Compositions and methods of combination

Abstract

The application belongs to the technical field of biological medicine, and relates to application of a new target in tuberous sclerosis complex related epilepsy, a composition and a combined medication mode. The application proves that when a combination of a high-dose SLC6A1 inhibitor + a medium-dose GAD1 function enhancer + a low-dose SLC1A2 activator is adopted, the average seizure frequency of experimental animals is lower than that of a combined medication group with the same dose or close to the same dose, and is also lower than that of a single-drug experimental group, proving the effectiveness of the combination of SLC6A1 as a core and GAD1 and SLC1A2 as synergistic targets.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of new targets in tuberous sclerosis-related epilepsy, compositions, and combination therapy methods; specifically, it relates to new uses of SLC6A1, GAD1, and SLC1A2 genes as potential therapeutic targets for tuberous sclerosis-related epilepsy, as well as compositions and combination therapy methods based on these targets. Background Technology

[0002] Tuberous sclerosis complex (TSC) is a rare autosomal dominant inherited disease caused by mutations in the TSC1 or TSC2 gene, leading to abnormal activation of the mTOR signaling pathway. Epilepsy is the most common neurological manifestation of TSC. TSC-related epilepsy typically has an early onset and frequent seizures, making it more likely to cause neurodevelopmental delays and cognitive impairment. The pathogenesis of TSC-related epilepsy is closely related to abnormal activation of the mTOR pathway, ultimately leading to hyperexcitability and abnormal discharge of the cerebral cortex network, causing epileptic seizures.

[0003] mTOR inhibitors (such as sirolimus and everolimus) have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of TSC-related epilepsy, but their clinical application still has significant limitations: the response rate is low, with only about 40% of TSC patients experiencing effective seizure control after treatment, and there are significant individual differences in efficacy among different patients; the safety risks are prominent, as these drugs may cause adverse reactions such as immunosuppression, metabolic disorders, and stomatitis. In addition, since they directly act on cell proliferation and growth regulation pathways, long-term use in infants and young children will significantly increase the risk of tumor development, limiting their long-term use in young patients. Furthermore, traditional antiepileptic drugs (such as levetiracetam and oxcarbazepine) mostly target neurotransmitter receptors or ion channels, and about 70% of patients do not respond to drug treatment, developing drug-resistant epilepsy (the refractory rate is twice that of ordinary epilepsy).

[0004] Furthermore, current research on mTOR pathway inhibitors mainly focuses on single-target interventions and has not yet developed a synergistic regulatory strategy targeting the entire neurotransmitter synthesis-transportation-clearance chain.

[0005] In summary, exploring new targets for tuberous sclerosis (TSC)-related epilepsy and developing new drugs or novel drug combinations are urgent needs in the current treatment of TSC-related epilepsy. Summary of the Invention

[0006] The purpose of this invention is to provide a new strategy for treating tuberous sclerosis-related epilepsy based on multi-target synergistic effects, in order to partially solve or alleviate the problems of limited efficacy, large side effects, and insufficient control of refractory epilepsy caused by single-target drugs in the prior art.

[0007] This invention targets the SLC6A1, GAD1, and SLC1A2 genes simultaneously to synergistically regulate the homeostasis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and glutamate clearance, aiming to more comprehensively and effectively restore the excitation-inhibition balance of the central nervous system, thereby controlling epileptic seizures. The specific technical solution employed in this invention is as follows.

[0008] One aspect of this invention is the application of the SLC6A1 gene, GAD1 gene, and SLC1A2 gene as combined biomarkers in the preparation of drugs or reagents for tuberous sclerosis (TSC)-related epilepsy.

[0009] Furthermore, the combined biomarkers are used to guide the development of drugs or agents for tuberous sclerosis-related epilepsy. By simultaneously targeting the SLC6A1, GAD1, and SLC1A2 genes, it is possible to simultaneously intervene in GABA synthesis, transport, and glutamate clearance, producing a synergistic antiepileptic effect, which is of great value in guiding the development of novel antiepileptic drugs.

[0010] Another aspect of the present invention is to provide a novel target composition for targeting TSC-related epilepsy.

[0011] A combination of biomarkers targeting tuberous sclerosis-associated epilepsy, the biomarker combination being a combination of the SLC6A1 gene, the GAD1 gene, and the SLC1A2 gene.

[0012] Another aspect of the present invention is to provide a composition for targeted treatment of TSC-related epilepsy and a combination of medications.

[0013] A pharmaceutical composition for targeted treatment of tuberous sclerosis-associated epilepsy, the pharmaceutical composition comprising an SLC6A1 inhibitor, a GAD1 function enhancer, and an SLC1A2 activator.

[0014] Furthermore, the SLC6A1 inhibitor is a selective GAT-1 inhibitor.

[0015] Furthermore, the GAD1 function enhancer includes pyridoxal phosphate (PLP) and its precursor compounds. The precursor compounds can be metabolized in vivo to produce vitamin B6 compounds of PLP. Furthermore, the SLC1A2 activator includes β-lactam compounds.

[0016] Furthermore, the pharmaceutical composition may also include other pharmaceutically acceptable excipients or adjuvants for preparation into a pharmaceutical formulation; the dosage form of the pharmaceutical formulation may include injections, tablets, capsules, or solutions.

[0017] In one specific embodiment, the SLC6A inhibitor may be selected from tiagabin, EF1502, NO-711, CL-966, etc.; the GAD1 activator may be selected from pyridoxal phosphate (PLP), pyridoxine (vitamin B6), etc.; and the SLC1A2 activator may be selected from ceftriaxone or its derivatives, etc.

[0018] The use of a combination of drugs in the preparation of a drug for targeted treatment of tuberous sclerosis-related epilepsy, wherein the combination of drugs is a combination of an SLC6A1 inhibitor, a GAD1 enhancer, and an SLC1A2 activator; wherein the dose of the SLC6A1 inhibitor is greater than the dose of the GAD1 enhancer; and the dose of the GAD1 enhancer is greater than the dose of the SLC1A2 activator (SLC6A1 inhibitor dose > GAD1 enhancer dose > SLC1A2 activator dose).

[0019] Furthermore, the SLC6A1 inhibitor is tiagabe; the GAD1 function enhancer is vitamin B6; and the SLC1A2 activator is ceftriaxone.

[0020] Furthermore, the dosage ratio of the SLC6A1 inhibitor, GAD1 function enhancer, and SLC1A2 activator includes 3:2:1, 4:2:1, or 5:3:1.

[0021] As a preferred embodiment, the dosage ratio of the SLC6A1 inhibitor, GAD1 enhancer, and SLC1A2 activator is 3:2:1.

[0022] Beneficial technical effects:

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] A multi-target synergistic mechanism significantly enhances antiepileptic effects: This invention is the first to propose a therapeutic combination using SLC6A1 (GABA transporter 1) as the core, and GAD1 (glutamate decarboxylase 1) and SLC1A2 (glutamate transporter EAAT2) as synergistic targets. By simultaneously inhibiting GABA reuptake (SLC6A1 inhibitor), promoting GABA synthesis (GAD1 function enhancer), and strengthening glutamate clearance (SLC1A2 activator), this synergistic effect, acting from both "enhanced inhibition" and "reduced excitation," can more effectively improve the neuronal excitation-inhibition imbalance, and is expected to produce a stronger and broader antiepileptic effect than single-target drugs.

[0025] Reduced risk of side effects: Through the synergistic effect of the primary target + auxiliary target and low dose, better efficacy can be achieved while reducing the required dosage of a single component. This may reduce specific side effects caused by high-dose monotherapy (such as drowsiness that may be caused by high doses of SLC6A1 inhibitors), improve the safety window of treatment, and enhance patient tolerability. In one specific embodiment of the invention, it was verified that when a combination of high-dose SLC6A1 inhibitor + medium-dose GAD1 function enhancer + low-dose SLC1A2 activator was used, the average number of seizures in experimental animals was lower than that in the combination therapy group with the same or nearly the same dose, and also lower than that in the monotherapy group, demonstrating the effectiveness of the treatment combination with SLC6A1 as the core and GAD1 and SLC1A2 as synergistic targets.

[0026] In summary, the three-target synergistic regulation strategy of SLC6A1-GAD1-SLC1A2 proposed in this invention can effectively enhance inhibitory neurotransmission and improve excitatory neurotransmitter imbalance, thereby achieving better anti-epileptic effects. The combination formulation has a low cost, providing a new, effective, and low-cost potential drug for the treatment of tuberous sclerosis-related epilepsy, and has significant innovation and practical value. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0028] Figure 1 The data quality assessment results of snRNA-seq in one embodiment of the present invention (AD: The violin plot shows the quality distribution of single-cell RNA sequencing data, including the number of genes in each cell nucleus, the number of transcripts (UMI), the percentage of mitochondrial gene expression, and the predicted duplex score in different samples).

[0029] Figure 2 This is a UMAP visualization result of cell type clustering patterns of snRNA-seq data in one embodiment of the present invention (cell types are annotated according to the expression of known marker genes and marked with different colors; A shows the brain tissue cell type clustering analysis after integrating the two sets of data; B shows the distribution comparison of lesions and normal brain tissue (red and green); C on the left shows the cell clustering of normal brain tissue, and C on the right shows the cell clustering of patients with TSC and drug-resistant epilepsy).

[0030] Figure 3 This is an example of differentially expressed genes (DEGs) found in different cell types from snRNA-seq data in one embodiment of the present invention (the volcano plot shows the DEGs between epileptogenic foci and normal controls in astrocytes (Ast), endothelial cells (End), interneurons In_PV, interneurons In_SST, excitatory neurons Ex_L5-6, and excitatory neurons Ex_NRGN_2 from snRNA-seq data; each point represents a DEG, red indicates high expression in epileptogenic foci of TSC with drug-resistant epilepsy, blue indicates high expression in normal controls, and genes with log2FC>0.5 are marked).

[0031] Figure 4 In one embodiment of the present invention, TSC2 after intervention in each drug treatment group GFAP Results of the average daily seizure frequency variation in CKO model mice (box plot showing TSC2) GFAP (mean number of seizures per day in CKO model mice after intervention with SLC6A1 inhibitor, GAD1 enhancer, SLC1A2 activator and combination therapy groups, respectively).

[0032] Figure 5 TSC2 is one embodiment of the present invention. GFAP CKO model H&E and immunofluorescence staining results (A shows TSC2) GFAP The CKO cortex contains numerous atypical neurons and balloon-like cells; B shows TSC2. GFAP CKO mice showed increased astrocyte density compared to wild-type normal controls (white arrows indicate astrocytes, yellow arrows indicate neurons). Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0035] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0036] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0037] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0038] Example 1

[0039] Based on single-nucleus RNA sequencing (snRNA-seq) data of epileptogenic focus brain tissue from TSC patients with drug-resistant epilepsy and normal controls, core genes were screened as therapeutic targets. The specific screening process is as follows.

[0040] Four patients with TSC and drug-resistant epilepsy who underwent epileptogenic focus resection surgery were included. Whole-genome sequencing confirmed pathogenic mutations in the TSC2 gene. Patients were diagnosed with TSC according to the 2021 International TSC Consensus Group diagnostic criteria, and with epilepsy according to the ILAE criteria. Their epilepsy was not controlled after regular and full-course treatment with two or more antiepileptic drugs, and they experienced more than two seizures per month. Informed consent was obtained from all participants before surgery, and basic clinical information (including gender, age, family history, age of onset of epilepsy, seizure type, frequency, and treatment history) was collected, along with video EEG and imaging results. Neurosurgeons performed epileptogenic focus resection surgery, collecting eight brain tissue samples.

[0041] Single-cell nucleus suspension preparation and transcriptome sequencing: Brain tissue surgically removed from TSC patients was washed with PBS until free of blood and impurities. After being minced on ice, the tissue was dissociated using tissue dissociation buffer (containing collagenase IV and DNase I) at 37°C with constant temperature shaking. Pre-cooled washing buffer was then added, and the tissue fragments were removed by filtration through a 70 μm cell sieve. Single-cell nuclei were obtained by Percoll density gradient centrifugation. Total RNA was extracted after treatment with nuclear lysis buffer. After passing quality testing (RNA Integrity Number ≥ 7.0), the single-nucleus suspension was loaded onto the Chromium™ controller (10×Genomics), with a target cell recovery rate of 10,000 cells per reaction. cDNA library preparation was performed according to the manufacturer's instructions for the v3.1 Chromium Single-Cell 3' Library and Gel Bead Kit (10×Genomics1000121). Quality control of cDNA and constructed libraries was performed using a 4200 Bioanalyzer (Agilent), and transcriptome sequencing was performed using an Illumina NovaSeq 6000.

[0042] Raw snRNA-seq data were preprocessed using 10×Cell Ranger software (version 6.0.1) to map samples to the human GRCh38 reference genome. Double cells were removed using scrublet software (version 0.1). High-quality data were then selected using Seurat R package (version 4.1.1) based on nFeature_RNA counts of 300-6000 and percent.mt < 10. After normalization using SCTransform R package, the dataset was integrated using Seurat's RPCA method. Dimensionality reduction using PCA, UMAP visualization, and cluster analysis were performed. Cell type annotation was completed by combining published brain tissue marker genes, scCATCH database annotation, Metascape software gene ontology enrichment analysis, and tag migration results. Low-quality cells and unknown cell types were removed. Cell composition statistical analysis was then performed using tools such as scCODA, and cluster resolution was optimized using Clutree and the SC3 stability index. Finally, marker genes for each cell subpopulation were selected. The snRNA-seq data quality assessment results are shown below. Figure 1 . Figure 1 The data quality of all eight samples was good. The number of genes, transcripts, and mitochondrial gene expression percentages in each cell nucleus were within a reasonable range in each sample, and the proportion of two cells in all samples was low.

[0043] Using published snRNA-seq data from normal human brain tissue samples in public databases as a control (Science. 2019;364(6441):685-689.), the clustering information was readjusted after data integration, resulting in a total of 16 cell types, including excitatory neurons (Ex), interneurons (In), astrocytes (Ast), microglia (Mic), oligodendrocytes (Oli), endothelial cells (End), and oligodendrocyte precursor cells (OPC). The cell clustering results are shown in […]. Figure 2 . Figure 2 The results showed that, compared with normal brain tissue, the epileptogenic focus of patients with TSC-related epilepsy exhibited a significant increase in excitatory neuronal subsets (Ex_L2 / 3, Ex_L4, and Ex_L5 / 6_CC), abnormal proliferation of astrocytes, and a decrease in inhibitory neuronal subsets (In_VIP, In_SST, and In_PV) and oligodendrocytes, indicating a disordered cellular subset population.

[0044] Further differentially expressed genes (DEGs) analysis was performed using two different methods: (1) all cell types were divided into epileptogenic focus and normal control groups, and the 'FindMarker' command (Wilcoxon rank-sum test) was used to find differentially expressed genes for each cell type between the two groups (P<0.05); (2) the Libra package (version=1.7) was used to divide the single-cell nucleus data in the two groups into pesudodebulks according to the samples, and then the edgeR algorithm was used to find DEGs (P<0.05). This method can effectively avoid negative signals due to individual differences. Subsequently, only the common set of differentially expressed genes found by the Wlicox and edgeR methods was selected for downstream analysis. Finally, the DEGs between the epileptogenic focus group and the normal control group in different cell types were obtained from the snRNA-seq data. Figure 3 The results showed that the SLC6A1 (GABA transporter) gene was highly expressed in astrocytes (Ast), interneurons (In_PV), and In_SST) of the epileptogenic focus. Compared with normal controls, the GAD1 (a key enzyme in GABA synthesis) gene in the epileptogenic focus was lowly expressed in interneurons (In_SST) and excitatory neurons (EX_NRGN_2). The SLC1A2 (amino acid transporter) gene was also reduced in astrocytes (Ast), endothelial cells (End), and excitatory neurons (EX_L5-6) of the epileptogenic focus.

[0045] Based on the DEGs results in the snRNA-seq data above, a treatment combination with "SLC6A1 as the core and GAD1 and SLC1A2 as synergistic targets" was selected. Further analysis of its expression characteristics and the direct association with the occurrence of TSC-related epilepsy is as follows.

[0046] SLC6A1 (GABA transporter, core target) encodes the major GABA transporter 1 (GAT-1) on the presynaptic membrane, responsible for re-uploading GABA from the synaptic cleft back into the neuronal cell body, terminating the inhibitory effect of GABA. This gene directly determines the rate of GABA clearance from the synaptic cleft and is a key control point for the termination of inhibitory neurotransmission. In TSC patients with epilepsy, SLC6A1 is significantly overexpressed in astrocytes (Ast), interneurons (In_PV), and In_SST. Its overexpression leads to abnormally rapid clearance of GABA from the synaptic cleft, causing premature termination of inhibitory signals, resulting in insufficient inhibition and instability of the excitation-inhibition network, ultimately triggering epileptic discharges. Therefore, SLC6A1 occupies a "core braking point" position in the excitation-inhibition imbalance and has a decisive influence on neural network stability, making it suitable as a core therapeutic target.

[0047] GAD1 and SLC1A2 respectively fill the defects at the GABA synthesis end and glutamate scavenging end, acting as co-regulatory sites.

[0048] GAD1 (a key enzyme in GABA synthesis and a co-target) encodes glutamate decarboxylase 67 (GAD67), the rate-limiting enzyme in the conversion of glutamate to GABA. In the interneuron In_SST of the TSC, GAD1 is significantly underexpressed, and GAD67 activity is weakened, leading to insufficient GABA synthesis and decreased reserves of inhibitory neurotransmitters, further exacerbating the excitation-inhibition imbalance. As a key node in the inhibitory neurotransmitter synthesis process, the regulation of GAD1 is functionally complementary to that of SLC6A1.

[0049] SLC1A2 (an amino acid transporter and co-target) encodes the EAAT2 (GLT-1) protein, a key transporter for glutamate reuptake in the synaptic cleft of the central nervous system, responsible for over 90% of glutamate reuptake. In TSC brain tissue samples, SLC1A2 is expressed at low levels in astrocytes (Ast), endothelial cells (End), and excitatory neurons (EX_L5-6). EAAT2 dysfunction leads to the accumulation of glutamate excitatory neurotransmitters in the synaptic cleft, resulting in overactivation of AMPA / NMDA receptors, causing neuronal calcium overload and abnormal discharge, thus triggering epilepsy. SLC1A2 regulation targets defects in excitatory neurotransmitter clearance, complementing the inhibitory regulation of SLC6A1 and GAD1.

[0050] In summary, this invention proposes that SLC6A1, GAD1, and SLC1A2 act on three key nodes: GABA clearance, GABA synthesis, and glutamate clearance, respectively, and the synergistic imbalance of these three constitutes the core molecular mechanism of TSC-related epilepsy.

[0051] This invention uses the aforementioned three genes as joint intervention targets to achieve bidirectional regulation of enhanced inhibition and weakened excitation, while simultaneously restoring GABA supply, normalizing GABA clearance rhythm, and reducing synaptic glutamate accumulation, achieving synergistic and non-linear improvement effects that cannot be achieved by a single target. While regulating SLC6A1 alone can enhance inhibition, it cannot correct the insufficient GABA synthesis caused by GAD1 downregulation, nor can it alleviate glutamate overload caused by SLC1A2 deficiency. Joint regulation, however, can establish a transcellular homeostatic recovery effect between neurons and astrocytes, producing a comprehensive network stabilizing effect that transcends simple superposition. The synergistic effect of these three genes can simultaneously regulate the three major cellular systems—astroglia, interneurons, and excitatory neurons—significantly reducing the threshold for abnormal synchronous discharge, resulting in a non-linear decrease in the frequency and intensity of epileptic seizures. This is something that a single-target strategy cannot achieve. Synergistic regulation can significantly reduce the threshold for abnormal synchronous discharge, manifested as a non-linear decrease in the frequency and intensity of epileptic triggers. Furthermore, by simultaneously repairing the dual closed-loop regulation of the inhibitory end (SLC6A1, GAD1) ​​and the excitatory end (SLC1A2), this invention unexpectedly achieves a systematic rebalancing of the glutamate-GABA metabolic circuit, which is a synergistic effect with outstanding but not obvious characteristics.

[0052] Example 2

[0053] Based on the synergistic imbalance of SLC6A1, GAD1, and SLC1A2 as the core molecules of TSC-related epilepsy proposed in Example 1, a drug development and application is proposed.

[0054] Example 1 provides a potential novel therapeutic target combination (SLC6A1, GAD1, SLC1A2) with synergistic effects. The synergistic mechanism of this target combination is as follows: through a combined strategy of "GAD1 function enhancer promoting GABA synthesis + SLC6A1 inhibitor reducing GABA reuptake + SLC1A2 agent accelerating glutamate clearance," it simultaneously regulates three dimensions: "increased synthesis, reduced consumption, and antagonist clearance," rapidly restoring the "excitation-inhibition neurotransmitter balance" in the epileptogenic focus area. This can significantly improve the seizure control rate, especially suitable for refractory cases that do not respond well to mTOR inhibitors.

[0055] This invention protects the use of a combination of SLC6A1 inhibitors, GAD1 enhancers, and SLC1A2 activators in the preparation of TSC-related epilepsy treatment drugs or related drug combinations.

[0056] SLC6A1 inhibitors include, but are not limited to, selective GAT-1 inhibitors (tiagabin, EF1502, NO-711, CL-966), which can specifically inhibit the activity or expression of GABA transporter 1 and reduce GABA reuptake.

[0057] GAD1 function enhancers include, but are not limited to, pyridoxal phosphate (PLP) and its precursor compounds. Pyridoxine (vitamin B6) serves as a precursor of PLP and promotes GAD1-mediated GABA synthesis by converting it into PLP, an essential coenzyme for GAD1, in vivo.

[0058] SLC1A2 activators include, but are not limited to, β-lactam derivatives (ceftriaxone or its derivatives), which can specifically enhance the transport function or expression level of SLC1A2 and accelerate glutamate clearance.

[0059] Specifically, a combination therapy is proposed: SLC6A1 activator (tiagabin), functional enhancer (vitamin B6), and SLC1A2 activator (cefotaxime sodium) are mixed in a dosage ratio (3:2:1), and pharmaceutically acceptable excipients such as lyophilization protectant (e.g., mannitol), solubilizer (e.g., polysorbate 80), and solvent (e.g., physiological saline) are added to prepare an injectable formulation.

[0060] Tiagabine has central nervous system targeting (blood-brain barrier penetration ≥40%) and reduces off-target effects in peripheral tissues. The ratio of this combination therapy (3:2:1) is based on drug synergy and dosage optimization. Tiagabine, as the core drug, works by enhancing GABA inhibitory signaling and has the largest dosage proportion; Vitamin B6 promotes GAD1-mediated GABA synthesis by being converted into PLP, an essential coenzyme for GAD1, and has the second largest dosage proportion; Ceftriaxone sodium plays an auxiliary role by enhancing SLC1A2 expression and regulating glutamate clearance, and has the smallest dosage proportion. This ratio maximizes the synergistic effect of the three drugs through low-dose combination and avoids the side effects that may occur with high-dose single-drug use, such as drowsiness caused by high doses of tiagabine. This ratio effectively reduces the occurrence of adverse reactions while ensuring efficacy. The above drug combination can significantly improve the treatment effect of TSC-related epilepsy by synergistically regulating neurotransmitter balance, and compared with single-target drugs, it can reduce the dosage of each drug and reduce the incidence of adverse reactions. This combination strategy targets the specific regulation of neurotransmitter metabolic pathways, avoiding the direct impact of mTOR inhibitors on cell proliferation and reducing the potential tumor risk associated with long-term use.

[0061] Those skilled in the art will understand that the specific types and proportions of SLC6A1 activator, GAD1 function enhancer, and SLC1A2 activator used in this embodiment are merely examples and not limitations.

[0062] Example 3

[0063] Animal model validation of the combined drug dosage ratio provided in Example 2.

[0064] This embodiment uses TSC2. GFAP CKO mice were housed in an SPF-grade animal facility at 22±2℃ and 50±5% humidity, with a 12-hour light / dark cycle and free access to food and water, in accordance with the Guidelines for Ethical Review of Laboratory Animal Welfare (GB / T35892-2018). Three-week-old TSC2 mice were... GFAP CKO mice were randomly divided into 6 groups, with 3 mice in each group, as follows:

[0065] (1) Control group: TSC2 GFAP CKO mice + physiological saline;

[0066] (2) Combination drug ratio Group 1: TSC2 GFAP CKO mice + combination therapy group (tiagabin: vitamin B6: ceftriaxone sodium = 1:1:1);

[0067] (3) Combination drug ratio in group 2: TSC2 GFAP CKO mice + combination therapy group (tiagabin: vitamin B6: ceftriaxone sodium = 2:1:1);

[0068] (4) Combination drug ratio in 3 groups: TSC2 GFAP CKO mice + combination therapy group (tiagabin: vitamin B6: ceftriaxone sodium = 3:2:1);

[0069] (5) Combination drug ratio in 4 groups: TSC2 GFAP CKO mice + combination therapy group (tiagaben:vitamin B6:ceftriaxone sodium = 4:2:1);

[0070] (6) Combination drug ratio in 5 groups: TSC2 GFAP CKO mice + combination therapy group (tiagabin: vitamin B6: ceftriaxone sodium = 5:3:1).

[0071] Dosing regimen and monitoring methods: In each combination therapy group, the total dose of the compound preparation was 5 mg / kg, and the components were allocated according to the above dosage ratio. All groups were administered intraperitoneally starting at 3 weeks of age, with a dosage volume of 10 μL / g, once daily for 2 weeks, at a fixed time of 8:00-9:00 AM daily. The blank control group received an equal volume of physiological saline. During the administration period, mice in each group were continuously monitored using an animal behavior video monitoring system to record the number of seizures per day. The general condition of the mice was observed daily during the administration period, including food intake, water intake, activity level, and mental state. Adverse reactions (such as lethargy, reduced activity, and worsening of seizures) and mortality were recorded.

[0072] The results showed that the average number of seizures per day in the blank control group was 3.65±4. The average number of seizures per day in group 1 (1:1:1) was 2.81±2.1, and in group 2 (2:1:1) it was 2.54±1.63. Group 3 (3:2:1) showed the best seizure control, with an average of 1.56±1.8 seizures per day, and no significant adverse reactions were observed. In group 4 (4:2:1), the average number of seizures per day was 1.63±1.9, with two mice showing reduced activity and one mouse showing lethargy. In group 5 (5:3:1), the average number of seizures per day was 1.91±3.2, with one mouse dying and two mice showing varying degrees of lethargy during the treatment period.

[0073] The above results indicate that all dosage ratios of the combined medication (1:1:1 to 5:3:1) significantly reduced the number of seizures in mice. The dosage ratio of tiagabe:vitamin B6:ceftriaxone sodium = 3:2:1 showed the best efficacy and safety, suggesting that this ratio has a better synergistic anti-epileptic effect.

[0074] Example 4

[0075] Animal model validation of the combined drug use provided in Example 2.

[0076] This embodiment uses TSC2. GFAP CKO mice (specifically, the Tsc2 gene is knocked out in astrocytes; this model exhibits stable spontaneous seizures for approximately 3 weeks, with a Racine grade ≥3, and is an internationally recognized classic model of TSC-related epilepsy); wild-type C57BL / 6J mice were used as normal controls. All mice were housed in an SPF-grade animal facility at 22±2℃ and 50±5% humidity, with a 12-hour light / dark cycle and free access to food and water, in accordance with the "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T 35892-2018).

[0077] 3-week-old TSC2 GFAP CKO mice were randomly divided into 5 groups, with wild-type mice serving as the normal control group. Each group consisted of 6 mice (half male and half female, n=6), as follows:

[0078] (1) Control group: wild-type C57BL / 6J mice + physiological saline;

[0079] (2) Model group: TSC2 GFAP CKO mice + physiological saline;

[0080] (3) SLC6A1 inhibitor group: TSC2 GFAP CKO mice + tiagabin;

[0081] (4) GAD1 functional enhancer group: TSC2 GFAP CKO mice + Vitamin B6;

[0082] (5) SLC1A2 activator group: TSC2 GFAP CKO mice + ceftriaxone sodium;

[0083] (6) Combination therapy group: TSC2 GFAP CKO mice + compound preparation (tiagabin + vitamin B6 + ceftriaxone sodium, mixed in a dosage ratio of 3:2:1).

[0084] Dosage regimen: All groups received intraperitoneal injection starting at 3 weeks of age (the age of seizure onset), with a dosage of 5 mg / kg and a volume of 10 μL / g, once daily for 2 weeks, at a fixed time of 8:00-9:00 AM daily. The dose for each single-drug group (tiagabe, vitamin B6, or ceftriaxone sodium) was 5 mg / kg. In the combination therapy groups, the total dose of the compound preparation remained 5 mg / kg, but based on a 3:2:1 dose ratio, the actual doses of tiagabe, vitamin B6, and ceftriaxone sodium in the compound were 2.5 mg / kg, 1.67 mg / kg, and 0.83 mg / kg, respectively.

[0085] Seizure monitoring: The Videomex-V behavioral analysis system (Columbus Instruments) was used for continuous monitoring for 12 hours daily (9:00-21:00) to record the frequency of seizures (number of seizures per day). The seizure determination criteria were based on the Racine classification (≥3 grade was defined as an effective seizure: forelimb clonus with orthostatic-generalized tonic-clonic-fall). At the end of the second week of drug administration, cortical electrodes (Pinnacle Technology) were implanted through a burr hole in the skull to record the electroencephalogram (EEG) of mice in the awake state at a sampling frequency of 1 kHz. The number of epileptiform discharges (number of spikes / sharp waves per hour) and the discharge amplitude (peak voltage) were analyzed.

[0086] Tissue sample collection: After EEG monitoring, mice were anesthetized intraperitoneally with 10% chloral hydrate (300 mg / kg), and some mice were fixed by cardiac perfusion with 4% paraformaldehyde (PFA). Cerebral cortex tissue was taken to prepare paraffin sections (5 μm thick).

[0087] Detection indicators and methods:

[0088] (1) Histopathological examination (H&E staining):

[0089] After dewaxing and hydration, paraffin sections were stained with hematoxylin for 5 minutes and eosin for 3 minutes, followed by gradient dehydration, clearing, and mounting. The proliferation of atypical neurons, balloon cells, and astrocytes in the cortical tissue was observed under an optical microscope (Olympus BX53), and cell density was calculated using ImagePro Plus software.

[0090] (2) Immunofluorescence detection:

[0091] 2-1: Dewaxing and hydration: The sections were dewaxed by washing with xylene three times (10 minutes each time), then hydrated by incubating with gradient concentrations of ethanol (100%, 95%, 80%, 70%) for 5 minutes each, and finally washed twice with phosphate-buffered saline (PBS) (5 minutes each time).

[0092] 2-2: Antigen retrieval: Immerse the slides in citrate buffer (Sigma-Aldrich) at pH 6.0 and incubate at 95°C for 30 minutes for antigen retrieval. After naturally cooling to room temperature, wash twice with PBS (5 minutes each time).

[0093] 2-3: Blocking and permeabilization: The sections were placed in PBS blocking and permeabilization solution containing 10% normal donkey serum (NDS) and 0.3% Triton X-100 and incubated at room temperature for 1 hour to block non-specific binding and enhance cell membrane permeability.

[0094] 2-4: Primary antibody incubation: Discard the blocking solution, add the specific primary antibody diluted with the above blocking permeation solution, and incubate overnight in a humidified chamber at 4°C; the primary antibodies used and the dilution ratios are as follows: NeuroTrace (Thermo Fisher Scientific, catalog number N21483, 1:300), GFAP (Cell Signaling Technology, catalog number 3670S, 1:200).

[0095] 2-5: Secondary antibody incubation: The next day, take out the slides, wash them 3 times with PBS (5 minutes each time), add species-matched AlexaFluor conjugated secondary antibody (Life Technologies, 1:500), and incubate at room temperature in the dark for 1 hour.

[0096] 2-6: Nuclear staining: After the secondary antibody incubation, wash three times with PBS (5 minutes each time), add 1:1000 diluted DAPI staining solution (Sigma-Aldrich), incubate at room temperature in the dark for 10 minutes to label the cell nuclei, and then wash once quickly with PBS.

[0097] 2-7: Imaging and Analysis: After the slides were mounted with anti-fluorescence quenching mounting medium, fluorescence images were acquired using the LAS X Navigator system, and image processing and quantitative analysis were performed using ImageJ software (Fiji version).

[0098] (3) Safety assessment:

[0099] 3-1: General condition observation: Record the weight, diet and water intake of mice daily, and observe for adverse reactions such as lethargy, ataxia, and respiratory depression.

[0100] 3-2: Organ pathological examination: After the administration of the drug, heart, liver and kidney tissues were taken, paraffin sections were prepared and H&E stained, and the tissue morphology was observed to see if there were any abnormalities.

[0101] 3-3: Blood biochemistry test: Blood was drawn from the orbital cavity, serum was separated, and ALT, AST, BUN, Cr and other indicators were tested (fully automated biochemical analyzer, Hitachi 7600). Reference range: ALT 20-60U / L, AST 40-100U / L, BUN 3.5-8.0mmol / L, Cr 30-80μmol / L.

[0102] Statistical analysis: All data were analyzed using GraphPad Prism 10.1.2 software. Quantitative data were expressed as mean ± standard deviation (x ± s). One-way ANOVA with Tukey multiple comparisons was used for comparisons among multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly significant.

[0103] Experimental results:

[0104] Effect on improvement of epileptic seizures: The combination therapy group showed the most significant effect in improving epileptic seizures. Figure 4 Regarding seizure frequency, during the 2-week intervention period, the model group mice experienced an average of 3.8 seizures per day, while the combination therapy group mice experienced only 1.43 seizures per day, a reduction of 62.4% compared to the model group (P<0.001). The SLC6A1 inhibitor group mice experienced an average of 2.19 seizures per day, a reduction of 42.4% compared to the model group (P<0.05); the GAD1 enhancer group mice experienced an average of 2.78 seizures per day, a reduction of 26.8% compared to the model group (P<0.05); and the SLC1A2 activator group mice experienced an average of 2.82 seizures per day, a reduction of 25.8% compared to the model group (P=0.083, no statistically significant difference). Therefore, although both single-drug groups reduced seizure frequency compared to the model group, the combination therapy group showed the most significant reduction. The statistics of the average number of seizures per day for each drug group are shown below. Figure 4 .

[0105] (2) Safety assessment results: No significant weight loss or abnormal immune function was observed in the mice in the combined drug group, while 33.3% of the mice in the SLC6A1 inhibitor group experienced mild drowsiness (without serious adverse reactions such as respiratory depression or ataxia), confirming that the combined drug group had better safety. Organ pathology and blood biochemistry results showed that the heart, liver, and kidney tissues in the combined drug group had no abnormalities in morphology, and ALT, AST, BUN, Cr, and other indicators were all within the normal reference range, with no peripheral organ damage observed.

[0106] H&E staining shows TSC2 GFAP The cortex of the CKO model group contained a large number of atypical neurons and balloon-like cells (see...). Figure 5 A), Immunofluorescence shows TSC2 GFAP CKO mice had increased astrocyte density compared to the normal control group (see...). Figure 5 B), compared with snRNA-seq results ( Figure 2 The conclusion regarding the increase in astrocytes is consistent with this.

[0107] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0108] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for targeted treatment of tuberous sclerosis-related epilepsy, characterized in that, The pharmaceutical composition comprises an SLC6A1 inhibitor, a GAD1 function enhancer, and an SLC1A2 activator; The SLC6A1 inhibitor is tiagabin; The GAD1 functional enhancer is vitamin B6; The SLC1A2 activator is ceftriaxone sodium; The dosage ratio of the SLC6A1 inhibitor, GAD1 enhancer, and SLC1A2 activator is 3:2:1, 4:2:1, or 5:3:

1.

2. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition includes other pharmaceutically acceptable excipients.

3. The application of a drug combination in the preparation of a drug for targeted treatment of tuberous sclerosis-related epilepsy, characterized in that, The drug combination is a combination of an SLC6A1 inhibitor, a GAD1 enhancer, and an SLC1A2 activator; the SLC6A1 inhibitor is tiagabe, the GAD1 enhancer is vitamin B6, and the SLC1A2 activator is ceftriaxone sodium; the dose ratio of the SLC6A1 inhibitor, GAD1 enhancer, and SLC1A2 activator is 3:2:1, 4:2:1, or 5:3:1.

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