Application of Compound C108 in the preparation of drugs for treating autism spectrum disorders

Abstract

This invention discloses the application of Compound C108 in the preparation of drugs for treating autism spectrum disorder, belonging to the biomedical field. Experimental studies have demonstrated that SGs (sigma cells) are key factors involved in the formation of autism caused by genetic mutations and environmental factors. Compound C108 can inhibit SG levels by intervening in G3BP2 protein expression, ultimately alleviating autistic social behaviors. Therefore, Compound C108 can serve as a novel compound for preparing drugs to treat autistic social disorders.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Compound C108 in the preparation of drugs for treating autism spectrum disorder. Background Technology

[0002] Autism spectrum disorder (ASD) is a neurodevelopmental mental disorder characterized by social impairment and repetitive, stereotyped behaviors, often accompanied by language and communication difficulties and cognitive deficits. The number of people with ASD is increasing annually, placing a significant burden on families and society. However, ASD has an insidious onset, its pathogenesis is not fully understood, and current treatments, primarily focused on rehabilitation training, have extremely limited effectiveness. Therefore, researching effective new methods for treating ASD has significant scientific and clinical value.

[0003] According to relevant literature, stress granules (SGs) are involved in the formation of various neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, but there are no reports of SGs being associated with ASD.

[0004] Compound C108, molecular formula C 15 H 14 N2O3, with a molecular weight of 270.28, is a white or off-white powder that is readily soluble in DMSO. It can significantly reduce the overexpression of G3BP2 (a key factor in the formation of SGs) and has been used to inhibit the metastasis of esophageal squamous cell carcinoma (ESCC) cells. Currently, there are no reports on the application of Compound C108 in alleviating social impairments in autistic individuals. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide the application of Compound C108 in the preparation of drugs for treating autism spectrum disorder.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] The first aspect of the present invention discloses the use of Compound C108 in the preparation of a drug for treating autism spectrum disorder.

[0008] Preferably, Compound C108 is a drug that reduces SG content.

[0009] Preferably, Compound C108 is a drug that inhibits G3BP2 expression.

[0010] Preferably, Compound C108 is a drug that alleviates social impairment by inhibiting G3BP2 and regulating the content of SGs.

[0011] Preferably, Compound C108 is a drug that can increase social frequency.

[0012] Preferably, Compound C108 is a drug that can increase social time.

[0013] Preferably, the drug contains one or more pharmaceutically acceptable carriers or excipients.

[0014] Preferably, the dosage form of the drug is powder, tablet, capsule or injection.

[0015] Preferably, the Compound C108 is administered at a concentration of 10 μM, with a unilateral dose of 500 nL, and bilaterally administered to the ACC brain regions.

[0016] A second aspect of the invention discloses a composition for treating autism spectrum disorder, comprising Compound C108 and other medicaments for treating social impairments in autism.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The application of Compound C108 provided by this invention in the preparation of drugs for treating autism spectrum disorder, through... Shank3 Mutations (genetic factors) and prenatal VPA exposure (environmental factors) significantly increased SG content in relevant brain regions and primary neurons of ASD mice. Compound C108 can reduce SG content by inhibiting G3BP2 (a key factor in SG formation), thereby alleviating autism symptoms in mice and improving the social abilities of autistic mice, which strongly supports the application of Compound C108 in the preparation of drugs for treating autism and social impairment. Attached Figure Description

[0019] Figure 1 The image shows the results of SGs content measurement in neurons of ASD mice; where A represents... Shank3 Mouse immunofluorescence staining results (upward) and Western blotting results (downward), B is... VPA Mouse immunofluorescence staining results (upward) and Western-Blotting results (downward), n=15 (neurons from 3 mice), Bar=4 μm;

[0020] Figure 2Figure 1 shows the experimental results of Compound C108 intervention in G3BP2 to regulate SGs; where A is a schematic diagram of the localized and uniform injection of Compound C108 into the bilateral ACC brain regions of mice, B is the Western-Blotting results (left) and the statistical results (right), and C is the immunofluorescence staining results.

[0021] Figure 3 The diagram shows the results of the Compound C108 study on behavioral representation of autism. In the diagram, A represents the results of the three-box experiment, with the trajectory of social preference, the interval time, and the social preference score from left to right. B represents the results of the exploration time of the social novelty mouse (S2), with the trajectory of social preference, the interval time, and the social score of the novelty mouse from left to right. C represents the results of the inhabit-invasion experiment, with the social time on the left and the contact frequency on the right. D represents the duration of social contact per unit time. Detailed Implementation

[0022] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0023] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0024] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0025] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0026] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0027] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0028] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0029] 1. Determine the content of SGs in relevant brain regions of two ASD mouse models and normal mice:

[0030] 1) Select commonly used genetic factors that lead to ASD Shank3 Mice, and environmental factors leading to ASD during gestation VPA The mouse model was used as an ASD mouse model (6-week-old young male mice). [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Shank3 mouse model, VPA Key ASD anterior cingulate cortex (ACC) brain tissue or neurons from primary cultured fetal rat cortex for 7 days were isolated from mouse models and normal mice (C57BL6J mice).

[0031] 2) Take 2×10 6 The protein components of SGs from primary neurons or 0.2 g mouse ACC tissue were purified in an RNase-free environment, and the mRNA components of SGs were then purified by Trizol extraction.

[0032] 3) Immunofluorescence was used to detect the co-labeling of the SGs marker protein TIA-1 with different cellular markers in the ACC; the expression of TIA-1 / Tuj1 in primary neurons was detected to reflect the distribution of SGs, and the data were analyzed using Imgae J software to analyze the changes in the number and area of ​​SGs.

[0033] Shank3 and VPA Results of SGs content measurement in neurons of two types of ASD mice are as follows: Figure 1 As shown, Figure 1 China A Shank3 Mouse immunofluorescence staining results showed Shank3 Mutation (genetic factor) induced the formation of typical SGs in primary neurons of ASD mice (arrow), and Western blotting results showed that G3BP2 was present in... Shank3 The mutant mice showed a significant increase in cortical levels. Figure 1 B VPA Immunofluorescence staining in mice showed typical SGs formation in primary neurons of ASD mice induced by VPA exposure during pregnancy (an environmental factor) (arrows), and Western blotting results showed a significant increase in G3BP2 in the cortex of VPA-exposed mice. These results indicate an abnormal increase in the number and area of ​​SGs in neurons of both ASD mouse species.

[0034] 2. Compound C108 regulates the formation of SGs:

[0035] 1) such as Figure 2 As shown in Figure A, two types of ASD mice ( Shank3 Mice, VPA In a mouse model, Compound C108 reagent (10 μM, 500 nL, administered for 3 consecutive days) or placebo (physiological saline) was injected into the bilateral ACC brain regions.

[0036] 2) Three weeks later, the protein expression level of G3BP2, a key factor in SG formation, and the SG content were detected using SG extraction technology and Western blot. The specific steps of the SG extraction technology were as follows: 2 × 10⁻⁶ oz. 6 Primary neurons or 0.2g of mouse ACC tissue were used to extract SGs components under RNase-free conditions using SGs lysis buffer (50 mM Tris-HCl, 100 mM KOAc, 2 mM MgOAc, and 0.5 mM DTT). 1 mL of SGs lysis buffer was added to each sample, and the tissue was physically ground and digested. The samples were then incubated on ice for 15 min, followed by centrifugation at 2000×g for 5 min at 4°C. 50 μL of the supernatant was used for total protein or total RNA extraction. 950 μL of the supernatant was centrifuged at 18000×g for 20 min at 4°C, repeated twice to obtain a precipitate. The precipitate was resuspended in 200–300 μL of SGs lysis buffer and centrifuged at 850×g for 2 min at 4°C. This supernatant is the protein component of the SGs and is used for subsequent RNA extraction or protein experiments.

[0037] 3) Immunofluorescence technique was used to detect the formation of SGs in cortical neurons.

[0038] Experimental results of Compound C108 regulating SGs through intervention of G3BP2 are as follows: Figure 2 As shown, Western blotting results revealed a significant decrease in the expression of G3BP2, a key factor in SG formation, indicating that Compound C108 has an inhibitory effect on G3BP2 expression. Figure 2(B) Immunofluorescence staining results showed a decrease in SGs content in the ACC brain region, indicating that Compound C108 has an inhibitory effect on SGs formation. Figure 2 (C)

[0039] 3. Testing the social behavior of ASD mice after injection of Compound C108 reagent:

[0040] 1) To Shank3 Mice were injected bilaterally into the ACC brain region with Compound C108 reagent (10 μM, 500 nL, for 3 consecutive days) or placebo (physiological saline).

[0041] 2) The three-box test and the dwell-invasion test in animal behavioral techniques were used to observe changes in social behavior and other ASD-like behaviors (such as repetitive stereotypes, anxiety, etc.) in mice.

[0042] Compound C108 alleviates autism behavioral characteristics as shown in the following results Figure 3 As shown, in the three-box experiment, ASD mice injected with Compound C108 spent significantly more time exploring socially-biased mice (S1). Figure 3 The exploration time of the social novelty mouse (S2) was also significantly increased (in the middle A). Figure 3 (B). In the inhabitation-invasion experiment, ASD mice injected with Compound C108 showed significantly increased contact time and frequency with same-sex stranger mice. Figure 3 (C and D in the middle).

[0043] The above results indicate that Compound C108 can intervene in G3BP2 expression, thereby inhibiting SG formation and ultimately effectively alleviating social impairment in ASD mice.

[0044] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. Application of compound C108 in the preparation of drugs for treating autism spectrum disorder.

2. The use of compound C108 according to claim 1 in the preparation of drugs for treating autism spectrum disorder, characterized in that, Compound C108 is a drug that alleviates social anxiety by inhibiting G3BP2 and regulating the content of SGs.

3. The use of compound C108 according to claim 1 in the preparation of drugs for treating autism spectrum disorder, characterized in that, Compound C108 is a drug that can increase social frequency.

4. The use of compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorder, characterized in that, Compound C108 is a drug that can increase social time.

5. The use of compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorder, characterized in that, The drug contains one or more pharmaceutically acceptable carriers.

6. The use of compound C108 according to claim 1 in the preparation of a drug for treating autism spectrum disorder, characterized in that, The dosage form of the drug is powder, tablet, capsule or injection.

7. The use of compound C108 according to any one of claims 1 to 6 in the preparation of a drug for treating autism spectrum disorder, characterized in that, The dosage of compound C108 was 10 μM, the dose for unilateral administration was 500 nL, and the administration was performed bilaterally in the ACC brain region.

Images