A short peptide SP2 and its application in the preparation of drugs for treating childhood autism

Abstract

The present invention provides a short peptide SP2 (MWPLKTILPLGTA) and its application in the preparation of products for preventing and / or treating childhood autism, belonging to the technical field of prevention and treatment of childhood autism. The present invention discovers for the first time that the short peptide SP2 achieves good effects in preventing or treating childhood autism, and significantly improves childhood autism after treatment. Developing the short peptide SP2 into a product for preventing and protecting childhood autism is of great significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of prevention and treatment of childhood autism, and particularly relates to a short peptide SP2 and its application in the preparation of drugs for treating childhood autism. Background Art

[0002] Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders mainly characterized by social impairment, language communication disorder, restricted interests or activities, and repetitive stereotyped behaviors. There have been many reports on the causes of autism spectrum disorder worldwide, and the causes involve genetics, immunity, neuroendocrine and neurotransmitter dysfunction, and environmental factors, etc. ASD often occurs in infancy, and in recent years, the incidence survey figures have been rising globally. Currently, the number of autistic children in China is in the millions, and there are still many potential patients who have not been identified. Establishing a clinical research cohort for childhood autism and conducting in-depth research on the etiology mechanism, diagnosis and treatment, intervention, and prognosis will provide more possibilities for more effectively diagnosing and treating such disorders and reducing the social and economic burden.

[0003] Because the etiology of autism is not yet clear, the current treatment plan for autism can only target its core behaviors. Common treatment methods include early behavioral influence, haloperidol, risperidone, and aripiprazole, citalopram, fluoxetine, fluvoxamine, memantine, and riluzole, abaclofen, baclofen and other drugs to inhibit some behavioral characteristics, psychological intervention, hyperbaric oxygen therapy, etc. These treatment methods have high treatment costs but little benefit, with poor curative effects and many adverse reactions, such as extrapyramidal reactions, drowsiness, fatigue, and decreased appetite, weight gain, and a series of problems such as being more prone to chronic diseases.

[0004] Although a large number of chemical drugs have been applied to the treatment of childhood autism, considering that biomedicine, especially protein drugs, has become the future development direction, the inventor has obtained a new short peptide compound with therapeutic activity against childhood autism through the screening of a high-throughput polypeptide library. Summary of the Invention

[0005] In view of this, one of the purposes of the present invention is to provide a short peptide SP2, which is characterized in that the sequence of the short peptide SP2 is MWPLKTILPLGTA, and the sequence is shown as SEQ ID NO: 1. The short peptide SP2 of the present invention is obtained through the screening of a high-throughput polypeptide library and is synthesized by a conventional solid-phase synthesis method in the art.

[0006] Another object of the present invention is to provide a preparation that ultimately contains the short peptide SP2, and the dosage forms of the preparation include but are not limited to powder, tablet, pill, granule, capsule, solution, emulsion, suspension, and oil. More specifically, the dosage form of the preparation is powder. Specifically, the preparation includes a medically acceptable carrier. More specifically, the carrier includes but is not limited to excipients, buffers, emulsifiers, stabilizers, diluents, binders, and preservatives.

[0007] Another object of the present invention is to provide the use of the above-mentioned short peptide SP2 or preparation in the preparation of a drug for preventing and / or treating autism in a subject.

[0008] The subject can be any mammal. Preferably, the subject is a rat, mouse, rabbit, monkey or human, and the human is a child.

[0009] The prevention and / or treatment of the present invention is achieved by improving one or more of learning and memory ability, social interaction, etc.

[0010] The dosage used for the prevention and / or treatment of the present invention can be adjusted according to the subject. The recommended dosage of the SP2 short peptide is 0.001 - 8 μg / d, and specifically, 0.001 μg / d, 0.005 μg / d, 0.05 μg / d, 0.1 μg / d, 0.5 μg / d, 1 μg / d, 2 μg / d, 3 μg / d, 4 μg / d, 5 μg / d, 6 μg / d, 8 μg / d can be selected. Beneficial effects

[0011] The present invention first obtains a short peptide SP2 through the screening of a high-throughput polypeptide library, and its amino acid sequence is MWPLKTILPLGTA. It is found that it has good curative effects in preventing or treating childhood autism. Developing SP2 into a product with the function of preventing and / or treating childhood autism is of great significance, and it provides more candidate compounds for the new drug research and development of drugs for treating childhood autism. Specific embodiments

[0012] The content of the present invention will be described more specifically below in conjunction with the embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any formal modification and / or change made to the present invention fall within the protection scope of the present invention. The implementation methods without specific conditions noted in the embodiments are usually carried out according to the conditions described in the conventional conditions or according to the conditions recommended by the manufacturer. The raw materials, reagents, and reagent kits used in the embodiments are all obtained through commercial channels.

[0013] 1. Establish an autism SD rat model: 60 SPF-grade SD rats, including 40 females and 20 males, with a body weight range of 165 - 215 g. The rats were housed in a clean-grade rat breeding room in the experimental animal breeding center for one week, provided with sufficient feed and water. The light was maintained at 12 h per day by fluorescent lamps, the environment was quiet, and the room temperature was about 25°C. Administration route: Oral gavage. Administration frequency and time: Once a day. Administration time: 14 days. Preparation and grouping of autism model mice. Male and female rats were caged together overnight according to a ratio of 2:1. Female rats with vaginal plugs were marked as the first day of pregnancy (E1). Pregnant female rats were separated and housed individually. The pregnant female rats were randomly divided into a control group and a model group: In the

[0014] model group (n = 30), 650 mg / kg VPA was intraperitoneally injected at E12.5; the offspring were autism pups; in the control group (n = 10), an equal volume of 0.9% sodium chloride solution was intraperitoneally injected at E12.5, and the offspring were normal pups, and the day of birth was defined as the first day after birth. The pups were weaned 21 days after birth, and male offspring were selected for experimental grouping. The neonatal pups of female rats in the model group were randomly divided into 2 groups: SP2 treatment group (n = 10, 5 μg / d, × 14 d); VPA group (n = 10, no drug administration). The neonatal pups of female rats in the control group were set as the blank group (n = 10, injected with an equal volume of 0.9% sodium chloride solution).

[0015] Statistical analysis: GraphPad Prism 9.0 software was used to analyze the experimental data. The Mann-Whitney Test method was used to compare the significant differences between groups. A P value less than 0.05 indicated a statistical difference, and the experimental results were expressed as mean ± SD.

[0016] Experimental grouping and design

[0017] Detection of learning and memory ability - Morris water maze test: The pups in the blank group, VPA group, and SP2 treatment group were subjected to the water maze experiment to detect their spatial learning and memory ability. The water maze was a circular pool with a diameter of 160 cm and a height of 60 cm. The inner wall was black, the depth was 50 cm, and the water temperature in the pool was maintained at (25 ± 0.5)°C. The pool was evenly divided into four quadrants, namely SW, NW, SE, and NE. The escape platform was in the center of one quadrant of the pool, 15 cm away from the pool edge, and submerged 1 cm below the water surface.

[0018] Place navigation experiment: The neonatal mice were placed in one of the quadrants, and the time from entering the water to climbing onto the platform was recorded as the escape latency. Training was conducted continuously for 6 days, and the escape latency of each group of neonatal mice on the 6th day of training was recorded. Spatial exploration experiment: The spatial exploration experiment was carried out 24 hours after the last training. The platform in the pool was removed, and the quadrant where the platform was originally placed was used as the target quadrant. The neonatal mice were randomly placed in the other 3 quadrants except the target quadrant, and the number of times the neonatal mice crossed the target quadrant within 60 seconds was recorded.

[0019] Learning and memory ability detection in the water maze experiment - Morris water maze test Neonatal mice in the 6-week-old blank group, VPA group, and SP2 treatment group were subjected to the water maze experiment to detect their spatial learning and memory ability. The water maze is a circular pool with a diameter of 160 cm and a height of 60 cm. The inner wall is black, the depth is 50 cm, and the water temperature in the pool is maintained at (25 ± 0.5) °C. The pool is evenly divided into four quadrants, namely SW, NW, SE, and NE. The escape platform is placed at the center of one quadrant of the pool, 15 cm away from the edge of the pool, and submerged 1 cm below the water surface. The experiment includes two stages: ① Place navigation test: The experiment lasted for 6 days, and each neonatal mouse was trained 5 times a day. During training, the neonatal mouse was placed into the water facing the pool wall from a specified position, and the time it took to find the platform within 90 seconds (escape latency) was recorded. If the neonatal mouse failed to find the platform within 90 seconds, the tester guided it onto the platform, let it stay on the platform for 15 seconds and then removed it, and the latency was recorded as 90 seconds. The escape latency of each group of neonatal mice on the 6th day of training was recorded. ② Spatial exploration experiment: The spatial exploration experiment was carried out 24 hours after the last training. The platform in the pool was removed, and the quadrant where the platform was originally placed was used as the target quadrant. The neonatal mice were randomly placed in the other 3 quadrants except the target quadrant, and the number of times the neonatal mice crossed the target quadrant within 60 seconds was recorded by the camera system and computer software.

[0020] Table 1 Comparison of learning and memory abilities of neonatal mice in each group (mean ± SD, n = 10)

[0021]

[0022] Compared with the blank group, in the VPA group: "#", P < 0.05; compared with the VPA group: "*", P < 0.05

[0023] Results: Under the conditions of this experiment, compared with the blank group, the escape latency time of the VPA group was significantly longer than that of the blank group, and the difference was statistically significant (P < 0.05). The number of times the VPA group passed through the target quadrant was significantly less than that of the blank group, and the difference was statistically significant (P < 0.05), indicating that the model was successfully established. Compared with the VPA group, the escape latency time of the SP2 treatment group was significantly reduced, and the difference was statistically significant (P < 0.05). Compared with the VPA group, the SP2 treatment group could significantly reduce the escape latency time and increase the number of times passing through the target quadrant, and the difference was statistically significant (P < 0.05). It shows that the SP2 treatment group can effectively improve the learning and memory ability of VPA offspring rats.

[0024] Behavioral test - social interaction experiment: 35 days after the offspring rats were born, the offspring rats in the blank group, VPA group, and SP2 treatment group were subjected to a three - chamber experiment to detect the social interaction ability of the offspring rats. Prepare a transparent box with a size of 60 cm × 60 cm × 60 cm. The partition between each box is made of transparent glass, and the middle part is open, allowing free entry into each box. Place a cage of the same specification on the left and right sides of the box for placing unfamiliar rats. Put the test rat into the middle box to adapt for 6 minutes; randomly put the unfamiliar rat into the cage in the left or right box, and the cage on the other side box is empty; remove the glass resin board so that the test rat can move freely in the three boxes for 10 minutes; turn on the image automatic acquisition system to record the contact time between the test rat and the unfamiliar rat or the empty cage.

[0025] Table 2 Comparison of "sociality" of offspring rats in each group (mean ± SD, n = 10)

[0026]

[0027] Compared with the blank group: "#", P < 0.05; compared with the VPA group: "*", P < 0.05; for the same group, the contact time with the unfamiliar rat and the contact time with the empty cage: "&", P < 0.05.

[0028] Results: Under the conditions of this experiment: The time that the pups in the blank group spent contacting the strange mouse was longer than that of contacting the empty cage, and the difference was statistically significant (P < 0.05); There was no statistically significant difference in the time that the pups in the VPA group spent contacting the strange mouse and that of contacting the empty cage. Compared with the blank group, the time that the VPA group spent contacting the strange mouse was shorter than that of the blank group, and the time of contacting the empty cage was longer than that of the blank group, and the difference was statistically significant (P < 0.05), indicating that the model was successfully established. The time that the pups in the SP2 treatment group spent contacting the strange mouse was longer than that of contacting the empty cage, and the difference was statistically significant (P < 0.05); Compared with the VPA group, the time that the SP2 treatment group spent contacting the strange mouse was longer than that of the VPA group, and the time of contacting the empty cage was shorter than that of the blank group, and the difference was statistically significant. The above results show that the SP2 treatment group can significantly improve the sociability of VPA pups.

[0029] Anxiety detection open field test: The device for the open field test is a black uncovered square box with a size of 36 cm × 36 cm × 36 cm, its bottom plate is white, and the bottom of the box is divided into 9 equal small grids by black lines. The experimental time was the 35th day after the mice were born. One day before the experiment, the open field box was placed under the camera to ensure that the images could be clearly collected. The image automatic collection system was turned on, and the blank group, VPA group, and SP2 treatment group were placed in the central small square respectively, and the number of grid crossings and the number of upright postures of the pups within 30 s were observed. The interest activities of the pups were detected.

[0030] Table 3: Comparison of the open field test of pups in each group (mean ± SD, n = 10)

[0031]

[0032] Compared with the blank group: "#", P < 0.05; Compared with the VPA group: "*", P < 0.05

[0033] Results: Under the conditions of this experiment: Compared with the blank group, the number of upright postures and the number of grid crossings in the VPA group were significantly lower than those in the blank group, and the difference was statistically significant (P < 0.05), indicating that the model was successfully established. Compared with the VPA group, the number of upright postures and the number of grid crossings in the SP2 treatment group were higher than those in the VPA group, and the difference was statistically significant (P < 0.05), indicating that the SP2 treatment group can effectively increase the number of upright postures and the number of grid crossings of VPA pups.

[0034] Twenty-five patients who met the diagnostic criteria for autism were selected, including 15 males and 10 females, aged 5 to 12 years, with an average age of (8.72 ± 2.42) years. Informed consent was signed by the families of all the subjects. Inclusion criteria: meet the diagnostic criteria for childhood autism in the ICD-10 diagnostic criteria, jointly diagnosed by more than two attending physicians; aged 5 to 12 years; normal blood routine, liver and kidney functions, and electrocardiogram; no history of food or drug allergies; no history of surgery; the patients and their families voluntarily signed the informed consent form. Exclusion criteria: severe somatic diseases and brain organic diseases; patients with allergic constitution; those with a history of other mental diseases; those who were receiving one or more treatments before enrollment; those with a bleeding tendency or a history of abnormal bleeding.

[0035] Research methods: The research was divided into three stages: a screening period (3 - 6 d), a treatment period (8 weeks), and a maintenance period (4 weeks). The subjects who passed the screening entered the treatment period and received SP2 treatment (2 μg / d, intravenous injection) for 8 weeks, and then entered the 4-week (1 μg / d, intravenous injection) SP2 maintenance treatment period.

[0036] Efficacy evaluation: Trained researchers performed the scoring of the Autistic Behavior Checklist (ABC) before treatment and at 4, 8, and 12 weeks after treatment. The ABC includes five major factors and 57 items, namely 9 items of sensation, 12 items of communication, 12 items of physical movement, 13 items of language, and 11 items of self-care. Scores of 4, 3, 2, and 1 points were given according to their loadings in the scale respectively. With a critical value of 53 points, a total score greater than 67 points can be diagnosed as autism, and the higher the score, the more severe the symptoms. During the follow-up, it was filled out by the parents. After all were filled out, the specialist physician gave the corresponding loading scores to the items answered "yes" according to the scale regulations, and the sum of the loading scores of each item was calculated to obtain the total score. In this study, the ABC scale was used to evaluate the changes before and after treatment. The reliability and validity of the ABC scale were both good, and it can be used for the symptom assessment of autism.

[0037] Table 4: Comparison of ABC scores before and after treatment (points)

[0038]

[0039] *: Comparison result between 4 weeks of treatment and before treatment; #: Comparison result between 8 weeks of treatment and 4 weeks of treatment; &: Comparison result between 12 weeks of treatment and 8 weeks of treatment.

[0040] Results: The total ABC scores at the 4th, 8th, and 12th weeks of treatment decreased compared with those before treatment, and the differences were statistically significant (P < 0.05). This indicates that SP2 has the effect of treating childhood autism, and the longer the treatment duration, the better the curative effect.

[0041] In summary, the SP2 short peptide can significantly treat childhood autism, and it is of great significance to develop the SP2 short peptide into a product for preventing and protecting childhood autism.

[0042] The applicant declares that the products, uses and usage methods of the present invention are illustrated by the above-mentioned implementation examples. However, the present invention is not limited to the above-mentioned detailed uses or usage methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed uses and usage methods to be realized. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention and the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A short peptide SP2 with the function of treating childhood autism, characterized in that, The sequence of the short peptide SP2 is MWPLKTILPLGTA, and its sequence is shown in SEQ ID NO:

1.

2. A preparation comprising the short peptide SP2 described in claim 1, and the dosage form of the preparation includes powder, tablet, solution or emulsion.

3. Use of the short peptide SP2 described in claim 1 or the preparation described in claim 2 in the preparation of a drug for preventing and / or treating autism in a subject.

4. The application according to claim 3, characterized in that, The subject is a mammal.

5. The application according to claim 4, characterized in that The subject is selected from rats, mice, rabbits, monkeys or humans.

6. The application according to claim 5, wherein The human is a child.

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