CN109675037B

CN109675037B - Application of ATP and receptor thereof in preparation of drugs for treating autism

Info

Publication number: CN109675037B
Application number: CN201910074968.1A
Authority: CN
Inventors: 高天明; 王倩; 孔颖; 尤强龙; 黄浪; 杨建明
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2019-01-25
Filing date: 2019-01-25
Publication date: 2021-09-14
Anticipated expiration: 2039-01-25
Also published as: CN109675037A

Abstract

The invention discloses application of ATP and an ATP receptor in preparation of a medicament for treating autism. The invention proves that ATP and ATP gamma s which are analogues thereof have the function of treating the autism through experimental research, so that the ATP and ATP gamma s which are analogues thereof can be used for preparing the medicine for treating the autism, and ATP receptors can be used as targets for screening the medicine for preventing and treating the autism, and the invention has wide application prospect.

Description

Application of ATP and receptor thereof in preparation of drugs for treating autism

The technical field is as follows:

the invention belongs to the field of medicines, and particularly relates to application of ATP or ATP gamma s which is a derivative thereof in preparation of drugs for treating autism and application of an ATP receptor serving as a target for screening drugs for preventing and treating autism.

Background art:

autism seriously jeopardizes human life and health. The typical core symptom of autism is a neurodevelopmental disorder with social communication and social interaction disorder and stereotyped and repeated behaviors and interests. The prevalence rate in European and American areas is about 1/110, and the prevalence rate of infants in China is about 4/1000. Although autism continues to be "epidemic," the etiology and pathogenesis of autism have been poorly understood by prior studies.

Treatment of autism presents serious challenges. Because the pathogenesis and mechanism of autism are not completely understood, the current treatment of autism spectrum disorder is mainly behavior intervention and behavior correction. Although behavioral therapy can partially improve symptoms of patients with autism and improve life treatment, the treatment is still palliative and not permanent, so that many researchers are dedicated to the research of drugs for autism. Drug treatment can significantly improve the patient's hyperactivity and mood problems, but there is no evidence that drugs can alleviate the core symptoms of autism. With the deep understanding of genetic and environmental influence factors of autism spectrum disorders and the effective use of animal models, the basic research on autism will strongly advance the process of searching for reasonable therapeutic drugs.

Adenosine triphosphate, abbreviated as ATP. ATP consists of adenosine and three phosphate groups, the chemical formula is C10H16N5O13P3, the structural formula is C10H8N4O2NH2(OH)2(PO3H)3H, and the molecular weight is 507.184. Is an organic compound containing high-energy phosphate bonds, and a large amount of chemical energy is stored in the high-energy phosphate bonds.

ATP is a direct source of energy for all vital activities of tissue cells in the body and is also an important signal molecule in cells. It has been shown that extracellular ATP has a role as a neurotransmitter and in regulating nerves in the central nervous system, and can be used as a growth stimulator and a trophic factor to promote proliferation, development and death of neurons and glial cells via P2 receptors. Therefore, it is believed that extracellular ATP plays a very complex role in the central nervous system, and after binding to the specific P2 receptor, it can play an important physiological role in the nervous system.

At present, ATP is mainly used for treating craniocerebral trauma posttraumatic syndrome, muscular atrophy, hepatitis, arrhythmia and other heart diseases in the aspect of clinical application.

ATP is widely distributed in the organism, and has no toxic or side effect when being used in conventional dosage.

The invention content is as follows:

the first purpose of the invention is to provide the application of ATP or ATP gammas which are derivatives of ATP in preparing the medicine for treating the autism.

The invention discovers that:

ATP and ATP gamma s which are derivatives of ATP can effectively improve the self-closed behavior of experimental mice. In order to detect the effect of ATP and ATP gammas which are derivatives of ATP, the inventor finds that an IP3R2 whole-body knockout mouse which silences astrocytes shows self-closing-like behaviors, detects that the social ability of the mouse is damaged through a three-box experiment, and finds that stereotypical behaviors are existed in a bead burying experiment and a hair care experiment, because the mouse can be used as a model for the research of treating the self-closing diseases; to further optimize the model, excluding the effect of whole body knockout mice on development, mice specifically expressing Cre recombinase from astrocytes introduced with Aldh1l1-CreER and IP3R 2-conditional knockout mice were mated to obtain astrocyte-specific knockout IP3R2 mice (i.e., IP3R 2-conditional knockout mice). On an IP3R2 mouse with an astrocyte specific knockout obtained, three cases of experiments are screened to find that the social ability of the mouse is damaged; meanwhile, a bead burying experiment and a hair trimming experiment are screened to find that the mouse also shows stereotypy behavior, so that the IP3R2 mouse with the astrocyte specific knockout can also be used as a model for autism research.

To further investigate the effect of ATP in autism, the inventors investigated the level of neurotransmitters in the brains of IP3R2 full-body knockout mice, where ATP concentrations were found to be significantly increased in prefrontal cortex release; at the same time, we took the astrocytes and neurons in the brain bag of the mouse for primary culture, and found that the decrease in ATP level was derived from astrocytes rather than neurons. Neurotransmitter levels were also examined in astrocyte-specific knockout IP3R2 mice, and a significant increase in ATP concentration released in the prefrontal cortex was also found.

To determine that ATP and its derivative ATP γ s are able to treat autism, the inventors further validated by pharmacological experiments. Carrying out abdominal cavity ATP treatment on the IP3R2 full-knock mouse, and finding that the self-imposed behavior of the model mouse is reversed; meanwhile, the inventor carries out abdominal cavity ATP treatment on IP3R2 mice with astrocyte specific knockout, and the result shows that ATP can well treat the core symptom of autism, namely social impairment. To rule out the effects of ATP rapid metabolism, the inventors also used the derivative ATP γ s lateral ventricle administration to treat self-closing-like behavior in astrocyte-specific knockout IP3R2 mice, and found that it could also act therapeutically.

Therefore, the invention provides the application of the ATP receptor stimulant in preparing the medicine for treating the autism.

The second purpose of the invention is to provide the application of the ATP receptor as a target for screening drugs for preventing and/or treating autism.

The third purpose of the invention is to provide the application of ATP level as a biomarker for screening autism, or an early warning or clinical diagnosis reagent for screening autism.

The fourth object of the present invention is to provide a medicament for treating autism, which is characterized by comprising an effective amount of an ATP receptor agonist, and a pharmaceutically acceptable carrier.

The ATP receptor agonist is ATP, derivative ATP gammas, or other compound or polypeptide.

The ATP or the derivative ATP gamma s can be replaced by compounds or polypeptides designed aiming at the ATP or the derivative ATP gamma s receptor, and the ATP or the derivative ATP gamma s receptor is mainly stimulated to achieve the same effect as the ATP or the derivative ATP gamma s receptor. The medicament for treating the autism can be in a liquid dosage form or a solid dosage form.

The liquid preparation can be injection, solution, suspension, emulsion or aerosol.

The solid dosage form is tablet, capsule, pill, powder injection, sustained release preparation or various microparticle drug delivery systems.

The invention proves that ATP and ATP gamma s which are analogues thereof have the function of treating the autism through experimental research, so that the ATP and ATP gamma s which are analogues thereof can be used for preparing the medicine for treating the autism, and ATP receptors can be used as targets for screening the medicine for preventing and treating the autism, and the invention has wide application prospect.

The ATP and ATP gamma s are commonly used clinically, and the medicament has small toxic and side effects and low price.

Description of the drawings:

FIGS. 1a and b are graphs showing the results of a three-box social behavior experiment in IP3R2 full-knock mice and wild-type mice; FIG. 1c is a graph of the results of a full knock-out IP3R2 mouse and a wild type mouse in a bead burying experiment; FIG. 1d is a graph of the results of hairstyling experiments in IP3R2 full knock-out mice and wild type mice; FIG. 1e, f is a graph of the results of IP3R2 conditional knockout mice and control mice in a three-box social behavior test; FIG. 1g is a graph of the results of a conditional knockout IP3R2 mouse and a control mouse in a bead burying experiment; FIG. 1h is a graph of the results of conditioned knockout IP3R2 mice and control mice in hairstyling experiments;

FIG. 2a is a graph showing the results of collecting cerebrospinal fluid from brain regions of mPFC using microdialysis technique in IP3R2 full knock-out mice and wild type mice, followed by determination of neurotransmitter using high performance liquid chromatography; FIG. 2b is a graph showing the results of determining ATP levels in brain regions of mPFCs in IP3R2 full knock-out mice and wild type mice; FIG. 2c is a graph of the results of IP3R2 full knock-out mouse phase and wild type mice in determining the ATP levels released by primary cultured astrocytes in mPF brain regions; FIG. 2d is a graph of the results of IP3R2 full knock-out mice and wild type mice determining ATP levels released from primary cultured neurons in mPF brain regions; FIG. 2e is a graph of the results of IP3R2 conditioned knockout and control mice collecting cerebrospinal fluid from the brain region of mPFC using microdialysis techniques followed by neurotransmitter determination using high performance liquid chromatography; FIG. 2f is a graph of the results of determining ATP levels in brain regions of mPF in IP3R2 conditioned knockout and control mice;

3a and b are graphs showing the results of three cases of social behavior tests detected after ATP is administered to the abdominal cavity of IP3R2 full knock-out mice and wild-type mice; FIG. 3c is a graph showing the results of a detection beading experiment after ATP is administered to the abdominal cavity of IP3R2 full knock-out mice and wild-type mice; FIG. 3d is a graph showing the results of a hair care assay performed after ATP is administered to the abdominal cavity of IP3R2 full-knock mice and wild-type mice; FIG. 3e, f is a graph showing the results of three-box social behavior test performed after ATP was administered to the abdominal cavity of an IP3R2 conditional knockout mouse and a control mouse; FIG. 3g is a graph showing the results of a detection beading experiment after ATP is administered to the abdominal cavity of an IP3R2 conditional knockout mouse and a control mouse; FIG. 3h is a graph showing the results of a hairiness test performed after ATP is administered to the abdominal cavity of an IP3R2 conditioned knockout mouse and a control mouse; 3i, j are graphs of the results of experiments for detecting three cases of social behaviors after ATP γ s administration to lateral ventricles of IP3R2 conditional knockout mice and control mice; FIG. 3k is a graph showing the results of a detection beading experiment after lateral ventricle administration of ATP γ s in IP3R2 conditioned knockout and control mice; FIG. 3l is a graph showing the results of hair care experiments performed after administering ATP γ s to lateral ventricle of IP3R2 conditioned knockout and control mice, and FIG. 3 represents that SALINE (in case of intraperitoneal administration) or ACSF (in case of lateral ventricle administration) is a solvent for ATP and + represents that ATP/ATPrs is administered.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1 behavioral Studies of autism model IP3R2 full-knock mice and IP3R2 full-knock mice

1. Three-box experiment

The three-box experiment is a mature experiment widely applied to mouse social behavior detection. The experimental equipment is a transparent box with the thickness of 45cm multiplied by 25cm multiplied by 38cm, the box is divided into three box chambers by two transparent partition plates in the middle, and the partition plates are provided with doors and can be opened and closed freely. In addition, a small metal cage camera is arranged above the box and used for collecting videos and recording mouse behaviors.

The experimental process is divided into three stages, namely an adaptation stage, wherein the experimental mouse is placed in a middle box room, two side doors are opened, and the mouse can freely pass through the three box rooms for exploration for 10 minutes; then in the social stage, the experimental mouse is put back into the middle box room, the two side doors are closed, a strange mouse (the age and the sex of the mouse are matched with those of the experimental mouse) is put into the small metal cage on one side of the box room and then put into the box room on one side, and then the two side doors are opened to allow the experimental mouse to explore and socialize for 10 minutes; and finally, in a social new and different stage, the experimental mouse is put back to the middle, the door is closed, a new strange mouse is put into the small metal cage on the other side and placed in the box room on the other side, and the door is opened to allow the experimental mouse to freely explore and socialize for 10 minutes. The retention time of the laboratory mouse in each chamber and the time for smelling the small metal cage are manually recorded by the experimenter according to the video for statistics.

2. Experiment of embedding beads

The experimental mice were placed in the experimental room for adaptation 1 hour in advance. Fresh padding 5cm thick was spread in a mouse cage, and 20 black glass beads (14 mm in diameter) were gently placed on the padding surface in a 4X 5 manner. The mouse is gently placed in the middle of the mouse cage, and the cage cover is covered. The mice were removed after 30 minutes and the number of buried glass beads was recorded (greater than 1/2 volumes of buried glass beads counted as buried beads).

3. Hair-conditioning experiment

Normal mice usually have spontaneous stereotypical behaviors such as circling in place, jumping, combing, etc., but if a certain behavior is repeated for a long time, it is considered to be an abnormal behavior. The experimental mice are independently placed into a mouse cage paved with a thin layer of fresh padding, the cage is covered to adapt for 10 minutes, and then the total time shared by the hair-care behaviors of the mice in the next 10 minutes is recorded.

4. Experimental animals: an IP3R2 full knockout mouse (namely an IP3R2 full body knockout mouse) experimental group and a littermate wild type mouse are used as a control group; an IP3R2 conditional knockout mouse experimental group and a littermate control mouse are used as control groups;

IP3R2 full knockout mice, IP3R2 conditional knockout mice were generously given by professor chenopodium, the manufacturing process was reported, specific references: li, X.D., Zima, A.V., Sheikh, F., Blatter, L.A. & Chen, J.Endothelin-1-induced arhytogenic Ca2+ signalling is inactive in alkaline microorganisms of inositols-1, 4, 5-trisphosphine (IP3) -receptor type 2-specific mice. circ Res 96, 1274-and 1281, doi:10.1161/01.Res.0000172556.05576.4C (2005) which is a mouse obtained after mating a 539bp loxp site-containing fragment in exon 3, C57 chimera 57BL/6J mother mouse and SWISS mother mouse to obtain IP3R2 floxed. IP3R2Floxed mice were mated with Pro-cre mice to obtain IP3R2 full knock-out progeny (IP3R2 full knock-out mice). The Aldh1l1-CreER mouse (IP3R2 conditional knockout mouse) can be customized from Nanjing biomedical research institute of Nanjing university, the mouse utilizes CRISPR/Cas9 technology to transfer an exogenous fusion protein Aldh1l1 promoter, the fusion protein is fused from Cre recombinase and estrogen receptor, and can activate the expression of Cre recombinase in the presence of estrogen. The Cre recombinase can specifically recognize loxp sites, and the purpose of knocking out a certain gene is achieved.

The experimental method comprises the following steps: carrying out autism-like ethological detection on two groups of IP3R2 full-knock mice and wild-type mice; IP3R2 conditional knockout mice and control mice were evaluated for autism-like behavior.

The experimental results are as follows:

the social phase of the IP3R2 full knock-out mice in the three-box experiment was significantly decreased in the time for mice to socialize compared to the wild-type mice, which indicates that the social capacity of the IP3R2 full knock-out mice was impaired compared to the wild-type mice (fig. 1 a); in the social neo-phase, the search time for newly-placed mice was not significantly different between IP3R2 full-knock mice and wild-type mice, indicating that there was no difference in social neo-potency between the two (fig. 1 b). It can be seen that the IP3R2 full-knock mice exhibited the core symptoms of autism: social interaction and social interaction disability.

IP3R2 full knock-out mice had a significant increase in the number of buried beads compared to wild type mice in the buried bead experiment (fig. 1 c); in hair grooming experiments, the hair grooming time of the IP3R2 full-knock mice was significantly increased compared to wild-type mice (fig. 1d), both experiments indicating that IP3R2 full-knock mice exhibit another core symptom of autism: the stereotypy was repeated.

The results show that the IP3R2 full-knock mouse can be used as a model book for the study of the autism and can be used for the study of the autism drugs.

The social phase of the IP3R2 conditional knockout mice in the three-box experiment was significantly reduced in mice social time compared to the control mice, which indicates that the IP3R2 conditional knockout mice were impaired in social ability compared to the control mice (fig. 1 e); in the social neo-phase, the IP3R2 conditional knockout mice and the control mice had no significant difference in the exploration time for the newly placed mice, indicating that there was no difference in the social neo-potency (fig. 1 f). Thus, the IP3R2 conditional knockout mice also exhibited the core symptoms of autism: social interaction and social interaction disability.

In the bead burying experiment, the number of buried beads was significantly increased in IP3R2 conditional knockout mice compared to control mice (fig. 1 g); in hair grooming experiments, the hair grooming time of the IP3R2 conditional knockout mice was significantly increased compared to control mice (fig. 1h), both experiments indicating that the IP3R2 conditional knockout mice also exhibit another core symptom of autism: the stereotypy was repeated.

The above results all show that the IP3R2 conditional knockout mouse can be used as a model for the study of autism and can be used for the study of autism drugs.

In conclusion, the mice of two strains, namely the IP3R2 full-knockout mouse and the IP3R2 conditional knockout mouse, can be used as animal models for researching the autism, can be used for researching the occurrence and development of the autism, and can also be used for researching the autism drugs.

Example 2: study of transmitter levels in autism model IP3R2 full knockout mice and IP3R2 conditional knockout mice

1. Microdialysis experiments

A semipermeable membrane catheter (CMA7, Sweden) is implanted into a specific brain area 12h in advance through a stereotactic technique, a probe connected with a microdialysis system is placed into a catheter at the head of a conscious mouse during an experiment, and the mouse is placed into a free-moving device to move freely. The perfusion pump (CMA402, Sweden) injects filtered artificial cerebrospinal fluid to replace extracellular fluid, and the dialyzed fluid is collected by an automatic sample collector (CMA820, Sweden) and stored at 4 degrees.

2. Pre-column derivatization reversed-phase high performance liquid chromatography determination

I.e. aspartic acid, glutamic acid, serine, glutamine, glycine, r-aminobutyric acid, were pre-column derivatised by OPA-FMOC and then measured by means of an RP-HPLC system equipped with a fluorescence detector (Agilent 1200, Waldbronn, Germany). The pre-column derivatizing agent was an o-phthalaldehyde/mercaptoethanol containing solvent (containing 0.4M borate, 0.04M o-phthalaldehyde, 0.4M mercaptoethanol, PH 10.4), mobile phase a was containing 0.5% by mass tetrahydrofuran (PH 7.2), and mobile phase B was PB (phosphate solution) -methanol-acetonitrile (50:35:15, v/v). The elution procedure was 100% solution A + 0% solution B (0min, start), 0% solution A + 100% solution B (25min, end).

Experimental animals: an IP3R2 full-knock mouse experimental group and a littermate wild type mouse are used as control groups; an IP3R2 conditional knockout mouse experimental group and a littermate control mouse are used as control groups;

the experimental method comprises the following steps: embedding sleeves in an IP3R2 full-knock mouse and a wild mouse, connecting a microdialysis system after rest, and collecting cerebrospinal fluid for high performance liquid chromatography detection.

And burying a sleeve in the IP3R2 condition knockout mouse and the control mouse, resting, connecting a microdialysis system, and collecting cerebrospinal fluid to perform high performance liquid chromatography detection.

The experimental results are as follows:

as shown in fig. 2a and b, no difference was found in neurotransmitter levels in prefrontal cortex of IP3R2 full-knock-out mice compared with wild-type mice; but ATP levels were significantly reduced. Further primary cultures of neurons and astrocytes from mouse brains were performed, and it was found that this reduced ATP levels was due to a reduced release of ATP from astrocytes, while the ATP levels released from neurons were unchanged (FIG. 2c, d). The result shows that the reduction of ATP level can be used as an index for screening the autism and participate in the generation and development process of the autism.

To rule out the effect of full knockout mice during development, the inventors further examined neurotransmitter levels using IP3R2 conditional knockout mice. As shown in fig. 2e, f, IP3R2 conditional knockout mice showed no change in prefrontal cortex neurotransmitters compared to control mice, but also found significantly reduced levels of ATP. The results further indicate that the reduction of ATP level can be used as an index for screening the autism and participate in the generation and development process of the autism.

Example 3: pharmacological study of ATP and ATP gamma s derivative in treatment of autism

Experimental animals: IP3R2 full knockout mice and IP3R2 conditional knockout mice

Stereoscopic positioning buried pipe

Fixing a needle holder on a stereotaxic apparatus, fixing the holder with a tube on a stereotaxic arm, positioning and marking according to the coordinates of a target nucleus, drilling a hole by using a handheld rotary head, slowly inserting the needle to the positioning depth of a target lateral ventricle, modulating the glass ionomer cement to be semi-solidified, carefully coating the glass ionomer cement on an insertion site, carefully removing the needle holder after the cement is solidified, keeping the catheter in the brain of the mouse, and sealing the plastic tube to avoid causing infection. For pre-experimental dosing, an injection tube was gently passed into the catheter and 0.5ul of drug was administered by a microinjection pump (QSI, N53311, STOELING, USA) using a 5ul Hamilton needle.

The experimental method comprises the following steps:

IP3R2 full knock-out mice and wild type mice were administered to the abdominal cavity with ATP therapy or solvent control groups for detection of self-imposed behavior.

IP3R2 conditional knockout mice and control mice were administered to the abdominal cavity with ATP therapy or a solvent control group for detection of self-imposed behavior.

Implanting micro-dose cannulas into the IP3R2 conditional knockout mice and the control mice by operation, recovering for seven days, performing single-dose ATP gammas or solvent control group by using a micro-dose system, and performing behavioral detection after 30 min.

The experimental results are as follows:

as shown in fig. 3a, b, IP3R2 full knock mice improved impaired social ability in a three-box experiment 30 minutes after intraperitoneal ATP treatment; the drug had no effect on wild type mice. As shown in fig. 3c, d, IP3R2 full knock mice also exhibited improved stereotypy behavior in the beading experiment and grooming use after intraperitoneal ATP treatment.

As shown in fig. 3e, f, IP3R2 conditional knockout mice improved impaired social ability in three-box experiments 30 minutes after intraperitoneal ATP treatment; the drug had no effect on control mice. As shown in fig. 3g, h, IP3R2 conditioned knockout mice also demonstrated improved stereotypical behavior in bead burying experiments and hair grooming following intraperitoneal ATP therapy.

As shown in fig. 3i, j, IP3R2 conditional knockout mice also improved impaired social ability in three-box experiments 30 minutes after lateral ventricle ATP γ s treatment; the drug had no effect on control mice. As shown in fig. 3k, l, IP3R2 conditioned knockout mice also demonstrated improved stereotypical behavior in bead burying experiments and hair grooming following lateral ventricle ATP γ s treatment.

The experiments show that ATP and ATP gamma s which are derivatives of the ATP have the effect of treating autism, and have the characteristics of quick response, less side effect and the like. ATP receptor can be used as target for screening new drugs for preventing and treating autism, ATP and ATP receptor level can be used as autism biomarker and detection target for developing autism early warning and clinical diagnosis reagent (kit).

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

The application of ATP gamma s in preparing the medicine for treating autism.