Application of Yinyanghuoalkaloid in preparing drugs for treating stroke

Abstract

The present invention belongs to the technical field of stroke drug research, and particularly relates to the application of yindichin in the preparation of drugs for treating stroke. The present invention provides the application of yindichin in the preparation of drugs for treating stroke. In vitro and in vivo experiments show the definite neuroprotective activity of yindichin, and for the first time verify the protective effect of yindichin as a novel positive allosteric modulator targeting GluN1 / 2A NMDAR on stroke, and specifically proposes the following functions: 1) enhancing agonist-induced calcium influx of GluN1 / 2A NMDAR; 2) reducing the volume of cerebral infarction after stroke; 3) improving the cognitive function deficit caused by stroke.

Description

Technical Field

[0001] The present invention belongs to the technical field of stroke drug research, and specifically relates to the application of aegeline in the preparation of drugs for treating stroke. Background Art

[0002] Stroke, also known as "apoplexy" and "cerebrovascular accident", is an acute cerebrovascular disease, a group of diseases caused by sudden rupture of blood vessels in the brain or blockage of blood vessels leading to inability of blood to flow into the brain, resulting in brain tissue damage, including ischemic and hemorrhagic strokes.

[0003] In scientific research on stroke, excitotoxicity mediated by N-methyl-D-aspartic acid (NMDA) type glutamate receptors has been at the center stage. According to the "NMDAR subtype" hypothesis, activation of NMDARs containing GluN2A promotes neuron survival, while activation of NMDARs containing GluN2B induces neuron death. Targeting allosteric sites to enhance receptor function can avoid the neurotoxicity induced by direct and irregular overactivation of NMDARs and achieve better pharmacological control. Therefore, developing new positive allosteric modulators (PAMs) targeting GluN2A-containing NMDARs is considered a promising neuroprotection strategy.

[0004] Aegeline is an α-hydroxyamide compound isolated from the leaves of the Rutaceae plant Aegle marmelos. This structure was first isolated and identified in 1952. The leaves of Aegle marmelos are widely used in India to treat diabetes. Research shows that aegeline is the active source of blood sugar and lipid lowering in the leaves of Aegle marmelos, and relevant literature points out that this compound has antioxidant and anti-obesity activities. However, there is currently no report on whether aegeline has an effect on the prognosis of ischemic stroke and the regulation of NMDA receptors. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the application of aegeline in the preparation of drugs for treating stroke. Through in vitro and in vivo experiments, the clear neuroprotective activity of aegeline is shown, and the protective effect of aegeline on stroke is verified for the first time, providing a new reference for treating stroke with natural products.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides the application of aegeline in the preparation of drugs for treating stroke.

[0008] The present invention also provides the application of aegeline in the preparation of neuroprotective drugs.

[0009] Preferably, the stroke is ischemic stroke.

[0010] The structural formula of the aegeline is shown in I:

[0011]

[0012] Preferably, the drug is a drug mixture or a pharmaceutical composition.

[0013] Preferably, the drug mixture or the pharmaceutical composition is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions and solutions.

[0014] The present invention provides a drug mixture or a pharmaceutical composition, the active ingredient of which includes aegeline.

[0015] The drug mixture or the pharmaceutical composition has at least one of the following functions 1)-3):

[0016] 1) Enhancing agonist-induced calcium influx of GluN1 / 2A NMDAR;

[0017] 2) Reducing the volume of cerebral infarction after stroke;

[0018] 3) Improving the cognitive function deficit caused by stroke.

[0019] The drug mixture or the pharmaceutical composition is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions and solutions.

[0020] Compared with the prior art, the beneficial effect of the present invention is that:

[0021] The present invention reports for the first time that aegeline is a novel positive allosteric modulator targeting GluN1 / 2A NMDAR, and its significant neuroprotective effect provides a potential opportunity for the development of drugs for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 When compound Aegeline is co-applied with Glu / Gly, it selectively increases the intracellular calcium of cell lines expressing GluN1 / 2A (A) and GluN1 / 2B (B). The data are expressed as the mean ± SD of at least three independent experiments, ***P < 0.005, **P < 0.01, *P < 0.05. Compared with the Glu / Gly group, one-way ANOVA and Dunnett's test are used for multiple comparisons. Glu, glutamate; Gly, glycine;

[0023] Figure 2, Aegeline alleviated nerve injury in tMCAO mice. (A) Experimental protocol and timeline for in vivo neuroprotective activity. (B) Representative brain slices of mice in the control group, model / vehicle, edaravone (10 mg / kg), and Aegeline (1, 5, and 10 mg / kg) treatment groups stained with TTC by intraperitoneal injection; white: damaged tissue. (C) Quantification and statistical analysis of infarct volume after tMCAO in different groups. (D) Statistical analysis of neurological scores after tMCAO in different groups. Data are presented as mean ± SD, with 6 replicates for each measurement, ***P < 0.005, **P < 0.01, compared with the vehicle-treated MCAO group, one-way ANOVA, Dunnett's test for multiple comparisons;

[0024] Figure 3 , Docking pose of aegeline in the NMDAR binding pocket (PDB code: 7EOS). (A) Ligand binding pocket vertically cut by molecular lipophilic potential. (B) Top view of local information in the receptor binding pocket. Hydrogen bond interactions are shown as dashed lines;

[0025] Figure 4 , Analysis of molecular dynamics simulation results. The RMSD trajectories of each chain (A) and the NTD tetramer or two NTD dimers (B) were calculated for Ca atoms over the entire simulation time of 100 ns based on the initial model coordinates (docking complex). (C) Change in the average centroid distance between two chains of the GluN2A subunit and the R2-R2 lobe. (D) Top-down view showing the conformational change of the NTD tetramer by aligning the initial receptor structure (bound to agonist but not to Aegeline). (E) After binding to Aegeline, the MD simulation was run for 100 ns, and the distance between the Ca atoms of two V217s was shortened;

[0026] Figure 5 , Under glutamate exposure, Aegeline increased the phosphorylation of CREB in PC-12 cells. Glutamate (Glu; 20 mM) was used to induce excitotoxicity, and PC-12 cells were treated for 24 hours in the presence and absence of the GluN2A-preferred antagonist NVP-AAM077 (NVP; 0.4 μM) or Aegeline (1 μM). (A) Protein levels of phosphorylated CREB (p-CREB), CREB, and tubulin were measured. (B) Statistical analysis of the densitometry data for the indicated experimental groups. Data obtained from 3 separate experiments are shown as mean ± SD, normalized to total CREB protein or normalized to tubulin. *P < 0.05, one-way ANOVA, Dunnett's test for multiple comparisons. Detailed implementation

[0027] The present invention will be further described below in conjunction with embodiments. Those skilled in the art can understand that these embodiments are only used to illustrate the present invention and do not limit the scope of the present invention in any way. All raw materials used in the embodiments are commercially available unless otherwise specified.

[0028] Example 1: Aegeline selectively enhances agonist-induced calcium influx through GluN1 / 2A NMDAR

[0029] NMDA receptors have several distinct features from other glutamate receptors, including voltage-dependent inhibition by extracellular Mg 2+ , high permeability to Ca 2+ , and the requirement for binding of an antagonist, glutamate, and glycine (or D-serine) to open the channel. To verify whether the hit compound as a PAM can enhance agonist-induced calcium influx into NMDA receptors, the compound was detected by calcium fluorometry in HEK-293 cells expressing GluN1 / GluN2A or GluN1 / GluN2B receptors, respectively.

[0030] Human embryonic kidney 293 cells (HEK293 cells) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO 2 . When the HEK293 cells reached 90% confluence, the plasmids of GluN1 and GluN2A or GluN1 and GluN2B were co-transfected into the cells using Sinofection (Sino Biological). The transfection ratio for these NMDAR subunit combinations was 1:1 (GluN1 / GluN2A or GluN1 / GluN2B).

[0031] The calcium indicator Cal-520 AM (AAT Bioquest, USA) was used to detect changes in intracellular calcium in cell populations using a multimode microplate reader (Molecular Devices) in a FlexStation 3 assay. HEK293 cells were transiently transfected with the cDNA of NMDAR and then seeded at a density of 40,000 cells per well on poly-D-lysine-treated black-bottom clear 96-well plates (Corning Costar) and cultured overnight at 37 °C and 5% carbon dioxide. The cells were incubated with the calcium fluorescent dye for 1.5 hours at 37 °C, and the Glu(2 mM) / Gly(100 μM)-induced response was measured in modified HEPES-buffered Tyrode's solution (HBTS) containing (mM): 135 NaCl, 5 KCl, 2.5 CaCl2, 10 HEPES, 10 glucose, pH = 7.2. The values of relative fluorescence units (RFU) were measured by FlexStation 3 at wavelengths of 485 nm (excitation) and 515 nm (emission) at an interval of 1.6 seconds.

[0032] The results are expressed as the area under the curve (F MAX / F 0 ) of the fluorescence values. At a concentration of 10 μM, Aegeline significantly increased the intracellular Ca 2+ levels induced by Glu / Gly on GluN1 / N2A receptors (P < 0.05). In contrast, there was no detectable Ca 2+ enhancement signal on the GluN1 / GluN2B receptors in response to agonists ( Figure 1 B). The results indicate that these compounds have good selectivity for the GluN2B subunit.

[0033] Example 2: The ADME characteristics of Aegeline meet the criteria for central nervous system drugs

[0034] ADME analysis was performed to examine the druggability and biological properties of the newly discovered GluN1 / GluN2A positive allosteric modulator. The properties predicted by Qikprop are listed in Table 3. Colorectal cancer cells are a model of the intestinal blood barrier. The predicted Caco-2 cell permeability values of the compound were 1359.356 nm / s, indicating that the compound has extremely high cell permeability (>1000). In addition, most central nervous system drugs are small molecule compounds that cross the BBB through the passive transcellular diffusion pathway. MDCK cells are considered to be a good mimic of the BBB (blood-brain barrier). The predicted cell permeability value based on MDCK cells was 689.394, indicating that the compound may have good brain exposure (>500). At the same time, their affinity for brain tissue (QPLogBB) and the ability to be active in the central nervous system were predicted. The results showed that the compound has a good brain / blood partition coefficient, and the corresponding predicted brain / blood partition coefficient was -0.842.

[0035] Example 3: Aegeline reduces the cerebral infarction volume and improves the neurological deficit in tMCAO mice

[0036] C57BL / 6 mice were housed under standard conditions with a 12 / 12 hour light / dark cycle and had free access to food and water. Male C57BL / 6 mice, 6 to 8 weeks old and weighing 18 to 22 g, were used in the study and randomly assigned to each group. Aegeline and edaravone were dissolved in 5% DMSO, 20% PEG300, and 5% Tween-80 and diluted with saline for intraperitoneal treatment. All animal use procedures complied with the university's policy on the use and care of animals.

[0037] The mice were anesthetized with isoflurane, and the left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were exposed through a midline neck incision. A monofilament nylon suture was introduced into the left internal carotid artery, and the origin of the middle cerebral artery (MCA) was occluded by positioning it proximal to the external carotid artery. After 1.5 hours of occlusion, the suture was withdrawn to restore blood flow (reperfusion). After the surgery, the mice were transferred to the intensive care unit, and the temperature was maintained at 37 °C until the animals were fully awake. During the entire surgery and recovery process, the body temperature was maintained at 37.0 ± 0.5 °C.

[0038] Experimental groups and drug administration: The mice were randomly divided into the following four groups: sham operation group (saline), model / vehicle group (5% DMSO, 20% PEG300, and 5% Tween-80 in saline), compound 15 (Aegeline) group (1, 5, and 10 mg / kg), and edaravone group (1, 5, and 10 mg / kg). These drugs were administered by intraperitoneal injection within 2 hours after the start of reperfusion.

[0039] Assessment of neurological score: At 24 hours after reperfusion, the neurological score was evaluated by an examiner who was blinded to the treatment conditions to assess the neurological deficits in the mice. The score was calculated using the following criteria: 0 points: no neurological dysfunction; 1 point: unable to fully extend the right forepaw; 2 points: turning to the right; 3 points: falling to the right; 4 points: no spontaneous locomotion and low level of consciousness.

[0040] Infarct volume analysis: After the neurological score was evaluated, the mice were sacrificed by injection of 10% chloral hydrate. The brain tissues were dissected and stored at -80 °C for 10 minutes. If subarachnoid hemorrhage was observed, the mice were excluded from the study. Then, the brain tissues were transferred to a brain slicer matrix and cut into 7 coronal slices, 1.0 mm thick. The brain slices were incubated with 2% TTC solution at 37 °C for 20 minutes and then soaked in 4% paraformaldehyde overnight. Digital images were taken after fixation and analyzed using an image analysis system (Image-Pro Plus).

[0041] As Figure 2 shown, at 24 hours after tMCAO, the infarct volume was calculated on 1-mm thick brain slices stained with 2,3,5-triphenyltetrazolium chloride (TTC). The white areas on the brain slices of the model mice represented the infarcts caused by MCAO. Compared with the vehicle group (130.60 ± 9.93 mm 3 ), after treatment with 5 mg / kg (90.53 ± 4.92 mm 3 ) and 10 mg / kg Aegeline (74.83 ± 10.29 mm 3 ), a significant reduction in infarct volume was observed (about 30.68% and 42.70%, P < 0.005). Meanwhile, edaravone (10 mg / kg) as a positive drug significantly reduced ischemic cerebral infarction (68.04 ± 4.53 mm 3 , P < 0.005 compared with the vehicle). At the same dose (10 mg / kg), the in vivo neuroprotective effects of edaravone and Aegeline were comparable. Matching the histological staining results, we observed that the neurological deficit scores in both the 10 mg / kg Aegeline-treated group and the 10 mg / kg edaravone-treated group were restored (P < 0.005 vs vehicle). The 5 mg / kg Aegeline-treated group also reduced the neurological score to some extent (P < 0.01 vs vehicle). Therefore, Aegeline is beneficial for the improvement of cerebral infarction and neurological deficits.

[0042] Example 4: Aegeline allosterically modulates the receptor by bringing GluN2A NTDs spatially closer

[0043] Use The Induced Fit Docking module of Glide44 docked aegeline to the GluN1 / N2A NMDAR active site. Side chains within 5.0 Å of the docked ligand were refined with Prime. In the final re-docking step, XP Precision was used to score the poses. The optimized docked complex was submitted to the Desmond (version 3.8) module of the software package for MD simulations. A cubic box shape was used to build the system for the complex, with an edge distance of 10 Å from the protein, and the box was solvated using the TIP3P water model. The system was neutralized with sodium ions. Molecular dynamics was used to set the simulation parameters. First, the steepest descent algorithm was used to minimize the energy of the system for 50,000 steps; then the positions of the heavy atoms were constrained to run NVT equilibration and NPT equilibration for 50,000 steps; the system temperature was maintained at 300 K and the system pressure was maintained at 1 atm. After completing these two equilibration phases, an unrestricted molecular dynamics simulation of 100 ns was performed. We set the energy and the coordinates of the trajectory to be saved every 10 ps. Then, the RMSD was calculated for each chain, the entire NTD of the protein, and the AB and CD dimers, respectively, and then the R2-R2 centroid distance of GluN2A was calculated.

[0044] Compared with the rigid receptor docking, the induced fit docking (IFD) method can explain the more reasonable spatial conformation of the receptor in the presence of the ligand and further evaluate a more reliable complex. The docking pose with the highest score for aegeline was used to analyze the binding mode of the ligand to the GluN1 / N2A receptor (PDB ID: 7EOS). As Figure 3 shown, the hydroxyanisole and styrene groups are linked by an amide bond and occupy a protein pocket that is hydrophobic on both sides and hydrophilic in the middle. A large number of hydrophobic interactions with residues including Ile 204, Leu219, Ile222, Ile 227, and Ile 239 fix aegeline in the pocket. More importantly, aegeline forms a hydrogen bond with serine at position 224 as a hydrogen bond donor and a hydrogen bond acceptor.

[0045] Starting from the docking results, Aegeline was placed in a 100-ns all-atom explicit water MD simulation. The molecular dynamics was established from the docked protein-ligand complex corresponding to the best docking score. Interactions throughout the trajectory showed conserved interactions of Aegeline with residues of the GluN2A subunit. Trp222 and Ile255 were very important for hydrophobic interactions, Ser171, Val226, and Ser224 mainly contributed to water bridges, while Ser224 also contributed to hydrogen bond interactions.

[0046] Molecular dynamics (MD) simulation results showed that large conformational changes occurred in all NTDs of GluN1 and GluN2A ( Figure 4 B). The chain C (GluN2A-NTD) bound to Aegeline experienced greater fluctuations than other NTD chains ( Figure 4 A). Notably, the conformations within the two heterodimers were relatively stable, initially showing significant fluctuations compared to the entire tetrameric NTD and then tending to impede large-scale movements ( Figure 4 B).

[0047] After the MD simulation, we measured the changes in the centroid (COM) distances between the two GluN2A chains and between the GluN2A-R2 lobes. The distance between the NTDs of the GluN2A subunit remained unchanged, while the distance between their respective R2 lobes decreased by 2 Å ( Figure 4 C). Then, in Figure 4 D, the receptor-bound Aegeline was superimposed with the apo state. We found that the binding of Aegeline caused the R2 lobe to flip inwards and upwards, resulting in spatial proximity to GluN2A. At the same time, the distance between the Val217s on α5 in the two GluN2A chains decreased by 1.52 Å, which was also consistent with the decrease in the distance between the entire R2 lobes ( Figure 4 E). In summary, the perturbation of the receptor by Aegeline promoted the opening of the GluN2A clamshell, leading to a more planar alignment between the GluN2A NTDs. The GluN2A NTDs approached each other spatially, which was beneficial for the stabilization of the receptor activation state. Therefore, we speculate that Aegeline improves receptor function through the allosteric regulation mechanism described above.

[0048] Example 5: Aegeline exerts neuroprotective effects by enhancing the phosphorylation of CREB downstream of the GluN2A subunit

[0049] CREB (cAMP response element-binding protein) is a key downstream prosurvival molecule of GluN2A. CREB-mediated transcriptional activation is a prerequisite for NMDAR-mediated neuronal survival. Activation of GluN2A protects neurons from ischemic injury by upregulating CREB signaling and subsequent expression of CREB target genes, including the anti-apoptotic BTG2, the apoptotic P53 inhibitor BCL6, and the prosurvival neurotrophic factor BDNF (brain-derived neurotrophic factor).

[0050] To determine the activity of CREB, we examined CREB phosphorylated at Ser-133 (pCREB-S133), an activated form of CREB. Proteins from cell samples were extracted on ice in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Next, protein concentration was determined using a BCA protein assay kit. Protein samples were electrophoretically separated in SDS-polyacrylamide gels and transferred to nitrocellulose filters. Five percent non-fat milk or bovine serum albumin (BSA) diluted in TBST (20 mM Tris-Cl, 140 mM NaCl, pH 7.5, 0.05% Tween-20) was used for blocking. Then, primary antibodies were added and the membranes were incubated overnight at 4 °C. The primary antibodies were as follows: rabbit anti-pCREB (Ser133) (1:1,000, Cell Signaling Technology, 9198) or rabbit anti-CREB (1:1,000, Cell Signaling Technology, 9197). Rabbit anti-Tubulin beta antibody (1:5,000, Affinity Biosciences, AF7011) was used as an internal control. After washing three times in TBST, the membranes were incubated with the secondary antibody (goat anti-rabbit IgG (H+L) HRP) for 1 h at room temperature. Finally, the membranes were detected using a ChampChemi 610 chemiluminescence imager (SinSage Technology). Band density was analyzed using ImageJ software.

[0051] The results are shown in Figure 5 . NVP-AAM077, a highly selective antagonist of the NMDA receptor GluN2A, blocked intracellular phosphorylation of CREB under glutamate-induced excitotoxicity (P < 0.05 vs Glu alone). Administration of Aegeline significantly enhanced phosphorylation of CREB (P < 0.05 vs Glu alone), and NVP-AAM077 reversed this increased effect (P < 0.05 vs Glu+NVP), indicating that the neuroprotective effect of Aegeline is achieved by activating the GluN2A subunit and enhancing downstream phosphorylation of CREB.

Claims

1. Application of yindazoline in the preparation of a therapeutic drug for ischemic stroke.

2. Application of yindazoline in the preparation of a neuroprotective drug for ischemic stroke.

3. The application according to claim 1 or 2, wherein, the drug is a drug mixture or a drug composition.

4. The application according to claim 3, wherein, the drug mixture or the drug composition is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions and solutions.

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