WO2019037225A1 - P2y1受体及其阻断剂在预防和治疗抗抑郁症和/或抗焦虑症中的应用 - Google Patents

P2y1受体及其阻断剂在预防和治疗抗抑郁症和/或抗焦虑症中的应用 Download PDF

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WO2019037225A1
WO2019037225A1 PCT/CN2017/107289 CN2017107289W WO2019037225A1 WO 2019037225 A1 WO2019037225 A1 WO 2019037225A1 CN 2017107289 W CN2017107289 W CN 2017107289W WO 2019037225 A1 WO2019037225 A1 WO 2019037225A1
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receptor
mice
depression
anxiety
antidepressant
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French (fr)
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高天明
王倩
孔颖
吴定宇
杨建明
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南方医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates to the field of biopharmaceutical medicine technology, in particular to screening an antidepressant and/or anti-anxiety disease and a medicament thereof with the P2Y1 receptor as a target, or using a P2Y1 receptor blocker for preparing an antidepressant And/or anti-anxiety drugs.
  • Depression is the most common mood disorder, which is a serious threat to human psychology and physical health. Depression is a type of mood disorder syndrome characterized by low mood, cognitive impairment, and decreased will activity. It has the characteristics of high incidence, high disability rate and heavy burden. According to the World Health Organization, the estimated incidence rate is about It is 15-17%, and there is a rising trend. It is predicted to become the world's first cause of disability in 2020. Depression not only seriously threatens the health and life of patients, but also imposes enormous economic and living burdens on society and families. Therefore, prevention and treatment of depression has become a major public health problem.
  • the current treatment of depression mainly relies on drug treatment, but still faces many serious challenges: First, the drug efficacy is low, about 35% of patients are not sensitive to existing antidepressant drugs; secondly, drug compliance is poor, existing Antidepressants have a slow onset of action and take at least 3 to 6 weeks to work. Many patients will give up halfway. Third, the side effects of the drug are large and the recurrence rate occurs after stopping the drug. Therefore, finding effective targets and investigating new drugs with fast onset, long-lasting effects and few side effects has become an urgent problem to be solved.
  • anxiety disorders and depression are two different mental disorders, they are closely related and often merge. Studies have shown that anxiety symptoms are similar to depression symptoms; they are the same reaction to pathogenic factors, and characteristic symptoms vary from person to person; others believe that as the disease worsens and the disease migrates, either one can appear another. Secondary symptoms of the species. Because there are so many similarities, the two diseases are difficult to identify, so it is recommended that the two be combined.
  • Adenosine triphosphate also known as ATP
  • the ATP receptor is called the P2 receptor and is divided into a gated ion channel P2X receptor family and a G protein coupled P2Y receptor family.
  • the P2X receptor is an ion channel receptor, which can open the channel when activated, change the ion concentration on both sides and play a signal transduction role;
  • P2Y receptor is a G protein coupled receptor, and different subtypes of P2Y receptor activate different cells through G protein.
  • Internal signal Transduction pathways perform specific physiological functions.
  • P2Y receptors There are 8 subtypes of P2Y receptors in mammals, namely P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14, which are composed of 308-379 amino acids. It is a typical G protein with 7 Transmembrane domain, the N-terminus is extracellular and the C-terminus is intracellular.
  • P2Y receptors are widely distributed and have a high density of distribution in the nervous system, and both neurons and glial cells are expressed.
  • P2Y1, P2Y2, P2Y4, and P2Y12 receptors are expressed in the cerebral cortex and hippocampus and cerebellar cortex neurons; ATP, ADP, adenosine and other steroidal transmitters can regulate neuronal excitability and neuronality through different P2Y receptors. Synaptic transmission between them, which in turn regulates local brain circuit activity.
  • glial cells astrocytes, oligodendrocytes, microglia
  • P2Y receptors are involved in pathological pain in the nervous system, play a key role in the production and persistence of pain sensation, and are expected to become new therapeutic targets in therapy. It has also been reported that P2Y receptors are involved in neurodegenerative diseases. Among them, P2Y1 receptor may directly participate in the pathological process of degeneration of the nervous system. It has been reported that P2Y receptors participate in the process of glial cell activation in various ways during brain injury, inhibiting glial cell activity in the early stage of disease, and may become a site of action for treating epilepsy, chronic pain, brain tumors and the like in the future. .
  • agonists and blockers of P2Y1 receptors are mainly used for the treatment of platelet coagulation in the cardiovascular system and immunoinflammatory injury.
  • the object of the present invention is to overcome the above-mentioned deficiencies in the existing drugs for treating depression and anxiety, and to provide a P2Y1 receptor blocker for treating depression and/or anxiety, and also to P2Y1 receptor.
  • Targets are used to screen for antidepressant and/or anti-anxiety diseases and their drugs.
  • P2Y1 receptor systemic knockout mice were introduced from JAX, USA. It has been verified that P2Y1 receptors are no longer expressed in whole body knockout mice (as shown in Figure 4); in forced swimming experiments, P2Y1 receptor systemically knockout mice have significantly reduced immobility time compared to P2Y1 wild type mice.
  • the blocker of the P2Y1 receptor is a compound or polypeptide which inhibits the P2Y1 receptor.
  • the present invention produces an adeno-associated virus specifically interfered by the P2Y1 receptor to observe whether or not it Affect depression-like behavior.
  • the mice were given microinjection of P2Y1 receptor into the ventral dorsal region of the brain to interfere with RNA adeno-associated virus, and then forced swimming test was performed. The results showed that the P2Y1 receptor interfering RNA significantly decreased the expression of P2Y1 receptor in the experimental group compared with the control group.
  • the reduced saccharide preference in socially depressed depressive mice was reversed (as shown in Figure 11).
  • the above results indicate that the P2Y1 receptor is blocked by a blocker or the P2Y1 receptor is down-regulated to exert antidepressant. effect. Therefore, the P2Y1 receptor can be used as a target for screening drugs and clinical diagnostic agents for preventing, treating, and/or anti-anxiety disorders; it can also be used as a biomarker for screening depression and/or anxiety disorders. Early warning of depression and/or anxiety.
  • the invention has found through pharmacodynamic and pharmacological experiments that the specific blocker P2Y1 receptor MRS2500 and its derivatives, MRS2179 and its derivatives can effectively improve the depression state of experimental mice.
  • To detect the antidepressant effect of the specific blocker PRSY1 receptor MRS2500 2.5 mg/kg of MRS2500 was administered intraperitoneally, and a forced swimming test was performed for 6 minutes after 30 minutes. The anti-depressant was evaluated after 4 minutes of swimming.
  • the results showed that administration of MRS2500 and the administration of the traditional antidepressant fluoxetine showed the same antidepressant-like behavior (as shown in Figure 1).
  • the main pathogenesis of depression is thought to be caused by neurological abnormalities such as neuroendocrine and nerve regeneration.
  • the present invention provides 0.1 uM of MRS2500 and 0.2 uM of MRS2179 in the lateral ventricle of the mouse.
  • a forced swimming test lasting 6 minutes was performed after 30 minutes.
  • MRS2500 and MRS2179 significantly reduced the immobility time of mice during forced swimming, indicating that the blocker of P2Y1 receptor has antidepressant effect (as shown in Figure 2).
  • the present invention tested the antidepressant effects of P2Y1 receptor blockers on a socially depressed chronic depression model.
  • C57BL/6J mice received 10 minutes of daily invasiveness from long-term separate feeding of CD1 mice, and were separated by transparent perforated plexiglass plates after body contact.
  • C57BL/6J mice received 24-hour visual and olfactory responses. Excited, successful modeling for 10 consecutive days.
  • a catheter is implanted in the lateral ventricle. After a single administration, the state of the MRS 2500 group of depressed mice was significantly improved. The above results further indicate that MRS2500 has an antidepressant effect after blocking the P2Y1 receptor (as shown in Figure 3).
  • the blocker of the P2Y1 receptor of the present invention can be used for the preparation of an antidepressant and/or anti-anxiety drug comprising an effective amount of a blocking agent for the P2Y1 receptor, and a pharmaceutically acceptable carrier.
  • the blocker of the P2Y1 receptor is a compound or polypeptide that inhibits the P2Y1 receptor.
  • the blocker of the P2Y1 receptor is MRS2179 and its derivatives, or MRS2500 and its derivatives.
  • a preferred pharmaceutical dosage form is a liquid or a solid.
  • the liquid dosage form may be an injection, a solution, a suspension, an emulsion or an aerosol; the solid dosage form may be a tablet, a capsule, a pill, a powder injection, a sustained release preparation or various microparticle delivery systems.
  • the present invention has the following advantages and beneficial effects:
  • the present invention provides a target P2Y1 receptor associated with depression and anxiety, and a blocking agent for the P2Y1 receptor for use in the preparation of an antidepressant and/or anxiolytic agent for depression and
  • a blocking agent for the P2Y1 receptor for use in the preparation of an antidepressant and/or anxiolytic agent for depression and
  • the diagnosis and treatment of anxiety disorders provide new targets and drugs with good application prospects and research value.
  • Figure 1 is a graph showing the results of a forced swimming test after intraperitoneal administration of MRS2500 in C57BL/6J mice;
  • Figure 2 is a graph showing the results of a forced swimming test after MRS2500 and MRS2179 were administered to the lateral ventricle of C57BL/6J mice;
  • Figure 3 is a graph showing the results of a forced swimming test after MRS2500 was administered to mice modeled by a social failure depression model
  • Figure 4 is a graph showing the results of an experiment for detecting the expression level of P2Y1 receptor protein in P2Y1 receptor systemic knockout mice
  • Figure 5 is a graph showing the results of a forced swimming test in a P2Y1 receptor systemic knockout mouse
  • Figure 6 is a graph showing the results of a test of a tail suspension test in a P2Y1 receptor systemic knockout mouse
  • Figure 7 is a map showing the location of interfering RNA molecules involved in interfering RNA in CbBL/6J mice;
  • Figure 8 is a graph showing the results of a forced swimming test in which the interfering RNA molecule of the C57BL/6J mouse was injected into the ventral dorsal region of the brain;
  • Figure 9 is a graph showing the results of a social interaction experiment after a P2Y1 shRNA was administered to a mouse modeled by a social failure depression model
  • Figure 10 is a graph showing the results of a forced swimming test in mice given a social failure depression model after administration of P2Y1 shRNA;
  • Figure 11 is a graph showing the results of a saccharification water preference test in mice given a social failure depression model after administration of P2Y1 shRNA.
  • Accumulated immobility time within 4 minutes after recording (mainly characterized by stopping struggling, floating or erect, or only small limb movements to keep the head out of the water) is an indicator for detecting depression-like behavior.
  • the onset of antidepressant drugs shortens the immobility time of experimental mice.
  • mice C57BL/6J mice, SPF grade, male, weighing 20-24 g, animals were treated by Southern Medicine University of Experimental Animal Center (Guangzhou, China).
  • the mice were placed in SPF-class animal breeding room in cages, 3 to 4 per cage, plastic cage size 300mm ⁇ 170mm ⁇ 120mm, room temperature: 24 ⁇ 1 ° C; light and dark cycle: 12h / 12h; light time: 7:00am ⁇ 7:00 pm; relative humidity 70 ⁇ 85%; indoor environment is quiet, mice can freely eat and drink.
  • the experiments were carried out from 1:00 to 5:00 pm, and the mice were admitted to the experimental environment before the experiment and were adapted by the experimenter.
  • mice In the forced swimming test of C57BL/6J mice, the animals were randomly divided into 5 groups of 10 animals each. Each group of animals was implanted with a drug perfusion cannula in the lateral ventricle. After 7 days of feeding, the four groups of animals were treated as follows: The corresponding reagent was administered using a micro-drug system: control solvent (artificial cerebrospinal fluid, 1 ul, 1 ul/min), MRS 2500 (0.1 uM, 1 ul, 1 ul/min), MRS 2179 (0.2 uM, 1 ul, 1 ul/min), after each group was injected, forced swimming was performed 10 minutes later.
  • control solvent artificial cerebrospinal fluid, 1 ul, 1 ul/min
  • MRS 2500 0.1 uM, 1 ul, 1 ul/min
  • MRS 2179 0.2 uM, 1 ul, 1 ul/min
  • MRS2500 was administered intraperitoneally at 2.5 mg/kg, and forced swimming was performed for 6 minutes after 30 minutes. The time of swimming was measured 4 minutes after the measurement to evaluate the effect of antidepressants.
  • MRS2500 was given the same antidepressant-like behavior as the traditional antidepressant fluoxetine (shown in Figure 1).
  • MRS2500 and MRS2179 in the lateral ventricle of C57BL/6J mice significantly shortened the swimming inactivity time of mice, indicating that the blocker of P2Y1 receptor has a clear antidepressant effect.
  • the social failure model is carried out in accordance with Golden's method.
  • C57BL/6J mice were randomly divided into control group and stress group.
  • the stressed group of C57BL/6J mice were placed in the screened CD-1 mouse cage daily for 10 min.
  • the C57BL/6J will show signs of escape, fear, and obedience after being attacked.
  • C57BL/6J mice can smell, see, and hear the opposite CD-1 within 24 hours after contact with the transparent perforated septum for psychological stress.
  • the C57BL/6J is rotated every day to ensure a balanced stimulus.
  • the experiment was carried out for 10 days.
  • CD-1 in the control mice was replaced by another C57BL/6J, rotating daily, but avoiding physical contact. After the modeling was completed, the mice were housed in a single cage and subjected to lateral ventricular catheter implantation.
  • control group and the experimental group were given the corresponding reagents: control solvent (artificial cerebrospinal fluid, 1 ul, 1 ul / min), MRS 2500 (0.1 uM, 1 ul, 1 ul / min), and the level of depression in the mice was measured by social interaction experiment after the end of the administration. .
  • the social interaction experiment is divided into two phases, each of which is 2.5 min.
  • No target CD-1 mice were not placed in a clear-pore plexiglass box, and C57BL/6J mice were placed distally, and the residence time in the interaction area and corners within 2.5 minutes was recorded.
  • First Target CD-1 mice were placed in a transparent plexiglass box and C57BL/6J was placed distally, and the residence time in the interaction area and corners within 2.5 minutes was recorded.
  • mice C57BL/6J mice, SPF grade, male, weighing 20-24 g, animals were provided by Experimental Animal Center of Southern Medical University (Guangzhou, China). The mice were placed in SPF-class animal breeding room in cages, 3 to 4 per cage, plastic cage size 300mm ⁇ 170mm ⁇ 120mm, room temperature: 24 ⁇ 1 ° C; light and dark cycle: 12h / 12h; light time: 7:00am ⁇ 7:00pm; relative humidity 70-85%; indoor environment is quiet, mice can eat and drink freely. The experiments were carried out from 1:00 pm to 5:00 pm, and the mice entered the experimental environment before the experiment and were touched by the experimenters.
  • mice in the artificial cerebrospinal fluid group compared with the artificial cerebrospinal fluid group control group significantly reduced the time in the interaction area, indicating successful modeling.
  • Intracerebral perfusion of MRS2500 can increase the time of the experimental group of mice in the interaction area, and the results show that the P2Y1 receptor has an antidepressant effect after specific blockade.
  • mice were purchased from Jax (strain number: 009131). This gene has only one coding exon, and most of the coding regions were interfered by PGK neo cassette. The obtained 129 chimeric mice were backcrossed with C57BL/6J. generation. The experimental animals were kept in a 12/12 hour light-dark conversion environment and were free to eat and drink.
  • mice showed a significant decrease in immobility time in forced swimming experiments compared with wild type mice. The results showed that the mice exhibited antidepressant-like behavior after down-regulation of P2Y1 receptor expression.
  • the experiment used the MED Associates tail suspension device to monitor the movement status of mice in real time.
  • the hanging tail box is open in the front and the other side is closed.
  • a pressure sensor is fixed at the bottom end of the suspension tail rod, and the movement condition of the mouse is input into a computer through a pressure sensor for data processing.
  • 2 cm of the tip of the mouse was fixed in a hanging box (ENV-505TS, Med associates Inc.) so as to be upside down, keeping the head about 10 cm from the bottom of the box.
  • the mouse will struggle exclusively at first, and will find a state of inactivity when it is found that it cannot be undone. That is, the mouse stops struggling or passively swings in a small amplitude.
  • the software Med Suspension Tail automatically records and analyzes the time of immobility in the mouse within 6 min, and the immobility time below the set lower threshold is recorded as an indicator for detecting depression-like behavior.
  • mice were purchased from Jax (strain number: 009131). This gene has only one coding exon, and most of the coding regions were interfered by PGK neo cassette. The obtained 129 chimeric mice were backcrossed with C57BL/6J. generation. The experimental animals were kept in a 12/12 hour light-dark conversion environment and were free to eat and drink.
  • mice showed a significant decrease in immobility time in the tail suspension experiment compared with the wild type mice. The results showed that the mice exhibited antidepressant-like behavior after down-regulation of P2Y1 receptor expression.
  • mice C57BL/6J mice, SPF grade, male, weighing 20 ⁇ 2 g, animals were provided by Experimental Animal Center of Southern Medical University (Guangzhou, China). The mice were placed in SPF animal breeding room in cages, 3 to 4 per cage, room temperature: 24 ⁇ 1 °C; light and dark cycle: 12h/12h; illumination time: 7:00am to 7:00pm; relative humidity 70 ⁇ 85%; the indoor environment is quiet, the mice are free to eat and drink. The experiments were carried out from 1:00 to 5:00 pm, and the mice were admitted to the experimental environment before the experiment, and the experimenters were adapted to adjust.
  • RNA molecule adeno-associated virus 0.5 ul (0.1 ul) in the ventral dorsal region of the brain of each group (angle 7°, AP: -3.2 mm, ML: 1.0 mm, DV: -4.6 mm) / min, after the completion of the indwelling needle for 5 minutes), after 14 days of virus expression recovery, forced swimming test.
  • the P2Y1 specific interference shRNA titer was 1 ⁇ 10 13 vg/ml, 9.55E+08 copy/ul, the packaged virus was adeno-associated virus type 9, the promoter was U6, and the serotype was AAV9. (Packaged by the company, Shenzhen, China); sequence information: the forward sequence is shown as SEQ ID NO: 1; the reverse sequence is shown in SEQ ID NO: 2.

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Abstract

公开了P2Y1受体及其阻断剂在抗抑郁症和/或抗焦虑症中的应用。通过实验研究证实，P2Y1受体可用于筛选抗抑郁症和/或抗焦虑症的药物，还可用于筛选抑郁症和/或焦虑症，及抑郁症和/或焦虑症的早期预警；且P2Y1受体的阻断剂具有抗抑郁和抗焦虑作用，可用于制备抗抑郁症和/或抗焦虑症的药物。

Description

P2Y1受体及其阻断剂在预防和治疗抗抑郁症和/或抗焦虑症中的应用 技术领域
本发明涉及生物药物医学技术领域，具体涉及一种以P2Y1受体为靶点筛选抗抑郁症和/或抗焦虑症疾病及其药物，或者将P2Y1受体的阻断剂用于制备抗抑郁症和/或抗焦虑症的药物。
背景技术
抑郁症是最常见的心境障碍疾病，严重威胁着人类的心理和身体健康。抑郁症是以心境低落、认知功能损伤、意志活动下降为主要临床特征的一类心境障碍综合症，具有发病率高、致残率高、负担重等特点，据世界卫生组织估计发病率约为15～17％，且有不断上升的趋势，预测在2020年会成为全球首位致残原因。抑郁症不仅严重威胁了患者个人的健康和生活，也对社会和家庭造成了极大的经济和生活负担。因此，抑郁症的防治已经成为一个重大公共卫生问题。
现有的抑郁症的治疗主要是依靠药物治疗，但仍然面临诸多严峻的挑战：首先，药物效能低，约35％的病人对现有的抗抑郁药物不敏感；其次，药物依从性差，现有抗抑郁药物起效慢，需至少3～6周才起效，很多患者会半途放弃；第三，药物副作用大且停药后出现高复发率。因此，寻找有效靶点，研究起效快、作用持久、副作用少的新型药物成为了亟需解决的问题。
焦虑症和抑郁症虽然是两种不同的精神障碍疾病，但两者关系密切，经常合并存在。有研究指出焦虑症与抑郁症症状近似；是对致病因素的同一反应，而特征性症状是因人而异；也有人认为是随着病情加重和病情迁徙，任一种都可以出现另一种的继发症状。因为有如此多的相同点，两种疾病很难鉴定，因此建议二者合并治疗。
三磷酸腺苷，又称ATP，是生命体内最直接的能量来源，同时也作为一种重要的神经递质被认识，生物体内广泛存在其相应的受体。ATP的受体叫做P2受体，分为门控离子通道P2X受体家族和G蛋白偶联P2Y受体家族。P2X受体为离子通道受体，在激活时可开启通道，改变两侧离子浓度和发挥信号传递作用；P2Y受体为G蛋白偶联受体，不同亚型P2Y受体通过G蛋白激活不同胞内信号 转导通路，执行特定的生理功能。P2Y受体在哺乳动物中有8个亚型，分别为P2Y1、P2Y2、P2Y4、P2Y6、P2Y11、P2Y12、P2Y13、P2Y14，由308～379个氨基酸组成，是一种典型的G蛋白，含有7个跨膜结构域，N端在胞外，C端在胞内。
P2Y受体分布广泛，且在神经系统分布密度较高，神经元和胶质细胞均有表达。P2Y1、P2Y2、P2Y4、P2Y12受体在大脑皮层及海马、小脑皮层神经元均有表达；ATP，ADP，腺苷等嘌呤类递质可通过不同P2Y受体调节神经元的兴奋性和神经元之间的突触传递，进而调控局部脑环路活动。在胶质细胞(星形胶质细胞、少突胶质细胞、小胶质细胞)上亦有大量P2Y受体表达，参与神经元-胶质细胞相互作用。有研究报道，P2Y受体参与神经系统病理性疼痛，在痛觉的产生和持续过程中起关键作用，有望在治疗中成为新的治疗靶点；也有报道，P2Y受体参与神经系统退行性病变，其中P2Y1受体可能直接参与神经系统退行性变的病理过程。更有报道，P2Y受体在脑损伤过程中以多种途径参与胶质细胞活化过程，在疾病早期抑制胶质细胞活性，将来可能会成为治疗癫痫、慢性痛、脑肿瘤等药物的作用位点。
目前临床应用方面P2Y1受体的激动剂和阻断剂主要用于心血管系统血小板凝血方面以及免疫炎症损伤的治疗。
发明内容
本发明的目的在于：克服现有治疗抑郁症和焦虑症的药物中存在的上述不足，提供一种以P2Y1受体的阻断剂用于治疗抑郁症和/或焦虑症，还以P2Y1受体为靶点用于筛选抗抑郁症和/或抗焦虑症疾病及其药物。
本发明为了确定P2Y1受体的整体水平效应，从美国JAX引进了P2Y1受体全身敲除小鼠。经过验证，在全身敲除小鼠中P2Y1受体不再表达(如图4所示)；在强迫游泳实验中，P2Y1受体全身敲除小鼠相对于P2Y1野生型小鼠不动时间明显降低，表现出抗抑郁样的行为(如图5所示)；在悬尾实验中，P2Y1受体全身敲除小鼠相对于P2Y1野生型小鼠不动时间也明显降低，表现出抗抑郁样的行为(如图6所示)；以上结果进一步证明P2Y1受体阻断可以发挥抗抑郁效应。因此，P2Y1受体的阻断剂可以用于抗抑郁症和/或抗焦虑症。
作为本发明一种进一步的优选技术方案，所述P2Y1受体的阻断剂为抑制P2Y1受体的化合物或多肽。
为了进一步验证P2Y1受体可否作为抗抑郁和/或抗焦虑的靶点，避免全身敲除小鼠可能出现的代偿作用，本发明制作了P2Y1受体特异性干扰的腺相关病毒来观察其是否影响抑郁样行为。给予小鼠脑内腹侧背盖区微量注射P2Y1受体干扰RNA腺相关病毒后，进行强迫游泳实验检测。结果发现，相对于对照组小鼠，实验组小鼠中P2Y1受体干扰RNA显著降低了P2Y1受体的表达；强迫游泳实验中，实验组小鼠的不动时间显著减少，具有明显的抗抑郁效果(如图7～8所示)。发明人进一步使用了社交失败慢性抑郁模型来验证P2Y1受体特异性干扰的腺相关病毒的效果。在抑郁模型造模完成后，小鼠休息一天，给予P2Y1受体特异性干扰的腺相关病毒，两周后进行行为学检测。在社会交互实验中，社交失败的抑郁小鼠状态被逆转(如图9所示)；在强迫游泳实验中，社交失败的抑郁小鼠本来升高的不动时间回到了对照组水平(如图10所示)。在糖水偏好实验中，社交失败的抑郁小鼠降低的糖水偏好被逆转(如图11所示)，以上结果表明P2Y1受体被阻断剂阻断或者P2Y1受体表达水平下调均可以发挥抗抑郁作用。因此，P2Y1受体可作为靶点，用于筛选预防、治疗抗抑郁症和/或抗焦虑症的药物及临床诊断试剂；还可作为生物标志物，用于筛选抑郁症和/或焦虑症以及抑郁症和/或焦虑症的早期预警。
本发明通过药效和药理学实验发现：P2Y1受体的特异性阻断剂MRS2500及其衍生物、MRS2179及其衍生物能有效改善实验小鼠抑郁状态。为检测P2Y1受体的特异性阻断剂MRS2500的抗抑郁作用，腹腔给予2.5mg/kg的MRS2500，30分钟后进行时长6min的强迫游泳实验，测定后4min游泳不动的时间来评估抗抑郁药的效果，结果发现给予MRS2500和给予传统抗抑郁药氟西汀出现一样的抗抑郁样行为(如图1所示)。抑郁症主要的发病机制认为是神经内分泌，神经再生等神经系统异常导致的，因此为了进一步检测抗抑郁药的效果，本发明在小鼠侧脑室埋管给予0.1uM的MRS2500和0.2uM的MRS2179，30分钟后进行时长6min的强迫游泳实验。与对照组相比，MRS2500和MRS2179显著降低了小鼠强迫游泳时的不动时间，表明P2Y1受体的阻断剂具有抗抑郁作用(如图2所示)。
为了进一步检测P2Y1受体阻断剂的抗抑郁作用，本发明在社交失败慢性抑郁模型上检测了P2Y1受体阻断剂的抗抑郁效果。C57BL/6J小鼠作为侵入者每天接受10min来自长期单独饲养CD1小鼠的身体攻击，身体接触后使用透明有孔的有机玻璃板隔开，C57BL/6J小鼠接受24小时的视觉和嗅觉的应激，连续10天造模成功。在侧脑室植入导管。单次给药后，MRS2500组抑郁状态小鼠的状态能被显著的改善。以上结果进一步表明MRS2500阻断P2Y1受体后具有抗抑郁效应(如图3所示)。
综上所述，本发明P2Y1受体的阻断剂可用于制备抗抑郁症和/或抗焦虑症的药物，其含有有效剂量的P2Y1受体的阻断剂，以及药学上可以接受的载体。
优选地，所述P2Y1受体的阻断剂为抑制P2Y1受体的化合物或多肽。
更优选地，所述P2Y1受体的阻断剂为MRS2179及其衍生物，或MRS2500及其衍生物。
将本发明P2Y1受体的阻断剂用于制备抗抑郁症和/或抗焦虑症的药物时，优选的药物剂型为液体或固体。其中，液体剂型可以为注射剂、溶液剂、混悬剂、乳剂或气雾剂；固体剂型可以为片剂、胶囊、丸剂、粉针剂、缓释制剂或各种微粒给药系统。
相对于现有技术，本发明具有如下优点和有益效果：
本发明提供了一种与抑郁症和焦虑症相关的靶点P2Y1受体，以及将该P2Y1受体的阻断剂用于制备抗抑郁症和/或抗焦虑症的药物中，为抑郁症和焦虑症的诊断和治疗提供了新的靶点和药物，具有良好的应用前景和研究价值。
附图说明
图1为C57BL/6J小鼠腹腔给予MRS2500后检测强迫游泳实验的结果图；
图2为C57BL/6J小鼠侧脑室给予MRS2500和MRS2179后检测强迫游泳实验的结果图；
图3为社交失败抑郁模型造模的小鼠给予MRS2500后检测强迫游泳实验的结果图；
图4为P2Y1受体全身敲除小鼠检测P2Y1受体蛋白表达水平实验的结果图；
图5为P2Y1受体全身敲除小鼠检测强迫游泳实验的结果图；
图6为P2Y1受体全身敲除小鼠检测悬尾实验的结果图；
图7为C57BL/6J小鼠脑内腹侧背盖区注射干扰RNA分子腺相关病毒定位图；
图8为C57BL/6J小鼠脑内腹侧背盖区注射干扰RNA分子腺相关病毒检测强迫游泳实验的结果图；
图9为社交失败抑郁模型造模的小鼠给予P2Y1shRNA后检测社会交互实验的结果图；
图10为社交失败抑郁模型造模的小鼠给予P2Y1shRNA后检测强迫游泳实验的结果图；
图11为社交失败抑郁模型造模的小鼠给予P2Y1shRNA后检测糖水偏好实验的结果图。
具体实施方式
为了使本发明的目的、技术方案和有益技术效果更加清晰，以下结合实施例，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的实施例仅仅是为了解释本发明，并非为了限定本发明，实施例的参数、比例等可因地制宜做出选择而对结果并无实质性影响。
实施例1 P2Y1受体的特异性阻断剂MRS2500和MRS2179抗抑郁和抗焦虑的药理学实验研究
1.强迫游泳实验(Forced swimming test，FST)：
1977年由Porsolt提出强迫游泳实验，最早用于大鼠上，后扩展到小鼠。强迫游泳实验用来筛选抗抑郁药物，发现抗抑郁药物可以使强迫游泳小鼠的不动时间降低，该模型是现在筛选抗抑郁药最为常用的模型之一。实验步骤如下：实验小鼠于行为学实验室中至少提前适应60min后，开始进行如下实验：将单只小鼠置于水深23cm的圆柱形玻璃缸内(高：45cm；直径：19cm)，水温22～25℃，进行强迫游泳6min。记录后4min内累计不动时间(主要表现为停止挣扎，呈漂浮直立状态，或仅有细小的肢体运动以保持头部浮出水面)为检测抑郁样行为的指标。抗抑郁药物起效会使实验小鼠不动时间缩短。
实验动物：C57BL/6J小鼠，SPF级，雄性，体重20～24g，动物均由南方医 科大学实验动物中心(中国，广州)提供。小鼠置于SPF级动物饲养室内分笼饲养，每笼3～4只，塑料笼规格300mm×170mm×120mm，房间温度：24±1℃；明暗周期：12h/12h；光照时间：7:00am～7:00pm；相对湿度70～85％；室内环境安静，小鼠可自由进食饮水。实验均在下午1:00～5:00进行，小鼠在实验前都会提前进入实验环境适应并由实验人员进行抚摸适应。
在C57BL/6J小鼠强迫游泳实验中，把动物随机分成5组，每组10只。各组动物侧脑室手术植入药物灌流套管，饲养7日恢复后，4组动物分别进行以下处理：使用微量给药系统给予相应试剂：对照溶剂(人工脑脊液，1ul，1ul/min)，MRS2500(0.1uM，1ul，1ul/min)，MRS2179(0.2uM，1ul，1ul/min)，各组注射完毕后，10min后进行强迫游泳实验。
腹腔给予2.5mg/kg的MRS2500，30分钟后进行时长6min的强迫游泳实验,测定后4min游泳不动的时间来评估抗抑郁药的效果。
实验结果：给予MRS2500和给予传统抗抑郁药氟西汀出现一样的抗抑郁样行为(如图1所示)。
如图2所示，C57BL/6J小鼠侧脑室给予MRS2500和MRS2179均能够显著缩短小鼠的游泳不动时间，表明P2Y1受体的阻断剂具有明确的抗抑郁作用。
2.社会失败模型：
社会失败模型参照Golden的方法进行。
C57BL/6J小鼠随机分为对照组和应激组。每天将应激组的C57BL/6J小鼠放入筛选过的CD-1小鼠笼中，身体接触10min。C57BL/6J作为侵入者，被攻击后会表现出逃避、恐惧、顺从等行为。接触结束后用透明带孔隔板隔开后24h内，C57BL/6J小鼠可以闻到、看到、听到对面CD-1，进行心理应激。每天轮换C57BL/6J，保证受到均衡的刺激。实验进行10天。对照组小鼠中的CD-1被另外一只C57BL/6J替换，每天轮换，但是避免身体接触。造模完成后小鼠均单笼饲养，接受侧脑室导管植入手术。对照组和实验组分别给予相应试剂：对照溶剂(人工脑脊液，1ul，1ul/min)，MRS2500(0.1uM，1ul，1ul/min)，给药结束后通过社会交互实验来检测小鼠的抑郁水平。社会交互实验分为两个阶段，每个阶段2.5min。第一阶段(no target)：透明有孔有机玻璃盒中不放置CD-1小鼠，在远侧放入C57BL/6J小鼠，记录其在2.5分钟内交互区域和角落的停留时间。第 二阶段(target)：透明有机玻璃盒中放入CD-1小鼠，在远侧放入C57BL/6J，记录其在2.5分钟内交互区域和角落的停留时间。
实验动物：C57BL/6J小鼠，SPF级，雄性，体重20～24g，动物均为由南方医科大学实验动物中心(中国，广州)提供的。小鼠置于SPF级动物饲养室内分笼饲养，每笼3～4只，塑料笼规格300mm×170mm×120mm,房间温度：24±1℃；明暗周期：12h/12h；光照时间：7:00am～7:00pm；相对湿度70-85％；室内环境安静，小鼠可自由进食饮水。实验均在下午1:00～5:00进行，小鼠在实验前都会提前进入实验环境，并由实验人员进行抚摸适应。
实验结果：如图3所示，人工脑脊液组实验组小鼠相对人工脑脊液组对照组小鼠在交互区域时间明显减少，说明造模成功。脑内灌流MRS2500能增加实验组小鼠在交互区域的时间，结果表明，P2Y1受体的特异性阻断后具有抗抑郁的效果。
实施例2 P2Y1受体全身敲除小鼠的抑郁样行为筛选
1.强迫游泳实验
实验方法：将P2Y1受体全身敲除小鼠和野生型小鼠进行6分钟的强迫游泳实验检测，记录后4分钟内的游泳不动时间。
实验动物：P2Y1小鼠购自Jax(品系编号：009131)，此基因只有一个编码外显子，通过PGK neo cassette来干扰了大部分编码区，得到的129嵌合鼠与C57BL/6J回交12代。实验动物饲养于12/12小时明暗转换的环境，自由摄食饮水。
实验结果：如图5所示，P2Y1受体全身敲除小鼠与野生型小鼠比较，在强迫游泳实验中不动时间明显减少。结果表明，P2Y1受体表达水平下调后，小鼠表现出抗抑郁样的行为。
2.悬尾实验
该实验采用MED Associates悬尾设备对小鼠的运动状态进行实时监测。悬尾箱采用前面开放，其余面封闭的形式。悬尾杆底端固定着压力传感器，小鼠的运动情况通过压力传感器输入计算机进行数据处理。将小鼠尾尖部2cm处固定悬于悬尾箱内(ENV-505TS，Med associates Inc.)，使其呈倒挂状态，保持头部离箱底约10cm。小鼠起初会拼命挣扎，在发现无法摆脱后会出现间断的不动状态， 即小鼠停止挣扎或被动小幅度摆动状态。软件Med Suspension Tail自动记录和分析处理小鼠6min内出现不动状态时间，低于设定下限阈值的不动时间记录为检测抑郁样行为的指标。
实验动物：P2Y1小鼠购自Jax(品系编号：009131)，此基因只有一个编码外显子，通过PGK neo cassette来干扰了大部分编码区，得到的129嵌合鼠与C57BL/6J回交12代。实验动物饲养于12/12小时明暗转换的环境，自由摄食饮水。
实验结果：如图6所示，P2Y1受体全身敲除小鼠与野生型小鼠比较，在悬尾实验中不动时间明显减少。结果表明，P2Y1受体表达水平下调后，小鼠表现出抗抑郁样的行为。
实施例3 使用RNA干扰技术验证P2Y1受体作为抗抑郁新靶点的实验
实验动物：C57BL/6J小鼠，SPF级，雄性，体重20±2g，动物均为由南方医科大学实验动物中心(中国，广州)提供的。小鼠置于SPF级动物饲养室内分笼饲养，每笼3～4只，房间温度：24±1℃；明暗周期：12h/12h；光照时间：7:00am～7:00pm；相对湿度70～85％；室内环境安静，小鼠可自由进食饮水。实验均在下午1:00～5:00进行，小鼠在实验前都会提前进入实验环境适应，并由实验人员进行抚摸适应。
实验方法：各组动物脑腹侧背盖区(角度7°，AP：-3.2mm，ML：1.0mm，DV：-4.6mm)微量注射干扰RNA分子腺相关病毒(shRNA)0.5ul(0.1ul/分钟，完成后留置针5分钟)，饲养14天病毒表达恢复后进行强迫游泳实验。P2Y1特异干扰shRNA滴度为1×1013v.g/ml，9.55E+08copy/ul，包装病毒是用腺相关病毒9型，启动子是U6，血清型为AAV9。(由百恩维公司包装，中国深圳)；序列信息：正向序列如SEQ ID NO:1所示；反向序列如SEQ ID NO:2所示。
实验结果：C57BL/6J小鼠接受腺相关病毒注射14天后，shRNA病毒感染大量神经元，使其腹侧背盖区表达绿色荧光蛋白，使用激光共聚焦显微镜观察发现shRNA主要表达在小鼠的腹侧背盖区。结果发现P2Y1shRNA小鼠相较于对照病毒组小鼠在强迫游泳实验中不动时间明显降低，表明P2Y1受体阻断后可出现抗抑郁的效应。
社交失败抑郁小鼠在造模结束后休息一天，注射shRNA病毒14天后检查抑 郁小鼠的抑郁状态是否改变。在社会交互实验中，注射shRNA能增加抑郁小鼠在交互区域的时间，表现出抗抑郁样的行为；在强迫游泳实验中，注射shRNA能降低抑郁小鼠增加的不动时间，表现出抗抑郁样的行为；在糖水偏好实验中，注射shRNA能增加抑郁小鼠降低的血糖偏好。此结果更加证明阻断P2Y1受体具有抗抑郁的效应(如图7～11所示)。
根据上述说明书的揭示和教导，本发明所属领域的技术人员还可以对上述实施方式进行适当的变更和修改。因此，本发明并不局限于上面揭示和描述的具体实施方式，对本发明的一些修改和变更也应当落入本发明的权利要求的保护范围内。此外，尽管本说明书中使用了一些特定的术语，但这些术语只是为了方便说明，并不对本发明构成任何限制。
Figure PCTCN2017107289-appb-000001

Claims (10)

  1. P2Y1受体作为靶点，在筛选预防、治疗抗抑郁症和/或抗焦虑症的药物中的应用。
  2. P2Y1受体作为生物标志物，在筛选抑郁症和/或焦虑症，及抑郁症和/或焦虑症的早期预警中的应用。
  3. P2Y1受体在筛选抑郁症和/或焦虑症的临床诊断试剂中的应用。
  4. P2Y1受体的阻断剂在抗抑郁症和/或抗焦虑症中的应用。
  5. 根据权利要求4所述的P2Y1受体的阻断剂的应用，其特征在于，所述P2Y1受体的阻断剂为抑制P2Y1受体的化合物或多肽。
  6. 根据权利要求5所述的P2Y1受体的阻断剂的应用，其特征在于，所述P2Y1受体的阻断剂为MRS2179及其衍生物，或MRS2500及其衍生物。
  7. 一种抗抑郁症和/或抗焦虑症的药物，其特征在于，含有有效剂量的P2Y1受体的阻断剂，以及药学上可以接受的载体。
  8. 根据权利要求7所述的抗抑郁症和/或抗焦虑症的药物，其特征在于，所述P2Y1受体的阻断剂为抑制P2Y1受体的化合物或多肽。
  9. 根据权利要求8所述的抗抑郁症和/或抗焦虑症的药物，其特征在于，所述P2Y1受体的阻断剂为MRS2179及其衍生物，或MRS2500及其衍生物。
  10. 根据权利要求7所述的抗抑郁症和/或抗焦虑症的药物，其特征在于，所述药物的剂型为液体或固体。
PCT/CN2017/107289 2017-08-21 2017-10-23 P2y1受体及其阻断剂在预防和治疗抗抑郁症和/或抗焦虑症中的应用 WO2019037225A1 (zh)

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