions (50 mM, pH 7.2) with or without 5 μM analyte, by adjusting the ratio of pump A and pump B, at 5.0 μM, 4.0 μM, 2.0 μM, 1.0 μM, 0.5 μM, and 0.25 μM.

[0037] 1 / mLapp = (K D / m L ) / [A]+1 / mL (Formula 1) In Equation 1, m LappK represents the number of analyte moles at the midpoint of the breakthrough curve under each concentration condition. [A] represents the molar concentration of the analyte. D denoted as m, which is the dissociation equilibrium constant of the analyte. L This represents the number of moles of receptor binding sites. The Kinteraction parameter for drug-receptor interaction is obtained by measuring the ratio of the slope to the intercept of the regression curve using a linear regression method. D value.

[0038] (3) Determination of drug binding sites by cell membrane chromatographic zone elution method The mobile phase consisted of two streams, A and B. Mobile phase A was composed of 50 mM PBS, while mobile phase B contained the same concentration of Na₂HPO₄ and 5 μM clozapine. By adjusting the ratio of mobile phases A and B, the peak shape of 10 μL and 500 μM analytes on cell membrane chromatography was detected under competition with clozapine at concentrations of 5.0 μM, 4.0 μM, 3.0 μM, 2.0 μM, 1.0 μM, 0.5 μM, and 0.25 μM. The flow rate was 0.4 mL / min, the temperature was 15 ℃, and the mobile phase was maintained at 4 ℃. All experiments were repeated three times. The binding sites of the analytes were analyzed using regression curves of the analyte capacity factor versus clozapine concentration.

[0039] 1 / k=(K aD *V M *[A]) / (K D *m L )+V M / (K D *mL) (Formula 2) In Equation 2, k is the capacity factor of the analyte, K aD and K D V is the dissociation equilibrium constant between clozapine and the analyte. M Let m be the volume of the column voids. L Let A represent the number of moles at the binding site, and A be the concentration of clozapine used in the chromatographic column. The method analyzes whether the drug and its site-competing agent have the same binding site by measuring the change in the drug's retention time on the chromatographic column.

[0040] (4) Molecular docking The affinity binding sites of clozapine and (R)-cocourine with 5HT2AR, D2R, and H1R were simulated using Autodock, Chem3D, Open Babel GUI, and PyMOL software, respectively. The structures of clozapine and (R)-cocourine were obtained from the PubChem database, and their differential energy conformations were obtained using Chem3D. The protein structures 5HT2AR (PDB: 7RAN), D2R (PDB: 6LUQ), and H1R (PDB: 8YN2) were exported from the PDB database. Existing ligands and water molecules were removed from the protein structures using PyMOL software. Drug and protein structures in molecular docking format (.pdbqt) were prepared using Open Babel GUI software. The prepared drug and protein molecules were imported into Autodock according to the program requirements. Before running the docking program, the protein molecules were hydrogenated, charged, and subjected to atomic rigidity selection. The charge was set to the computer gaseiger, and the atoms were set to assign AD4 type. The docking search parameters were set to a genetic algorithm. The default values ​​were selected for the detailed parameters of the genetic algorithm. The default settings were selected for the running options. The final docking results were sorted by energy. The docking results were visualized using PyMOL software.

[0041] (5) Molecular dynamics simulation of drug / receptor interaction To further investigate the molecular mechanism of binding of clozapine and cauliflower to 5HT2AR and H1R proteins, molecular dynamics simulations of the screened receptor protein-small molecule complexes were performed using the Gromacs 2020 software package. AMBER99SB-ILDN force field parameters were used for the proteins, while GAFF universal force field parameters were used for the small molecule ligands. The small molecule topology was constructed using the Sobtop program, and charge fitting was performed using RESP. The TIP3P dominant water model was selected, with a minimum distance of 1.0 nm between the protein atoms and the edge of the water box. Sodium or chloride ions were used to neutralize the system charge based on the docking results. The molecular dynamics simulation workflow consisted of four steps: energy minimization, heating, equilibrium, and production kinetics simulation. First, the heavy atoms of the protein (and small molecules) were constrained, and energy minimization was performed on the water molecules for 10,000 steps (including 5,000 steps of steepest descent and 5,000 steps of conjugate gradient). Then, the constraints were lifted, and energy minimization was performed on the entire system for another 10,000 steps (including 5,000 steps of steepest descent and 5,000 steps of conjugate gradient). After energy optimization, the system was slowly heated to 300 K over 50 ps. After heating, the system was equilibrated for 50 ps under the npt ensemble. Finally, a 100 ns molecular dynamics simulation was performed under the npt ensemble. Trajectory data was saved every 10 ps and related analyses were performed using the trjconv module. The binding free energy of the ligand and protein was calculated using the gmxMMPBSA method in Gromacs 2020.

[0042] 2. Experimental Results (1) Analysis of the interaction between linderine and 5HT2AR See appendix Figure 2 The experiment used frontier analysis to determine the relative activity of uredinyl (R)-coclaurine and clozapine with 5HT2AR K. D Value, K of clozapine bound to 5HT2AR D The value is (3.75±0.60)×10 -6 mol / L (e.g.) Figure 2 A-2B); linderine and 5HT2AR K D The value is (7.04 ± 0.82) × 10 -6 mol / L ( Figure 2 C-2D).

[0043] The binding sites of caudaline (R)-coclaurine and 5HT2AR were determined using a competitive displacement assay, and the binding sites of caudaline and its receptor were determined using clozapine as a site competitor. The competitive displacement results showed that with increasing clozapine concentration in the mobile phase, clozapine significantly reduced its retention time on D2R-CMC. Figure 2 E-2F). The results of clozapine competitive displacement of linderine showed that the retention time of linderine on 5HT2AR-CMC did not change significantly with increasing clozapine concentration, indicating that the competitive effect of clozapine on linderine in 5HT2AR was not significant, and that the two drugs had different sites of action. Figure 2 G-2H).

[0044] Computer-aided drug / receptor molecular docking and molecular dynamics simulations were used to analyze the binding site of caudacin(R)-coclaurine to the 5HT2AR protein. Molecular dynamics simulation results showed that the RMSD fluctuation of the 5HT2AR-caudacin complex was higher than that of the 5HT2AR-clozapine complex, suggesting that clozapine may bind slightly better to the protein. Figure 2 L). For example, clozapine can form hydrogen bond interactions with protein site residue ASN363, and the compound can also form hydrophobic interactions with PHE-339, LEU-362, ILE-358, TYR-139, and LEU-228. In particular, the benzene ring of the compound can also form a pi-pi conjugation interaction with the TYR-139 residue. Figure 2 J); according to Figure 2 Although the lindera alkaloid compound formed hydrogen bonds with ASP231, it had significantly fewer binding sites on the 5HT2AR protein, and the binding was shallow, with the methoxy group exposed in the solvent, which was unfavorable for the binding. This indicates that the binding between the two is slightly weak. Figure 2 K). In comparison, the protein amino acid fluctuation of 5HT2AR-clozapine is slightly smaller ( Figure 2 M). The Rg values ​​of both the 5HT2AR-codone complex and the 5HT2AR-clozapine complex decreased, which may be due to the conformational changes induced by the binding of the protein and compounds, leading to more effective interactions within the protein and promoting complex stability. The trend of change in solvent polarizable surface area (SASA) was consistent with that of Rg. Figure 2 N and Figure 2 O). Clozapine forms hydrogen bonds with a significantly higher occupancy rate of pocket amino acids in the 5HT2AR protein, and these hydrogen bonds play an important role in the stable binding of small molecules to proteins; while the hydrogen bonds formed between linderaine and the 5HT2AR protein are intermittent and have a low occupancy rate, which is detrimental to the stable binding of the two. Figure 2The binding free energies of linderaine and clozapine to the 5HT2AR protein are -35.06 + / -1.2 kcal / mol and -15.48 + / -3.12 kcal / mol, respectively. Clozapine forms effective hydrogen bonds with the protein pocket, resulting in a significant contribution from electrostatic interactions to the stability of the small molecule (-25.75 + / -0.85 kcal / mol). Linderaine, on the other hand, has fewer hydrogen bonds with the protein, resulting in a very low electrostatic contribution (-0.49 + / -2.72 kcal / mol). These results indicate that linderaine and clozapine interact at different sites on 5HT2AR. Linderaine primarily interacts with the non-positive phase sites of 5HT2AR through van der Waals forces and electrostatic forces.

[0045] (2) Analysis of the interaction between linderine and D2R See appendix Figure 3 The K+ binding of caustic somatic alkaloid (R)-coclaurine and clozapine to D2R was determined using frontier analysis. D Value, K value of clozapine binding to D2R D The value is (4.75±1.77)×10 -6 mol / L, such as Figure 3 As shown in A-3B. The K-type compound formed by the binding of linderine and D2R. D The value is (5.92±0.57)×10 -6 mol / L, Figure 3 As shown in C-3D.

[0046] The binding sites of linderaine and D2R were determined using a competitive displacement method, and the binding sites of linderaine and its receptor were determined using clozapine as a site competitor. The competitive displacement results showed that with increasing clozapine concentration in the mobile phase, clozapine significantly reduced its retention time on D2R-CMC, as shown in the following figures. Figure 3 As shown in E-3F, the results of clozapine competitive substitution of linderine indicate that the retention time of linderine on the D2R-CMC significantly decreases with increasing clozapine concentration, suggesting that clozapine has a significant competitive effect on the linderine action site on the D2R, and that there may be a common action site, such as... Figure 3 G-3H.

[0047] Molecular dynamics simulations of the binding of (R)-coclaurine to D2R showed that the RMSD fluctuation of the D2R-coclaurine complex was slightly higher than that of the D2R-clozapine complex, suggesting that clozapine may bind slightly better to the protein. For example, clozapine can form hydrogen bonds with protein residue CYS-107, and the compound can also form hydrophobic interactions with TRP-100, LEU-94, PHE-110, VAL-111, ILE-184, and PHE-102; coclaurine compounds can also form hydrogen bonds (PRO-426, GLU-99) and hydrophobic interactions with D2R proteins, exhibiting stronger protein binding. Figure 3 I-3L). According to the RMSF diagram, the amino acid conformational changes in the complex are significant and follow a regular pattern, which is largely related to the protein's own conformation. Figure 3 M). According to the Rg plot, the Rg values ​​of the D2R-codonopsis alkaloid complex and the D2R-clozapine complex initially increased and then slightly decreased, indicating the formation of more effective interactions within the proteins. The solvent polarizable surface area (SASA) did not show significant fluctuations during the kinetic process, further suggesting good overall protein stability. Figure 3 N and Figure 3 O). Clozapine and linderaine have a slightly lower occupancy rate in forming hydrogen bonds with D2R protein pocket amino acids. Figure 3 While both compounds (P) are stable within the protein pocket and form effective hydrophobic interactions, they exhibit strong binding free energies with the D2R protein: -18.89 + / -1.2 kcal / mol and -22.38 + / -1.68 kcal / mol, respectively. Due to the effective hydrogen bonding between the two compounds and the protein pocket, electrostatic interactions significantly contribute to the stability of the small molecule (-6.43 + / -0.85 kcal / mol and -12.76 + / -0.8 kcal / mol, respectively). In summary, uredinin and clozapine have the same interaction site on D2R. uredinin primarily interacts with the positive phase point of D2R through van der Waals forces and electrostatic forces, with the D2R-uredinin complex showing slightly better performance.

[0048] Example 3 This embodiment investigates the antagonistic activity of linderaine (R)-coclaurine against the 5-HT2AR receptor. Cellular calcium imaging was used to determine the antagonistic effect of linderaine on calcium influx induced by the 5HT2A receptor agonist TCB2 in SHSY-5Y cells. The specific experimental procedure is as follows: Cell culture: SHSY-5Y cell line was cultured in DMEM / F12 medium containing 10% FBS. All cells were cultured at 37°C and 5% CO2.

[0049] Live-cell imaging: The antagonistic effect of the 5HT2AR agonist TCB2 on calcium mobilization activity in SHSY-5Y cells was assessed using a Cytation5 imaging recorder. Approximately 1×10 6 SHSY-5Y cells were seeded into 96-well plates to a final volume of 200 μL and incubated overnight at 37 °C and 5% CO2. 100 μL of cell culture medium was discarded, and 100 μL of FLIPR Calcium 5 assay kit and 50 μL of drug were added to the cell culture system. The cells were incubated at 37 °C and 5% CO2 for 1 hour. 150 μL of supernatant was discarded, ensuring a final volume of 100 μL. Cell calcium mobilization in the presence of different concentrations of antagonist was captured using a 4x field of view. The agonist was automatically added to 100 μL test wells using the instrument. The imaging time for each field of view was set to 40 seconds. The experimental results are shown in the appendix. Figure 3 As shown.

[0050] Analysis of the antagonistic activity of linderine against 5HT2AR Live-cell imaging was used to investigate the effect of lindera (R)-coclaurine on calcium mobilization activity in SHSY-5Y cells by blocking 5HT2AR antagonism and inhibiting the selective 5HT2AR agonist TCB2 to activate the 5HT2A receptor. Different concentrations of lindera (increasing from 0.1 nM to 100 μM) were co-treated with 18 nM TCB2. Changes in calcium fluorescence intensity, shifts in the calcium flux-induced dose-response curve, and changes in EC50 values ​​were observed. The IC50 value of lindera antagonism against TCB2 was calculated. 50 The results showed that, compared with the TCB2 control group (vehicle + TCB2), both 10 mM caustic solanine (R-coclaurine + TCB2) and 10 mM 5HT2A receptor selective antagonist M100907 (M100907 + TCB2) significantly inhibited TCB2 activation of 5HT2AR. Figure 4 A), the cellular calcium flux fluorescence intensity in the linderine and M100907 groups was significantly lower than that in the TCB2 control group ( Figure 4 (C and 4D). Simultaneously, with increasing doses of linderine and M100907, the calcium mobilization activity of TCB2 continuously decreased, yielding an IC50 of linderine antagonizing 5HT2AR. 50 Curves, such as Figure 4 As shown in B, the results of linderaine antagonizing TCB2 on SHSY5Y calcium mobilization indicate that linderaine can antagonize the calcium mobilization effect of 5HT2AR agonists on cells by blocking 5HT2AR.

[0051] Example 4 This embodiment investigates the antagonistic activity of linderine (R)-coclaurine on dopamine D2 receptor (D2R). The level of cAMP, a second messenger in D2R-HEK293 cells regulated by dopamine activation of the D2 receptor, was measured using ELISA to analyze the antagonistic activity of linderine on the D2 receptor. The specific experimental procedure is as follows: Cell culture: D2R-HEK293 cell line was cultured in DMEM medium containing 10% FBS. All cells were cultured at 37°C and 5% CO2.

[0052] ELISA assay: The regulatory level of cAMP after D2 receptor activation in D2R-HEK293 cells was measured by ELISA to assess the antagonistic effect of linderine on dopamine-induced D2 receptor agonism. Approximately 1×10 6 SHSY-5Y cells were seeded into 24-well plates and cultured overnight. The supernatant was discarded, and 50 μM of an antagonist (codonopsis or haloperidol) and 100 μM of the phospholipase inhibitor IBMX were added to each well. After incubation with the antagonist for 60 min, 5 μL of 10 nM dopamine was added to the corresponding well, and incubation was continued for 5 min. The cells were thoroughly pipetted to detach the D2R-HEK293 cells. All cell suspension was collected into 1.5 mL centrifuge tubes and centrifuged at 5000 rpm at 4°C for 5 min. Cells were lysed using 100 μL of 0.5% Triton-X100 (diluted in PBS) per well, and the reaction was carried out at 4°C for 5 min. The cell lysate was then transferred to EP tubes and centrifuged at 12000 rpm at 4°C for 15 min. 80 μL of the supernatant was collected as the experimental sample, and 50 μL of the sample was used for ELISA. The OD value was measured at 450 nM according to the kit instructions. See the appendix for experimental results. Figure 4 As shown.

[0053] Analysis of linderine's antagonistic D2R activity The regulatory effect of linderaine on cAMP, the second messenger in D2R-HEK293 cells, was determined by ELISA. After the D2R agonist dopamine (DA) activates the receptor, it inhibits intracellular cAMP synthesis by inhibiting adenylate cyclase. Linderaine's blockade of D2R antagonism antagonized the dopamine's inhibitory effect on cAMP through D2R activation. The results showed that, compared with the DA control group, both 50 μM linderaine (R-coclaurine) and 10 mM 5HT2A receptor antagonist haloperidol (Halo) significantly inhibited DA activation of the D2 receptor and upregulated intracellular cAMP levels. Figure 5 ).

[0054] Example 5 This embodiment constructs a MK801-induced C57BL / 6 mouse model of schizophrenia and uses the three-box social test and forced swimming test to evaluate the effect of linderaine ((R)-coclaurine) on improving social preference behavior and forced swimming stillness time in schizophrenic mice, and to preliminarily explore the anti-schizophrenic activity of linderaine.

[0055] (1) Three-box social experiment Seventy C57BL / 6 mice (18–22 g) were selected and divided into seven groups of ten each. The mice were administered MK801 + saline, 20 mg / kg lindera alkaloid + MK801, 40 mg / kg lindera alkaloid + MK801, and saline + saline, respectively. The experimental dose of MK801 was 0.025 mg / kg. In the experimental groups, 0.2 mL of saline or lindera alkaloid was injected intraperitoneally using a 1 mL syringe. After 10 minutes of observation, 0.2 mL of 0.025 mg / mL MK801 was injected. In the control group, 0.2 mL of saline was injected intraperitoneally first, followed by another 0.2 mL of saline after 10 minutes of observation. Before the first stage of the three-box socialization experiment, the mice were placed in the three-box socialization experiment (socialization box dimensions: 60×40×20 cm) for 10 minutes to familiarize themselves with the environment. At the start of the first phase, one social mouse was placed in the left social cage, and an empty social cage of the same size was placed in the right cage. The experiment lasted for 10 minutes. In the second phase, a familiar social mouse was placed in the left social cage, and an unfamiliar social mouse was placed in the right social cage. The experiment lasted for 10 minutes. The mouse's motor behavior, social preferences, and social novelty preferences were measured using ANY maze (version 7.33) animal behavior analysis software. The experimental results are shown in the appendix. Figure 6 - As shown in Figure 8.

[0056] From the appendix Figure 6 Data shows that a schizophrenia model was established in C57 / BL6 mice using MK801 (0.25 mg / kg), allowing for a preliminary evaluation of the anti-schizophrenic activity of linderaline ((R)-coclaurine). In the three-box social behavior test, distance results showed that 20 mg / kg linderaline had no significant inhibitory effect on the MK801-induced increase in motor activity in MK801-induced schizophrenia mice; however, 40 mg / kg linderaline showed a significant inhibitory effect on the MK801-induced increase in motor activity. Figure 6 This indicates that linderma can significantly improve the overall motor ability of mice under the experimental conditions.

[0057] In the first phase of the three-box socialization experiment, the social index results showed that, in the koclaurine-treated MK801-induced schizophrenic mouse experimental group, compared with the MK801 model group (saline + MK801), the koclaurine treatment group ((R)-coclaurine + MK801) significantly improved the social index of schizophrenic mice. The social index of the koclaurine treatment group was the same as that of the saline control group (saline + saline). Figure 7 This indicates that uredinin can effectively improve MK801-induced social withdrawal in mice.

[0058] In the second phase of the three-box social interaction, the social novelty index results showed that, compared with the MK801 model group (saline + MK801) mice, cauliflower alkaloids significantly improved the decrease in social novelty caused by MK801. The social novelty index of the cauliflower alkaloid treatment group ((R)-coclaurine + MK801) was the same as that of the saline group (saline + saline). Figure 8 ).

[0059] In conclusion, in the three-box social experiment, cauliflower alkaloids can significantly improve the social behavior of MK801-induced schizophrenic mice.

[0060] (2) Forced swimming experiment Seventy C57BL / 6 mice (18-22 g) were selected and divided into seven groups of ten each. The mice were administered MK801 + saline, 2.5 mg / kg + MK801, 5 mg / kg + MK801, 10 mg / kg + MK801, clozapine 2.5 mg + MK801, and saline + saline, respectively. The experimental dose of MK801 for inducing schizophrenia in mice was 0.02 mg / kg. The control group and each experimental group received two intraperitoneal injections of the experimental drug, MK801, or saline, respectively, with a 10-minute interval between the two injections. Twenty minutes after the second injection, the mice were placed in a swimming tub (12 cm in diameter, 25 cm in height, and 15 cm in depth) for a forced swimming experiment. The immobility time within 5 minutes in different drug administration groups was measured using ANY Maze (version 7.33) animal behavior analysis software. The experimental results are shown in the appendix. Figure 8 As shown.

[0061] In the forced swimming test, immobility time was used as the indicator. Compared with the MK801 monotherapy group, the 10 mg / kg cauliflower alkaloid treatment group had a significantly increased immobility time, which was consistent with the 1.25 mg / kg clozapine group. Figure 9Furthermore, linderaine showed a significant dose-dependent effect in improving MK801-induced immobility time. This indicates that linderaine has a significant antagonistic effect on the MK801-induced reduction in immobility time in mice during the forced swimming experiment, meaning that linderaine can improve the behavioral abnormalities in mice under forced swimming conditions caused by MK801.

[0062] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. Application of linderine in the preparation of drugs for treating schizophrenia.

2. The application according to claim 1, characterized in that, The lindera alkaloid is selected from lindera alkaloid itself or its pharmaceutically acceptable salt, ester, or prodrug form.

3. The application according to claim 2, characterized in that, The acceptable salt is selected from any one of hydrochloride, phosphate, sulfate, maleate and oxalate.

4. The application according to claim 1, characterized in that, The drug is used to improve both positive and negative symptoms of schizophrenia.

5. The application according to claim 4, characterized in that, The drug is one that antagonizes the 5-HT2AR and D2R receptors.

6. The application according to claim 5, characterized in that, The drug described herein exerts its effect through interaction with the non-positive phase point of 5-HT2AR via van der Waals forces and electrostatic forces.

7. The application according to claim 5, characterized in that, The drug exerts its effect through interaction with the positive phase point of D2R via van der Waals forces and electrostatic forces.

8. The application according to claim 1, characterized in that, The drug works by improving social preferences and social novelty behaviors.

9. A drug for treating schizophrenia, characterized in that, The drug includes linderaine and pharmaceutically acceptable carriers or excipients.

10. A medicament for treating schizophrenia according to claim 9, characterized in that, The dosage form of the drug is any one of tablets, capsules, sustained-release formulations, and injections.

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