Application of CCR2 antagonist in preparation of medicine for treating and / or improving epilepsy

Abstract

The invention discloses an application of a CCR2 antagonist in preparation of a medicine for treating and / or improving epilepsy. The key effect of CCR2 specific antagonism in treating GABRG2 mutation related epilepsy is disclosed for the first time, and it is clear that improvement of neuroinflammation by inhibiting CCL2 / CCR2 axis is an effective new strategy for treating the type of epilepsy; the selective CCR2 antagonist INCB3344 has the advantages that the effect of the selective CCR2 antagonist INCB3344 in the aspects of reducing the epileptic seizure grade and reducing the motion activity is obviously superior to that of a CCR2 / 5 dual antagonist Cenicriviroc (CVC), and clear directional selection is provided for clinical medication; the INCB3344 gene has the advantages of high transformation value, effectiveness of INCB3344 is verified in hereditary epilepsy models of two different species of zebra fish and mouse, epileptic seizure score and frequency are obviously reduced, and curative effect universality and high clinical transformation potential of the INCB3344 gene are proved.

Description

Technical Field

[0001] This invention relates to the application of a CCR2 antagonist in the preparation of drugs for treating and / or improving epilepsy, and belongs to the field of biomedicine. Background Technology

[0002] Epilepsy is one of the most common chronic neurological disorders in childhood, characterized by recurrent, unprovoked seizures (intervals of more than 24 hours), the unpredictability of seizures, and the long-term nature of medication. Neuroinflammation and abnormal neuronal discharge are important pathological mechanisms of epilepsy. Epilepsy is classified into primary and secondary types based on etiology, with primary epilepsy often related to gene defects. The GABRG2 gene is located on chromosome 5q34 and is a GABAergic gene. A GABRG2 is a member of the receptor heteropentamer ligand-gated ion channel gene family. GABRG2 mutations are commonly associated with autosomal dominant epilepsy. Missense mutations in GABRG2 (P282S, I107T, and F343L) have been found in clinically affected children, most of whom develop seizures in childhood. Zebrafish carrying wild-type GABRG2 (WT), mutant GABRG2 (P282S), GABRG2 (I107T), and GABRG2 (F343L) have been constructed using the Tol2kit transgenic system and the Gateway method. These zebrafish models exhibit spontaneous seizures and convulsive behavior in the juvenile stage.

[0003] However, current research on the pathogenic mechanisms of GABRG2 mutations largely focuses on electrophysiological dysfunction, neglecting the synergistic effect of neuroinflammation. Therefore, clarifying the relevant targets of neuroinflammation and finding suitable inhibitors is crucial. Summary of the Invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of a CCR2 antagonist in the preparation of drugs for treating and / or improving epilepsy.

[0005] Technical solution: To solve the above-mentioned technical problems, the present invention provides the application of a CCR2 antagonist in the preparation of drugs for treating and / or improving epilepsy.

[0006] The CCR2 antagonist is INCB3344.

[0007] The epilepsy mentioned here refers to primary epilepsy.

[0008] The epilepsy mentioned is GABRG2 mutation-related epilepsy.

[0009] One method is to treat and / or improve epilepsy by reducing neuroinflammation.

[0010] The application also provides a drug or a pharmaceutical composition for treating and / or improving epilepsy, which contains a CCR2 antagonist.

[0011] Beneficial effects: Compared with the prior art, the application has the following remarkable advantages:

[0012] 1. Target precision: The application first discloses the key role of CCR2 specific antagonism in treating GABRG2 mutation related epilepsy, and clearly defines that improving neural inflammation by inhibiting the CCL2 / CCR2 axis is an effective new strategy for treating this type of epilepsy.

[0013] 2. Significant therapeutic effect: The selective CCR2 antagonist INCB3344 is significantly better than the CCR2 / 5 dual antagonist Cenicriviroc (CVC) in reducing seizure grade and reducing motor activity, providing a clear directional choice for clinical medication.

[0014] 3. Strong model applicability: High conversion value, the effectiveness of INCB3344 is verified in genetic epilepsy models of two different species of zebrafish and mice, significantly reducing seizure score and frequency, proving the universality of its efficacy and high clinical conversion potential. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 For Gabrg2 + / A105T Phenotypic characterization of the knock-in mouse model of Gabrg2 A (A) Structural modeling of the human full-length heteromeric α1β3γ2L GABA A R) complex, left panel: interaction analysis of γ2A106 with residues within the γ2 subunit (γ2L192 and γ2P327), middle panel: structural model of the wild-type (WT) GABA A R complex, right panel: predicted structural changes of the γ2T106 variant, showing a weakened interaction between γ2T106 and γ2P327 near the transmembrane domain; (B) Genotyping results of Gabrg2 + / A105T mice, showing a mutation from "G" to "A"; (C) Representative gel image of genotyping PCR products, heterozygous Gabrg2 + / A105T mice show two bands (309 bp and 190 bp), while WT mice show a single band (190 bp); (D) Percentage of postnatal WT and heterozygous Gabrg2 + / A105T mice; (E) Morphology of Gabrg2 + / A105T mice at postnatal day 7 (P7) compared to WT mice; (F) Gabrg2 + / A105TWeight analysis of WT mice (n=9–10); (G) survival curves showed that, compared with WT littermates, Gabrg2... + / A105T Early mortality was increased in mice, with approximately 75.1% dying before one week of age. The peak mortality occurred around day 7 after birth. Data are expressed as mean ± standard deviation (mean ± SD). Two-way ANOVA and Sidak multiple comparison test were used. **P < 0.01.

[0016] Figure 2 For Gabrg2 + / A105T Transcriptome analysis of brain tissue from gene knock-in mice: (A) Volcano plot showing Gabrg2 + / A105T Differentially expressed genes (DEGs) in mouse hippocampus tissue compared to WT controls: a total of 154 differentially expressed genes were identified (corrected P value < 0.05), of which 112 genes were upregulated and 42 genes were downregulated; (B) Heatmap of the 154 differentially expressed genes, showing Gabrg2 + / A105T Significant differences in gene expression patterns between the mice and WT mice; (C) GO functional analysis of differentially expressed genes showed that upregulated and downregulated genes were significantly enriched in cellular processes, developmental processes, and immune system processes.

[0017] Figure 3 The KEGG pathway analysis was conducted for differentially expressed genes, with key pathways including signal transduction, the immune system, development and regeneration, and nervous system function.

[0018] Figure 4 For Gabrg2 + / A105T Transcriptome analysis of brain tissue from gene knock-in mice: (A) Gabrg2 + / A105T The top 20 most significantly upregulated genes in mouse hippocampus; (B) Gabrg2 + / A105T The top 20 most significantly downregulated genes in mouse hippocampus; (C) qRT-PCR validation of immune-related genes in hippocampus (n=3 per group). Data are expressed as mean ± standard deviation. Two-way ANOVA and Sidak multiple comparison test were used. **P<0.01 indicates a significant difference compared with the WT group.

[0019] Figure 5 For Gabrg2 + / A105T Changes in brain tissue inflammation and microglia activation in gene knock-in mice: (AB)Gabrg2 + / A105TThe expression levels of inflammatory factors Ccl2 and Ccl5 in the hippocampus of WT mice on day 1 (P1) (n=6 per group) and day 7 (P7) (n=3 per group) after birth; (CD) Expression levels of inflammatory factors IL-1β, IL-6 and TNF-α in the hippocampus (n=3 per group); (E) Immunofluorescence staining of the CA3 region of the hippocampus in P7 and P28 mice, with neurons labeled with NeuN (red), microglia labeled with Iba1 (green), and cell nuclei stained with DAPI (blue); scale bar = 50 μm; (F) Quantitative analysis of NeuN-positive cells in the hippocampus at P7 (n=3 per group); Data are expressed as mean ± standard deviation, and two-way ANOVA and Sidak multiple comparison test were used. *P<0.05, **P<0.01 indicated significant differences compared with the WT group.

[0020] Figure 6 For Gabrg2 + / - Expression of inflammatory factors in the brain tissue of gene knockout mice: Gabrg2 + / - The expression levels of inflammatory factors Ccl2(A), Ccl5(B), TNF-α(C), IL-1β(D), and IL-6(E) in the hippocampus of gene knockout mice were measured (n=3 per group). Data are expressed as mean ± standard deviation. Two-way ANOVA and Sidak multiple comparison test were used. *P<0.05 and **P<0.01 indicate significant differences compared with the WT group.

[0021] Figure 7 A comparison of the effects of the CCR2 antagonist INCB3344 and the CCR2 / 5 dual antagonist Centicriviroc on improving kinesiology in epileptic zebrafish: Tg(hGABRG2) mutant GABRG2 transgenic zebrafish were cultured at 5 days postnatal time (5 dpf). F343L ), Tg(hGABRG2) P282S ) and Tg(hGABRG2 I107T (A) Expression levels of Ccl2 (B) and Ccl5 (C) in brain tissue (n=5 per group); (C) At 5 dpf, INCB3344 and Centicriviroc inhibited the expression of Tg(hGABRG2) I107T (D) Quantitative analysis of total distance traveled in each group (n=22 per group); Data are expressed as mean ± standard deviation. One-way ANOVA and Tukey's multiple comparison test were used. *P<0.05, **P<0.01 indicate a significant difference compared with the I107T group without medication; ## indicates a significant difference compared with the WT group.

[0022] Figure 8 INCB3344, a CCR2 antagonist, is effective against Gabrg2. + / A10T5 and Gabrg2+ / - Comparison of therapeutic effects in gene knockout mice: (A) Intraperitoneal injection of INCB3344 (10 mg / kg) on ​​Gabrg2 + / A10T5 and Gabrg2 + / - Effects of gene knockout mice on epilepsy scores (n=10 per group); (B) INCB3344 on Gabrg2 + / A10T5 and Gabrg2 + / - The effect of gene knockout mice on the number of epileptic seizures (n=4 per group); data are expressed as mean ± standard deviation, and one-way ANOVA and Tukey's multiple comparison test were used. **P<0.01 indicates a significant difference compared with the WT group. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0024] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0025] The experimental animals used in the following examples were zebrafish carrying wild-type (WT) GABRG2, mutant GABRG2 (P282S), GABRG2 (F343L), and GABRG2 (I107T) previously constructed in the laboratory (Differential inflammation responses determine the variable phenotypes of epilepsy induced by GABRG2 mutations. CNS Neurosci Ther, 2024, 30(2):e14583.), and healthy wild-type zebrafish (AB) provided by Nantong University. The transgenic zebrafish hearts expressed green fluorescence, which could be used for subsequent screening. Zebrafish embryos were collected, and zebrafish embryos with positive green fluorescence in the heart were screened. The zebrafish embryos were cultured in E3 medium at 28°C. The GABRG2 used... + / A105T The gene knock-in mouse (catalog number TIS191206JN1) was provided by Cyagen Biosciences Co., Ltd., and was constructed using C57BL / 6J mice via ES gene targeting technology; it is SPF grade. Gabrg2 + / - Gene knockout mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (product number T003794).

[0026] Example 1: Effect of GABRG2 (A105T) mutation on receptor structural stability

[0027] To elucidate the structural effects of the GABRG2(A105T) mutation, a study was conducted based on the human full-length heteropentamer α1β3γ2LGABA. A Receptor structural modeling ( Figure 1 A) Utilizing human pentameric GABA A High-resolution cryo-electron microscopy (PDB: 6HUO 16) of the receptor, combined with calculations using DynaMut 20 software, showed that the ΔΔG stability of GABRG2(T106) was -1.2 kcal / mol, indicating that the A→T mutation reduces the overall stability of the receptor complex. Further structural analysis revealed that GABRG2(T106) interacts only with residues (L192 and P327) within the γ2 subunit, while the interaction between T106 and P327 is weakened. Figure 1 A) suggests that this mutation affects receptor membrane localization and assembly by disrupting the stability of the transmembrane region of the γ2 subunit.

[0028] Example 2 Gabrg2 + / A105T Construction and phenotypic analysis of gene knock-in mouse models

[0029] By introducing a targeting vector containing a Gabrg2 genomic homologous sequence and a p.(A105T) mutation into embryonic stem cells using homologous recombination technology, Gabrg2 was obtained. + / A105T Heterozygous knock-in mice ( Figure 1 B). Sequencing confirmed the successful introduction of the c.G313A (p.A105T) mutation. Figure 1 B). The genotype ratio of the offspring conforms to Mendelian inheritance laws. Figure 1 CD). Compared with wild-type (WT) mice, Gabrg2 + / A105T Mice showed no difference in birth weight, but experienced stunted growth after birth and a significant decrease in weight in adulthood. Figure 1 EG). Anatomical analysis showed that the brain tissue morphology of 10-12 week old mutant mice was not significantly different from that of WT mice, and no obvious pathological changes were observed in organs such as heart, liver, spleen, lungs, and kidneys.

[0030] Example 3: Effects of GABRG2 (A105T) mutation on hippocampal gene expression profile

[0031] RNA-seq analysis of hippocampal tissue from 2-month-old mice revealed Gabrg2. + / A105T The group contained 154 differentially expressed genes (DEGs, 112 upregulated and 42 downregulated). Figure 2 AB). GO analysis showed that upregulated genes were enriched in cellular processes (100), developmental processes (57), and the immune system (52); KEGG pathway analysis indicated that DEGs were involved in signal transduction (37), the immune system (35), and nervous system function (5). Figure 2C and Figure 3 The 20 genes most significantly upregulated and the 20 genes most significantly downregulated were shown in [the diagram]. Figure 4 A and Figure 4 B.

[0032] qRT-PCR (primer sequences are shown in Table 1; reaction system includes: 2×Mix 5μL, Primer F / Primer R 0.5μL / 0.5μL, H2O 3μL, cDNA 1μL; reaction procedure is shown in Table 2) was used for verification. The results showed that the expression of immune-related genes (such as Ccl5, Ccl2, and Clec4d) in hippocampal tissue was significantly increased. Figure 4 C) indicates that the mutation affects neuronal development and the occurrence of epilepsy through immune dysregulation.

[0033] Table 1 qRT-PCR primer sequences

[0034]

[0035]

[0036] Table 2 qPCR Procedure

[0037]

[0038] Example 4 Gabrg2 + / A105T Mutant mice and Gabrg2 + / - Hippocampal neuroinflammatory characteristics in gene knockout mice

[0039] Gabrg2 without epileptic symptoms + / A105T In newborn mice (P1, P7), qRT-PCR results showed that the expression of Ccl2 and Ccl5 in the mouse hippocampus was significantly increased. Figure 5 AB), while IL-1β and IL-6 only increased at P7, and TNF-α decreased at P1 (AB). Figure 5 CD). Immunofluorescence showed a significant increase in the density of microglia (Iba1 positive) in the CA3 region of P7 and P28 mutant mice. Figure 5 E), while neurons (NeuN positive) were significantly reduced ( Figure 5 F) suggests that the mutation directly drives neuroinflammation and promotes epilepsy through microglial cell activation.

[0040] In Gabrg2 + / - In (KO) newborn mice (P1), qRT-PCR results showed that only Ccl2 expression was slightly increased in the mouse hippocampus, but Ccl5 expression was not significantly increased. Figure 6 AB), TNF-α increased, while IL-1β and IL-6 showed no significant changes. Figure 6 CE).

[0041] Example 5: Comparison of the effects of CCR2 antagonist INCB3344 and CCR2 / 5 dual antagonist Centicriviroc (CVC) on improving motility in epileptic zebrafish.

[0042] Three types of mutant GABRG2 transgenic zebrafish Tg(hGABRG2) were cultured at 5 days after birth (5 dpf). F343L ), Tg(hGABRG2) P282S ) and Tg(hGABRG2 I107T In the brain tissue of all samples, ccl2 and its receptor ccr2 were expressed, but compared with the wild-type control group, ccl2 was only expressed in Tg(hGABRG2). I107T The expression level was significantly increased in zebrafish brain tissue. Figure 7 (AB). At 5 dpf, zebrafish were incubated for 30 min in E3 medium containing 50 μmol / L INCB3344 (5 mM NaCl, 0.33 mM CaCl2, 0.17 mM KCl, 0.33 mM MgSO4) or E3 medium containing 50 μmol / L CCR2 / 5 dual antagonist Centicriviroc (CVC). Basal swimming behavior was then measured for 30 min using a Danio Vision instrument. Behavioral data and trajectory maps were derived, and the swimming distance was statistically analyzed. Treatment with the CCR2 antagonist INCB3344 significantly reduced the seizure severity of mutant zebrafish (the proportions of stage II and III seizures decreased to 12.2% and 7.3%, and 10.4% and 5.6%, respectively), and reduced the swimming distance. Figure 7 The results (CD) indicate that INCB3344 treatment significantly reduced the swimming activity of transgenic zebrafish. Although treatment with the CCR2 / 5 dual antagonist CVC also reduced the swimming activity of transgenic zebrafish, the reduction in swimming distance was far less than that of INCB3344, indicating that INCB3344 was significantly more effective than CVC.

[0043] Example 6: CCR2 antagonist INCB3344 against Gabrg2 + / A10T5 and Gabrg2 + / - Comparison of therapeutic effects in gene knockout mice

[0044] According to the Racine scale (Grade 0: no response; Grade I: facial clonic seizures, including blinking, whisker twitching, rhythmic chewing, etc.; Grade II: Grade I plus rhythmic head nodding; Grade III: Grade II plus forelimb myoclonus, but no hindlimb upright position; Grade IV: Grade III plus hindlimb upright position; Grade V: generalized tonic-clonic seizures with loss of postural control), the epilepsy scores of 2-month-old mice were recorded. The results showed that most Gabrg2 mice... + / A10T5Point mutant mice exhibited forelimb spasticity and other symptoms, with epilepsy scores mostly ranging from Stage 2 to 3, displaying a human-like epilepsy phenotype. Gabrg2... + / - This phenomenon is rare in gene knockout mice; they only exhibit blinking, mild twitching, and rhythmic chewing, with scores mostly falling within Stage 1. Figure 8 A). Intraperitoneal injection of INCB3344 (10 mg / kg) significantly reduced Gabrg2 levels. + / A10T5 Epilepsy scores in point mutant mice, suppression of seizure frequency, but not Gabrg2 + / - Gene knockout mice had no significant effect. Figure 8 AB).

Claims

1. The use of a CCR2 antagonist in the preparation of drugs for treating and / or improving epilepsy.

2. The application according to claim 1, characterized in that, The CCR2 antagonist includes INCB3344.

3. The application according to claim 1, characterized in that, The epilepsy mentioned is primary epilepsy.

4. The application according to claim 1, characterized in that, The epilepsy in question is GABRG2 mutation-related epilepsy.

5. The application according to claim 1, characterized in that, Treating and / or improving epilepsy by reducing neuroinflammation.

6. A medicament or pharmaceutical composition for treating and / or improving epilepsy, characterized in that, It contains a CCR2 antagonist.

7. The drug or pharmaceutical composition according to claim 6, characterized in that, The CCR2 antagonist includes INCB3344.

8. The drug or pharmaceutical composition according to claim 6, characterized in that, The epilepsy mentioned is primary epilepsy.

9. The drug or drug composition according to claim 6, characterized in that, The epilepsy in question is GABRG2 mutation-related epilepsy.

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