Use of betaine in preventing autism spectrum disorder, pharmaceutical composition comprising same, nutritional supplement for pregnant women, and food product composition

Abstract

Provided in the present application is the use of betaine in preventing the occurrence of Autism Spectrum Disorder (ASD). Specifically, provided in the present application is the use of betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof in the preparation of a drug. By using the betaine or the pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof as an active ingredient, the occurrence of ASD can be effectively prevented, fetal neurodevelopment can be optimized, or the risk of fetal neurodevelopmental disorders can be reduced.

Description

Uses of betaine in the prevention of autism spectrum disorder, including pharmaceutical compositions thereof, nutritional supplements for pregnant women, and food compositions thereof.

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411502933.0, filed on October 25, 2024, entitled “Use of betaine in the prevention of autism spectrum disorder”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application 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 a medicament for the prevention of ASD, optimization of fetal neurodevelopment, or reduction of the risk of fetal neurodevelopmental disorders. Background Technology

[0004] 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

[0005] This application aims to propose a means to effectively prevent the occurrence of ASD.

[0006] This application 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 method is to have pregnant women take betaine.

[0007] In view of this, in a first aspect of this application, 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.

[0008] 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 Not Otherwise Specified (PDD-NOS).

[0009] 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.

[0010] 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.

[0011] 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.

[0012] According to embodiments of this application, the excipients include, but are not limited to, fillers, disintegrants, binders, lubricants, sweeteners, or flavorings.

[0013] According to embodiments of this application, the drug is formulated for oral administration, injection administration, transdermal administration, or as a food additive.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] In a fifth aspect, this application also provides a method for preventing the occurrence of ASD, optimizing fetal neurodevelopment, or reducing the risk of fetal neurodevelopmental disorders, including administering betaine to a person in need.

[0018] In some specific embodiments, the ASD includes at least one of Autistic Disorder, Asperger's Syndrome, Childhood Disintegrative Disorder (CDD), and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS).

[0019] In some specific embodiments, the betaine is formulated for oral administration, injection administration, transdermal administration, or as a food additive.

[0020] In some specific embodiments, the betaine is administered to pregnant women or women preparing for pregnancy. Optionally, the recipients are exposed to the risk of ASD.

[0021] In some specific embodiments, the dosage of betaine is 0.12-1.50 g / d.

[0022] In some specific embodiments, the dosage of betaine is 0.12 g / d, 0.13 g / d, 0.14 g / d, 0.15 g / d, 0.16 g / d, 0.17 g / d, 0.18 g / d, 0.19 g / d, 0.2 g / d, 0.3 g / d, 0.4 g / d, 0.5 g / d, 0.6 g / d, 0.7 g / d, 0.8 g / d, 0.9 g / d, 1.0 g / d, 1.10 g / d, 1.20 g / d, 1.30 g / d, 1.40 g / d, and 1.50 g / d.

[0023] In some specific implementations, when the target of application is a human, the dosage is 1.50 g / d.

[0024] 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

[0025] Figure 1 shows a technical roadmap according to an embodiment of this application;

[0026] Figure 2 shows a line graph and a bar graph of the weight of the offspring mouse and the eye-opening score according to an embodiment of the present application;

[0027] Figures 3-6 show schematic diagrams of behavioral assessment results according to an embodiment of this application;

[0028] Figure 7 shows the Nissl staining results according to an embodiment of this application. Detailed Implementation

[0029] Definitions and general terms

[0030] 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. 11NO2 has the following structure:

[0031] 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.

[0032] 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:

[0033] 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.

[0034] Communication barriers: These may include delayed language development, difficulty using language, difficulty in initiating or maintaining conversations, and a lack of nonverbal communication skills.

[0035] Repetitive 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.

[0036] Narrow interests: They may show excessive interest or focus on certain specific topics or items.

[0037] Sensory processing problems: Individuals with ASD may have unusual reactions to auditory, tactile, gustatory, olfactory, or visual stimuli.

[0038] 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.

[0039] 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.

[0040] According to embodiments of this application, ASD is a series of neurodevelopmental disorders with similar characteristics, and ASD may include the following:

[0041] Autistic Disorder (AD): Also known as classic autism, it is characterized by severe social impairments, communication difficulties, and repetitive and stereotyped behaviors.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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 modeling ASD and provides a powerful tool for exploring the pathological mechanisms and potential treatment strategies of ASD.

[0046] The compounds of this application 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 application, 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 application.

[0047] Unless otherwise stated, the structures described in this application also represent all isomers (e.g., enantiomers, diastereomeric atropisomers, and geometric (or conformational) forms) including this structure; for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, individual stereochemical isomers of the compounds of this application, as well as mixtures of enantiomers, diastereomeric mixtures, and mixtures of geometric isomers (or conformational isomers), are all within the scope of this application.

[0048] 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.

[0049] As used in this application, "nitrogen oxide" refers to a compound containing several amine functional groups, in which one or more nitrogen atoms can be oxidized to form an N-oxide. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen-containing heterocyclic nitrogen atoms. The corresponding amines can be treated with oxidizing agents, such as hydrogen peroxide or peracids (e.g., peroxycarboxylic acids) to form N-oxides (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages). In particular, N-oxides can be prepared using the LWDeady method (Syn. Comm. 1977, 7, 509-514), in which the amine compound is reacted with m-chloroperbenzoic acid (MCPBA), for example, in an inert solvent, such as dichloromethane.

[0050] In this application, "solvent" refers to an association formed by one or more solvent molecules and a compound of this application. Solvents that form 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 by solvent molecules that are water.

[0051] "Metabolic products" refer to the products obtained from the metabolism of a specific compound or its salt in the body. 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 application. Such products can be obtained by subjecting the compound to oxidation, reduction, hydrolysis, acylation, deacylation, esterification, defatting, enzymatic cleavage, etc. Accordingly, this application includes the metabolites of compounds, including metabolites produced by sufficiently exposing the compounds of this application to mammals for a period of time.

[0052] As used in this application, "pharmaceutically acceptable salts" refers to the organic and inorganic salts of the compounds of this application. Pharmaceutically acceptable salts are well-known in the field, as described in the literature: SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts formed by reactions with amino groups, such as hydrochlorides, hydrobromic acids, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or these salts obtained by 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.

[0053] As used in this application, the term "prodrug" 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 in this application can be esters; among existing inventions, esters that can serve as prodrugs include phenyl esters and aliphatic (C14) esters. 1-24 Esters, acyloxymethyl esters, carbonates, carbamates, and amino acid esters. For example, one compound in this application 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 compound with a hydroxyl group. For a complete discussion of prodrugs, please refer to the following literature: T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the ACSSymposium Series; Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; J. Rautio et al., Prodrugs: Design and Clinical Applications, Nature Review Drug Discovery, 2008, 7, 255-270; and SJ Hecker et al., Prodrugs of Phosphates and Phosphonates, Journal of Medicinal Chemistry, 2008, 51, 2328-2345.

[0054] Any asymmetric atom (e.g., carbon, etc.) in the compounds of this application may exist in a racemic or enantiomerically enriched form, such as (R)-, (S)-, or (R,S)- configuration. In some embodiments, each asymmetric atom has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% enantiomeric excess in the (R)- or (S)- configuration. If possible, substituents on atoms having unsaturated double bonds may be present in cis-(Z)- or trans-(E)- form.

[0055] Therefore, as described in this application, the compounds of this application may 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.

[0056] 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.

[0057] 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); and Wilen, SHTables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).

[0058] 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.

[0059] The embodiments of this application are described in detail below, with examples of these embodiments illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0060] Example

[0061] 1. Laboratory animals and betaine dosage

[0062] 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 a stable and comfortable living environment for the mice.

[0063] 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.

[0064] 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%.

[0065] 2. Experimental grouping and modeling methods

[0066] Referring to Figure 1, after purchasing the mice, they were acclimatized for one week and then caged together at a female:male ratio of 2:1. The female mice found to have vaginal plugs were recorded as day 0.5 of pregnancy 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 day 12.5 of pregnancy. 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 the 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. A total of 22 offspring mice (11 males + 11 females) were born. The offspring mice were weaned 3 weeks after birth, and 10 male offspring mice were selected as 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. A total of 31 offspring mice (16 males + 15 females) were born. The offspring mice were weaned 3 weeks after birth, and 10 male offspring mice were randomly selected as 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.

[0067] Table 1. Weight and Eye-Opening Score of Baby Rats

[0068] 3. Behavioral assessment

[0069] 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.

[0070] (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), which mice could freely enter and exit. 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): A strange mouse (S1, Stranger 1) was placed in one box, while an empty cage (E, Empty) 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 ).

[0071] (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, each 1cm in diameter, were evenly placed on the bedding. The experiment lasted 30 minutes. Afterward, the number of embedded marble beads was counted; embedding more than two-thirds of the marble's size was considered valid. Images were taken after embedding. The counting was performed by two non-experimental technicians. If there were any discrepancies, a third researcher would discuss and determine the final number of embedded beads to ensure the objectivity and accuracy of the data.

[0072] (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.

[0073] (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 the time was recorded 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).

[0074] The results of the behavioral assessment are shown in the table below and Figures 3-6.

[0075] Table 2. Results of the Three-Box Social Experiment

[0076] Table 3. Results of the bead embedding experiment

[0077] Table 4. Results of the open field experiment

[0078] Table 5. Experimental Results of Novel Object Recognition

[0079] 4. Obtaining, sectioning, and testing mouse brain tissue

[0080] 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 in Figure 7.

[0081] 5. Statistical methods

[0082] 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.

[0083] Results and Discussion

[0084] Figure 2 shows the line graph and bar graph of the weight of the offspring and the eye-opening score. As shown in Figure 2 and 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 less weight at 4 and 8 weeks of age than 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 dose of VPA mothers who were given betaine had normal weight at 4 and 8 weeks: At 4 and 8 weeks of age, the offspring of the betaine + VPA group had significantly more 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.

[0085] 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.

[0086] Figure 3 and Table 2 show 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 defects, indicating that the ASD model was successfully established.14 This result suggests that the social ability of offspring in the VPA group is severely defective, 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 (p>0.05). This result suggests that the novelty preferences of offspring mice in the betaine + VPA group are normal.

[0087] 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 shortened 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 and betaine concurrently, 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.

[0088] 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.

[0089] Figure 4 and Table 3 illustrate 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.

[0090] 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.

[0091] Figure 5 and Table 4 illustrate 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 the 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 the mother rats were given VPA and betaine simultaneously, 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.

[0092] Figure 6 and Table 5 illustrate 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 statistical 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.

[0093] 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.

[0094] Figure 7 shows the Nissl staining results according to the embodiments of this application, which shows 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 was observed 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 in the betaine + VPA group had significantly more neurons in the prefrontal cortex and CA1 region of the hippocampus than those in the VPA-only group (p<0.05), and no statistically significant difference compared to the control group (p>0.05). Regarding neuronal morphology, no neuronal apoptosis was observed in the prefrontal cortex and CA1 region of the hippocampus in 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.

[0095] 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.

[0096] Therefore, we used Nissl staining to assess the effects of VPA on neurons in offspring rat brains, 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 suggests 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 had significantly more neurons in the prefrontal cortex and hippocampal CA1 region than the VPA-only group (p<0.05), and no significant difference compared to 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 CA1 region of the hippocampus in the betaine + VPA group of offspring mice, 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 areas of the offspring brain and leads to abnormal neuronal morphology, while betaine supplementation effectively prevents these adverse effects and maintains normal neuronal number and morphology in the offspring brain. These findings further support the potential application value of betaine in preventing VPA-induced neurodevelopmental disorders.

[0097] Conclusion: Betaine administration to pregnant mice can prevent the occurrence of ASD, and its mechanism is to maintain the normal function of brain neurons.

[0098] 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 this application. 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.

[0099] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. Use of betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitride, solvate, metabolite or prodrug thereof in the preparation of a medicament for the prevention of ASD, optimization of fetal neurodevelopment or reduction of the risk of fetal neurodevelopmental disorders.

2. The use according to claim 1, characterized in that, The ASDs include at least one of Autistic Disorder, Asperger's Syndrome, Childhood Disintegrative Disorder (CDD), and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS).

3. The use according to claim 1, characterized in that, The drug is formulated for oral administration, injection administration, transdermal administration, or as a food additive.

4. The use according to claim 1, characterized in that, The drug is administered to pregnant women and women who are trying to conceive. Optionally, the recipients may be exposed to the risk of ASD.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition is used to prevent the occurrence of ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders, and the pharmaceutical composition comprises: The active ingredient includes betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug, and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that, The excipients include, but are not limited to, fillers, disintegrants, binders, lubricants, sweeteners, or flavorings.

7. The pharmaceutical composition according to claim 5, characterized in that, The drug is formulated for oral administration, injection administration, transdermal administration, or as a food additive.

8. A nutritional supplement for pregnant women, characterized in that, It contains betaine or its pharmaceutically acceptable salts, stereoisomers, tautomers, nitrogen oxides, solvates, metabolites or prodrugs, and optionally vitamins and minerals.

9. The nutritional supplement according to claim 8, characterized in that, The nutritional supplement is intended to prevent the occurrence of ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders.

10. A food composition, characterized in that, The food composition is used to prevent the occurrence of ASD, optimize fetal neurodevelopment, or reduce the risk of fetal neurodevelopmental disorders. The food composition contains betaine or a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, a nitrogen oxide, a solvate, a metabolite, or a prodrug.

11. A method for preventing the occurrence of ASD, optimizing fetal neurodevelopment, or reducing the risk of fetal neurodevelopmental disorders, characterized in that, This includes applying betaine to those in need.

12. The method according to claim 11, characterized in that, The ASDs include at least one of Autistic Disorder, Asperger's Syndrome, Childhood Disintegrative Disorder (CDD), and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS).

13. The method according to claim 11, characterized in that, The betaine is formulated for oral administration, injection administration, transdermal administration, or as a food additive.

14. The method according to claim 11, characterized in that, The betaine is administered to pregnant women and women who are trying to conceive. Optionally, the recipients may be exposed to the risk of ASD.

15. The method according to claim 11, characterized in that, The dosage of betaine is 0.12-1.50 g / d.

16. The method according to claim 15, characterized in that, When applied to humans, the dosage is 1.50 g / day.

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