Short peptide SP2 and application thereof in preparation of medicine for treating autism of children

Abstract

The invention provides an oligopeptide SP2 (MWPLKTILPLGTA) and application of the oligopeptide SP2 in preparation of a product for preventing and / or treating autism of children, and belongs to the technical field of prevention and treatment of autism of children. It is found for the first time that the oligopeptide SP2 has a good effect on prevention or treatment of children autism, and children autism is obviously improved after treatment. The development of the oligopeptide SP2 into a product for preventing and protecting children autism has important significance.

Description

Technical Field

[0001] The invention belongs to the technical field of prevention and treatment of childhood autism, and specifically relates to a short peptide SP2 and application thereof in preparing a drug for treating childhood autism. Background Art

[0002] Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by social impairment, language communication impairment, narrow interests or activities, and repetitive stereotyped behaviors. There have been many reports on the causes of autism spectrum disorder worldwide, and the causes involve genetic, immune, neuroendocrine and neurotransmitter dysfunction and environmental factors. ASD often occurs in infancy and childhood, and its incidence survey figures have continued to rise worldwide in recent years. Currently, there are millions of children with autism in China, 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, diagnosis and treatment, intervention and prognosis will provide more possibilities for more effective diagnosis and treatment of such disorders and reducing the socioeconomic burden.

[0003] Because the cause of autism is still unclear, the current treatment plan for autism can only target its core behaviors. Common treatments include early behavioral influences, haloperidol, risperidone and aripiprazole, citalopram, fluoxetine, fluvoxamine, memantine and riluzole, drugs such as abaclofen and baclofen to inhibit some behavioral characteristics, psychological intervention, hyperbaric oxygen therapy, etc. These treatments are very expensive but have little benefit, poor efficacy and more adverse reactions such as extrapyramidal reactions, drowsiness, fatigue and loss of appetite, weight gain, and a greater susceptibility to chronic diseases.

[0004] Although a large number of chemical drugs have been used to treat autism in children, considering that biomedicine, especially protein drugs, will become the future development direction, the inventors obtained new short peptide compounds with therapeutic activity for autism in children through screening of high-throughput peptide libraries. Summary of the invention

[0005] In view of this, one of the purposes of the present invention is to provide a short peptide SP2, characterized in that the sequence of the short peptide SP2 is MWPLKTILPLGTA, as shown in SEQ ID NO: 1. The short peptide SP 2 of the present invention is screened through a high-throughput polypeptide library and 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, wherein the dosage form of the preparation includes but is not limited to powder, tablet, pill, granule, capsule, solution, emulsion, suspension, oil. More specifically, the dosage form of the preparation is a 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 short peptide SP2 or the preparation in the preparation of a drug for preventing and / or treating autism in a subject.

[0008] The subject may be any mammal, preferably, the subject is a rat, a mouse, a rabbit, a monkey or a 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 abilities, improving social interaction, and the like.

[0010] The dosage used for prevention and / or treatment of the present invention can be adjusted according to the subject. The recommended dosage of 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 obtains a short peptide SP2 for the first time through screening of a high-throughput peptide library, and its amino acid sequence is MWPLKTILPLGTA, and it is found that it has a good effect in preventing or treating autism in children. It is of great significance to develop SP2 into a product for preventing and / or treating autism in children, and provide more candidate compounds for the research and development of new drugs for treating autism in children. DETAILED DESCRIPTION

[0012] The content of the present invention is described in more detail below in conjunction with the examples. It should be understood that the implementation of the present invention is not limited to the following examples, and any formal modifications and / or changes made to the present invention fall within the scope of protection of the present invention. The implementation methods for which specific conditions are not specified in the examples are usually based on the conditions described in the normal conditions or the conditions recommended by the manufacturer. The raw materials, reagents, and test kits used in the examples are all purchased through commercial channels.

[0013] 1. Construction of an autism SD rat model: 60 SPF-grade SD rats, 40 females and 20 males, with a weight range of 165-215g; raised in the clean-grade rat breeding room of the experimental animal breeding center for one week, and given sufficient feed and drinking water. Fluorescent lamps maintain 12 hours of light per day, the environment is quiet, and the room temperature is about 25°C. Route of administration: oral administration. Frequency and time of administration: 1 time / day. Duration of administration: 14 days. Preparation and grouping of autism model mice: The number of male and female rats were caged together overnight according to a 2:1 ratio. Female rats with vaginal plugs were recorded as the first day of pregnancy (E1), and pregnant female rats were raised separately in separate cages. Pregnant female mice were randomly divided into a control group and a model group: the model group The model group (n = 30) was intraperitoneally injected with 650 mg / kg VPA at E12.5; the offspring born were autistic mice; the control group (n = 10) was intraperitoneally injected with an equal amount of 0.9% sodium chloride solution at E12.5, and the offspring born were normal mice, and the day of birth was the first day after birth. The pups were weaned 21 days after birth, and male offspring were selected for experimental grouping. The newborns of female mice in the model group were randomly divided into 2 groups: SP2 treatment group (n = 10, 5μg / d, × 14d); VPA group (n = 10, no drug administration). The newborns of female mice in the control group were set as blank group (n = 10, injected with an equal amount of 0.9% sodium chloride solution).

[0014] Statistical analysis: GraphPad Prism 9.0 software was used to analyze the experimental data, and the Mann-Whitney Test method was used to compare and analyze the significant differences among the groups. A P value less than 0.05 indicated a statistically significant difference. The experimental results were expressed as mean±SD.

[0015] Experimental grouping and design Learning and memory ability test - Morris water maze test: The blank group, VPA group, and SP2 treatment group mice were subjected to a water maze experiment to test the spatial learning and memory ability of the mice. The water maze is a circular pool with a diameter of 160 cm and a height of 60 cm. The inner wall is black and the depth is 50 cm. The water temperature in the pool is maintained at (25±0.5)℃. The pool is evenly divided into four quadrants, namely SW, NW, SE, and NE. The escape platform is placed in 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.

[0016] Positioning navigation experiment: Put the mice in one of the quadrants, and the time from entering the water to climbing onto the platform is recorded as the escape latency. Train for 6 consecutive days, and record the escape latency of the mice in each group on the 6th day of training. Space exploration experiment: 24 hours after the last training, the space exploration experiment is carried out, the platform in the pool is removed, and the quadrant where the platform was originally placed is used as the target quadrant. The mice are randomly placed in the other 3 quadrants except the target quadrant, and the number of times the mice cross the target quadrant within 60 seconds is recorded.

[0017] Water maze experiment to detect learning and memory ability - Morris water maze test The 6-week-old blank group, VPA group, and SP2 treatment group mice were subjected to a water maze experiment to detect the spatial learning and memory ability of the mice. The water maze is a circular pool with a diameter of 160 cm and a height of 60 cm. The inner wall is black and the depth is 50 cm. The water temperature in the pool is maintained at (25±0.5)℃. The pool is evenly divided into four quadrants, namely SW, NW, SE and NE. The escape platform is placed in the center of one quadrant of the pool, 15 cm away from the edge of the pool, and immersed 1 cm below the water surface. The experiment includes two stages: ① Positioning navigation test: The experiment lasted for 6 days, and each mouse was trained 5 times a day. During training, the mice were placed in the water from the specified position facing the pool wall close to the water surface, and the time it took to find the platform within 90 seconds (escape latency) was recorded. If the pups fail to find the platform within 90 seconds, the tester will guide them onto the platform and make them stay on the platform for 15 seconds before moving away. The latency is recorded as 90 seconds. The escape latency of the pups in each group on the 6th day of training was recorded. ② Spatial exploration experiment: The spatial exploration experiment was conducted 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 pups were randomly placed in the other three quadrants except the target quadrant. The camera system and computer software recorded the number of times the pups crossed the target quadrant within 60 seconds.

[0018] Table 1 Comparison of learning and memory abilities of mice in each group (mean±SD, n=10)

[0019] Compared with the blank control group, the VPA group had a significant difference in the number of lesions: “#”, P<0.05; compared with the VPA control group, the VPA group had a significant difference in the number of lesions: “*”, P<0.05 Results: Under the experimental conditions, the VPA group was compared with the blank group. The escape latency time in the VPA group was significantly longer than that in the blank group, and the difference was statistically significant (P<0.05). The number of crossing the target quadrant in the VPA group was significantly less than that 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 escape latency time in 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 crossing 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 mice.

[0020] Behavioral testing of social interaction experiment: 35 days after the birth of the offspring, the blank group, VPA group and SP2 treatment group were subjected to a three-box experiment to test the offspring's social interaction ability: a transparent box of 60cm×60cm×60cm was prepared, with the partition between each box made of transparent glass, and the middle part was open to allow free entry into each box. A cage of the same specifications was placed on the left and right sides of the box to place the strange mice. The test mouse was placed in the middle box to adapt for 6 minutes; the strange mouse was randomly placed in the cage in the left or right box, and the cage in the other box was empty; the glass resin board was removed so that the test mouse could move freely in the three boxes for 10 minutes; the automatic image acquisition system was turned on to record the contact time between the test mouse and the strange mouse or the empty cage.

[0021] Table 2 Comparison of “sociality” of mice in each group (mean±SD, n=10)

[0022] Compared with the blank control group, the VPA group: “#”, P<0.05; compared with the VPA control group: “*”, P<0.05; the time of contacting with strange mice and the time of contacting with the empty cage in the same group: “&”, P<0.05.

[0023] Results: Under the experimental conditions, the time that the blank group mice spent in contact with the stranger mice was longer than that of the empty cage, and the difference was statistically significant (P < 0.05); the time that the VPA group mice spent in contact with the stranger mice was not statistically significant compared with the time that they spent in contact with the empty cage. Compared with the blank group, the VPA group spent less time in contact with the stranger mice than the blank group, and spent more time in contact with the empty cage than the blank group, and the difference was statistically significant (P < 0.05), indicating that the model was successfully established. The time that the SP2 treatment group mice spent in contact with the stranger mice was longer than that of the empty cage, and the difference was statistically significant (P < 0.05); Compared with the VPA group, the SP2 treatment group spent more time in contact with the stranger mice than the VPA group, and spent less time in contact with the empty cage than the blank group, and the difference was statistically significant. The above results show that the SP2 treatment group can significantly improve the sociality of VPA mice.

[0024] Anxiety detection open field test: The device for the open field test is a 36cm×36cm×36cm black square box without a top, with a white bottom plate and black lines dividing the bottom into 9 equally divided small grids. The experimental time is the 35th day after the mice are born. The day before the experiment, the open field box is placed under the camera to ensure that the image can be clearly collected. The automatic image acquisition system is turned on, and the blank group, VPA group, and SP2 treatment group are placed in the central small square respectively. The number of times the mice cross the grid and stand upright within 30 seconds is observed. The interest activities of the mice are detected.

[0025] Table 3: Comparison of open field test in each group of mice (mean±SD, n=10)

[0026] Compared with the blank control group, the VPA group had a significant difference in the number of lesions: “#”, P<0.05; compared with the VPA control group, the VPA group had a significant difference in the number of lesions: “*”, P<0.05 Results: Under the experimental conditions, the VPA group had significantly lower standing times and crossing grids than the blank group, with a statistically significant difference (P<0.05), indicating that the model was successfully established. The SP2 treatment group had higher standing times and crossing grids than the VPA group, with a statistically significant difference (P<0.05), indicating that the SP2 treatment group can effectively increase the standing times and crossing grids of VPA mice.

[0027] Twenty-five patients who met the diagnostic criteria for autism were selected, including 15 males and 10 females, aged 5 to 12 years old, with an average age of (8.72±2.42) years old. All family members of the subjects signed informed consent. Inclusion criteria: Meet the ICD-10 diagnostic criteria for autism in children, diagnosed by two or more attending physicians; age 5 to 12 years old; normal blood routine, liver and kidney function, and electrocardiogram; no history of food or drug allergies; no history of surgery; patients and their families voluntarily signed informed consent. Exclusion criteria: severe physical diseases and organic brain diseases; allergic constitution; previous history of other mental illnesses; receiving one or more treatments before enrollment; bleeding tendency or abnormal bleeding history.

[0028] Research methods: The study was divided into three phases: screening phase (3-6 days), treatment phase (8 weeks) and maintenance phase (4 weeks). Qualified subjects entered the treatment phase and received 8 weeks of SP2 treatment (2 μg / d, intravenous injection), followed by a 4-week SP2 maintenance treatment phase (1 μg / d, intravenous injection).

[0029] Efficacy evaluation: Trained researchers scored the Autistic Behavior Checklist (ABC) before treatment and 4, 8, and 12 weeks after treatment. ABC includes 57 items in five major factors, including 9 items for sensation, 12 items for communication, 12 items for physical movement, 13 items for language, and 11 items for self-care. The scores are 4, 3, 2, and 1 points respectively according to the load size in the scale. 53 points are the critical value, and a total score greater than 67 points can be diagnosed as autism. The higher the score, the more severe the symptoms. Parents fill in the form during follow-up. After all the forms are filled in, specialists will give the appropriate load points to the items that are answered "yes" according to the scale regulations, and the total score will be added up. In this study, the ABC scale was used to evaluate the changes before and after treatment. The reliability and validity of the ABC scale are good, and it can be used for the symptom assessment of autism.

[0030] Table 4: Comparison of ABC scores before and after treatment (points)

[0031] *: Results after 4 weeks of treatment compared with those before treatment; #: Results after 8 weeks of treatment compared with those after 4 weeks of treatment; &: Results after 12 weeks of treatment compared with those before 8 weeks of treatment.

[0032] Results: The total ABC scores decreased in the 4th, 8th, and 12th weeks of treatment compared with those before treatment, and the difference was statistically significant (P < 0.05). This shows that SP2 is effective in treating autism in children, and the longer the duration of treatment, the better the effect.

[0033] In summary, SP2 short peptide can significantly treat autism in children, and it is of great significance to develop SP2 short peptide into a product that can prevent and protect children from autism.

[0034] The applicant declares that the above embodiments are used to illustrate the product, use and use of the present invention, but the present invention is not limited to the above detailed uses or use, that is, it does not mean that the present invention must rely on the above detailed uses and use to be realized. Personnel in the relevant technical field should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention and addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A short peptide SP2 with the function of treating autism in children, 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 according to claim 1, wherein the preparation is in the form of powder, tablet, solution or emulsion.

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

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

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

6. The use according to claim 5, characterized in that The person is a child.

Images