Application of lamotrigine in preparation of anti-depression drugs

Abstract

The invention belongs to the technical field of medical artificial intelligence, and particularly relates to a method for searching a target spot of a drug for treating diseases through network pharmacology and computer-aided drug design and application of the target spot. Epidemiological studies show that the treatment of depression is mainly psychological guidance and drug therapy, however, different patients have different reactions to drugs, and unexpected adverse reactions are generated. Through network pharmacology, molecular docking and Mendel randomization technologies, the application of lamotrigine in preparation of the anti-depression medicine through acting on one or more target spots in GABRA1, GABRB2, GABRA6, GABRD, GABRG2, GABRG1, GABRA5, GABRA4, GABRB3 and GABRA2 is found and verified. The lamotrigine provides new dawn for safely, economically and effectively improving clinical treatment of depression patients based on the safe and economical'old medicine 'characteristics and the potential effective targeting activation effect of the lamotrigine on GABA receptors.

Description

Technical Field

[0001] The present invention belongs to the field of medical artificial intelligence technology, and specifically relates to conducting "new uses of old drugs" research through network pharmacology and computer-aided drug design methods. Background Art

[0002] According to the World Health Organization (WHO), approximately one billion people worldwide suffer from mental disorders. Treatment for depression primarily relies on a combination of psychological counseling and medication. However, different patients respond differently to medications, often leading to unpredictable adverse reactions. Target screening through network pharmacology, combined with molecular docking technology and Mendelian randomization for validation, is developing new uses for old drugs for new antidepressant indications, which holds significant social significance and clinical need.

[0003] Currently, the label instructions for lamotrigine dispersible tablets marketed domestically and internationally list epilepsy and bipolar disorder as indications. They are suitable for use during pregnancy and have a good safety profile. Lamotrigine has the highest treatment retention index among new-generation antiepileptic drugs and is a first-line and standard-of-care treatment for patients with focal epilepsy. Clinically, lamotrigine tablets are used off-label for the treatment of bipolar disorder (mania plus depression), suggesting that lamotrigine may have antidepressant effects. Studies have found that the combination of lamotrigine and duloxetine is more effective in improving the therapeutic efficacy of refractory depression than duloxetine alone. However, whether lamotrigine has antidepressant effects and its mechanism of action remain to be elucidated. This study, using network pharmacology, molecular docking, and Mendelian randomization techniques, explored and validated the potential roles and targets of lamotrigine in the treatment of depression. This study provides data support and research direction for further research into new indications for lamotrigine and promotes its rational clinical use in the treatment of depression. Summary of the Invention

[0004] Due to the complex pathogenesis of depression, there is currently no effective drug for the treatment of depression. Lamotrigine can be used clinically to treat bipolar disorder, but there is no clear report on whether it can be used to treat depression. Its efficacy and mechanism of action remain to be elucidated and verified.

[0005] The present invention uses network pharmacology and computer-aided drug design methods to search for targets for lamotrigine in the treatment of depression. Molecular docking and Mendelian randomization validation experiments are used to reveal the use of lamotrigine in the preparation of antidepressant drugs by acting on one or more targets among GABRA1, GABRB2, GABRA6, GABRD, GABRG2, GABRG1, GABRA5, GABRA4, GABRB3, and GABRA2. Experimental data are further used for verification, demonstrating the use of lamotrigine in the preparation of antidepressant drugs.

[0006] Preferably, lamotrigine is used in the preparation of antidepressant drugs by acting on the GABRB3 receptor target.

[0007] The depression is preferably major depressive disorder.

[0008] According to the above application, the antidepressant drugs prepared by lamotrigine include tablets, capsules, sustained-release preparations, solutions, injections, nasal preparations, and patches prepared with lamotrigine as the active ingredient.

[0009] Application of GABRB3 receptor as a target in screening antidepressant drugs.

[0010] Beneficial Effects: This invention uses lamotrigine as a research target and employs network pharmacology and computer-aided drug design methods to identify drug targets for treating diseases, effectively reducing the time and financial costs of drug development. This invention uses the drug's pharmacophore to predict all potential targets, rather than just a few known targets studied through specific mechanisms of action. This method helps us identify more new drug targets and screen for optimal targets. Through network pharmacology, simulated molecular docking, and Mendelian randomization, this invention reveals that lamotrigine produces its antidepressant effects by acting on GABA receptor-related proteins, particularly the GABRB3 receptor protein. Experimental data further confirms that lamotrigine can activate GABA receptors and increase GABRB3 receptor protein expression, resulting in its antidepressant effects. Lamotrigine, based on its safe and economical "old drug" status and its potential for effective targeted activation of the GABA receptor family, offers new hope for safely, economically, and effectively improving the clinical prognosis of patients with depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a cross-sectional diagram of drug targets and disease targets.

[0012] Figure 2 This is the PPI chart of the STRING platform.

[0013] Figure 3 GO enrichment analysis histogram.

[0014] Figure 4 This is a bubble diagram of KEGG pathway enrichment.

[0015] Figure 5 This is the molecular docking result diagram.

[0016] Figure 6 This is the result of Mendelian randomization DETAILED DESCRIPTION

[0017] Our team's previous investigations and studies have found that lamotrigine may lead to an increase in plasma GABA, but there are also reports that lamotrigine has no correlation with plasma GABA. However, the targets of antidepressant action should focus more on brain-related tissues, cells, proteins, and molecular targets. There are also reports that depression may be related to the activity of GABA-A or GABA-B receptors, but the specific receptor subtypes are still unknown. Although lamotrigine is used off-label in clinical practice for the treatment of bipolar disorder, there are no clear reports on whether it is effective for depression, and its possible mechanism of action is even more unclear. Our research found that lamotrigine may produce antidepressant effects by acting on brain GABA receptors, especially GABRB3 receptors. The specific research process is as follows:

[0018] 1. Identify target genes at the intersection of lamotrigine and depression

[0019] By using the SwissTargetPrediction database ( http: / / swisstargetprediction.ch / )、Comparative Toxicogenomics Database ( https: / / ctdbase.org / )、DrugBank Database( https: / / go.drugbank.com / ) and the TargetNet (http: / / targetnet.scbdd.com / ) prediction website using default parameters to identify targets associated with lamotrigine. A GSE217811 dataset, representing the phenotype of major depressive disorder (MDD), was identified in the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) to identify targets for depression. Using GEO2R analysis, the GEO database's built-in tool, with default parameters and a Log 2-fold change threshold of 1.5, differentially expressed genes were identified as targets for major depression. Genes resulting from the intersection of the differentially expressed genes identified above and the lamotrigine target genes were used as target genes for subsequent analysis.

[0020] Research results: 266 possible target genes of lamotrigine were obtained; after normalization and standardization of the GSE217811 dataset with the phenotype of MDD, 2488 differentially expressed genes were confirmed as MDD-related target genes. By taking the intersection of drug target genes and disease target genes, 48 intersection target genes were finally obtained. (See Figure 1 ).

[0021] 2. Protein-protein interaction (PPI) screening of core target genes

[0022] The target gene results obtained above were imported into the STRING platform for analysis. The STRING platform results were then imported into Cytoscape (version 3.9.0) and clustered using the MCODE plug-in. The filtering conditions were set as follows: node score cutoff = 0.2, K core value = 2, maximum depth = 100, and node degree cutoff = 2. The maximum depth and K core value were used to control the search range, while the node score and node degree cutoffs were used to ensure accuracy, thereby better identifying biologically significant core target interaction networks.

[0023] The PPI of the intersection target genes was processed by STRING platform, and the minimum interaction score required was set with the highest confidence (0.900) to construct the PPI network (see Figure 2 ), the network had 48 nodes, 191 edges, and an average node degree of 7.96. Cluster analysis was then performed using the MCODE plugin in Cytoscape (version 3.9.0) (see Table 2). Key targets in cluster 1 were GABRP, GABRA6, GABRA5, GABRA4, GABRG2, GABRB3, GABRA1, GABRD, GABRA2, GABRQ, GABRB1, GABRE, GABRG3, GABRG1, GABRA3, and GABRB2 (see Table 1).

[0024] Table 1 Results of cluster analysis

[0025] Table 1 The results of cluster analysis

[0026]

[0027] 3. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis

[0028] To further explore the biological processes involved in the target genes, cluster-Profiler software in the R software package was used to perform functional enrichment analysis including molecular function (MF), cellular components (CC), and biological process (BP), as well as signaling pathway analysis. Bubble plots and result histograms were visualized using the clusterProfiler software in R, and the screening criteria were adjusted to p ≤ 0.05.

[0029] GO enrichment analysis results showed that lamotrigine's anti-MDD effects in BP were mainly enriched in the regulation of postsynaptic membrane potential, gamma-aminobutyric acid (GABA) signaling pathway, and membrane potential regulation; in CC, it was mainly involved in GABA-A receptor complex, GABA receptor complex, transmembrane transporter complex, and postsynaptic membrane; in MF, it was mainly involved in GABA-A receptor activity, GABA receptor activity, GABA-gated chloride channel activity, and neurotransmitter receptor activity; GABA-related functions were mentioned many times.

[0030] The most relevant biological pathways for MDD in KEGG enrichment analysis included GABAergic synapses, nicotine addiction, glutamatergic synapses, retrograde endocannabinoid signaling, morphine addiction, and neuroactive ligand and receptor interactions, and the results were consistent with the core target genes of the PPI network.

[0031] GO analysis diagram is shown in Figure 3 , KEGG enrichment analysis visualized the top 20 KEGG pathways see Figure 4 .

[0032] 4 Molecular docking verification

[0033] Based on the PPI analysis described above, the protein encoded by the core target gene was selected as the molecular docking protein, and lamotrigine was selected as the binding ligand for molecular docking validation. The protein structure encoded by the core target gene was obtained from the UniProt database (https: / / www.uniprot.org / ), and the molecular structure of lamotrigine was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Molecular docking was performed using Autodock and Vina software. The binding energy was used to evaluate the binding activity of the lamotrigine molecule with the target. A lower binding energy indicates a more stable docking. The molecular docking results were visualized using Pymol software.

[0034] The results showed that the binding energy of lamotrigine to GABRA1, GABRB2, GABRA6, GABRD, GABRG2, GABRG1, GABRA5, GABRA4, GABRB3 and GABRA2 receptors was ≤-5.8 kCal·mol-1 (see Table 2). Lamotrigine can stably bind to the protein receptors encoded by the core target genes, among which the binding between GABRB3 receptor and lamotrigine is the most stable, with a binding energy of -9.5 kCal·mol-1, suggesting that lamotrigine may produce anti-MDD effects by acting on GABA receptors.

[0035] Table 2 Molecular docking binding energy results (kCal·mol -1 )

[0036] Table 2 Results of molecular docking binding energy(kCal·mol -1 )

[0037]

[0038] The results of molecular docking (see Figure 5 ), where the cyan compound is the ligand, the green compound is the amino acid residue, and the yellow dashed line represents a hydrogen bond. As can be seen from the figure, lamotrigine can stably bind to the core target, potentially acting as a potential target for its therapeutic effects against major depressive disorder.

[0039] Molecular docking analysis revealed strong binding affinity between lamotrigine and GABRB3, a subtype of the GABA-α receptor that plays a key role in inhibitory neurotransmission. Lamotrigine may exert its antidepressant effects by acting on GABA receptors, specifically by binding to GABRB3 receptors to modulate inhibitory neurotransmission. This mechanism of action may be the key role of lamotrigine in the treatment of depression.

[0040] 5 Two-sample Mendelian randomization verification

[0041] Based on the molecular docking analysis results, the search term "GABA" was entered into the OpenGWAS (https: / / gwas.mrcieu.ac.uk / ) database to obtain the genome-wide association analysis (GWAS) data of γ-aminobutyric acid receptor-associated protein-like 1 (GABARAPL1) (GWAS ID: prot-a-1161, sample size 3301) and the GWAS data of major depressive disorder (GWASID: ieu-a-1188, sample size 173 005). Two-sample Mendelian randomization causal analysis was performed using MR Egger, weighted median, inverse variance weighting, simple model, and weighted model [7]. The instrumental variables for analysis were single nucleotide polymorphisms (SNPs). The selection criteria for each instrumental variable associated with the exposure factor (core target gene) were as follows: ① Correlation hypothesis, SNPs were strongly correlated with the core target gene; ② Exclusivity hypothesis, SNPs were unrelated to the outcome factor; ③ Independence hypothesis, SNPs were unrelated to the confounder. The tool for Mendelian randomization analysis was the Two-Sample (version 0.5.6) package in R (4.2.2) software.

[0042] Results of a two-sample Mendelian randomization analysis: In the GABARAPL1 GWAS data, 303 SNPs were associated with GABARAPL1 (P < 5 × 10-6), of which 256 SNPs were eligible for MDD GWAS analysis. After excluding SNPs with linkage disequilibrium (r2 < 0.001, kb = 1.0 × 104) and three unaligned palindromic SNPs, 15 SNPs remained available for Mendelian randomization analysis. The results showed that the causal effect estimates of GABARAPL1 on MDD maintained consistent trends and were of similar magnitude in inverse variance weighted, MR Egger, weighted median, simple model, and weighted pattern analyses, supporting a protective effect of GABARAPL1 on MDD.

[0043] See the results Figure 6 . Figure 6A shows forest plots of the risk estimates for GABARAPL1 and MDD using each outcome measure using different MR methods. In the IVW analysis, the odds ratio (OR) for GABARAPL1 to MDD was 0.97 (95% confidence interval [CI], 0.95-0.99; P = 0.045), and the estimates remained consistent and of similar magnitude in the MR Egger (OR = 0.983; 95% CI = 0.947-1.02), weighted median (OR = 0.978; 95% CI = 0.952-1.00), simple model (OR = 0.967; 95% CI = 0.903-1.035), and weighted mode (OR = 0.978; 95% CI = 0.951-1.007) analyses, supporting a protective effect of GABA against MDD. Figure 6 B shows a scatter plot between the SNP outcome association and the SNP GABARAPL1 association to visualize the causal effect estimate of each SNP on MDD. Figure 6 C also shows the estimated causal effect of each individual SNP on MDD.

[0044] This study used Mendelian randomization to confirm the causal relationship between GABARAPL1 and MMD, indirectly supporting the potential anti-MDD effect of lamotrigine. First, using SNPs as instrumental variables for Mendelian randomization analysis fully utilized known genotypes to reduce potential confounding factors. Second, this study used five methods to estimate causal effects, combined with three sensitivity analyses, to provide precise quantitative causal estimates while minimizing the potential influence of pleiotropic effects within the instrumental variables, confirming that GABARAPL1 may be a protective factor for MMD. This indirectly demonstrates that lamotrigine, which can bind to GABA receptors, may have anti-MDD effects.

[0045] 6. Verification of drug efficacy and target in animals

[0046] The experimental plan was designed with reference to the open literature [Effects of total iridoid terpenes from Gardenia jasminoides on neurotransmitters in depression model mice_Qu Shuyue_Chinese Patent Medicine_2021 Vol. 43 No. 4] as follows:

[0047] Grouping, Dosing, and Modeling: After 7 days of adaptive feeding, mice were randomly divided into 10 groups, each with 10 mice: a control group, a positive drug group, a model group, and a lamotrigine-treated group (divided into high, medium, and low dose groups). The positive drug group and the lamotrigine-treated group were gavaged continuously for more than 7 days, 1-2 times daily. The control group and the model group were given the vehicle solution. One hour after the last dose, the control group mice received an intraperitoneal injection of normal saline, while the remaining mice received an intraperitoneal injection of 0.8 mg / kg lipopolysaccharide (LPS). Three hours after LPS injection, the control group, the positive drug group, the lamotrigine-treated group, and the model group underwent a forced swim test.

[0048] Forced swimming test: Mice were placed in a transparent cylinder approximately 12 cm in diameter and 25 cm high. The water in the cylinder was 15 cm deep and kept at a temperature of (25 ± 1)°C. The mice were unable to touch the bottom of the cylinder. A camera was used to record the mice's swimming behavior for 6 minutes, and the duration of immobility during the next 4 minutes was recorded.

[0049] Hippocampal processing: After forced swimming, mice were immediately sacrificed, and hippocampal tissue was isolated on ice and quickly frozen in liquid nitrogen. Accurately weigh the hippocampal tissue and add 10-fold volume of ice-cold 0.1% formic acid. Homogenize for 3 minutes. Take 100 μL of the homogenate and add 200 μL of ice-cold 0.2% formic acid-acetonitrile. Vortex for 3 minutes and centrifuge at 12,000 rpm for 10 minutes in a refrigerated centrifuge at 4°C. The supernatant was aspirated and transferred to a centrifugal concentrator, evaporated to dryness, and reconstituted with 0.1% formic acid for injection.

[0050] The GABA content in the hippocampus was detected by HPLC, and the GABRB3 receptor protein expression in the hippocampus tissue was detected by Western-Blot.

[0051] Statistical analysis: Experimental data are expressed as (x±s) and analyzed using SPSS 17.0 software. One-way ANOVA was used for comparisons among multiple groups. P<0.05 was considered statistically significant.

[0052] The study found that the forced swimming immobility time of mice in the lamotrigine-treated group was significantly reduced compared to the model group, indicating that lamotrigine can effectively improve depressive symptoms in mice. Three hours after LPS injection, the hippocampal GABA content and GABRB3 receptor protein expression in the treated group were significantly increased compared to the model group, indicating that lamotrigine can act on GABRB3 receptors in the brain to regulate the amount of GABA neurotransmitters and thus control depressive symptoms. The results of the mouse animal experiments were consistent with the predicted results.

[0053] 7. Cell Experiment Verification

[0054] Further verification at the cell level was performed.

[0055] Primary hippocampal cells of newborn mice were cultured and routine operations were performed according to cell culture experiments. The MTT method was used to explore the dosage range of lamotrigine and design the dosage. The experiment was divided into a blank control group (without lamotrigine), a high-dose lamotrigine group, a medium-dose lamotrigine group, and a low-dose lamotrigine group. The hippocampal cells cultured normally were cultured for 24 hours after administration, and Western-Blot experiments were performed to detect the expression of GABRB3 protein in the groups. The expression of GABRB3 receptor protein in the high, medium, and low-dose lamotrigine groups was significantly increased compared with the blank control group and showed a dose-positive relationship. The higher the lamotrigine concentration, the more obvious the expression of GABRB3 receptor protein, indicating that lamotrigine can produce anti-depressant effects by acting on GABA receptors such as GABRB3.

[0056] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Obviously, other relevant modifications may be made based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of lamotrigine in the preparation of antidepressant drugs.

2. The use according to claim 1, characterized in that: Lamotrigine is used in the preparation of antidepressant drugs by acting on one or more receptor targets among GABRA1, GABRB2, GABRA6, GABRD, GABRG2, GABRG1, GABRA5, GABRA4, GABRB3, and GABRA2 receptors.

3. The use according to claim 2, characterized in that: Application of lamotrigine in the preparation of antidepressant drugs by acting on the GABRB3 receptor target.

4. The use according to claim 1, characterized in that: The depression is major depressive disorder.

5. The use according to any one of claims 1 to 4, characterized in that: The antidepressant drugs prepared from the lamotrigine include tablets, capsules, sustained-release preparations, solutions, injections, nasal preparations and patches prepared with lamotrigine as the active ingredient.

6. Application of GABRB3 receptor target in screening antidepressant drugs.

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