Use of betaine in prevention of autism spectrum disorders

Abstract

The invention provides a use of betaine in the prevention of the occurrence of an ASD (autism spectrum disorder), in particular to a use of betaine in the prevention of the occurrence of ASD. Specifically, the invention provides application of betaine or pharmaceutically acceptable salts, stereoisomers, tautomers, nitrogen oxides, solvates, metabolites or prodrugs thereof in preparation of drugs, and the drugs are used for preventing ASD, optimizing fetal neurodevelopment or reducing the risk of fetal neurodevelopment disorder. According to the present invention, the betaine or the pharmaceutically acceptable salt, the stereoisomer, the tautomer, the nitrogen oxide, the solvate, the metabolite or the prodrug thereof is adopted as the active component, such that ASD can be effectively prevented, fetal neurodevelopment can be optimized, or the risk of fetal neurodevelopment disorder can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and specifically, the present invention relates to use of betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof in preparing a drug, wherein the drug is used to prevent the occurrence of ASD, optimize fetal neurodevelopment or reduce the risk of fetal neurodevelopmental disorders. Background Art

[0002] In the field of biomedicine, Autism Spectrum Disorder (ASD), also known as autism, autism, etc., is widely recognized as a multifaceted neurodevelopmental disorder. The main characteristics of this disorder include obstacles to social interaction, limited range of interests, and repetitive stereotyped behaviors. According to the latest statistics, the total number of disabled people in China is as high as 85 million, of which the number of ASD patients has reached 13 million, and the number of new cases is close to 200,000 each year, making the incidence of ASD the highest among mental disabilities. This situation not only puts heavy psychological and economic pressure on the families of patients, but also poses a significant challenge to the national public health system and social economy. The exact cause of ASD has not yet been fully revealed, but it is generally believed that its pathogenesis is 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 the condition of patients, especially in preventing the occurrence of ASD, and there is currently a lack of effective strategies. Therefore, developing methods to prevent the occurrence of ASD will be of great significance for reducing its incidence, alleviating social and family burdens, 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] The present invention is based on the inventor's discovery in animal experiments that feeding mother mice betaine can prevent the occurrence of ASD in offspring mice, and the mechanism of the intervention method proposed in this application is to maintain the normal neurons in brain tissue. Therefore, the inventor proposed a method for preventing ASD to eliminate or reduce the occurrence of ASD in offspring. Specifically, the prevention method is to let pregnant women take betaine.

[0005] In view of this, in the first aspect of the present invention, the present invention proposes the use of betaine or its pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug in the preparation of a drug, which is used to prevent the occurrence of ASD, optimize fetal neurodevelopment or reduce the risk of fetal neurodevelopmental disorders. Thus, by using betaine or its pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug 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 an embodiment of the present application, the ASD may include at least one of typical autism (Autistic Disorder), Asperger's Syndrome (Asperger's Syndrome), Childhood Disintegrative Disorder (CDD) and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS).

[0007] According to the embodiments of the present application, the drug is formulated for oral administration, injection administration, transdermal administration or as a food additive. Thus, the compliance of the subjects can be improved through a convenient administration method, thereby improving the efficiency of preventing ASD.

[0008] According to an embodiment of the present application, the drug is administered to pregnant women, women preparing for pregnancy, and newborns. Optionally, the drug is administered to subjects who are exposed to the risk of ASD.

[0009] In the second aspect of the present application, the present application proposes a pharmaceutical composition for preventing the occurrence of ASD, optimizing fetal neurodevelopment, or reducing the risk of fetal neurodevelopmental disorders, and the pharmaceutical composition comprises: an active ingredient, the active ingredient comprising betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof, and a pharmaceutically acceptable excipient. Thus, by using betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, 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.

[0010] According to an embodiment of the present application, the auxiliary materials include but are not limited to fillers, disintegrants, binders, lubricants, sweeteners or flavoring agents.

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

[0012] In the third aspect of the present application, the present application proposes a nutritional supplement for pregnant women, characterized in that it contains betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof, as well as vitamins and minerals. Thus, the nutritional supplement can use betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof as an active ingredient, which can effectively prevent the occurrence of ASD, optimize fetal neurodevelopment or reduce the risk of fetal neurodevelopmental disorders.

[0013] According to an embodiment of the present 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 the fourth aspect of the present application, the present application proposes a food composition, the food composition is used to prevent the occurrence of ASD, optimize fetal neurodevelopment or reduce the risk of fetal neurodevelopmental disorders, and the food composition contains betaine or its pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug. Thus, the food composition can use betaine or its pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug as an active ingredient, which can effectively prevent the occurrence of ASD, optimize fetal neurodevelopment or reduce the risk of fetal neurodevelopmental disorders.

[0015] According to the embodiments of the present application, under the premise that there is no effective treatment for ASD at present, the social significance of preventing the occurrence of ASD by using the method of the present application is multifaceted. ① Preventing the occurrence of ASD can significantly reduce its incidence worldwide. Since ASD is a common neurodevelopmental disorder that affects millions of children and adults, effective preventive measures will significantly reduce its incidence, thereby eliminating the huge social and family burden caused by it. ② Preventing the occurrence of ASD can avoid the huge economic burden of long-term care and nursing for ASD patients. ③ Preventing the occurrence of ASD means eugenics, so that our next generation can grow healthier and stronger, and make positive contributions to the continuation of high-quality human beings. ④ Preventing the occurrence of ASD can reduce the number of individuals with social and communication disorders caused by ASD in society, thereby promoting a more inclusive and understanding social environment. ⑤ Preventing the occurrence of ASD can reduce the huge costs of the public health system and social services caused by it, so that they can be reallocated to other important social and public health fields. ⑥ Preventing the occurrence of ASD can reduce the demand for special education, make educational resources more evenly distributed to all students, and improve the overall quality and efficiency of education. ⑦ Preventing the occurrence of ASD can promote more research to better understand the causes, development mechanisms and possible treatments of ASD. ⑧ Preventing the occurrence of ASD can provide a deeper understanding of the occurrence mechanisms and possible prevention and intervention methods of other neurodevelopmental disorders, and play a very positive role in further reducing the incidence of neurodevelopmental disorders. In short, the use of the scheme of this application to prevent the occurrence of ASD will not only have a profound impact on patients and their families, but also have important significance for the health, economy and cultural development of the entire society. By preventing the occurrence of ASD, we can build a healthier, inclusive and prosperous society. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A technical roadmap according to an embodiment of the present application is shown; Figure 2 A line graph and a bar graph showing the weight and eye-opening score of offspring mice according to one embodiment of the present application are shown; Figure 3~Figure 6 A schematic diagram of behavioral assessment results according to an embodiment of the present application is shown; Figure 7 The Nissl staining result according to the example of the present application is shown. DETAILED DESCRIPTION

[0017] Definitions and general terms Betaine is an alkaloid with the chemical name N,N,N-trimethylglycine. Its chemical structure is similar to that of amino acids and it belongs to the quaternary ammonium base class. Its molecular formula is C5H 11NO2, which has the following structure:

[0018] Betaine is naturally present in a variety of foods, especially in foods such as beetroot, spinach, whole wheat, wheat germ, shrimp and crab. Betaine is stable under normal storage conditions and is not easily decomposed. Betaine is absorbed in the small intestine and participates in metabolic processes in the liver. As a methyl donor for methylation reactions in the body, betaine participates in a variety of biochemical processes, including the synthesis of DNA, protein and certain hormones. In addition, betaine plays a key role in homocysteine ​​metabolism, helping to convert it into methionine, thereby reducing the level of homocysteine ​​in the blood. And within the cell, betaine helps maintain osmotic pressure balance and protects cells from damage 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 the growth performance and health level of animals. Betaine is generally considered safe and well tolerated by most people. In some cases, betaine is also used as a drug, such as in the treatment of certain types of hyperhomocysteinemia.

[0019] The term "Autism Spectrum Disorder (ASD)" used in this article refers to a group of complex neurodevelopmental disorders that affect an individual's social interactions, communication abilities, and behavior patterns. Characteristics of ASD generally include the following: Social impairments: Individuals with ASD may have difficulty understanding nonverbal cues of social interactions, establishing and maintaining relationships, and understanding the emotions and perspectives of others.

[0020] Communication disorders: This may include delayed language development, difficulty using language, difficulty initiating or maintaining a conversation, and a lack of nonverbal communication skills.

[0021] Repetitive behaviors: Individuals with ASD may display repetitive body movements (such as hand flapping or rocking), persistence in the details of daily activities, and an intense, restricted focus on specific interests or activities.

[0022] Narrow interests: They may show excessive interest or preoccupation with certain subjects or objects.

[0023] Sensory processing problems: Individuals with ASD may have unusual responses to sound, touch, taste, smell, or visual stimuli.

[0024] Cognitive and learning differences: While individuals with ASD have varying levels of intelligence, they may demonstrate exceptional skills in some areas and have challenges in others.

[0025] 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 an individual's life. Currently, the exact cause of ASD is not fully understood, but research suggests that both genetic and environmental factors may be involved in its development.

[0026] According to an embodiment of the present application, ASD is a series of neurodevelopmental disorders with similar characteristics, and ASD may include the following: Autistic Disorder: Also known as typical autism, it is characterized by severe social disorders, communication disorders and repetitive and stereotyped behaviors.

[0027] Asperger's Syndrome: Patients usually have normal or above-normal intelligence and language ability, but have social difficulties and stereotyped behavior patterns.

[0028] Childhood Disintegrative Disorder (CDD): This is a rare disorder in which children show normal development until at least age two and then gradually lose previously acquired skills, including social, language, and play skills.

[0029] Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS): A term used to describe conditions that present with some autistic features but do not meet the criteria for other specific diagnoses.

[0030] According to the embodiments of the present application, although the pathogenesis of ASD is still unclear, there are signs that pregnancy may be a critical stage for the occurrence of ASD. A study published in JAMA confirmed that exposure to valproic acid (VPA) during pregnancy was significantly associated with an increased incidence of ASD in offspring. Intraperitoneal injection of VPA in pregnant mice at 12.5 days of gestation can simulate the behavioral and neurobiological characteristics of ASD. For this reason, when preparing an ASD animal model, intraperitoneal injection of VPA in pregnant mice causes them to produce ASD offspring, which show reduced social behavior, increased repetitive stereotyped behavior, and decreased adaptability to new environments, which are highly similar to human ASD patients. This method has become one of the classic ASD modeling methods and provides a powerful tool for exploring the pathological mechanisms and potential treatment strategies of ASD.

[0031] The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers and atropisomers and geometric (or conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention.

[0032] Unless otherwise indicated, structures depicted herein are also meant to include all isomeric (eg, enantiomers, diastereomeric atropisomers, and geometric (or conformational)) forms of the structure; for example, each asymmetric center R and S Configuration, ( Z ) and( E ) double bond isomers, and ( Z ) and( E Therefore, single stereochemical isomers as well as enantiomeric mixtures, diastereomeric mixtures, and geometric isomer (or conformational isomer) mixtures of the present compounds are within the scope of the present invention.

[0033] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions that occur via proton migration, such as keto-enol isomerizations and imine-enamine isomerizations. Valence tautomers include interconversions that occur via reorganization of some of the bonding electrons.

[0034] The term "nitrogen oxide" as used herein refers to a compound containing several amine functional groups which can oxidize one or more nitrogen atoms to form N -Oxide. N - A special example of an oxide is a tertiary amine N - oxide or nitrogen-containing heterocyclic nitrogen atom N -oxides. The corresponding amines may be treated with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., a peroxycarboxylic acid) to form N -oxides (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages). In particular,N The -oxides can be prepared by the method of LW Deady (Syn. Comm. 1977, 7, 509-514), wherein, for example, the amine compound is reacted with m-chloroperbenzoic acid (MCPBA) in an inert solvent such as dichloromethane.

[0035] The "solvate" of the present invention refers to an association formed by one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, aminoethanol. The term "hydrate" refers to an association formed by the solvent molecule being water.

[0036] "Metabolite" refers to a product obtained by the metabolism of a specific compound or salt thereof in vivo. The metabolites of a compound can be identified by techniques known in the art, and their activity can be characterized by experimental methods as described herein. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, etc. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting a compound of the present invention with a mammal for a period of time.

[0037] The "pharmaceutically acceptable salt" used in the present invention refers to organic salts and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, such as: SM Berge et al., describepharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences,1977, 66: 1-19. Pharmaceutically acceptable salts formed by non-toxic acids include, but are not limited to, inorganic acid salts formed by reaction with amino groups such as hydrochlorides, hydrobromides, phosphates, sulfates, 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 methods to obtain these salts. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1-4 Alkyl)4 salt.

[0038] The term "prodrug" as used in the present invention refers to a compound that is converted into betaine in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or by the enzymatic conversion of the prodrug into the parent structure in the blood or tissue. The prodrug compound of the present invention can be an ester. In the prior invention, esters that can be used as prodrugs include phenyl esters, aliphatic (C 1-24) esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters. For example, a compound of the present invention contains a hydroxyl group, which can be acylated to obtain a prodrug form of the compound. Other prodrug forms include phosphate esters, such as these phosphate ester compounds are obtained by phosphorylation of the hydroxyl group on the parent. For a complete discussion of prodrugs, please refer to the following documents: 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.

[0039] Any asymmetric atom (e.g., carbon, etc.) of the compounds of the present invention may exist in a racemic or enantiomerically enriched form, for example ( R )-、( S )- or (R, S)- configuration. In certain embodiments, each asymmetric atom is present in ( R )-or( S In some embodiments, the present invention relates to a 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in terms of the )-configuration. Substituents on atoms with unsaturated double bonds may be present in cis-(Z)- or trans-(E)-form, if possible.

[0040] Thus, as described herein, the compounds of the invention may exist in the form of one or a mixture of possible isomers, rotamers, atropisomers, tautomers, for example, in the form of substantially pure geometric (cis or trans) isomers, diastereomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0041] Any resulting mixtures of isomers can be separated on the basis of the physicochemical differences of the components into the pure or substantially pure geometric or optical isomers, diastereomers, racemates, for example, by chromatography and / or fractional crystallization.

[0042] Any racemates of the resulting final products or intermediates can be separated into optical antipodes by known methods by methods familiar to those skilled in the art, such as by separation of the diastereomeric salts obtained. The racemic products can also be separated by chiral chromatography, such as high pressure liquid chromatography (HPLC) using a chiral adsorbent. 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);

[0043] In the fourth aspect of the present application, the present application proposes a food composition, the food composition is used to prevent the occurrence of ASD, optimize fetal neurodevelopment or reduce the risk of fetal neurodevelopmental disorders, and the food composition contains betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof. Thus, the food composition can be used to use betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug as an active ingredient, which can effectively prevent the occurrence of ASD, optimize fetal neurodevelopment or reduce the risk of fetal neurodevelopmental disorders.

[0044] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0045] Example 1. Experimental animals and betaine dosage This study has been reviewed by the Capital Medical University Experimental Animal Welfare Ethics Committee (AEEI-2024-266). In this study, 8-week-old C57BL / 6 mice were selected, including 18 females and 9 males, all of which were housed in the SPF-level animal breeding room of the Chinese Institute of Rehabilitation Science. The breeding environment was carefully designed to ensure the health and welfare of the animals: the breeding room was equipped with a 100W fluorescent lamp, providing a light intensity of 150-200 Lux, and lighting was provided from 7 am to 7 pm every day to simulate the natural light cycle; the rest of the time was kept dark to maintain the biological rhythm of the mice. The indoor ambient temperature was strictly controlled at 20 to 24 degrees Celsius, and the humidity was maintained at 40% to 70%, providing a stable and comfortable living environment for the mice.

[0046] All mice were fed a regular maintenance feed with corn, beans and flour as the main ingredients. The fat content of the feed was less than 40 g / kg, ensuring that the mice were nutritionally balanced and met the experimental needs. When conducting animal experiments, the "International Guiding Principles for Animal Biomedical Research" formulated by the World Health Organization was strictly followed to ensure the ethics and scientificity of the experiments. Through these meticulous feeding management and experimental operations, we are committed to providing mice with an optimal experimental environment in order to obtain accurate and reliable research results.

[0047] According to the 2023 edition of the "Reference Intake of Dietary Nutrients for Chinese Residents", the Chinese Nutrition Society pointed out that the specific recommended value (Specific Physiological Level, SPL) of betaine is 1.50 g / d. The dosage conversion factor between humans and mice is 12.3, and the daily betaine dosage for mice is: 1.50 g / d ÷ 12.3 = 0.12g / d. Assuming that mice drink 6ml of water per day, the daily betaine concentration of mice is: 120mg ÷ 6ml = 20 mg / ml = 2.0% (mg / 100mL). Therefore, the betaine concentration was set to 2.0% in this study.

[0048] 2. Experimental grouping and modeling method refer to Figure 1As shown, the mice were purchased and fed adaptively for 1 week and then caged in a ratio of 2:1. Female mice with vaginal plugs were recorded as 0.5 days of pregnancy and were housed individually. (1) Betaine + VPA group: 6 pregnant female mice were fed 2.0% betaine solution (Betaine, Sigma, purity > 98%) 1 week before conception, during pregnancy and lactation, and injected intraperitoneally with 600 mg / 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 research subjects. (2) VPA group: 6 pregnant mice were fed with pure water 1 week before conception, during pregnancy and lactation, and intraperitoneally injected with 600 mg / kg VPA on 12.5 days of pregnancy. 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 randomly selected as research subjects. (3) Control group: 6 pregnant mice were fed with pure water 1 week before conception, during pregnancy and lactation, and intraperitoneally injected with an equal amount of saline on 12.5 days of pregnancy. 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 offspring mice in the three groups were observed and the eye opening score was recorded from 12 to 16 days after birth. No eye opening was scored as 0, one eye open was scored as 1, and two eyes open were scored as 2. Body weight was measured and recorded at 4 and 8 weeks after birth. Exemplary results are shown in Table 1.

[0049]

[0050] 3. Behavioral assessment Two days before all behavioral experimental evaluations, the mice to be tested were placed in the behavioral testing room to familiarize them with the testing environment to reduce the impact of environmental differences on the experimental results.

[0051] (1) Three-box social experiment: used to evaluate the social ability and novelty preference of offspring mice. Eight weeks after the offspring mice were born, their social ability was evaluated using a YH-HB three-box device with a box size of 60cm×40cm×20cm. The three-box system consists of three equally divided rectangular areas (20cm×40cm), and mice can enter and exit freely. Phase I (adaptation phase): The offspring mice were placed in the central box of the three-box device and the baffle was opened to allow them to freely explore the three-box area for 10 minutes. Phase II (social ability test): A stranger mouse 1 (S1, Stranger 1) was placed in one box, and an empty cage (E, Empty) was placed in the other box. The offspring mice were placed in the central box, the baffle was opened to allow them to move freely for 10 minutes, and the number of offspring mice contacting the empty cage in the second phase (T E ) and stranger mouse 1 (TS1 ) time. The third stage (novel preference test): Stranger 2 (S2) was placed in the box on the empty side of the cage, and Stranger 1 was placed on the other side. The offspring mice were placed in the central box, the baffle was opened to allow them to move freely for 10 minutes, and the offspring mice's third stage of Stranger 1 (T S1’ ) and stranger mouse 2 (T S2 ) time. Calculate the social function index of the second stage = T S1 / (T E +T S1 ) and the third stage novelty preference index = T S2 / (T S1’ +T S2 ).

[0052] (2) Bead burying experiment: used to evaluate the repetitive stereotyped behaviors of offspring mice. The offspring mice were placed in an experimental cage with a size of 30cm×50cm. The cage was covered with 5cm thick bedding and 20 (4×5) marble beads with a diameter of 1cm were evenly placed on the bedding. The experimental time was set to 30 minutes. After the experiment, the number of buried marble beads was calculated. If the burial degree exceeded 2 / 3 of the size of the marble beads, it was considered to be effectively buried. After the bead burying, pictures were taken. The counting was performed by 2 non-experimental professional technicians. If there was any objection, the number of buried beads was determined by a third researcher after discussion to ensure the objectivity and accuracy of the data.

[0053] (3) Open field test: used to evaluate the anxiety behavior of offspring mice. The offspring mice were placed in a square box (40 cm × 40 cm × 30 cm) and recorded and observed for 10 minutes. If the offspring mice were more active in the center area, it would indicate a lower anxiety level, while if they were more active at the edge, it might reflect a higher anxiety level.

[0054] (4) New object recognition experiment: used to evaluate the memory function of offspring mice. The first day is the adaptation period, and the offspring mice are placed in a square box (40cm×40cm×30cm) and allowed to freely explore in an object-free environment for 10 minutes. The second day is the familiarization period, and two identical objects are placed in the box, and the offspring mice are allowed to freely explore for 10 minutes. The third day is the test period, and one of the familiar objects is replaced with a new object. The offspring mice are placed in the box again, and video and timing are performed for 10 minutes. The time the offspring mice explore the new object and the old object is recorded, and the new object recognition index is calculated. The calculation formula is new object sniffing time / (new object sniffing time + old object sniffing time).

[0055] Exemplary results of behavioral assessments are shown in the following table and Figures 3 to 6 shown.

[0056]

[0057] 4. Obtaining, slicing and testing mouse brain tissue Three mice were randomly selected from each of the three groups of offspring mice and killed. After perfusion, the frontal and hippocampal regions were sliced ​​and prepared for Nissl staining. The number and morphology of neurons in the prefrontal cortex and hippocampal CA1 region were observed by Nissl staining. Figure 7 shown.

[0058] Results and Discussion Statistical methods All statistical analyses were performed by non-experimental personnel, and video analysis was completed by TopScanner software. SPSS 24.0 was used for statistical analysis, and GraphPad was used to present statistical results. Before statistical analysis, the data were tested for normality. One-way analysis of variance was used for normal distribution, LSD-T test was used for pairwise comparison between multiple groups, and rank sum test was used for non-normal distribution.

[0059] Figure 2 The line graph and bar graph of the weight and eye opening score of the offspring are shown in Figure 2 As shown in Table 1, the weight changes of offspring mice are as follows: (1) VPA can cause weight loss in offspring mice at 4 and 8 weeks of age: the weight of offspring mice in the VPA group alone at 4 and 8 weeks of age was significantly lower than that of offspring mice in the control group, with a statistically significant difference (p<0.01; p<0.001). This result suggests that the weight of offspring mice in the VPA group alone was significantly reduced. (2) The weight of offspring mice in the same dose of VPA mothers taking betaine at 4 and 8 weeks of age was normal: at 4 and 8 weeks of age, the weight of offspring mice in the betaine + VPA group was significantly higher than that of offspring mice in the VPA group alone (p<0.001), and there was no statistical difference compared with offspring mice in the control group (p>0.05). This result suggests that the weight of offspring mice in the betaine + VPA group is normal. Changes in the eye-opening scores of offspring: (1) VPA can cause a decrease in the eye-opening scores of offspring at 14 days of birth: the eye-opening scores of offspring in the VPA group alone were significantly lower than those in the control group at 14 days of birth, with a statistically significant difference (p<0.001). This result suggests that the eye-opening time of offspring in the VPA group alone is delayed. (2) The eye-opening scores of offspring in the same dose of VPA mothers taking betaine at 14 days of birth were normal: at 14 days of birth, the eye-opening scores of offspring in the betaine + VPA group were significantly higher than those in the VPA group alone (p<0.01), and there was no statistical 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.

[0060] Thus, we found that the weight of the offspring mice given VPA alone was significantly lower than that of the control group at 4 and 8 weeks after birth (p<0.01 and p<0.001), indicating that VPA caused a significant weight loss in the offspring mice. In contrast, the weight of the offspring mice given the same dose of VPA and betaine at 4 and 8 weeks after birth was not significantly different from that of the control group (p>0.05), and was significantly higher than that of the VPA group alone (p<0.001), indicating that betaine can effectively prevent the weight loss caused by VPA. At 14 days after birth, the eye opening score of the offspring mice given VPA alone was significantly lower than that of the control group (p<0.001), indicating that VPA caused a delay in the eye opening time of the offspring mice. However, when the mother mice were given VPA and betaine at the same time, the eye opening score of the offspring mice at 14 days after birth was significantly higher than that of the VPA group alone (p<0.01), and was not significantly different from the control group (p>0.05), indicating that betaine can effectively reverse the delay in eye opening time caused by VPA. These results indicate that VPA has a negative impact on the growth and development of offspring mice, including weight loss and delayed eye opening, and betaine supplementation can effectively prevent these adverse effects and maintain normal growth and development of offspring mice. These findings provide a scientific basis for the use of betaine in preventing developmental disorders caused by VPA.

[0061] Figure 3Table 2 shows the results of the three-box social experiment: Phase II (social ability test): (1) VPA can cause offspring to show social disorders: The sniffing time (TS1) of offspring in the VPA group was significantly shorter than that of offspring in the control group, and the social function index was significantly higher than that of offspring in the VPA group, with statistical differences (p<0.001; p<0.05). According to literature reports, offspring in the VPA group have social ability defects, indicating that the ASD model was successfully established14. This result suggests that offspring in the VPA group have serious social ability defects, and this study successfully established an ASD model. (2) The social function of offspring of mothers taking betaine at the same dose of VPA was normal: the sniffing time of offspring in the betaine + VPA group to stranger mouse 1 was significantly longer than that of offspring in the VPA group, and the social function index was significantly higher than that of offspring in the VPA group, with statistical differences (p<0.05; p<0.001), and there was no statistical difference compared with offspring in the control group (p>0.05). This result suggests that the social ability of the offspring in the betaine + VPA group is normal. Phase III (novelty preference test): (1) VPA can cause offspring to show abnormal novelty preference: the sniffing time (TS2) of the offspring in the VPA group alone to stranger mouse 2 is shorter than that of the offspring in the control group, but there is no statistical difference (p>0.05), and the novelty preference index is significantly higher than that of the VPA group alone, with a statistical difference (p<0.05). This result suggests that the novelty preference of the offspring in the VPA group alone is impaired. (2) The novelty preference of the offspring of the same dose of VPA mothers taking betaine is normal: the sniffing time of the offspring in the betaine + VPA group to stranger mouse is longer than that of the VPA group alone, but there is no statistical difference (p>0.05), and the novelty preference index is significantly higher than that of the VPA group alone, with a statistical difference (p<0.05), and there is no statistical difference compared with the offspring in the control group (p>0.05). This result suggests that the novelty preference of the offspring in the betaine + VPA group is normal.

[0062] The above results show that the sniffing time (TS1) of the offspring mice given VPA alone to stranger mouse 1 was significantly shortened, which was statistically different from the control group (p<0.001), indicating that the offspring mice in the VPA group had defects in social ability. In addition, the social function index was significantly higher in the VPA group than in the control group (p<0.05), further confirming that the social ability of the offspring mice in the VPA group was impaired. In contrast, when the mother mice were given the same dose of VPA and betaine at the same time, the sniffing time of the offspring mice to stranger mouse 1 was significantly longer than that of the simple VPA group (p<0.05), and the social function index was also significantly higher than that of the simple VPA group (p<0.001), and there was no significant difference with the control group (p>0.05), indicating that betaine can effectively improve the social ability defects caused by VPA. In the novelty preference test, the sniffing time (TS2) of the offspring mice given VPA alone to stranger mouse 2 was shortened, but there was no statistical difference compared with the control group (p>0.05). However, the novelty preference index in the VPA group was significantly higher than that in the control group (p<0.05), indicating that the novelty preference of the offspring in the VPA group was impaired. When the mother mice were given betaine at the same time as VPA, the sniffing time of the offspring to the strange mouse was longer than that in the VPA group alone, but there was no statistical difference compared with the control group (p>0.05). The novelty preference index in the betaine+VPA group was significantly higher than that in the VPA group alone (p<0.05), and there was no significant difference from the control group (p>0.05), indicating that betaine can effectively prevent the novelty preference disorder caused by VPA.

[0063] In summary, these results indicate that VPA has a negative impact on the social ability and novelty preference of offspring mice, and betaine supplementation can effectively prevent the occurrence of these adverse effects and maintain the normal social behavior and novelty preference of offspring mice. These findings provide a scientific basis for the potential application of betaine in preventing VPA-induced social disorders.

[0064] Figure 4 Table 3 shows the schematic results of the bead burying experiment: the number of bead burying in the VPA group alone was significantly higher than that in the control group (p<0.0001). This result suggests that the repetitive stereotyped behavior of the VPA group alone increased significantly; the number of betaine + VPA group bead burying was significantly lower than that in the VPA group (p<0.0001), but there was no statistical difference compared with the control group (p>0.05). This result suggests that the betaine + VPA group did not show repetitive stereotyped behavior.

[0065] Thus, it can be seen that the number of beads buried by the offspring of mice given VPA alone in the bead burying experiment was significantly higher than that of the control group (p<0.0001), indicating that the repetitive stereotyped behavior of the offspring of the VPA group increased significantly. When the mother mice were given VPA and betaine at the same time, the number of beads buried by the offspring was significantly lower than that of the VPA alone group (p<0.0001), and there was no significant difference with the control group (p>0.05), indicating that betaine can effectively prevent the repetitive stereotyped behavior caused by VPA.

[0066] Figure 5 Table 4 shows the schematic results of the open field test: the time spent in the central area by the offspring of the VPA group was significantly shorter than that of the offspring of the control group, with a statistical difference (p<0.01). This result suggests that the anxiety behavior of the offspring of the VPA group increased significantly; the time spent in the central area by the betaine + VPA group was significantly longer than that of the VPA group, with a statistical difference (p<0.05), but there was no statistical difference compared with the offspring of the control group (p>0.05). This result suggests that the offspring of the betaine + VPA group did not show anxiety behavior. Therefore, it can be seen that the time spent in the central area of ​​the open field test by the offspring of the VPA group was significantly shorter than that of the control group (p<0.01), indicating that the anxiety behavior of the offspring of the VPA group increased significantly. When the mother rats were given betaine at the same time as VPA, the time spent in the central area by the offspring of the VPA group was significantly longer than that of the VPA group (p<0.05), and there was no significant difference with the control group (p>0.05), indicating that betaine can effectively reduce the anxiety behavior caused by VPA.

[0067] Figure 6 Table 5 shows the schematic results of the novel object recognition experiment: the novel object recognition index of the VPA group alone was significantly higher than that of the control group, with a statistical difference (p<0.01). This result suggests that the VPA group alone has memory defects; the novel object recognition index of the betaine + VPA group was significantly higher than that of the VPA group alone, with a statistical difference (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 has no memory defects. The performance of the VPA group alone in the novel object recognition experiment was significantly worse than that of the control group (p<0.01), indicating that the VPA group had memory defects.

[0068] Therefore, it can be seen that when mother mice took betaine while being given VPA, the novel object recognition index of their offspring was significantly higher than that of the VPA group alone (p<0.01), and there was no significant difference with the control group (p>0.05), indicating that betaine can effectively prevent memory deficits caused by VPA.

[0069] Figure 7The Nissl staining results according to the embodiments of the present application are shown, which show that (1) VPA can cause a decrease in the number of neurons in the prefrontal cortex and hippocampal CA1 region of offspring mice and abnormal morphology: in terms of the number of neurons, the number of neurons (Nissl bodies) in the prefrontal cortex and hippocampal CA1 region of offspring mice in the VPA group alone was significantly reduced compared with that in the control group, with a statistically significant difference (p<0.05). In terms of neuronal morphology, neuronal cell apoptosis occurred in the prefrontal cortex and hippocampal CA1 region of offspring mice in the VPA group alone, while there was no neuronal cell apoptosis in the control group. This result suggests that VPA can cause a decrease in the number of neurons in the prefrontal cortex and hippocampal CA1 region of offspring mice and lead to neuronal cell apoptosis. (2) The number and morphology of neurons in the prefrontal cortex and hippocampus of the offspring of VPA mothers who took betaine at the same dose were normal: In terms of the number of neurons, the number of neurons in the prefrontal cortex and hippocampal CA1 region of the offspring of the betaine + VPA group was significantly greater than that of the offspring of the VPA group alone, with a statistical difference (p<0.05), and there was no statistical difference compared with the offspring of the control group (p>0.05). In terms of neuronal morphology, there was no neuronal apoptosis in the prefrontal cortex and hippocampal CA1 region of the offspring of the betaine + VPA group. This result suggests that the number and morphology of neurons in the brain tissue of the offspring born from VPA mothers who took betaine were normal.

[0070] In summary, these results indicate that VPA can significantly affect the repetitive stereotyped behaviors, anxiety behaviors, and memory functions of offspring mice, and betaine supplementation can effectively prevent the occurrence of these adverse effects and maintain the normal behavior and cognitive functions of offspring mice. These findings further support the potential application value of betaine in preventing VPA-induced behavioral and cognitive disorders.

[0071] Therefore, we used Nissl staining to evaluate the effects of VPA on neurons in the brains of offspring mice and the potential protective effect of betaine against these effects. The number of neurons in the prefrontal cortex and hippocampal CA1 region of offspring mice given VPA alone was significantly less than that of the control group (p<0.05), indicating that VPA caused a reduction in the number of neurons. Morphologically, neuronal apoptosis occurred in the prefrontal cortex and hippocampal CA1 region of offspring mice in the VPA alone group, while this phenomenon was not observed in the control group. This indicates that VPA not only reduced the number of neurons, but also affected the morphology and survival of neurons. For mothers given the same dose of VPA and betaine, the number of neurons in the prefrontal cortex and hippocampal CA1 region of their offspring mice was significantly greater than that of the VPA alone group (p<0.05), and there was no significant difference from the control group (p>0.05), indicating that betaine can effectively prevent the reduction in the number of neurons caused by VPA. Morphologically, no neuronal apoptosis was observed in the prefrontal cortex and hippocampal CA1 region of offspring mice in the betaine+VPA group, which further confirmed the protective effect of betaine on neuronal morphology and survival. These results show that VPA can significantly reduce the number of neurons in key areas of the brain of offspring mice and cause abnormal neuronal morphology, while betaine supplementation can effectively prevent these adverse effects and maintain the number and morphology of neurons in the offspring mice's brains. These findings further support the potential application value of betaine in preventing VPA-induced neurodevelopmental disorders.

[0072] Conclusion: Betaine administration to pregnant mice can prevent the occurrence of ASD, and its mechanism is to maintain the normal function of brain neurons.

[0073] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0074] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. Use of betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, 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.

2. The use according to claim 1, characterized in that The ASD includes at least one of typical autism (Autistic Disorder), Asperger's Syndrome (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 preparing for pregnancy. Optionally, the drug is administered to the subjects who are 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 comprises betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof, and a pharmaceutically acceptable excipient.

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

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 comprises betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, N-oxide, solvate, metabolite or prodrug thereof, and optionally vitamins and minerals.

9. The nutritional supplement according to claim 8, characterized in that The nutritional supplement is used 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, and the food composition contains betaine or a pharmaceutically acceptable salt, stereoisomer, tautomer, nitrogen oxide, solvate, metabolite or prodrug thereof.

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