Patent Description:
Autism spectrum disorder (Autism) is a kind of neurodevelopmental disorder syndrome, its symptoms include: social ability disorder, decreased verbal and non-verbal communication skill, narrow interest and repetitive stereotyped behavior. Professor Kanner in the United States observed the behavior of children with autism spectrum disorder in <NUM> and officially reported it to the world for the first time in <NUM>.

Autism spectrum disorder is a neurodevelopmental disorder with complex phenotypes in infancy or childhood, and for now there is no effective treatment. Autistic patients often have great defects in feeling and behavior, but they are normal or even better in other aspects. For example, the IQ of <NUM>% of autistic patient is significantly higher than that of the general population. There are great individual differences in the phenotype of autistic patient, which brings great difficulties to the diagnosis and identification of the disease. Autism spectrum disorder is often accompanied by a high degree of anxiety, for example, situations such as daily activities, environmental or character changes can make patients nervous, and often accompanied by self mutilation. Other common symptoms include abnormal eating, mental retardation, hyperactivity, distraction, lack of self-control, large emotional fluctuations. The symptoms of autistic patients appear very early. Generally, a clear phenotype can be observed within half a year of birth. The phenotype often lasts into adulthood and troubles the patient for life.

Therefore, there is an urgent need in this field to provide drugs that can effectively treat autism spectrum disorder.

<NPL>) discloses pharmaceutical compositions comprising ginkgolide A.

<NPL>) discloses Gingko Biloba Egb <NUM> extract to treat autism.

<CIT> describes compositions comprising Gingko Biloba extract for treating mild autism.

<NPL>) describes administration of Ginkgo biloba extract as an adjunctive agent to risperidone in the treatment of autism.

The invention aims to provide ginkgolide A for use in the treatment of autism spectrum disorder.

The first aspect of the present invention provides an active ingredient for use in a method of treatment of autism spectrum disorder.

The active ingredient is selected from the group consisting of ginkgolide A, an stereoisomer thereof, a crystal form thereof, a pharmaceutically acceptable salt thereof, a derivative thereof, an extract containing ginkgolide A, and a combination thereof, wherein the derivative is selected from the group consisting of <NUM> - (<NUM> '-dimethylaminoethoxy) - ginkgolide A, <NUM> - (<NUM> '- diethylaminoethoxy) - ginkgolide A, <NUM> - ((<NUM> '- methoxy-<NUM>', <NUM> '- dimethyl-<NUM>' - pyridyl) - methoxy) - ginkgolide A, and <NUM> - ((<NUM> '- pyridyl) - ethoxy) - ginkgolide A.

In another preferred example, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

In another preferred example, the active ingredient has a content of greater than 1wt%, preferably, greater than <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, or <NUM> wt%, based on the total weight of the pharmaceutical composition.

In another preferred example, the extract containing ginkgolide A is ginkgo biloba extract.

In another preferred example, in the extract containing ginkgolide A, the content of ginkgolide A C1 is greater than <NUM>%, preferably, greater than <NUM>%, more preferably, greater than <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, or <NUM> wt %, based on the dry weight of the extract.

In another preferred example, the pharmaceutical composition is not used with other medications for the treatment of autism spectrum disorder.

In another preferred example, the dosage form of the pharmaceutical composition is selected from the group consisting of liquid preparation (e.g., solutions, emulsions, suspensions), and solid preparation (e.g., lyophilized formulations).

In another preferred example, the dosage form is selected from the group consisting of injection (e.g., injection liquid or powder injection), oral preparation (e.g., capsule, tablet, pill, powder, granule, syrup, oral liquid or tincture), and more preferably the dosage form is oral preparation.

In another preferred example, the treatment includes reducing the severity of autistic behavior defect and/or shortening the duration of autistic behavior defect.

In another preferred example, the autistic behavior defect is selected from the group consisting of social behavior disorder, decreased verbal communication skill, narrow interest, repetitive stereotyped behavior, anger, anxiety, and a combination thereof.

In another preferred example, the autism spectrum disorder is an autism caused by point mutation of Nr2f1 gene.

In another preferred example, the autism spectrum disorder is an autism caused by NR2F1-R112K point mutation.

In another preferred example, the autism spectrum disorder is an autism resulting from a decrease in the number of excitatory neurons and/or a decrease in nerve conduction activity and an increase in the number of inhibitory neurons and/or an increase in nerve conduction activity.

Also described herein but not claimed is a pharmaceutical composition for the treatment of autism behavior defect, comprising:.

In another preferred example, the active ingredient is ginkgolide A.

In another preferred example, the active ingredient has a content of greater than 1wt%, preferably, greater than 5wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM> wt%, or <NUM> wt%, based on the total weight of the pharmaceutical composition.

In another preferred example, the content of the active ingredient in the pharmaceutical composition is based on the weight of ginkgolide A.

In another preferred example, the dosage form is selected from the group consisting of liquid formulations (e.g., solutions, emulsions, suspensions) and solid formulations (e.g., lyophilized formulations).

In another preferred example, the dosage form is selected from the group consisting of injection (e. , injection liquid or powder injection), oral preparation (e. , capsule, tablet, pill, powder, granule, syrup, oral liquid or tincture), and more preferably the dosage form is oral preparation.

In another preferred example, the pharmaceutical composition further comprises a therapeutic agent selected from the group consisting of antidepressant, antipsychotics, morphine blocker (e.g. Naltrexone), mood stabilizer (e.g. sodium valproate, carbamazepine, lithium carbonate, propranolol, clonazepam), central stimulant (e.g. methylphenidate (ritalin) ), dopamine (DA) antagonist, antiepileptics, drugs for the treatment of tourette syndrome (e.g. Tiapride), haloperidol, inosine, Pimozide, vitamin B6, clonidine hydrochloride, and a combination thereof.

In another preferred example, the antidepressant is selected from the group consisting of tricyclic antidepressant (e.g., amitriptyline, imipramine, doxepin and clomipramine), tetracyclic antidepressant (e.g. maprotiline, amoxapine), selective serotonin reuptake inhibitor (e.g., fluoxetine, escitalopram, citalopram, sertraline, fluvoxoxamine), selective serotonin and norepinephrine reuptake inhibitor (e.g., venlafaxine, duloxetine, milnacipran), noradrenergic and specific serotonin antidepressant (e.g., mirtazapine), norepinephrine and dopamine reuptake inhibitor (e.g., bupropion), serotonin balanced antidepressant (e.g., trazodone), selective norepinephrine reuptake inhibitor (e.g., reboxetine), monoamine oxidase inhibitor (e.g., moclobemide), and combinations thereof.

In another preferred example, the antipsychotics is selected from the group consisting of chlorpromazine, sulpiride, haloperidol, risperidone, clozapine, olanzapine, and a combination thereof.

In another preferred example, the antiepileptics is selected from the group consisting of sodium valproate, topiramate, clonazepam, nitrodiazepam, and a combination thereof.

Also described herein but not claimed is a method for treating autism spectrum disorder, comprising the step of:
administering a therapeutically effective amount of ginkgolide A, an stereoisomer thereof, a crystal form thereof, a pharmaceutically acceptable salt thereof, and a derivative thereof; an extract containing ginkgolide A, a combination thereof and the pharmaceutical composition described in the second aspect of the present invention to a subject in need thereof.

In another preferred example, the subject is mammal.

In another preferred example, the subject is human, rat or mouse.

In another preferred example, the subject carries a point mutation of NR2F1 gene.

In another preferred example, the subject carries NR2F1-R112K point mutation.

In another preferred example, the subject has an excitatory / inhibitory imbalance in the cerebral cortex, especially an increase in the inhibitory signal or / and a decrease in the excitatory signal.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as examples) can be combined with each other to form a new or preferred technical solution. Due to space limitations, it will not be repeated one by one here.

In each figure, +/+ refers to wild-type mice; +/m refers to heterozygous mutant mice.

After extensive and intensive research and through a large number of screening and testing, the inventor provided the use of ginkgolide A for the preparation of drugs for the treatment of autism spectrum disorder. Experiments have proved that ginkgolide A can significantly alleviate the behavioral defects of autism spectrum disorder in mice, and can be used as drugs for the treatment of autism spectrum disorder, providing a new medication choice for the treatment of autism spectrum disorder. On this basis, the present invention is completed.

Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" means that the value may change by no more than <NUM>% from the enumerated value when referred to a specific enumerated value. For example, as used herein, the expression "about <NUM>" includes all values between <NUM> and <NUM> (e. , <NUM>, <NUM>, <NUM>, <NUM>, etc.).

As used herein, the terms "contain" or "include (comprise)" may be open, semi-closed, and closed. In other words, the term also includes "basically consisting of" or "consisting of ".

The "treatment" of the present invention includes delaying and terminating the progression of the disease, or eliminating and regressing the disease. The regression of the disease is manifested by a decrease in the severity of the disease symptoms, an increase in the frequency and the duration of the asymptomatic period of the disease, or the prevention of the disorder or disability caused by the disease, and does not require <NUM>% inhibition, elimination and reversal.

As used herein, the terms "heterozygous mutant mouse" and "mutant mouse" are used interchangeably to refer to heterozygous mutant model mouse carrying NR2F <NUM> gene point mutation of the present invention.

The active ingredient of the invention is ginkgolide A, its stereoisomer, its crystal form, its pharmaceutically acceptable salt, its derivative, an extract containing ginkgolide A, or a combination thereof.

The structural formula of ginkgolide A is as follows:
<CHM>.

As used herein, the term "stereoisomer" is intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all the compounds of the present invention, each chiral carbon atom may optionally be in R configuration or S configuration, or mixtures thereof.

The inhibitors of the present invention may be used in the form of amorphous, crystalline or mixtures thereof.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention with an acid or base suitable to be used as drug. Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salt is the salt formed by the compound of the present invention and acid. Acids suitable for salt formation include but are not limited to: inorganic acid such as toluene sulfonic acid, hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid and the like; organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid and the like; and acidic amino acid such as aspartic acid, glutamic acid and the like. A preferred class of salt is the salt formed by the compound of the invention and a base. Bases suitable for salt formaion include but are not limited to inorganic base such as sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, sodium phosphate and the like; and organic base such as ammonia, triethylamine, diethylamine and the like.

In the present invention, the derivatives of ginkgolide A may be those disclosed in Chinese application <CIT>.

Ginkgolide A can be extracted from Ginkgo biloba leaves. Typically, extraction methods include, but are not limited to, solvent extraction, column extraction, solvent extraction-column extraction, supercritical extraction, chromatography or column chromatography and the like.

In another preferred example, in the extract containing ginkgolide A, the content of ginkgolide A C1 is greater than <NUM>%, preferably, greater than <NUM>%, more preferably, greater than <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, <NUM> wt %, or <NUM> wt %, base on the dry weight of the extract.

The pharmaceutical composition of the present invention comprises:(a) an active ingredient: a therapeutically effective amount of ginkgolide A, its stereoisomer, its crystal form, its pharmaceutically acceptable salt, its derivative, an extract containing ginkgolide A, or a combination thereof; and (b) a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention can improve/alleviate autism spectrum disorder. In particular, it alleviates autistic behavior defects, which typically include (but are not limited to): social behavior disorder, decreased verbal communication skill, repeated stereotyped behavior, anger, anxiety, or a combination thereof.

As used herein, the term "therapeutically effective amount" refers to any amount of a drug described below that, when used alone or in combination with another therapeutic agent, can promote regression of the disease, which manifests itself as a decrease in the severity of the disease symptoms, an increase in the frequency and the duration of the asymptomatic period of the disease, or the prevention of the disorder or disability caused by the disease. The "therapeutic effective amount" of the drug of the invention also includes "preventive effective dose". The "preventive effective dose" is any amount of the drug as described below. When this amount of the drug is administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering a recurrence of the disease, the occurrence or recurrence of the disease can be inhibited. Typically, the pharmaceutical composition contains <NUM>-<NUM> of the compound of the present invention/dose, and more preferably <NUM>-<NUM> of the compound of the present invention/dose. Preferably, "a dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" herein means that each component of the composition can be mixed with the compound of the invention and between them without significantly reducing the efficacy of the compound. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate), gelatin, talc, solid lubricants (such as stearic acid, Magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil), polyols (such as propylene glycol, glycerin, mannitol, sorbitol), emulsifiers (such as Tween ®), wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water.

There are no special restrictions on the administration of the compounds or pharmaceutical compositions of the invention. Representative methods of administration include (but are not limited to): oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients:(a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) adhesives, such as hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and Arabic gum; (c) humectants, e.g., glycerol;(d) disintegrants, e.g., agar, calcium carbonate, potato starch or tapioca starch, alginic acid, some composite silicates, and sodium carbonate;(e) slow solvents, e.g., paraffin;(f) absorption accelerators, e.g., quaternary amine compounds;(g) wetting agents, such as, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffers.

Solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be prepared using coatings and shell materials, such as casings and other materials well known in the art. They may contain opaque agents, and the release of active compounds or compounds in this composition may be released in a part of the digestive tract in a delayed manner. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound may also be in microencapsulated form with one or more of the excipients described above.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may include inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, <NUM>,<NUM>-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

In addition to these inert diluents, the composition may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices.

In addition to the active compounds, the suspension may contain suspending agents, such as ethoxylated isooctadecanol, polyoxyethylene sorbitol and dehydrated sorbitol ester, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.

The composition for external parenteral injections may include sterilized aqueous or anhydrous solutions, dispersions, suspensions or emulsions that are physiologically acceptable, and a sterile powder for re-dissolution into a sterile injectable solution or dispersion. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

The dosage forms of the compound of the invention for topical administration include ointment, powder, patch, spray and inhalant. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

The compound of the invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

In some embodiments, the Hedgehog signaling pathway inhibitors of the present invention are used in the same or separate formulations simultaneously with, or sequentially with, other agents that are part of the combination therapy regimen.

The general range of therapeutically effective amount of a compound of Formula I or a composition of the compound of Formula I will be about <NUM>-<NUM>/day, about <NUM>-<NUM>/day, about <NUM>-<NUM>/day, about <NUM>-<NUM>/day, about <NUM>-<NUM>/day, or about <NUM>-<NUM>/day. The therapeutically effective amount will be given in one or more doses. It should be understood that the specific dose of the compound of the invention for any particular patient will depend on a variety of factors, such as the age, gender, weight, general health status, diet, individual response of the patient to be treated, time of administration, severity of the disease to be treated, activity of the specific compound administered, dosage form, mode of application and concomitant drugs. The effective amount of treatment in a given situation can be determined by routine experiments and within the ability and judgment of clinicians or doctors. In any case, the compound or composition will be administered in multiple doses based on the individual condition of the patient and in a manner that allows delivery of a therapeutically effective amount.

The main advantages of the present invention include:.

The invention is further described below in combination with specific examples. It should be understood that these embodiments are only used to illustrate the invention and not to limit the scope of the invention. The experimental methods without specific conditions in the following embodiments are usually based on conventional conditions, such as <NPL>), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and portions are calculated by weight.

Wild type C57BL / <NUM> mice were bred in the experimental animal center of Shanghai Institute of Biochemistry and cell biology, Chinese Academy of Sciences by two pairs of mice purchased from slaker experimental animal center. By the beginning of this experiment, wild-type and mutant mice had been bred and passaged on for more than <NUM> generations in the Experimental Animal Center of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The feeding environment maintains a circadian rhythm of <NUM>:<NUM>, the temperature is maintained at <NUM>-<NUM>, and the air humidity is about <NUM>-<NUM>%. The animals were kept in a (<NUM> * <NUM> * <NUM>) cm<NUM> standard resin rat cage with shaving bedding, and were free to take in enough water and feed. Animal breeding and all experimental operations comply with the relevant regulations of the Experimental Animal Ethics Committee of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

Paraformaldehyde (PFA stock solution: <NUM>% in <NUM> × PBS, final concentration: <NUM>%); Normal goat serum (NGS, GIBCO); Normal mouse IgG (Santa Cruz); Normal rabbit IgG (Santa Cruz); DAPI (sigma, stock solution: <NUM> / ml, final concentration: <NUM>µg/ml-<NUM>µg/ml, reusable); The sealing agent mowil was purchased from calbiochem; OCT frozen section embedding agent was purchased from Leica company. ProteinA, ProteinG PLUS Agarose(Santa Cruz);Dynabeads Protein A/G(Invitrogen); Protein A/G Agarose/Salmon Sperm DNA(Upstate).

Ginkgolide A is purchased from Selleck (https://www. cn/products/ginkgolid e-a. html) with purity > <NUM>%.

Biorad PTC-<NUM>/S1000 PCR instrument, ABI <NUM> PCR instrument, Eppendorf Realplex2 quantitative PCR instrument, Beckman DU650 ultraviolet spectrophotometer, BioRad Pac <NUM> electrophoresis instrument, UVP M26 ultraviolet glue illuminator, SONY DP71 thermal printer, Eppendorf <NUM> refrigerated centrifuge, Thermo Pico17 room temperature centrifuge, Beckman Avanti J-E JSE 07B10 large refrigerated centrifuge, Beckman L-<NUM> ultra-speed freezing centrifuge, BayGene small centrifuge, Mettler Toledo pH meter, Eppendorf MixMate oscillator, Thermo forma - <NUM> refrigerator, cell incubator and ClassII biosafety cabinet, Su Jing Antai cell culture ultra-static stage, Aria flow cytometer, Olympus BX51/IX71 fluorescence microscope, Olympus SZX10/<NUM> Stereo microscope, BioRad Radiance <NUM> inverted laser confocal microscope, Leica SP2 ortho laser confocal microscope, Turner Design <NUM> Lumimetor,Leica CM1900 Cryostat Microtome, Samsung <NUM>°Cconstant temperature wine cabinet, bioRad Pac basic and Tanon EPS300 electrophoresis instrument, electrophoresis membrane transfer device, Shanghai Yiheng constant temperature biochemical incubator, Taicang Hualida HZ-<NUM> constant temperature shaker, DGX-<NUM> and <NUM> blast dryer, WH-<NUM> silent mixer, ZD9550 and ZD9556 shaker, Shanghai Anke TDL-4DB room temperature centrifuge, non-contact full-automatic ultrasonic crushing instrument of Bioruptor UCD-<NUM> of Belgium Diagenode Company.

The related anxiety state of mice were investigated by using the characteristics that mice like to explore in the new environment and the contradictory behavior of fear of heights with open arms at heights. In order to increase the total number of mice entering the arm and avoid hiding in the closed arm, the animals were usually placed in an open environment for <NUM> minutes and then placed in the maze. Similarly, the mice were placed in a room with an elevated experimental device to adapt to the environment one hour before the start of the experiment, with the red light turned on, and the room temperature was kept at <NUM>. At the beginning of the experiment, hold the mouse into the palm of the hand, place it in the middle of the cross facing the closed arm, and quickly leave this room. Then another person started recording and tracking the activity movement of the mouse in another room. In the process, the times of the experimental mouse entering the open arm and the closed arm was counted, and the time of staying in the open arm and the closed arm was counted respectively; after the whole process was recorded for <NUM> minutes for the experiment, the mice were gently stroked, and put back into the cage. The elevated-plus table was cleaned with <NUM>% alcohol for the experiment of the next mouse.

The experimental mice were placed in a feeding cage with the bottom covered with feed and the height was no more than <NUM>. The time of grooming behavior in mice in <NUM> minutes was recorded. The grooming position includes face, head, neck, ears, etc..

The experimental mice were placed in the Y-maze, and the times and order of entering the three arms of the Y-maze within <NUM> minutes were recorded. Entering into <NUM> different arms in succession is considered as one effective arm entry. Effective arm entry rate = effective arm entry times/(total arm entry times -<NUM>)%.

<NUM>×Taq Mixture: <NUM>µl; Evagreen: <NUM>µl; <NUM> primer (<NUM>'+<NUM>'): <NUM>µl; cDNA template: <NUM>µl; double distilled water: <NUM>µl.

The relative quantification based on the reference gene is adopted, i.e. <NUM>-ΔΔCT method, the CT values of the target gene and the reference gene of the defined experimental sample are CT(TARGET, TEST) and CT(REF, TEST) respectively, while the calibration samples are CT(TARGET, CAL) and CT(REF, CAL) respectively. The calculation steps are as followed:
For all experimental samples and calibration samples, the CT value of the target gene is normalized with the CT value of the reference gene: <MAT> <MAT>.

The ΔCT value of the calibration sample is used to normalize the ΔCT value of the experimental sample: <MAT>.

Calculate the expression level ratio of the target gene between the experimental sample and the calibration sample: <MAT>.

In order to study the effect and mechanism of NR2F1 gene point mutation in vivo, CRISPR/Cas9 system was used to edit the gene in mice with C57BL/<NUM> background. Two male Founder mice were obtained by directly injecting Cas9 mRNA and transcribed sgRNA into fertilized eggs. After identification, only the 109th locus (corresponding to the 112th locus of human NR2F1) was mutated (mutated from Arg to Lys), and they were all heterozygous mutations. The possible off-target sites were also sequenced, and no obvious off-target phenomenon was found. A male Founder mouse was selected, its sperm was extracted, fertilized with the eggs of wild-type female mice, and then injected back into the uterus of surrogate female mice to obtain F1 generation. The heterozygote in F1 generation was picked out for mating. The results of observation and statistics of F2 generation were as shown in <FIG>.

A total of three batches of <NUM> mice were collected, and the statistical results of their genotypes were identified. Nr2f1+/+, wild-type mice; Nr2f1+/m, heterozygous mutant mice; Nr2f1m/m, homozygous mutant mice.

There were <NUM> wild-type mice, <NUM> heterozygous mutant mice and <NUM> homozygous mutant mice. It is difficult to obtain homozygous mutant mice (m/m) on P0 days after birth, and the ratio of heterozygous mutant mice (+/m) to wild type mice(+/+) is close to <NUM>:<NUM>, which is in accordance with Mendelian inheritance law. This implies that mice with homozygous mutations are difficult to survive after birth, similar to the NR2F1 gene deletion mutant mice.

The brains of <NUM>-month-old wild-type and heterozygous mutant mice were collected, and the cerebral cortex was stripped and sliced for staining. The staining results are shown in <FIG>. In <FIG>, Cux1(A) and Ctip2(B) are markers of excitatory neurons. The results of immunostaining show that the number of excitatory neurons in the cerebral cortex of heterozygous mutant mice is reduced (C). In <FIG>, GAD1(A), PV(B) and SST(C) are markers of inhibitory neurons, and the results of immunostaining statistics show that the number of inhibitory neurons in the cerebral cortex of heterozygous mutant mice increases (D).

From the above results, it can be seen that the number of excitatory projection neurons in the superficial cortex of the brain of mutant mice decreases, while the number of inhibitory interneurons increases. Apparently, there is an imbalance in the ratio of excitatory/inhibitory neurons in the cortex of heterozygous mutant mice.

From Example <NUM>, it was found that the number of excitatory neurons in the cerebral cortex of the heterozygous mutant mouse decreased and the number of inhibitory neurons increased, and then the nerve conduction activity of the excitatory and inhibitory neurons was detected. The schematic diagram and statistical results (A,B) of micro excitatory potential synaptic current (mEPSC) are shown in <FIG>. It can be seen that the nerve conduction activity of excitatory neurons in the cerebral cortex of mutant mice is significantly lower than that of wild-type mice. The nerve conduction activity of inhibitory neurons is significantly higher than that of wild-type mice.

Wild-type mice are denoted by+/+, while heterozygous mutant mice are denoted by+/m. Three-box experiment, Y-maze experiment, self-grooming experiment and elevated-plus experiment were selected to test the behavioral characteristics of mice.

The three-box experiment mainly tested the social ability of mice. The experimental instrument is three transparent boxes, the boxes are connected together with a controllable gate in the middle. In the pre-experiment, the experimental mice were placed in the middle box, and the gates of the two boxes were opened allowing the mice to move to the boxes on both sides to get familiar with the environment. The formal experiment was divided into two rounds. The first round was to put the experimental mice in the middle box, with an unfamiliar stationary object in one box and an unfamiliar mouse in the other box. The mice were of the same sex, similar in age, and should not be in the same cage. After opening the gate, record the time spent by the experimental mice in the two boxes. Normal mice prefer to communicate with similar creature, but this preference is not found in autistic mice, as shown in <FIG>. The time spent by the experimental mice with the same kind is proportional to their social ability. The time spent by the heterozygous mutant mice with similar mice was significantly less than that of wild-type mice, indicating that the social ability of heterozygous mutant mice was impaired.

The second round is to put the experimental mice in the middle box, and put a brand-new unfamiliar mouse in the box where the static object was originally placed. This mouse was of the same sex and age as the experimental mice, and it cannot be the same cage. Familiar mice are still placed in the box where the same kind of mice were originally placed. Although wild-type mice can communicate with familiar mice, they prefer to communicate more with unfamiliar mice; this preference has not been found in autistic mice, as shown in <FIG>. The time spent by experimental mice with unfamiliar mice was proportional to their social ability. The time spent by heterozygous mutant mice with unfamiliar mice was significantly less than that of wild-type mice, indicating that the social ability of heterozygous mutant mice was impaired.

Then, the repetitive stereotyped behavior of mice was tested by Y-maze. The experimental instrument is a Y-shaped closed elevated rack. Put the mice into the maze and record the number and order of entering the arm. Only entering three different arms continuously can be counted as an effective score. Finally, the change rate of wild-type and heterozygous mutant mice (effective arm entry times/total arm entry times) was counted. The higher the change rate (alternation%), the less the repetitive stereotyped behavior. The experimental results showed that the change rate of heterozygous mutant mice was significantly lower than that of wild type, indicating that heterozygous mutant mice have obvious repetitive stereotyped behavior, as shown in <FIG>.

Subsequent self-grooming experiments were conducted to detect repetitive stereotyped behaviors. The experiment found that the self-grooming behavior of mutant mice was obvious, and the cumulative statistical time was significantly more than that of wild-type mice, as shown in <FIG>. This showed that mutant mice have obvious repetitive stereotyped behavior.

The elevated-plus experiment is mainly used to detect the anxiety behavior of mice. The instrument used was a cross-shaped shelf, half open and half closed. Although normal mice liked a closed environment, curiosity drives them to explore the open space, while anxious mice preferred to stay in a closed and safe environment. The experiment results showed that heterozygous mutant mice stayed in the closed arm much longer than wild-type mice, while wild-type mice stayed in the open arm longer than heterozygous mutant mice, as shown in <FIG>. This showed that heterozygous mutant mice were more anxious, tended to stay in a closed and safe environment, and did not like to explore new environments.

Ginkgolide A was administered to heterozygous mutant mice. The detailed administration scheme was as follows: the administration method was gavage treatment, the concentration of ginkgolide A was <NUM>/kg, the administration time is <NUM>-<NUM> weeks after the birth of mutant mice, and the administration frequency was once a day. The behavior of mutant mice was detected at <NUM> weeks after birth, as shown in <FIG>.

Firstly, the three-box experiment found that the time of interaction between mutant mice fed with ginkgolide A and similar mice or unfamiliar mice was significantly increased, as shown in <FIG>, which showed that the social defects of mutant mice were effectively alleviated.

Secondly, through the self-grooming experiment, it was found that the self-grooming time of point mutant mice fed with ginkgolide A was significantly reduced, as shown in <FIG>, indicating that the repetitive stereotyped behavior of mutant mice was effectively alleviated.

Finally, through the elevated-plus experiment, it was found that the time for mutant mice fed with ginkgolide A to explore the open arm increased significantly, as shown in <FIG>, indicating that the anxiety behavior of mutant mice was effectively alleviated.

To sum up, ginkgolide A can obviously relieve the social behavior disorder, repetitive stereotyped behavior and anxiety and other autistic behavior defects in mutant mice, and has a significant therapeutic effect on autism spectrum disorder, which can be used as a drug for autism spectrum disorder.

Autism spectrum disorder is also known as autistic disorder. Patients often have great defects in perception and behavior. Obvious phenotypes of autism spectrum disorder symptoms can be observed within half a year after birth and will accompany the patients for life. Autism spectrum disorder is a chronic disease and cannot be cured. Long-term administration is often required. The goal of drug treatment is to improve the patient's self-care ability and quality of life, so drugs for treating autism spectrum disorder cannot tolerate serious side effects.

Ginkgolide A is a natural terpenoid compound extracted from Ginkgo biloba leaves, which has a long history of medication. Its medication safety is high and it is well known by those skilled in the medical field. It is known that Ginkgolide A has anti-anxiety effects, but no research shows that it can be used to treat autism spectrum disorder. Among many behavioral experiments of the present invention, the elevated-plus experiment can also be used to detect anxiety-related behavior, it is confirmed that ginkgolide A can relieve anxiety, and the results of other behavioral experiments directly related to autism spectrum disorder, such as the three-box experiment, the Y-maze and the self-grooming experiment, further prove that ginkgolide A does have a therapeutic effect on autistic behavior defects (such as: social behavior disorder, repetitive stereotyped behavior, etc.), indicating that ginkgolide A can treat autism spectrum disorder.

Claim 1:
An active ingredient for use in a method of treatment of autism spectrum disorder, wherein the active ingredient is selected from the group consisting of ginkgolide A, an stereoisomer thereof, a crystal form thereof, a pharmaceutically acceptable salt thereof, a derivative thereof, or a combination thereof;
wherein the derivative is selected from the group consisting of <NUM>- (<NUM>'-dimethylaminoethoxy)- ginkgolide A, <NUM>- (<NUM>'- diethylaminoethoxy) - ginkgolide A, <NUM> - ((<NUM>'- methoxy-<NUM>', <NUM>'- dimethyl- <NUM>'- pyridyl) - methoxy) - ginkgolide A, and <NUM>-((<NUM>'-pyridyl) - ethoxy) - ginkgolideA.