Use of betaine in the prevention of autism spectrum disorders

Abstract

The present application provides the use of betaine in preventing the occurrence of autism spectrum disorder (ASD). Specifically, the present application provides the use of betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitroxide, solvate, metabolite or prodrug thereof in the preparation of a medicament for preventing the occurrence of ASD, optimizing fetal neural development or reducing the risk of fetal neural development disorder. By using betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitroxide, solvate, metabolite or prodrug thereof as an active ingredient, the occurrence of ASD can be effectively prevented, fetal neural development can be optimized, or the risk of fetal neural development disorder can be reduced.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the use of betaine or its pharmaceutically acceptable salts, stereoisomers, tautomers, nitrides, solvates, metabolites or prodrugs in the preparation of medicaments for the prevention of ASD, optimization of fetal neurodevelopment or reduction of the risk of fetal neurodevelopmental disorders. Background Technology

[0002] In the biomedical field, Autism Spectrum Disorder (ASD), also known as autism spectrum disorder, is widely recognized as a multifaceted neurodevelopmental disorder. Its main characteristics include impairments in social interaction, a limited range of interests, and repetitive, stereotyped behaviors. According to the latest statistics, China has a total of 85 million people with disabilities, of whom 13 million suffer from ASD, with nearly 200,000 new cases each year, making ASD the most prevalent mental disability. This situation not only places a heavy psychological and economic burden on patients' families but also poses a significant challenge to the national public health system and socioeconomic development. The exact cause of ASD is not yet fully understood, but its pathogenesis is generally believed to be related to the complex interaction of genetic and environmental factors. Although some progress has been made in the early diagnosis and behavioral treatment of ASD, these measures still have limitations in improving patients' conditions, especially in preventing the onset of ASD, where effective strategies are currently lacking. Therefore, developing methods to prevent ASD will be of great significance for reducing its incidence, alleviating the burden on society and families, and promoting the overall development of human society. Summary of the Invention

[0003] This application aims to propose a means to effectively prevent the occurrence of ASD.

[0004] This invention is based on the inventor's discovery in animal experiments that feeding betaine to female mice can prevent offspring mice from developing ASD. Furthermore, the mechanism of the intervention proposed in this application is to maintain the normal function of neurons in brain tissue. Therefore, the inventor proposes a method to prevent ASD in order to eliminate or reduce the occurrence of ASD in offspring. Specifically, the prevention method involves giving pregnant women betaine.

[0005] In view of this, in a first aspect of the present invention, the present invention provides the use of betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitride, solvate, metabolite, or prodrug thereof in the preparation of a medicament for preventing the occurrence of ASD, optimizing fetal neurodevelopment, or reducing the risk of fetal neurodevelopmental disorders. Thus, by using betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitride, solvate, metabolite, or prodrug thereof as an active ingredient, it is possible to effectively prevent the occurrence of ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders.

[0006] According to embodiments of this application, the ASD may include at least one of Autistic Disorder, Asperger's Syndrome, Childhood Disintegrative Disorder (CDD), and Pervasive Developmental Disorder NotOtherwise Specified (PDD-NOS).

[0007] According to embodiments of this application, the drug is formulated for oral administration, injection administration, transdermal administration, or as a food additive. This convenient administration method improves patient compliance, thereby increasing the efficiency of ASD prevention.

[0008] According to embodiments of this application, the drug is administered to pregnant women, women preparing for pregnancy, or newborns. Optionally, the recipients may be exposed to the risk of ASD.

[0009] In a second aspect, this application provides a pharmaceutical composition for preventing the occurrence of ASD, optimizing fetal neurodevelopment, or reducing the risk of fetal neurodevelopmental disorders. The pharmaceutical composition comprises: an active ingredient, including betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitride, solvate, metabolite, or prodrug thereof, and pharmaceutically acceptable excipients. Thus, by using betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitride, solvate, metabolite, or prodrug thereof as the active ingredient, it is possible to effectively prevent the occurrence of ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders.

[0010] According to embodiments of this application, the excipients include, but are not limited to, fillers, disintegrants, binders, lubricants, sweeteners, or flavorings.

[0011] According to embodiments of this application, the drug is formulated for oral administration, injection administration, transdermal administration, or as a food additive.

[0012] In a third aspect, this application proposes a nutritional supplement for pregnant women, characterized by comprising betaine or a pharmaceutically acceptable salt thereof, stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, or prodrugs, as well as vitamins and minerals. Thus, by using betaine or a pharmaceutically acceptable salt thereof, stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, or prodrugs as active ingredients, this nutritional supplement can effectively prevent the occurrence of ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders.

[0013] According to embodiments of this application, the nutritional supplement is used to prevent the occurrence of ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders.

[0014] In a fourth aspect, this application discloses a food composition for preventing ASD, optimizing fetal neurodevelopment, or reducing the risk of fetal neurodevelopmental disorders. The food composition comprises betaine or a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, a nitrogen oxide, a solvate, a metabolite, or a prodrug. Therefore, by using this food composition with betaine or a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, a nitrogen oxide, a solvate, a metabolite, or a prodrug as the active ingredient, it is possible to effectively prevent ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders.

[0015] According to the embodiments of this application, given the current lack of effective treatments for ASD, the social significance of using the methods described in this application to prevent ASD is multifaceted. ① Preventing ASD can significantly reduce its global incidence. Since ASD is a common neurodevelopmental disorder affecting millions of children and adults, effective prevention measures will drastically reduce its incidence, thereby eliminating the enormous social and family burden it causes. ② Preventing ASD can avoid the enormous economic burden of long-term care and nursing for individuals with ASD. ③ Preventing ASD means promoting healthy births and ensuring the healthier growth of future generations, making a positive contribution to the high-quality continuation of humanity. ④ Preventing ASD can reduce the number of individuals in society with social and communication impairments due to ASD, thereby promoting a more inclusive and understanding social environment. ⑤ Preventing ASD can reduce the enormous costs to public health systems and social services, allowing these costs to be redistributed to other important social and public health areas. ⑥ Preventing ASD can reduce the demand for special education, enabling a more equitable distribution of educational resources to all students, improving the overall quality and efficiency of education. ⑦ Preventing ASD can drive more research to better understand its etiology, development mechanisms, and potential treatments. ⑧ Preventing ASD can also deepen our understanding of the mechanisms of other neurodevelopmental disorders and possible prevention and intervention methods, playing a very positive role in further reducing the incidence of neurodevelopmental disorders. In conclusion, adopting the approach proposed in this application to prevent ASD will not only have a profound impact on patients and their families, but also have important significance for the health, economic, and cultural development of society as a whole. By preventing ASD, we can build a healthier, more inclusive, and prosperous society. Attached Figure Description

[0016] Figure 1 This illustrates a technical roadmap according to one embodiment of the present application;

[0017] Figure 2 The graphs shown are line graphs and bar graphs of the weight of offspring mice versus their eye-opening scores according to one embodiment of this application.

[0018] Figures 3-6 A schematic diagram showing the behavioral assessment results according to an embodiment of this application is displayed;

[0019] Figure 7 The results of Nissl staining according to an embodiment of this application are shown. Detailed Implementation

[0020] Definitions and general terms

[0021] Betaine is an alkaloid, chemically named N,N,N-trimethylglycine. Its chemical structure is similar to that of amino acids, belonging to the quaternary ammonium base class, with the molecular formula C5H2O. 11 NO2 has the following structure:

[0022]

[0023] Betaine is naturally found in many foods, especially in beets, spinach, whole wheat, wheat germ, shrimp, and crab. Betaine is stable under normal storage conditions and does not easily decompose. It is absorbed in the small intestine and participates in metabolic processes in the liver. As a methyl donor in methylation reactions in the body, betaine participates in various biochemical processes, including the synthesis of DNA, proteins, and certain hormones. Furthermore, betaine plays a crucial role in homocysteine ​​metabolism, helping to convert it to methionine, thereby lowering homocysteine ​​levels in the blood. Intracellularly, betaine helps maintain osmotic balance, protecting cells from damage caused by high osmotic pressure. As a dietary supplement, betaine is used to support liver health, heart health, and muscle function. In animal husbandry, betaine is used as a feed additive to help improve animal growth performance and health. Betaine is generally considered safe and well tolerated by most people. In some cases, betaine is also used as a medicine, such as in the treatment of certain types of hyperhomocysteinemia.

[0024] The term "Autism Spectrum Disorder (ASD)" as used in this article refers to a complex group of neurodevelopmental disorders that affect an individual's social interactions, communication abilities, and behavioral patterns. ASD is typically characterized by the following aspects:

[0025] Social impairment: Individuals with ASD may have difficulty understanding nonverbal cues in social interactions, establishing and maintaining interpersonal relationships, and understanding the feelings and perspectives of others.

[0026] Communication barriers: These may include delayed language development, difficulty using language, difficulty in initiating or maintaining conversations, and a lack of nonverbal communication skills.

[0027] Repetitive and stereotyped behaviors: Individuals with ASD may exhibit repetitive physical movements (such as clapping or swaying their hands), adherence to the same details of daily activities, and strong but limited attention to specific interests or activities.

[0028] Narrow interests: They may show excessive interest or focus on certain specific topics or items.

[0029] Sensory processing problems: Individuals with ASD may have unusual reactions to auditory, tactile, gustatory, olfactory, or visual stimuli.

[0030] Cognitive and learning differences: Although individuals with ASD have varying levels of intelligence, they may exhibit exceptional skills in some areas while facing challenges in others.

[0031] ASD is usually diagnosed in early childhood because early intervention can significantly improve outcomes. ASD is a lifelong condition, but its symptoms and severity can vary between individuals and at different stages of life. The exact causes of ASD are not fully understood, but research suggests that both genetic and environmental factors may be involved in its development.

[0032] According to embodiments of this application, ASD is a series of neurodevelopmental disorders with similar characteristics, and ASD may include the following:

[0033] Autistic Disorder (AD): Also known as classic autism, it is characterized by severe social impairment, communication difficulties, and repetitive and stereotyped behaviors.

[0034] Asperger's Syndrome: Individuals with Asperger's Syndrome typically have normal or above-normal intelligence and language abilities, but exhibit social impairments and stereotyped behavioral patterns.

[0035] Childhood Disintegrative Disorder (CDD): This is a rare disorder in which children exhibit normal development at least by age two, and then gradually lose previously acquired skills, including social, language, and play skills.

[0036] Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS): This term describes conditions that present with certain autistic characteristics but do not meet other specific diagnostic criteria.

[0037] According to embodiments of this application, although the pathogenesis of ASD is currently unclear, there are indications that pregnancy may be a critical stage in its development. A study published in JAMA confirmed that pregnant women's exposure to valproic acid (VPA) during pregnancy is significantly associated with an increased incidence of ASD in offspring. Intraperitoneal injection of VPA in pregnant mice at 12.5 days of gestation mimics the behavioral and neurobiological characteristics of ASD. Therefore, in the creation of animal models of ASD, intraperitoneal injection of VPA in pregnant mice resulted in offspring with ASD, exhibiting behaviors highly similar to those of human ASD patients, including reduced social behavior, increased repetitive and stereotyped behaviors, and decreased adaptability to new environments. This method has become one of the classic methods for ASD modeling and provides a powerful tool for exploring the pathological mechanisms and potential treatment strategies of ASD.

[0038] The compounds of this invention may contain asymmetric or chiral centers, and thus exist in different stereoisomer forms. It is contemplated that all stereoisomer forms of the compounds of this invention, including but not limited to diastereomers, enantiomers, atropisomers, and geometric (or conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of this invention.

[0039] Unless otherwise stated, the structures described in this invention also represent all isomers (e.g., enantiomers, diastereotropic atropisomers, and geometric (or conformational) forms including this structure); for example, each asymmetric center R and S Configuration, ( Z ) and( E Double bond isomers, and ( Z ) and( E Conformational isomers. Therefore, individual stereochemical isomers of the compounds of the present invention, as well as enantiomer mixtures, diastereomer mixtures, and mixtures of geometric isomers (or conformational isomers), are all within the scope of the present invention.

[0040] The terms "tautomer" or "tautomer form" refer to structural isomers with different energies that can interconvert through a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium can be achieved in the tautomer. For example, proton tautomers (also known as prototropic tautomers) involve interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers involve interconversions via the rearrangement of some bonding electrons.

[0041] As used in this invention, "nitrogen oxides" refers to compounds containing several amine functional groups that can oxidize one or more nitrogen atoms to form [a specific compound]. N -Oxides. N A special case of oxides is tertiary amines. N -Oxides or nitrogen-containing heterocyclic nitrogen atoms N -Oxides. The corresponding amines can be treated with oxidizing agents, such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids), to form amines. N -Oxides (see Advanced Organic Chemistry, Wiley Interscience, 4th Edition, Jerry March, pages). Especially, N -Oxides can be prepared by LWDeady's method (Syn.Comm.1977, 7, 509-514), in which the amine compound is reacted with m-chloroperbenzoic acid (MCPBA) in an inert solvent, such as dichloromethane.

[0042] In this invention, "solvent" refers to an association formed by one or more solvent molecules and the compound of this invention. Solvents forming solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association formed when the solvent molecules are water.

[0043] "Metabolic products" refer to the products obtained from the metabolism of a specific compound or its salt in vivo. The metabolites of a compound can be identified using techniques known in the art, and their activity can be characterized by experimental methods as described in this invention. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, acylation, deacylation, esterification, defatting, enzymatic cleavage, etc. Accordingly, this invention includes the metabolites of compounds, including metabolites produced by sufficiently exposing the compounds of this invention to mammals for a period of time.

[0044] The term "pharmaceutically acceptable salt" as used in this invention refers to the organic and inorganic salts of the compounds of this invention. Pharmaceutically acceptable salts are well-known in the field, as described in references such as SM Berge. et al. The text describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. These include, but are not limited to, inorganic acid salts formed by reactions with amino groups, such as hydrochlorides, hydrobroms, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioic acid, glucono-p-gluconate, glyceryl phosphate, gluconate, hemisulfate, heptanate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pyruvate, pectinate, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts obtained by means of appropriate bases include alkali metals, alkaline earth metals, ammonium, and nitrogen. + (C 1-4 Alkyl)4 salts.

[0045] The term "prodrug" as used in this invention refers to a compound that is converted into betaine in vivo. Such conversion is influenced by the hydrolysis of the prodrug in the blood or its enzymatic conversion into the parent structure in the blood or tissues. The prodrug compounds of this invention can be esters; among existing inventions, esters that can serve as prodrugs include phenyl esters and aliphatic (C) esters. 1-24Esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, one compound in this invention contains a hydroxyl group, meaning it can be acylated to yield a prodrug form. Other prodrug forms include phosphate esters, such as those obtained by phosphorylation of a parent hydroxyl group. A complete discussion of prodrugs can be found in the following literature: T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the ACS Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, J. Rautio et al. , Prodrugs: Design and ClinicalApplications, Nature Review Drug Discovery ,2008, 7, 255-270, and SJHecker et al. , Prodrugs of Phosphates and Phosphonates, Journal of Medicinal Chemistry ,2008, 51, 2328-2345.

[0046] Any asymmetric atom (e.g., carbon, etc.) in the compounds of the present invention can exist in racemic or enantiomerically enriched forms, for example ( R )-、( S It exists in (R, S)- or (R, S)- configuration. In some embodiments, the asymmetric atoms are in (R, S)- configuration. R )-or( S The configuration has at least 50% enantiomer excess, at least 60% enantiomer excess, at least 70% enantiomer excess, at least 80% enantiomer excess, at least 90% enantiomer excess, at least 95% enantiomer excess, or at least 99% enantiomer excess. If possible, the substituents on the atoms having unsaturated double bonds may be in cis-(Z)- or trans-(E)- form.

[0047] Therefore, as described in this invention, the compounds of this invention can exist in the form of one of the possible isomers, rotational isomers, tautomers, tautomers, or mixtures thereof, for example, in the form of essentially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates, or mixtures thereof.

[0048] Any mixture of isomers can be separated into pure or substantially pure geometric or optical isomers, diastereomers, and racemates based on the physicochemical differences of the components, for example by chromatography and / or stepwise crystallization.

[0049] Racemates of any resulting end product or intermediate can be separated into optical enantiomers using known methods, such as by separating their diastereomeric salts. Racemate products can also be separated by chiral chromatography, such as high-performance liquid chromatography (HPLC) using chiral adsorbents. In particular, enantiomers can be prepared by asymmetric synthesis (e.g., Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981). Principles of Asymmetric Synthesis (2 nd Ed. Robert E. Gawley, Jeffrey Aubé, Elsevier, Oxford, UK, 2012); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962);

[0050] In a fourth aspect, this application discloses a food composition for preventing the occurrence of ASD, optimizing fetal neurodevelopment, or reducing the risk of fetal neurodevelopmental disorders. The food composition comprises betaine or a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, a nitrogen oxide, a solvate, a metabolite, or a prodrug. Therefore, by using this food composition with betaine or a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, a nitrogen oxide, a solvate, a metabolite, or a prodrug as the active ingredient, it is possible to effectively prevent the occurrence of ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders.

[0051] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0052] Example

[0053] 1. Laboratory animals and betaine dosage

[0054] This study has been reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Capital Medical University (AEEI-2024-266). Eight-week-old C57BL / 6 mice, including 18 females and 9 males, were used in this study and housed in the SPF-grade animal housing at the Chinese Institute of Rehabilitation Sciences. The housing environment was carefully designed to ensure the health and welfare of the animals: the housing was equipped with 100W fluorescent lamps, providing a light intensity of 150-200 Lux, with lighting provided from 7:00 AM to 7:00 PM daily to simulate the natural light cycle; the remaining time was kept dark to maintain the mice's circadian rhythm. The indoor temperature was strictly controlled between 20 and 24 degrees Celsius, and the humidity was maintained between 40% and 70%, providing the mice with a stable and comfortable living environment.

[0055] All mice were fed a standard maintenance diet primarily composed of corn, soybeans, and flour, with a fat content below 40 g / kg, ensuring balanced nutrition that met experimental requirements. The animal experiments were conducted in strict accordance with the World Health Organization's "International Guidelines for Animal Biomedical Research," ensuring both ethical and scientific rigor. Through these meticulous feeding and experimental procedures, efforts were made to provide the mice with an optimal experimental environment to obtain accurate and reliable research results.

[0056] According to the 2023 edition of the *Chinese Dietary Reference Intakes*, the Chinese Nutrition Society recommends a specific physiological level (SPL) of 1.50 g / day for betaine. The dose conversion factor between humans and mice is 12.3. Therefore, the daily betaine dose for mice is 1.50 g / day ÷ 12.3 = 0.12 g / day. Assuming mice drink 6 ml of water daily, the daily betaine concentration fed to mice is 120 mg ÷ 6 ml = 20 mg / ml = 2.0% (mg / 100 mL). Therefore, this study sets the betaine concentration at 2.0%.

[0057] 2. Experimental grouping and modeling methods

[0058] refer to Figure 1As shown, after purchasing the mice, they were acclimatized for one week and then caged together at a female:male ratio of 2:1. Female mice with vaginal plugs were recorded as gestation day 0.5 and were raised separately. (1) Betaine + VPA group: Six pregnant female mice were fed 2.0% betaine solution (Betaine, Sigma, purity >98%) one week before conception, during pregnancy and lactation, and were injected intraperitoneally with 600mg / kg VPA (Valproic acid, Sigma) on 12.5 days of gestation. A total of 26 offspring mice (14 males + 12 females) were born. The offspring mice were weaned 3 weeks after birth, and 10 male offspring mice were randomly selected as research subjects. (2) VPA-only group: Six pregnant mice were fed purified water one week before conception, during pregnancy and lactation, and were injected intraperitoneally with 600 mg / kg VPA on day 12.5 of gestation, resulting in 22 offspring mice (11 males + 11 females). The offspring mice were weaned 3 weeks after birth, and 10 male offspring mice were selected as the research subjects. (3) Control group: Six pregnant mice were fed purified water one week before conception, during pregnancy and lactation, and were injected intraperitoneally with an equal volume of physiological saline on day 12.5 of gestation, resulting in 31 offspring mice (16 males + 15 females). The offspring mice were weaned 3 weeks after birth, and 10 male offspring mice were randomly selected as the research subjects. A total of 30 male mice were included in the three groups. The eye-opening scores of the offspring mice in the three groups were observed and recorded from day 12 to day 16 after birth. 0 points were scored for not opening eyes, 1 point for opening one eye, and 2 points for opening two eyes. Weight was measured and recorded at 4 and 8 weeks after birth. Exemplary results are shown in Table 1.

[0059]

[0060] 3. Behavioral assessment

[0061] Two days prior to all behavioral evaluations, the mice to be tested were placed in the testing room to familiarize themselves with and adapt to the testing environment, thereby reducing the impact of environmental differences on the experimental results.

[0062] (1) Three-box social test: used to assess the social abilities and novelty preferences of offspring mice. Eight weeks after birth, offspring mice were assessed for their social abilities using the YH-HB three-box system, with dimensions of 60cm × 40cm × 20cm. The three-box system consisted of three equally divided rectangular areas (20cm × 40cm), allowing mice free access. Phase 1 (Adaptation Phase): Offspring mice were placed in the central box of the three-box system with the barrier opened, allowing them to freely explore the three-box area for 10 minutes. Phase 2 (Social Ability Test): Stranger 1 (S1) was placed in one box, while an empty cage (E) was placed in the other box. Offspring mice were placed in the central box with the barrier opened, allowing them to move freely for 10 minutes, and their contact with the empty cage in Phase 2 (T) was recorded.E ) and Strange Mouse 1 (T S1 The third phase (novelty preference test): Stranger 2 (S2) was placed in one side of the empty cage, while Stranger 1 remained on the other side. The offspring mice were placed in the central cage, the barrier was opened to allow them free movement for 10 minutes, and the time of Stranger 1 (T) in the third phase was recorded. S1’ ) and Strange Mouse 2 (T S2 The time frame for calculating the social function index in the second stage is T. S1 / (T) E +T S1 ) and the novelty preference index of the third stage = T S2 / (T) S1’ +T S2 ).

[0063] (2) Marble embedding experiment: used to evaluate repetitive stereotyped behaviors in offspring mice. Offspring mice were placed in experimental cages measuring 30cm × 50cm, with a 5cm thick bedding layer. Twenty (4 × 5) marble beads with a diameter of 1cm were evenly placed on the bedding. The experiment lasted for 30 minutes. After the experiment, the number of embedded marble beads was counted. Embedding was considered valid if the embedding depth exceeded 2 / 3 of the marble bead size. After the embedding, pictures were taken. The counting was performed by two non-experimental technicians. If there were any discrepancies, a third researcher would discuss and determine the number of embedded beads to ensure the objectivity and accuracy of the data.

[0064] (3) Open field test: used to assess anxiety behavior in offspring mice. Offspring mice were placed in a square box (40cm×40cm×30cm) and observed for 10 minutes. More activity in the central area of ​​the offspring mice indicated a lower level of anxiety, while more activity at the periphery may reflect a higher level of anxiety.

[0065] (4) Novel Object Recognition Experiment: Used to assess the memory function of offspring mice. Day 1 was the adaptation period. Offspring mice were placed in a square box (40cm×40cm×30cm) and allowed to explore freely in an object-free environment for 10 minutes. Day 2 was the familiarization period. Two identical objects were placed in the box, and the offspring mice were allowed to explore freely for 10 minutes. Day 3 was the testing period. One of the familiar objects was replaced with a new object. The offspring mice were placed in the box again, and video was taken and timed for 10 minutes. The time taken for the offspring mice to explore the new object and the old object was recorded, and the novel object recognition index was calculated. The formula was: novel object sniffing time / (novel object sniffing time + old object sniffing time).

[0066] Exemplary results of the behavioral assessment are shown in the table below and Figures 3-6 As shown.

[0067]

[0068] 4. Obtaining, sectioning, and testing mouse brain tissue

[0069] Three mice were randomly selected from each of the three groups of offspring mice and sacrificed. After perfusion sampling to prepare paraffin blocks, sections of the forehead and hippocampus were prepared for Nissl staining. Nissl staining was used to observe the number and morphology of neurons in the prefrontal cortex and the CA1 region of the hippocampus. Exemplary results are shown below. Figure 7 As shown.

[0070] Results and Discussion

[0071] Statistical methods

[0072] All statistical analyses were performed by non-experimental personnel, and video analysis was completed using TopScanner software. Statistical analysis was conducted using SPSS 24.0, and the results were presented using GraphPad. Before statistical analysis, the data were tested for normality. One-way ANOVA was used for data conforming to a normal distribution, LSD-T tests were used for pairwise comparisons among multiple groups, and rank-sum tests were used for data not conforming to a normal distribution.

[0073] Figure 2 The graph shows line charts and bar charts of the relationship between the weight of the offspring mice and their eye-opening scores, such as... Figure 2As shown in Table 1, the changes in the weight of the offspring were as follows: (1) VPA caused the offspring to lose weight at 4 and 8 weeks: The offspring of the VPA-only group had significantly lower weight at 4 and 8 weeks compared with the control group, with statistical differences (p<0.01; p<0.001). This result suggests that the offspring of the VPA-only group had a significant decrease in weight. (2) The offspring of the same VPA-only mother mice had normal weight at 4 and 8 weeks after taking betaine: At 4 and 8 weeks after birth, the offspring of the betaine + VPA group had significantly higher weight than the offspring of the VPA-only group (p<0.001), and there was no statistical difference compared with the control group (p>0.05). This result suggests that the offspring of the betaine + VPA group had normal weight. Changes in eye-opening scores of offspring: (1) VPA caused a decrease in eye-opening scores of offspring at 14 days of age: The eye-opening scores of offspring in the VPA-only group at 14 days of age were significantly lower than those in the control group, with a statistically significant difference (p<0.001). This result suggests that the eye-opening time of offspring in the VPA-only group was delayed. (2) Offspring of mothers who received the same dose of VPA and betaine had normal eye-opening scores at 14 days of age: At 14 days of age, the eye-opening scores of offspring in the betaine + VPA group were significantly higher than those in the VPA-only group (p<0.01), and there was no statistically significant difference compared with the control group (p>0.05). This result suggests that the eye-opening time of offspring in the betaine + VPA group was normal.

[0074] Therefore, we found that offspring mice given only VPA had significantly lower body weights at 4 and 8 weeks after birth compared to the control group (p<0.01 and p<0.001), indicating that VPA caused a significant reduction in offspring weight. In contrast, offspring mice given the same dose of VPA and betaine had no significantly different body weights at 4 and 8 weeks after birth compared to the control group (p>0.05), but were significantly higher than the VPA-only group (p<0.001), indicating that betaine effectively prevented VPA-induced weight loss. At 14 days after birth, offspring mice given only VPA had significantly lower eye-opening scores than the control group (p<0.001), indicating that VPA caused a delay in eye-opening time. However, when mother mice were given VPA and betaine simultaneously, their offspring had significantly higher eye-opening scores at 14 days after birth than the VPA-only group (p<0.01), but no significant difference from the control group (p>0.05), indicating that betaine effectively reversed the VPA-induced delay in eye-opening time. These results indicate that VPA has a negative impact on the growth and development of offspring mice, including weight loss and delayed eye opening, while betaine supplementation can effectively prevent these adverse effects and maintain normal growth and development in offspring mice. These findings provide a scientific basis for the application of betaine in preventing developmental disorders caused by VPA.

[0075] Figure 3Table 2 shows the results of the three-box social experiment: Phase 2 (social ability test): (1) VPA can cause offspring to exhibit social impairment: the sniffing time (TS1) of offspring in the VPA group to stranger 1 was significantly shorter than that in the control group, and the social function index was significantly higher than that in the VPA group, with statistical differences (p<0.001; p<0.05). According to the literature, offspring in the VPA group have social ability deficits, indicating that the ASD model was successfully established.14 This result suggests that the social ability of offspring in the VPA group is severely deficient, and the ASD model was successfully established in this study. (2) Offspring of mother mice taking the same dose of VPA and betaine have normal social function: the sniffing time of offspring in the betaine + VPA group to stranger 1 was significantly longer than that in the VPA group, and the social function index was significantly higher than that in the VPA group, with statistical differences (p<0.05; p<0.001), and there was no statistical difference compared with the control group (p>0.05). This result suggests that the social abilities of offspring mice in the betaine + VPA group are normal. Phase 3 (Novelty Preference Test): (1) VPA can cause offspring mice to exhibit abnormal novelty preferences: the sniffing time (TS2) of offspring mice in the VPA group to unfamiliar mice 2 was shorter than that in the control group, but there was no statistical difference (p>0.05), and the novelty preference index was significantly higher than that in the VPA group, with a statistical difference (p<0.05). This result suggests that there is a novelty preference disorder in offspring mice in the VPA group. (2) Offspring mice of the same dose of VPA mother mice taking betaine have normal novelty preferences: the sniffing time of offspring mice in the betaine + VPA group to unfamiliar mice was longer than that in the VPA group, but there was no statistical difference (p>0.05), and the novelty preference index was significantly higher than that in the VPA group, with a statistical difference (p<0.05), and there was no statistical difference compared with the control group offspring mice (p>0.05). This result suggests that the novelty preferences of offspring mice in the betaine + VPA group are normal.

[0076] The results show that offspring mice given only VPA exhibited a significantly shorter sniffing time (TS1) for unfamiliar mouse 1 compared to the control group (p<0.001), indicating a deficiency in social skills. Furthermore, the social function index was significantly higher in the VPA group than in the control group (p<0.05), further confirming the impaired social skills of the offspring. In contrast, offspring mice given the same dose of VPA along with betaine exhibited significantly longer sniffing times for unfamiliar mouse 1 compared to the VPA-only group (p<0.05), and their social function index was also significantly higher (p<0.001), but not significantly different from the control group (p>0.05), indicating that betaine effectively improves the social skill deficits induced by VPA. In the novelty preference test, while the sniffing time (TS2) for unfamiliar mouse 2 was shorter in offspring mice given only VPA, there was no statistically significant difference compared to the control group (p>0.05). However, the novelty preference index was significantly higher in the VPA group than in the control group (p<0.05), indicating that the offspring in the VPA group had a novelty preference disorder. When mother mice were given VPA along with betaine, their offspring spent a longer time sniffing unfamiliar mice than in the VPA-only group, but there was no statistically significant difference compared to the control group (p>0.05). The novelty preference index was significantly higher in the betaine + VPA group than in the VPA-only group (p<0.05), but there was no significant difference compared to the control group (p>0.05), indicating that betaine can effectively prevent the novelty preference disorder induced by VPA.

[0077] In summary, these results indicate that VPA negatively impacts the social abilities and novelty preferences of offspring mice, while betaine supplementation effectively prevents these adverse effects and maintains normal social behavior and novelty preferences. These findings provide a scientific basis for the potential application of betaine in preventing VPA-induced social impairments.

[0078] Figure 4 Table 3 illustrates the schematic results of the bead embedding experiment: the number of beads embedded in the VPA-only group was significantly higher than that in the control group (p<0.0001). This result suggests that the repetitive stereotyped behaviors of the VPA-only group were significantly increased; the number of beads embedded in the betaine + VPA group was significantly lower than that in the VPA group (p<0.0001), while there was no statistically significant difference compared with the control group (p>0.05). This result suggests that the repetitive stereotyped behaviors were not observed in the betaine + VPA group.

[0079] Therefore, it can be seen that offspring mice given VPA alone buried significantly more beads in the bead-burying experiment than the control group (p<0.0001), indicating a significant increase in repetitive stereotyped behaviors in the VPA group offspring. Offspring mice given VPA and betaine simultaneously had significantly fewer beads buried than the VPA-only group (p<0.0001), but no significant difference compared to the control group (p>0.05), indicating that betaine can effectively prevent repetitive stereotyped behaviors induced by VPA.

[0080] Figure 5 Table 4 illustrates the schematic results of the open field experiment: The offspring of the VPA-only group spent significantly less time in the central zone than the control group (p<0.01). This result suggests that the offspring of the VPA-only group exhibited significantly increased anxiety behavior. The offspring of the betaine + VPA group spent significantly more time in the central zone than the VPA-only group (p<0.05), but there was no statistically significant difference compared to the control group (p>0.05). This result suggests that the offspring of the betaine + VPA group did not exhibit anxiety behavior. Therefore, it can be seen that offspring of VPA-only groups spent significantly less time in the central zone of the open field experiment than the control group (p<0.01), indicating a significant increase in anxiety behavior. When mother mice were given VPA and betaine concurrently, their offspring spent significantly more time in the central zone than the VPA-only group (p<0.05), but there was no significant difference compared to the control group (p>0.05), indicating that betaine can effectively reduce VPA-induced anxiety behavior.

[0081] Figure 6 Table 5 presents the schematic results of the novel object recognition experiment: the novel object recognition index of the VPA-only group was significantly higher than that of the control group (p<0.01). This result suggests that the VPA-only group had a memory deficit. The novel object recognition index of the betaine + VPA group was significantly higher than that of the VPA-only group (p<0.01), but there was no statistically significant difference compared with the control group (p>0.05). This result suggests that the betaine + VPA group did not have a memory deficit. The performance of the VPA-only group in the novel object recognition experiment was significantly worse than that of the control group (p<0.01), indicating that the VPA-only group had a memory deficit.

[0082] Therefore, it can be seen that when mother mice were given VPA and betaine at the same time, their offspring had a significantly higher new object recognition index than the VPA-only group (p<0.01), and no significant difference from the control group (p>0.05), indicating that betaine can effectively prevent VPA-induced memory deficits.

[0083] Figure 7The Nissl staining results according to the embodiments of this application are shown, which show that (1) VPA can cause a decrease in the number and morphological abnormalities of neurons in the prefrontal cortex and CA1 region of the hippocampus of offspring mice: In terms of the number of neurons, the number of neurons (Nissl bodies) in the prefrontal cortex and CA1 region of the hippocampus of offspring mice in the VPA-only group was significantly reduced compared with that in the control group, with a statistically significant difference (p<0.05). In terms of neuronal morphology, neuronal apoptosis occurred in the prefrontal cortex and CA1 region of the hippocampus of offspring mice in the VPA-only group, while no neuronal apoptosis occurred in the control group. This result suggests that VPA can cause a decrease in the number of neurons in the prefrontal cortex and CA1 region of the hippocampus of offspring mice and lead to neuronal apoptosis. (2) Offspring of VPA-treated mother mice given the same dose of betaine showed normal number and morphology of neurons in the prefrontal cortex and hippocampus: In terms of neuron count, offspring of the betaine + VPA group had significantly more neurons in the prefrontal cortex and CA1 region of the hippocampus than offspring of the VPA-only group (p<0.05), and there was no statistically significant difference compared with offspring of the control group (p>0.05). In terms of neuronal morphology, no neuronal apoptosis was observed in the prefrontal cortex and CA1 region of the hippocampus of offspring of the betaine + VPA group. This result suggests that offspring born to VPA-treated mother mice have normal number and morphology of neurons in their brain tissue.

[0084] In summary, these results indicate that VPA significantly affects repetitive and stereotyped behaviors, anxious behaviors, and memory function in offspring mice, while betaine supplementation effectively prevents these adverse effects and maintains normal behavior and cognitive function. These findings further support the potential application value of betaine in preventing VPA-induced behavioral and cognitive impairments.

[0085] Therefore, we assessed the effects of VPA on neurons in offspring rat brains using Nissl staining, and the potential protective effect of betaine against these effects. Offspring rats administered VPA alone had significantly fewer neurons in the prefrontal cortex and hippocampal CA1 region than the control group (p<0.05), indicating that VPA led to a reduction in neuronal number. Morphologically, neuronal apoptosis was observed in the prefrontal cortex and hippocampal CA1 region of offspring rats administered only VPA, while this phenomenon was not observed in the control group. This indicates that VPA not only reduces the number of neurons but also affects neuronal morphology and survival. For mother rats administered the same dose of VPA concurrently with betaine, their offspring rats had significantly more neurons in the prefrontal cortex and hippocampal CA1 region than the VPA alone group (p<0.05), and no significant difference from the control group (p>0.05), indicating that betaine can effectively prevent the reduction in neuronal number induced by VPA. Morphologically, no neuronal apoptosis was observed in the prefrontal cortex and hippocampal CA1 region of offspring rats in the betaine + VPA group, further confirming the protective effect of betaine on neuronal morphology and survival. These results indicate that VPA significantly reduces the number of neurons in key regions of the rat brain and leads to abnormal neuronal morphology, while betaine supplementation effectively prevents these adverse effects and maintains the normal number and morphology of neurons in the rat brain. These findings further support the potential application value of betaine in preventing VPA-induced neurodevelopmental disorders.

[0086] Conclusion: Betaine administration to pregnant mice can prevent the occurrence of ASD, and its mechanism is to maintain the normal function of brain neurons.

[0087] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0088] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of betaine for the manufacture of a medicament for preventing the occurrence of autism spectrum disorder in an offspring.

2. Use according to claim 1, characterized in that, The medicament is formulated as an oral administration, an injection administration, a transdermal administration preparation.

3. Use according to claim 2, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient.

4. Use according to claim 3, characterized in that, The pharmaceutically acceptable excipient is selected from a filler, a disintegrant, a binder, a lubricant, or a flavoring agent.

2. The use according to claim 1, wherein the medicament is administered to a pregnant female.

3. The use according to claim 1, wherein the medicament is administered to a pregnant female in the first trimester of pregnancy.

4. The use according to claim 1, wherein the medicament is administered to a pregnant female in the second trimester of pregnancy.

5. The use according to claim 1, wherein the medicament is administered to a pregnant female in the third trimester of pregnancy.

6. The use according to claim 1, wherein the medicament is administered to a pregnant female in the first trimester

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