WO2023202682A1 - Novel target for treating or diagnosing emotional disorders or stress disorders, and application thereof - Google Patents
Novel target for treating or diagnosing emotional disorders or stress disorders, and application thereof Download PDFInfo
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- WO2023202682A1 WO2023202682A1 PCT/CN2023/089623 CN2023089623W WO2023202682A1 WO 2023202682 A1 WO2023202682 A1 WO 2023202682A1 CN 2023089623 W CN2023089623 W CN 2023089623W WO 2023202682 A1 WO2023202682 A1 WO 2023202682A1
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- motor cortex
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
- A61N1/00—Electrotherapy; Circuits therefor
- A61N1/18—Applying electric currents by contact electrodes
- A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
- A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
Definitions
- the present invention relates to the field of disease treatment and medicine. Specifically, the present invention relates to methods for treating or diagnosing mood or stress disorders, as well as related pharmaceutical compositions, kits or medical devices.
- DSM-5 Diagnostic and Statistical Manual 5th Edition (DSM-5) of Mental Disorders (5th Edition) published by the American Psychiatric Association (APA) in 2013, mood disorders mainly include depressive disorders , bipolar disorder and their subtypes.
- Anxiety disorders mainly include social phobia, panic attacks, separation anxiety, etc.
- Stress disorders mainly include acute stress disorder and post-traumatic stress disorder.
- the prefrontal cortex is the main target of current electromagnetic stimulation therapy.
- the therapeutic effect is relatively clear in patients with depression, but there is not enough evidence in anxiety disorders, bipolar disorder, and stress disorders. Therefore, clinical There is still a lack of exploration into other possible brain regions for treating mental illnesses.
- This field also needs to explore the role of new brain regions in mood or stress disorder-like mental illnesses including anxiety, depression, panic, bipolar disorder and stress disorder, in order to break through the current regulation of single brain region activity.
- the idea is helpful for the clinical treatment of the above types of diseases, and may also provide clinical Clinical diagnosis of these psychiatric disorders provides more objective markers and predicts the effectiveness of treatment.
- the present invention first discovered that the motor cortex of mammals, especially the primary motor cortex, is involved in bipolar and related disorders, depressive disorders, anxiety disorders, stress disorders, etc.
- the pathogenesis of mental illness such as mood or stress disorders.
- the present invention has found that regulating the excitability of the motor cortex of mammals, especially the primary motor cortex, can treat mood or stress disorders such as bipolar and related disorders, depressive disorders, anxiety disorders and stress disorders. Animals suffering from mental disorders or symptoms.
- the present invention thus provides methods, medicines or devices for treating mood or stress disorders by regulating motor cortex excitability, as well as methods, kits or devices for diagnosing mood or stress disorders by detecting motor cortex excitability.
- the present invention provides methods of treating mood or stress disorders by modulating the excitability of the patient's motor cortex.
- the present invention provides an agent or a medical device for regulating the excitability of the motor cortex and a method for treating mood or stress disorders.
- the agent is locally applied to the motor cortex or the medical device is used to locally stimulate the motor cortex.
- Methods for treating mood or stress disorders The present invention provides drugs or medical devices that modulate the excitability of the motor cortex for the treatment of mood or stress disorders, particularly drugs that are locally applied to the motor cortex or medical devices that locally stimulate the motor cortex.
- the present invention provides the use of the reagent or medical device for preparing drugs or medical devices for treating depression, especially for preparing drugs or medical devices that act locally on the motor cortex.
- a method for treating mood or stress disorders which includes modulating the excitability of the motor cortex, especially the excitability of the primary motor cortex of the patient.
- the method for treating mood or stress disorders modulates the excitability of the motor cortex, especially the primary motor cortex, by increasing its excitability.
- a subject (patient) in need of the methods and medicaments (pharmaceutical compositions) described herein can be a mammal, including humans, or a non-human primate such as an orangutan or monkey. Mammals can also be other animals, such as rats, mice, rabbits, pigs, dogs, etc. The mammal may be a domestic animal, such as a cat or a dog.
- the motor cortex of mammals is also called the "precentral gyrus", or "the first somatic motor area”. Sports leather The layer is an area of the frontal lobe, a large mass of gray matter in the postcentral gyrus in front of the central sulcus. It has been found that the motor cortex of mammals is mainly responsible for controlling the movement of various parts of the body, and electrical stimulation of this part will cause motor responses.
- the mammalian motor cortex includes the primary motor cortex. Nerve impulses generated by the primary motor cortex are transmitted down to the spinal cord, controlling the execution of human movements.
- the motor cortex of primates also includes the premotor cortex and supplementary motor area. In other categories of mammals, in addition to the primary motor cortex, there is also a secondary motor cortex.
- the excitability of the motor cortex, especially the primary motor cortex can be adjusted by methods known in the art, including through physical, chemical or biochemical methods.
- the excitability of the motor cortex is adjusted by applying physical methods such as electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation, pressure stimulation, acoustic stimulation, and ultrasonic stimulation to the patient's motor cortex.
- electrical stimulation can be delivered through an internally implanted probe or through electrodes placed externally on the scalp.
- magnetic stimulation can be delivered by a directional magnetic field generated by an internally implanted probe or an external electrocoil.
- thermal stimulation may be performed using an implanted probe that generates or emits heat and/or cold temperatures.
- These stimulation devices or systems are operated to stimulate predetermined parts of the brain.
- These devices may include stimulation portions or probes, for example, electrodes, electrode assemblies (such as electrical stimulation leads), coils, drug delivery components (such as catheters) or combinations of these and/or signal generators or sources of signal or pulse generation (i.e. electrical signal source, chemical signal source (i.e. drug delivery pump) or magnetic signal source).
- the probe can be connected to a signal source, a drug delivery pump, or both, and the signal source and drug delivery pump are used to stimulate the intended treatment site.
- the probe and signal generator or source may be combined to form a unit or a single device, which may include one, two or more electrodes. These devices are known in the art as microstimulators.
- the excitability of the motor cortex is modulated by chemical or biochemical methods such as administering to the patient a preparation that modulates the excitability of cells (nerve cells) in the motor cortex.
- Preparations that can regulate the excitability of nerve cells include optogenetics preparations, chemogenetics preparations, chemical drugs or preparations, etc.
- the optogenetic preparation is capable of expressing photoreceptor genes in cells of the motor cortex.
- cells of the motor cortex e.g. ChR2, eBR, NaHR3.0, Arch or OptoXR.
- the chemical genetic preparation is an agent that can express a protein that regulates cell excitability in cells of the motor cortex, and activates/antagonizes an agent that regulates a protein that regulates cell excitability, such as expressing a neurotransmitter receptor or transporter. or agents for degrading enzymes, or for the neurotransmitters including acetylcholine (ACh), glutamate, ATP, adenosine, gamma-aminobutyric acid (GABA), norepinephrine, dopamine, endocannabinoids ( endocannabinoids), nitric oxide, histamine, etc.
- ACh acetylcholine
- GABA gamma-aminobutyric acid
- norepinephrine dopamine
- endocannabinoids endocannabinoids
- nitric oxide histamine, etc.
- chemical or biochemical agents capable of increasing cellular excitability of the motor cortex include, but are not limited to:
- Neurotransmitters such as acetylcholine (ACh), glutamate, ATP, adenosine, gamma-aminobutyric acid (GABA), norepinephrine, dopamine, endocannabinoids, nitric oxide, Histamine, etc.;
- Neurotransmitter receptor agonists such as Gq GPCR agonists, including endocannabinoid receptor (CB1Rs) agonists; metabotropic glutamate type I and II receptor agonists and AMPA receptor agonists ; Purinergic receptor agonist; GABAergic receptor agonist; alpha-adrenergic receptor agonist, dopaminergic receptor agonist; histaminergic receptor agonist; protease-activated receptor ) agonist;
- Gq GPCR agonists including endocannabinoid receptor (CB1Rs) agonists; metabotropic glutamate type I and II receptor agonists and AMPA receptor agonists ; Purinergic receptor agonist; GABAergic receptor agonist; alpha-adrenergic receptor agonist, dopaminergic receptor agonist; histaminergic receptor agonist; protease-activated receptor ) agonist;
- Neurotransmitter reuptake inhibitors such as norepinephrine, dopamine and serotonin reuptake inhibitors
- Agonists of ion channels such as agonists of transient receptor potential cation channel, subfamily V, TRPV1; agonists of Na + /Ca 2+ exchangers (NCXs) .
- “mood disorder or stress disorder” refers to patients who have destructive moods such as pain, sadness, loss of pleasure, emptiness, irritability, and irritability, and usually in the context of anxiety or fear, the patient's cognitive changes are accompanied by physical changes. Abnormal mental illness.
- “mood or stress disorders” include bipolar and related disorders, depression disorders, anxiety disorders and stress disorders. Mental illness. The above four types of mental disorders are classified in the American Classification and Diagnostic Criteria for Mental Disorders (DSM-5) and the World Classification and Diagnostic Criteria for Mental Disorders proposed by the American Psychiatric Association (APA). It is described and defined in the International Classification of Diseases, Eleventh Revision (ICD-11) published by the World Health Organization (WHO).
- “mood disorder or stress disorder” includes the following diseases (according to ICD-11 definition and numbering):
- Bipolar I disorder currently in partial remission, most recently in mixed episodes
- the method of treating a mood disorder or a stress disorder by modulating the excitability of the patient's motor cortex has a patient with one of the above mood or stress disorders or Various diseases or conditions.
- the patient has any two of bipolar and related disorders, depressive disorders, anxiety or fear-related disorders, and stress-related disorders.
- the patient has any three or four of bipolar and related disorders, depressive disorders, anxiety or fear-related disorders, and stress-related disorders.
- the method of treating mood disorders or stress disorders by modulating the excitability of the patient's motor cortex is particularly suitable for the treatment of stress disorders, especially the treatment of post-traumatic stress. inflammatory disorder and complex post-traumatic stress disorder.
- treatment includes: the ongoing process or results of improving, alleviating, reducing or preventing symptoms related to mental disorders such as mood disorders or stress disorders; the ongoing process of improving symptoms related to the mental diseases.
- the process or result the ongoing process or result of normalizing the functions of a body in a disease or condition that results in impairment of a specified body function; or the ongoing process or result of improving one or more clinically measurable parameters of the disease or result.
- the purpose of treatment is to prevent or slow down an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired result.
- the outcome may be, for example, medical, physiological, clinical, physiotherapy, occupational therapy, for healthcare providers or patients; or a parameter understood in the art as "quality of life" or activities of daily living.
- beneficial or desired clinical results include, but are not limited to, alleviating symptoms; reducing/narrowing the extent of the condition, disorder, or disease; stabilizing the condition, disorder, or disease; delaying the onset of the condition, disorder, or disease To start or slow down the progress of a condition, disorder or disease; to improve or alleviate a condition, disorder or disease.
- treatment involves eliciting a clinically effective response without undue levels of side effects.
- treatment also includes prolonging survival compared to expected survival if not receiving treatment.
- treatment may be prevention, cure, or improvement of the patient's clinical condition, including reducing the course or severity of the disease, or subjectively improving the patient's quality of life or prolonging the patient's survival period.
- the present invention discovered for the first time that the motor cortex of mammals, especially the primary motor cortex, is involved in biphasic As well as related disorders, depressive disorders, anxiety disorders and stress disorders and other mood disorders or stress disorder-type mental illness pathogenesis. More importantly, the present invention has discovered that regulating the excitability of the motor cortex of mammals, especially the primary motor cortex, can treat mood disorders or stress disorders such as bipolar and related disorders, depressive disorders, anxiety disorders and stress disorders. Animals suffering from irritable mental illness or symptoms. This is a mechanism known in the art for treating mental disorders such as mood disorders or stress disorders, and drugs fail to target the pathological mechanisms and target brain tissues for treatment. Therefore, the methods, medicines or devices provided by the present invention are suitable for treating mental disorders such as mood disorders or stress disorders.
- the present invention also provides pharmaceutical compositions or kits, or medical devices for treating mood or stress disorders in patients, which contain agents or devices that modulate the excitability of the motor cortex, especially the primary motor cortex, for example, including through physical, Agents and devices for chemically or biochemically modulating the excitability of the motor cortex, especially the primary motor cortex.
- a device for regulating the excitability of the patient's motor cortex is provided, such as applying electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation, pressure stimulation, acoustic stimulation, and ultrasonic stimulation to the patient's motor cortex. one or more devices.
- a pharmaceutical composition comprising an agent that modulates motor cortex excitability.
- the agent or device is as described above in the method of modulating the excitability of the motor cortex, especially the primary motor cortex.
- reagents and devices that apply electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation, pressure stimulation, acoustic stimulation, and ultrasonic stimulation to the patient's motor cortex to adjust the excitability of the motor cortex.
- they are optogenetics preparations, chemogenetics preparations or chemical drugs.
- the optogenetic agent is an agent that can express light-sensing genes (eg, ChR2, eBR, NaHR3.0, Arch or OptoXR) in cells of the motor cortex.
- the chemical genetic agent is an agent that can express a protein that regulates cell excitability in cells of the motor cortex, an agent that activates/antagonizes a protein that regulates cell excitability, or is a neurotransmitter.
- chemical drugs that can increase the excitability of cells in the motor cortex include, but are not limited to: a. neurotransmitters; b. neurotransmitter receptor agonists; c. neurotransmitter reuptake inhibitors; or d. Ion channel agonist.
- an agent or device containing an agent or device that modulates the excitability of the motor cortex, especially the primary motor cortex, in a medicament or device for treating mood or stress disorders in a patient in one aspect of the present invention.
- the drug is a dosage form that is administered locally in the motor cortex or nearby tissues.
- the effect of the drug can be limited to the target tissue by local administration, for example, by formulating the drug into a dosage form that can be implanted into the motor cortex through a cannula for local administration.
- Another example is making the drug into a dosage form that can be sustained-released after being implanted into the tissue.
- the above-mentioned drugs can also be formulated into tissue-specific targeted drug delivery systems.
- preparations that activate or enhance the excitability of motor cortex nerve cells including small molecule compounds or bioactive molecules (nucleic acids such as protein-coding DNA or mRNA molecules, proteins such as antibodies, etc.), can be combined with those that can specifically bind to the motor cortex.
- the sexually expressed protein-binding antibodies or antibody fragments are linked to form complex molecules capable of recognizing and binding to cells of the motor cortex.
- the medical device may be a device that acts locally in the motor cortex or nearby tissue. For example, through an external electric coil externally placed on the scalp, it is possible to set a directional magnetic field that acts locally in the motor cortex or nearby tissues.
- the active ingredients in the pharmaceutical compositions provided by the present invention can be administered in the form of raw materials.
- the active ingredients can also be administered, optionally in the form of physiologically acceptable salts, with one or more adjuvants, excipients, etc. excipients, carriers, buffers, diluents and/or other conventional pharmaceutical excipients.
- compositions of the present invention may be administered by any convenient route suitable for the desired therapy.
- routes of administration include oral administration, especially in the form of tablets, capsules, lozenges, powders and liquids; and parenteral administration, especially cutaneous, subcutaneous, intramuscular and intravenous injection.
- Pharmaceutical compositions of the present invention may be prepared by those skilled in the art using standard methods and conventional techniques suitable for the desired formulation. If desired, compositions suitable for sustained release of the active ingredient may be used.
- the carrier can be solid or liquid.
- Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
- a solid carrier may be one or more substances that may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.
- compositions suitable to provide sustained release of the active ingredient may be used.
- compositions are preferably in unit dosage form.
- the preparation is subdivided into unit doses containing appropriate quantities of the active ingredient.
- Unit dosage forms may be packaged preparations containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Additionally, the unit dosage form may be a capsule, tablet, cachet, or lozenge itself, or it may be the appropriate number of any of these in packaged form.
- a therapeutically effective dose means an amount of active ingredient that relieves a symptom or condition.
- Therapeutic efficacy and toxicity such as ED50 and LD50, can be determined by standard pharmacological procedures in cell culture or experimental animals. The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed by the ratio LD50/ED50.
- the dosage administered must of course be carefully tailored to the age, weight and condition of the individual treated, as well as the route, dosage form and regimen, and desired results, and the exact dosage should of course be determined by the physician.
- a method for diagnosing mood disorders or stress disorders by detecting the excitability of the subject's motor cortex is also provided.
- the present invention provides reagents or medical devices for detecting the excitability of the motor cortex and methods for diagnosing mood disorders or stress disorders, especially methods for detecting the excitability of the motor cortex in local brain areas.
- the present invention provides the use of reagents or devices for detecting the excitability of the motor cortex in preparing kits or equipment for diagnosing mood disorders or stress disorders, especially for detecting the excitability of the motor cortex in local brain areas. Use in kits or devices.
- the method for diagnosing sensory disorders or stress disorders includes comparing subjects at different stages (for example, at various stages of onset of different affective disorders or stress disorders, or before onset or treatment). (2) The excitability of the motor cortex was tested to see whether it was significantly down-regulated.
- the method for diagnosing mood disorders or stress disorders by detecting the excitability of the subject's motor cortex further includes the step of stimulating the subject's motor cortex.
- the subject can first stimulate the motor cortex using magnetic stimulation, electrical stimulation, light stimulation or ultrasonic stimulation, and then detect the excitability of the motor cortex and its relative changes.
- bioelectric signals related to motor cortex excitability are detected, such as resting membrane potential and action potential firing frequency of neurons, based on Detection of current or voltage thresholds, etc.
- bioelectrical signals can be detected by methods known in the art, including detection devices composed of single or multiple electrodes, or electroencephalography (EEG), etc. Whole or partial electrical signal detection and analysis can be performed on the brain area of concern.
- biomarkers related to motor cortex excitability such as neurotransmitters and signaling pathway-related proteins or nucleic acids. Examples include, but are not limited to, detection of glutamate AMPA or NMDA receptor subunits, glutamate transporter gene or protein expression, etc.
- the nerve cells or signal transmission pathways in the motor cortex can be detected by methods known in the art, including by detecting neurotransmitters in the nerve cells or signal transmission pathways, or their receptor proteins, Or detect the amount of neurotransmitter hydrolase. Examples include but are not limited to the use of PCR, ELISA, Western Blot, electrophysiology, microdialysis combined with high-performance liquid chromatography and other technologies to detect related gene and protein expression levels.
- Methods of detecting protein (expression) in a sample useful in the present invention include immunoassays. For example, ELISA or Western blotting can be performed using antibodies that specifically recognize the relevant proteins.
- Antibodies can be monoclonal or polyclonal.
- Antibodies can be humanized or chimeric.
- Methods of detecting protein (expression) in a sample useful in the present invention also include detecting the presence or amount of mRNA of the relevant gene, for example by RT-PCR to detect the amount of mRNA or fragments thereof in the sample.
- the detection sample is from an ex vivo sample.
- the detection may also include in vivo observation and detection of the motor cortex.
- the subject's motor cortex is observed and examined by imaging the subject's brain region.
- the subject's motor cortex is imaged, such as computed tomography (CT) or magnetic resonance imaging (MRI). wait.
- CT computed tomography
- MRI magnetic resonance imaging
- the imaging is PET imaging or SPECT.
- the signal of a combined positron-emitting radionuclide tracer can be identified and visualized by injecting it intravenously and then performing a PET scan of the relevant brain region.
- the imaging is performed in conjunction with magnetic resonance imaging (MRI) imaging.
- MRI magnetic resonance imaging
- the imaging is performed in conjunction with magnetic resonance imaging (MRI) imaging.
- MRI magnetic resonance imaging
- the motor cortex to observe the activity of the nuclei. This includes detecting tissue blood flow, metabolic status, etc., observing physiological and metabolic changes in local brain tissue in corresponding functional brain areas, and reflecting changes in nuclear activity.
- the present invention also provides a kit for diagnosing mood disorders or stress disorders through the above-mentioned method of the present invention.
- the present invention also provides a medical device for diagnosing mood disorders or stress disorders through the method of the present invention.
- Figure 1 shows the preparation of disease or disease-susceptible (diseased) and disease-resistant (resistant) mice through the fear-enhanced feel learning model (Stress-enhanced feel learning, SEFL).
- SEFL Stress-enhanced feel learning
- A. Use restraint stress combined with plantar electrical stimulation to create a model in mice, and detect the mice's anxiety, depression, fright and fear levels after modeling.
- BE. Record the four behavioral indicators of each modeled animal and compare them with the control group (fear conditioning, FC). Animals with abnormal behavior can be obtained for each behavioral method, which are marked with dotted boxes.
- B is the elevated-plus maze (EPM), used to detect anxiety levels
- C is the acoustic startle response (ASR), used to detect startle levels
- D is the forced-swim test (FST) ) is used to detect the level of depression
- E is fear recall (fear recall or fear memory recall), which is used to detect the level of fear memory.
- FI after comprehensively assessing the animal's anxiety, startle, depression and fear memory levels, animals with three or more behavioral abnormalities are defined as diseased, and animals showing at most one behavioral abnormality are defined as resistant.
- Figure 2 shows the excitability of brain regions in diseased mice, tolerant mice and control mice.
- A Brain c-fos staining pattern obtained by VS120 whole-brain scan. Neurons labeled with c-fos are excitable neurons. Scale bar: 1mm. The upper left corner of the figure is marked as the coordinates of the brain slice (relative to bregma).
- B-D Differences in the degree of neuronal activation in the cortex, amygdala, and hippocampus regions between mice in the diseased and resistant groups compared to mice in the Control group.
- B Brain c-fos staining pattern obtained by VS120 whole-brain scan. Neurons labeled with c-fos are excitable neurons. Scale bar: 1mm. The upper left corner of the figure is marked as the coordinates of the brain slice (relative to bregma).
- B-D Differences in the degree of neuronal activation in the cortex, amyg
- Control and disease/tolerant mice showed a significant decrease in c-fos in disease mice in the cingulate cortex (Cg), infralimbic cortex (IL) and prelimbic cortex (PrL) contained in the cortex, while the resistant mice showed a significant decrease in c-fos. Affected mice have a rebound trend.
- C Differences in the number of c-fos neurons in various subregions of the amygdala between control and disease/tolerant mice. In tolerant mice, c-fos neurons showed a relatively significant decrease compared with disease mice. Compared with the control group, the disease group showed a significant upward trend in the lateral amygdala (LA) area.
- D D.
- Figure 3 shows the regulation of activity of primary motor cortex (Primary motor cortex) and secondary motor cortex (Secondary motor cortex) in disease-susceptible and tolerant mice.
- A. The activity of the primary motor cortex was significantly down-regulated in diseased mice compared with controls, while the activity was restored in tolerant mice.
- B. The activity of the secondary motor cortex in diseased mice has a downward trend compared with the control and is significantly lower than that in the tolerant mice, while the activity in the tolerant mice is no different from the control.
- *P ⁇ 0.5, one-way ANOVA followed by Tukey multiple comparisons; Control group, disease susceptible group and tolerant group n 11,8and 9. All data are expressed as mean ⁇ SEM.
- Figure 4 shows functional abnormalities in synaptic transmission in diseased mice revealed by RNA sequencing analysis.
- Figure 4A shows that the M1 region of diseased mice mainly shows a trend of down-regulation of core genes in aspects such as associative learning, circadian sleep/wake cycle, and sleep.
- Figure 4B shows that through GSEA gene set enrichment analysis, it was found that diseased mice showed poor performance in dopamine synthesis and transport (dopamine transport), G protein-coupled receptor signaling pathway (G protein-coupled receptor signaling) and synaptic transmission (dopaminergic synaptic transmission).
- the downregulation of core genes indicates that synaptic signaling in the M1 region is impaired and key signaling pathways are inhibited.
- Figure 5 shows that activating the primary motor cortex of diseased mice significantly improved their levels of anxiety, depression, startle, and fear.
- Figure 5A shows injection of rAAV-CaMKIIa-hM3D(Gq)-mCherry excitovirus in bilateral M1 of control Con and diseased mice.
- Figure 5B shows that intraperitoneal injection of CNO (3 mg/kg) into mice before behavioral testing specifically activated the primary motor cortex of Con and diseased mice.
- Figure 5C-F shows the comparison and found that after activating the primary motor cortex, the anxiety level (C), depression level (D), startle level (E) and fear level (F) of the diseased mice were significantly improved compared with before activation.
- Figure 6 shows that clinically, transcranial magnetic stimulation of the primary motor cortex of patients has a significant effect on the core symptoms of PTSD, and also has a good therapeutic effect on the accompanying anxiety, depression, sleep abnormalities, social abnormalities, etc.
- Figure 6A and B shows a patient undergoing a motor cortex treatment protocol in the form of intermittent theta burst stimulation (iTBS). After treatment, the disease severity index (CAPS score) of PTSD patients decreased significantly (Figure 6C).
- the accompanying anxiety levels (Figure 6D), depression levels (Figure 6E), sleep quality (Figure 6F) and social performance (Figure 6G) all reflected significant improvements at different treatment stages.
- mice 6-8 week old male C57 mice, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., are used to construct an animal model of stress disorder in mice after being exposed to negative stimulation.
- the c-fos antibody used to label neuronal activity was purchased from Synaptic Systems (#226 008);
- PFA Paraformaldehyde
- OCT OCT
- the virus rAAV-CaMKIIa-hM3D(Gq)-mCherry-WPREs-pA for primary motor cortex activation and the virus rAAV-hSyn-hM4D(Gi)-EGFP-WPRE-hGH pA for inhibition were purchased from Wuhan Primus Brain Science and Technology Co., Ltd. .
- the reagent Clozapine N-oxide (CNO) used to activate Gq was purchased from Sigma-Aldrich (34233-69-7).
- Behavioral instruments for detecting animal anxiety, depression, startle and fear are Any-maze system (stoelting Any-maze Beijing Jingguan Zhuoyi Business Center), forced swimming (Shanghai Shishu), startle reflex system (San Diego) and Conditioned Fear System (Coulbourn Instruments).
- the microscope used to capture the fluorescence of mouse brain samples was the Virtual Digital Slice Scanning System VS120 (Olympus).
- a mouse brain stereotaxic instrument Reward, Shenzhen, model: 68030
- a microsyringe Stoelting
- Negative stimulus modeling method combine stress and conditioned fear to achieve excessive fear enhancement. Mice were subjected to restraint stress in 50 ml centrifuge tubes for 2 hours. Seven days later, all animals, including the control group, will be subjected to a conditioned fear experiment, which is divided into two stages: the training stage and the fear extinction stage. During the training phase, a sound signal was given in conjunction with the co-terminated foot shock training. After the training, the experimental mice were allowed to stay in the environment for 1 minute and then placed in the mouse cage. A fear extinction experiment was performed 24 hours later. During the sound extinction experiment, mice were placed in different environments with the same sound signal. Finally, the mice were also allowed to stay in the environment for 1 minute, and then returned to the original cage.
- the elevated plus maze places mice in a plus maze that is elevated approximately 0.5 m above the ground.
- the plus maze has two open arms (30x5cm 2 ) and two closed arms (30x5x20cm 3 ). Let the mice move freely in the maze for 5 minutes, and finally record the time the mice stay in the open and closed arms. The longer the mouse stayed in the open arm, the less anxious the mouse was.
- Forced swimming is to put the mouse into a transparent glass measuring cylinder containing 2L of water with a height of about 30cm.
- the mouse will continue to struggle in the water to keep swimming, and from time to time there will be a state of giving up where the mouse is completely motionless.
- the mice were taken out of the water, and the time the mice remained in a completely motionless floating state for the next 5 minutes was counted based on the captured video. The longer the mice remained completely still, the more depressed the mice were.
- ASR Acoustic startle reflex
- mice were placed in the same environment as when fear was extinguished and received sounds coupled with plantar stimulation four times in total, each lasting 30 seconds. The gap between each sound is random, roughly between 60s-90s. Finally, use software to count the average freezing time of the mouse during the four sounds. The longer the freezing time, the stronger the fear memory of the mice.
- mice were cardiac perfused with 4% paraformaldehyde dissolved in 0.1M PBS to fix the tissue. After the brain tissue was taken out, it was soaked in paraformaldehyde and sucrose solutions at 4°C for 24 hours. After fixation, 50 ⁇ m coronal sections were cut out with a vibrating microtome (VT 1000S). Immunohistochemical staining was then used to add antibodies identifying the immediate early gene c-fos to label activated neurons throughout the brain. Finally, the nuclei were stained with DAPI for 5 minutes and then mounted. Stained brain slices were captured using Olympus's VS120 digital sectioning workstation. The captured images were processed using imageJ and fitted to the corresponding brain atlas, and c-fos neurons were counted in each brain region of the whole brain.
- VT 1000S vibrating microtome
- mice aged 9-10 weeks were anesthetized with 1% sodium pentobarbital at 50 mg/kg, shaved to expose the scalp, and fixed on a stereotaxic apparatus.
- Use ophthalmic scissors to gently cut the skin along the midline of the mouse's head from the posterior edge of the line connecting the eyes to the front edge of the line connecting the ears to expose the top of the mouse skull.
- the mouse skull was leveled stereotactically with reference to the bregma (Bregma), the focal point of the median sagittal suture and coronal suture, and the posterior fontanel (lambda), the intersection point of the median sagittal suture and herringbone suture, on the mouse skull.
- a craniotomy is performed, and the skull directly above the target brain area is opened vertically with a cranial drill with a diameter of 0.5 mm to avoid puncturing the meninges and causing cerebral hemorrhage.
- AP +1.54mm
- ML ⁇ 1.30mm
- DV -1.60mm
- Inject the virus bilaterally at a rate of 20nl/min rAAV-CaMKIIa-hM3D(Gq)-mCherry or rAAV-hSyn-hM4D(Gi)-EGFP
- mice After the injection, the glass needle stayed for 7 minutes and then slowly lifted until it moved out of the skull. After the virus injection, the mice were sutured, and lincomycin ointment was applied to the sutures to reduce the risk of postoperative infection. After the operation, the mice need to be placed on a heating pad at 37°C. Wait for the animal to slowly regain consciousness, and observe the animal's health every day for 3 days after the operation.
- mice with symptoms of anxiety, depression, fright, and fear that were comprehensively screened through behavioral tests were injected with Gq excitatory virus in their bilateral primary motor cortex.
- the control group was injected with the same virus.
- the mice were tested for levels of anxiety, depression, fright and fear.
- mice were intraperitoneally injected with CNO (3 mg/kg) to activate the motor cortex, and behavioral indicators were measured half an hour later. Finally, behavioral testing technology was combined to evaluate whether the anxiety, depression, fright, and fear indicators of susceptible mice were significantly improved.
- Posttraumatic stress disorder (PTSD) model mice were prepared according to the method of fear-enhanced learning (SEFL) described in Figure 1A. This model is described in Sillivan SE et al, Biol Psychiatry. 2017 and is widely recognized in reviews of animal models of PTSD (e.g. Richter-Levin G et al, Mol Psychiatry. 2019). After modeling, mice were subjected to elevated-plus maze (EPM) to detect anxiety levels; acoustic startle response (ASR) to detect startle levels; forced-swim test (FST) to detect depression levels; and fear recall to detect fear memory. levels, thereby screening disease mice that are susceptible to negative stimuli and those that are resistant.
- EPM elevated-plus maze
- ASR acoustic startle response
- FST forced-swim test
- fear recall to detect fear memory. levels, thereby screening disease mice that are susceptible to negative stimuli and those that are resistant.
- Example 3 Research on neuronal excitability in animal brains shows significant changes in the activity of multiple important nuclei in the brain
- Example 4 The activity of the primary motor cortex of animals with disease is significantly down-regulated.
- Example 5 RNA sequencing analysis of primary motor cortex of animals with disease, indicating abnormal synaptic transmission function
- the disease-producing mice and control mice obtained in Example 2 were dissected from the primary motor cortex, and the tissues were lysed to extract RNA and conduct transcriptome sequencing analysis.
- Transcriptome sequencing analysis was entrusted to Kaitai Biotechnology Company to conduct RNA-seq.
- the experimental process mainly includes: sample quality inspection, double-stranded cDNA synthesis, transcriptome library construction and quality inspection, sample sequencing, and sequencing using Performed on Illumina Novaseq 6000. After sequencing obtains the raw sequencing sequence (Raw data), it enters the information analysis process. The process is divided into two stages: 1) Sequencing data quality assessment: By counting the sequencing error rate, data volume, comparison rate, etc., follow-up will be carried out if the standards are met.
- Example 6 Activating the activity of the primary motor cortex of diseased mice improves their disease symptoms
- the activating virus rAAV-CaMKIIa-hM3D(Gq)-mCherry was injected into the bilateral M1 of both control and disease mice.
- the virus is mainly expressed in the glutamatergic neurons of M1 and is activated by the injection of CNO in vitro. neurons (Fig. 5A).
- various indicators of the diseased mice were detected, and the mice were intraperitoneally injected with CNO (3 mg/kg) half an hour before the test.
- CNO 3 mg/kg
- mice which showed an increase in anxiety levels (accompanied by a decrease in the desire to explore after adapting to the environment), and did not show changes in depression, fright, and fear.
- the combined dosage should be maintained stably for more than four weeks;
- Treatment history Have taken antipsychotics and antidepressants; have used benzodiazepines or other drugs that affect the central nervous system one month before the MRI examination; have received continuous drug treatment for six months;
- TSS Adverse Effect Questionnaire
- the resting motor threshold is the minimum stimulation intensity that can elicit a motor evoked potential greater than 50 ⁇ V when the patient is in a relaxed state for 10 consecutive stimulations, of which at least 5 are. It is operated by professional therapists who have received TMS training to ensure the stability of the treatment area and treatment parameters.
- the statistical results of the scale showed that after iTBS stimulation of the motor cortex, the PTSD symptom severity index (CAPS scale) of PTSD patients showed a significant downward trend (Figure 6C).
- accompanying symptoms such as anxiety level (Figure 6D), depression level (Figure 6E), sleep quality (Figure 6F), and social performance (Figure 6G) all reflected significant improvements at different treatment stages.
- the specific manifestations include a decrease in the Hamilton Anxiety Scale, a decrease in the Hamilton Depression Scale, a decrease in the Sleep Quality Index, and an increase in the Social Performance Scale.
- transcranial magnetic stimulation of the primary motor cortex has a significant effect on the core symptoms of PTSD, and also has a good therapeutic effect on its accompanying anxiety, depression, sleep abnormalities, social abnormalities, etc.
- the unit "degree” for temperature appearing in this article refers to degrees Celsius, that is, °C.
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Abstract
The present invention provides a method for treating or diagnosing emotional disorders or stress disorders, comprising regulating the excitability of the motor cortex of a patient or detecting the excitability of the motor cortex of the patient. The present invention also provides a pharmaceutical composition for treating emotional or stress disorders, comprising a preparation for regulating the excitability of the motor cortex. The present invention also provides a device for treating emotional or stress disorders, comprising an apparatus for regulating the excitability of the motor cortex. The present invention also provides a kit or a medical apparatus for diagnosing emotional or stress disorder.
Description
本申请要求以下中国专利申请的优先权：申请日为2022年4月22日、申请号为202210428036.4、发明名称为“情绪障碍或应激障碍的治疗或诊断”，其全部内容通过引用结合在本申请中。This application claims priority from the following Chinese patent applications: the application date is April 22, 2022, the application number is 202210428036.4, and the invention title is "Treatment or Diagnosis of Mood Disorders or Stress Disorders", the entire contents of which are incorporated herein by reference. Applying.
本发明涉及疾病治疗和药物领域。具体的，本发明涉及情绪或应激障碍的治疗或诊断方法，以及相关的药物组合物、试剂盒或医疗器械。The present invention relates to the field of disease treatment and medicine. Specifically, the present invention relates to methods for treating or diagnosing mood or stress disorders, as well as related pharmaceutical compositions, kits or medical devices.
随着社会环境的复杂、人们物质水平的提高、生活习惯的改变，诸多精神健康的不利因素不断增多，各类精神疾病的患病率在逐步提升。目前包括焦虑症、抑郁症、惊恐障碍、双相情感障碍和应激障碍在内的情绪或应激障碍类的精神疾病，临床常用药物效果因人而异，并有不同程度的副作用。With the complexity of the social environment, the improvement of people's material standards, and changes in living habits, many negative factors for mental health are increasing, and the prevalence of various mental diseases is gradually increasing. Currently, for mental illnesses such as mood or stress disorders, including anxiety disorder, depression, panic disorder, bipolar disorder and stress disorder, the effects of commonly used clinical drugs vary from person to person and have varying degrees of side effects.
根据美国精神病学协会(The American Psychiatric Association,APA)于2013年出版的《精神障碍诊断与统计手册(第5版)》(Diagnostic and Statistical Manual 5th Edition,DSM-5)，情绪障碍主要包括抑郁障碍、双相情感障碍及二者的亚型，焦虑障碍主要包括社交恐惧、惊恐发作、分离性焦虑等，应激障碍主要包括急性应激障碍和创伤后应激障碍。According to the Diagnostic and Statistical Manual 5th Edition (DSM-5) of Mental Disorders (5th Edition) published by the American Psychiatric Association (APA) in 2013, mood disorders mainly include depressive disorders , bipolar disorder and their subtypes. Anxiety disorders mainly include social phobia, panic attacks, separation anxiety, etc. Stress disorders mainly include acute stress disorder and post-traumatic stress disorder.
近年来，电、磁刺激疗法因其无创、副作用小以及病人耐受程度高，在治疗精神类疾病方面的运用日趋增多。前额叶皮层是当下电、磁刺激疗法的最主要的靶点，治疗效果在抑郁症患者中较为明确，但在焦虑障碍、双相情感障碍以及应激类障碍中尚没有足够的证据，故临床上仍缺乏对其他可能脑区治疗精神类疾病的探索。In recent years, electrical and magnetic stimulation therapy has been increasingly used in the treatment of mental illnesses due to its non-invasiveness, minimal side effects and high patient tolerance. The prefrontal cortex is the main target of current electromagnetic stimulation therapy. The therapeutic effect is relatively clear in patients with depression, but there is not enough evidence in anxiety disorders, bipolar disorder, and stress disorders. Therefore, clinical There is still a lack of exploration into other possible brain regions for treating mental illnesses.
本领域还需要发掘新的脑区在包括焦虑症、抑郁症、惊恐、双相情感障碍和应激障碍在内的情绪或应激障碍类精神疾病中的作用，以突破目前单一脑区活性调控的思路，有助于以上各类疾病的临床治疗，也可能为临
床诊断这些精神类疾病提供更客观的标记物并预测疾病的治疗效果。This field also needs to explore the role of new brain regions in mood or stress disorder-like mental illnesses including anxiety, depression, panic, bipolar disorder and stress disorder, in order to break through the current regulation of single brain region activity. The idea is helpful for the clinical treatment of the above types of diseases, and may also provide clinical Clinical diagnosis of these psychiatric disorders provides more objective markers and predicts the effectiveness of treatment.
发明内容Contents of the invention
本发明首次发现哺乳动物的运动皮层，特别是初级运动皮层参与双相及相关障碍(Bipolar and related disorders)，抑郁障碍(Depression disorders)，焦虑障碍(Anxiety disorders)和应激障碍(Stress disorders)等情绪或应激障碍类精神疾病的发病过程。更重要的是，本发明发现了对哺乳动物的运动皮层，特别是初级运动皮层的兴奋性进行调节，可治疗具有双相以及相关障碍，抑郁障碍，焦虑障碍和应激障碍等情绪或应激障碍类精神疾病或病征的动物。本发明由此提供了调节运动皮层兴奋性来治疗情绪或应激障碍的方法和药物或设备，以及通过对运动皮层兴奋性的检测来诊断情绪或应激障碍的方法和试剂盒或设备。This invention first discovered that the motor cortex of mammals, especially the primary motor cortex, is involved in bipolar and related disorders, depressive disorders, anxiety disorders, stress disorders, etc. The pathogenesis of mental illness such as mood or stress disorders. More importantly, the present invention has found that regulating the excitability of the motor cortex of mammals, especially the primary motor cortex, can treat mood or stress disorders such as bipolar and related disorders, depressive disorders, anxiety disorders and stress disorders. Animals suffering from mental disorders or symptoms. The present invention thus provides methods, medicines or devices for treating mood or stress disorders by regulating motor cortex excitability, as well as methods, kits or devices for diagnosing mood or stress disorders by detecting motor cortex excitability.
本发明提供了通过对患者的运动皮层的兴奋性进行调节来治疗情绪或应激障碍的方法。本发明提供了调节运动皮层的兴奋性的试剂或医疗装置用于治疗情绪或应激障碍的方法，特别是将所述试剂局部施用于运动皮层或将所述医疗装置对运动皮层做局部刺激用于治疗情绪或应激障碍的方法。本发明提供了调节运动皮层的兴奋性的药物或医疗设备，其用于治疗情绪或应激障碍，特别是局部施用于运动皮层的药物或对运动皮层做局部刺激的医疗设备。本发明提供了所述试剂或医疗装置用于制备治疗抑郁症的药物或医疗设备的用途，特别是用于制备局部作用在运动皮层的药物或医疗设备。The present invention provides methods of treating mood or stress disorders by modulating the excitability of the patient's motor cortex. The present invention provides an agent or a medical device for regulating the excitability of the motor cortex and a method for treating mood or stress disorders. In particular, the agent is locally applied to the motor cortex or the medical device is used to locally stimulate the motor cortex. Methods for treating mood or stress disorders. The present invention provides drugs or medical devices that modulate the excitability of the motor cortex for the treatment of mood or stress disorders, particularly drugs that are locally applied to the motor cortex or medical devices that locally stimulate the motor cortex. The present invention provides the use of the reagent or medical device for preparing drugs or medical devices for treating depression, especially for preparing drugs or medical devices that act locally on the motor cortex.
在本发明的其中一个方面，提供了治疗情绪或应激障碍的方法，其包括对运动皮层的兴奋性进行调节，尤其是对患者的初级运动皮层的兴奋性进行调节。In one aspect of the present invention, a method for treating mood or stress disorders is provided, which includes modulating the excitability of the motor cortex, especially the excitability of the primary motor cortex of the patient.
在本发明的其中一个方面，所述治疗情绪或应激障碍的方法中对运动皮层特别是初级运动皮层的兴奋性的调节是增加其兴奋性。In one aspect of the present invention, the method for treating mood or stress disorders modulates the excitability of the motor cortex, especially the primary motor cortex, by increasing its excitability.
需要本文所述的方法和药物(药物组合物)的对象(患者)可以是哺乳动物，包括人或者非人灵长类如猩猩或猴。哺乳动物还可以是其它动物，例如大鼠、小鼠、兔、猪、狗等。所述哺乳动物可以是家养动物，例如猫或者狗。A subject (patient) in need of the methods and medicaments (pharmaceutical compositions) described herein can be a mammal, including humans, or a non-human primate such as an orangutan or monkey. Mammals can also be other animals, such as rats, mice, rabbits, pigs, dogs, etc. The mammal may be a domestic animal, such as a cat or a dog.
哺乳动物的运动皮层也称为“中央前回”，或“第Ⅰ躯体运动区”。运动皮
层是额叶的一个区域，是位于中央沟前的后中央回的一大块灰质。已发现哺乳动物的运动皮层主要负责支配躯体各部分的运动，电刺激该部位会引起运动反应。The motor cortex of mammals is also called the "precentral gyrus", or "the first somatic motor area". Sports leather The layer is an area of the frontal lobe, a large mass of gray matter in the postcentral gyrus in front of the central sulcus. It has been found that the motor cortex of mammals is mainly responsible for controlling the movement of various parts of the body, and electrical stimulation of this part will cause motor responses.
哺乳动物的运动皮层包括初级运动皮层。初级运动皮层产生的神经冲动向下传递到脊髓，控制着人体运动的执行。灵长类动物的运动皮层还包括前运动皮层和辅助运动区。在其它类别的哺乳动物中，除了初级运动皮层，还包括次级运动皮层。The mammalian motor cortex includes the primary motor cortex. Nerve impulses generated by the primary motor cortex are transmitted down to the spinal cord, controlling the execution of human movements. The motor cortex of primates also includes the premotor cortex and supplementary motor area. In other categories of mammals, in addition to the primary motor cortex, there is also a secondary motor cortex.
在本发明提供的方法中，可以通过本领域已知的方法对运动皮层特别是初级运动皮层的兴奋性进行调节，包括通过物理、化学或生物化学方法。In the method provided by the present invention, the excitability of the motor cortex, especially the primary motor cortex, can be adjusted by methods known in the art, including through physical, chemical or biochemical methods.
在本发明的其中一个方面，通过对患者运动皮层施加电刺激、磁刺激、光刺激、振动刺激、压力刺激、声刺激、超声刺激等物理方法来调节运动皮层的兴奋性。本领域已知的各种对脑部组织例如运动皮层进行电刺激、磁刺激、光刺激、振动刺激、压力刺激、声刺激、超声刺激的方法和装置都可以用于本发明的方法。例如，电刺激可以通过植入内部的探针或外置于头皮的电极来提供。例如，磁刺激可以通过植入内部的探头或外部电圈产生的定向磁场来提供。又例如，热刺激可以通过使用植入的可产生或发出热和/或冷的温度的探针来进行。In one aspect of the present invention, the excitability of the motor cortex is adjusted by applying physical methods such as electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation, pressure stimulation, acoustic stimulation, and ultrasonic stimulation to the patient's motor cortex. Various methods and devices known in the art for electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation, pressure stimulation, acoustic stimulation, and ultrasonic stimulation of brain tissues, such as motor cortex, can be used in the method of the present invention. For example, electrical stimulation can be delivered through an internally implanted probe or through electrodes placed externally on the scalp. For example, magnetic stimulation can be delivered by a directional magnetic field generated by an internally implanted probe or an external electrocoil. As another example, thermal stimulation may be performed using an implanted probe that generates or emits heat and/or cold temperatures.
这些刺激装置或******作以刺激预定的脑的部位。这些装置可包括刺激部分或探针，例如，电极、电极组件(如电刺激导线)、线圈、药物输送组件(如导管)或这些的组合和/或信号发生器或信号源或脉冲产生源(即电信号源、化学信号源(即药物输送泵)或磁信号源)。探头可与信号源、给药泵或两者相连接，而信号源和给药泵又被用来刺激预定的治疗部位。探针和信号发生器或信号源可以结合在一起，形成一个单元或单一装置，这种装置可以包括一个、两个或多个电极。这些装置在本领域中被称为微刺激器。These stimulation devices or systems are operated to stimulate predetermined parts of the brain. These devices may include stimulation portions or probes, for example, electrodes, electrode assemblies (such as electrical stimulation leads), coils, drug delivery components (such as catheters) or combinations of these and/or signal generators or sources of signal or pulse generation ( i.e. electrical signal source, chemical signal source (i.e. drug delivery pump) or magnetic signal source). The probe can be connected to a signal source, a drug delivery pump, or both, and the signal source and drug delivery pump are used to stimulate the intended treatment site. The probe and signal generator or source may be combined to form a unit or a single device, which may include one, two or more electrodes. These devices are known in the art as microstimulators.
在本发明的其中一个方面，通过对患者给予调节运动皮层的细胞(神经细胞)兴奋性的制剂等化学或生物化学方法来调节运动皮层的兴奋性。可调节神经细胞兴奋性的制剂包括光遗传(Optogenetics)制剂、化学遗传学(Chemogenetics)制剂或化学药物或制剂等。In one aspect of the present invention, the excitability of the motor cortex is modulated by chemical or biochemical methods such as administering to the patient a preparation that modulates the excitability of cells (nerve cells) in the motor cortex. Preparations that can regulate the excitability of nerve cells include optogenetics preparations, chemogenetics preparations, chemical drugs or preparations, etc.
在本发明中，所述光遗传制剂为可在运动皮层的细胞中表达光感基
因(例如ChR2，eBR，NaHR3.0，Arch或OptoXR)的试剂。In the present invention, the optogenetic preparation is capable of expressing photoreceptor genes in cells of the motor cortex. (e.g. ChR2, eBR, NaHR3.0, Arch or OptoXR).
在本发明中，所述化学遗传学制剂为可在运动皮层的细胞中表达调节细胞兴奋性蛋白的试剂，激活/拮抗调节细胞兴奋性蛋白的试剂，例如为表达神经递质受体或转运体或降解酶的试剂，或为所述神经递质包括乙酰胆碱(ACh)，谷氨酸，ATP，腺苷，γ-氨基丁酸(GABA)，去甲肾上腺素，多巴胺，内源性***素(endocannabinoids)，一氧化氮，组胺等。In the present invention, the chemical genetic preparation is an agent that can express a protein that regulates cell excitability in cells of the motor cortex, and activates/antagonizes an agent that regulates a protein that regulates cell excitability, such as expressing a neurotransmitter receptor or transporter. or agents for degrading enzymes, or for the neurotransmitters including acetylcholine (ACh), glutamate, ATP, adenosine, gamma-aminobutyric acid (GABA), norepinephrine, dopamine, endocannabinoids ( endocannabinoids), nitric oxide, histamine, etc.
在本发明中，能够增加运动皮层的细胞兴奋性的化学或生物化学制剂包括但不限于：In the present invention, chemical or biochemical agents capable of increasing cellular excitability of the motor cortex include, but are not limited to:
a.神经递质，例如乙酰胆碱(ACh)，谷氨酸，ATP，腺苷，γ-氨基丁酸(GABA)，去甲肾上腺素，多巴胺，内源性***素(endocannabinoids)，一氧化氮，组胺等；a. Neurotransmitters such as acetylcholine (ACh), glutamate, ATP, adenosine, gamma-aminobutyric acid (GABA), norepinephrine, dopamine, endocannabinoids, nitric oxide, Histamine, etc.;
b.神经递质受体激动剂，例如Gq GPCR的激动剂，包括内源性***素受体(CB1Rs)激动剂；代谢型谷氨酸I和II型受体激动剂以及AMPA受体激动剂；嘌呤能受体激动剂；GABA能受体激动剂；α-肾上素能受体激动剂，多巴胺能受体激动剂；组胺能受体激动剂；蛋白酶激活受体(protease-activated receptor)的激动剂；b. Neurotransmitter receptor agonists, such as Gq GPCR agonists, including endocannabinoid receptor (CB1Rs) agonists; metabotropic glutamate type I and II receptor agonists and AMPA receptor agonists ; Purinergic receptor agonist; GABAergic receptor agonist; alpha-adrenergic receptor agonist, dopaminergic receptor agonist; histaminergic receptor agonist; protease-activated receptor ) agonist;
c.神经递质再摄取抑制剂，例如去甲肾上腺素、多巴胺和5-羟色胺的再摄取抑制剂；c. Neurotransmitter reuptake inhibitors, such as norepinephrine, dopamine and serotonin reuptake inhibitors;
或or
d.离子通道的激动剂，例如瞬态电压感受器阳离子通道,子类V,成员1(transient receptor potential cation channel,subfamily V,TRPV1)的激动剂；Na+/Ca2+exchangers(NCXs)激动剂。d. Agonists of ion channels, such as agonists of transient receptor potential cation channel, subfamily V, TRPV1; agonists of Na + /Ca 2+ exchangers (NCXs) .
在本发明中，“情绪障碍或应激障碍”是指患者具有痛苦、悲伤、快感消失、空虚或烦躁易怒等破坏性心境，以及通常在焦虑或恐惧等背景下，患者认知改变伴随躯体功能异常的精神类疾病。In the present invention, "mood disorder or stress disorder" refers to patients who have destructive moods such as pain, sadness, loss of pleasure, emptiness, irritability, and irritability, and usually in the context of anxiety or fear, the patient's cognitive changes are accompanied by physical changes. Abnormal mental illness.
在本发明的其中一个方面，“情绪障碍或应激障碍”包括双相以及相关障碍(Bipolar and related disorders)，抑郁障碍(Depression disorders)，焦虑障碍(Anxiety disorders)和应激障碍(Stress disorders)类精神疾病。上述这四类精神疾病在美国精神病学会(American Psychiatric Association,APA)提出的美国精神疾病分类与诊断标准(DSM-5)和世
界卫生组织(WHO)发布的国际疾病分类第十一次修订本(ICD-11)中进行了描述和定义。在本发明的其中又一个方面，“情绪障碍或应激障碍”包括以下疾病(根据ICD-11定义和编号)：In one aspect of the invention, "mood or stress disorders" include bipolar and related disorders, depression disorders, anxiety disorders and stress disorders. Mental illness. The above four types of mental disorders are classified in the American Classification and Diagnostic Criteria for Mental Disorders (DSM-5) and the World Classification and Diagnostic Criteria for Mental Disorders proposed by the American Psychiatric Association (APA). It is described and defined in the International Classification of Diseases, Eleventh Revision (ICD-11) published by the World Health Organization (WHO). In yet another aspect of the invention, "mood disorder or stress disorder" includes the following diseases (according to ICD-11 definition and numbering):
L2-6A6双相及相关障碍L2-6A6 Bipolar and Related Disorders
6A60双相障碍I型6A60 Bipolar disorder type I
6A60.0双相I型障碍，目前为不伴精神病性症状的躁狂发作6A60.0 Bipolar I disorder, currently manic episode without psychotic symptoms
6A60.1双相I型障碍，目前为伴精神病性症状的躁狂发作6A60.1 Bipolar I disorder, currently manic episode with psychotic symptoms
6A60.2双相I型障碍，目前为轻躁狂发作6A60.2 Bipolar I disorder, current hypomanic episode
6A60.3双相I型障碍，目前为轻度抑郁发作6A60.3 Bipolar I disorder, current mild depressive episode
6A60.4双相I型障碍，目前为不伴精神病性症状的中度抑郁发作6A60.4 Bipolar I disorder, currently a moderate depressive episode without psychotic symptoms
6A60.5双相I型障碍，目前为伴精神病性症状的中度抑郁发作6A60.5 Bipolar I disorder, current moderate depressive episode with psychotic symptoms
6A60.6双相I型障碍，目前为不伴精神病性症状的重度抑郁发作6A60.6 Bipolar I disorder, currently a major depressive episode without psychotic symptoms
6A60.7双相I型障碍，目前为伴精神病性症状的重度抑郁发作6A60.7 Bipolar I disorder, current major depressive episode with psychotic symptoms
6A60.8双相I型障碍，目前为未特指严重程度的抑郁发作6A60.8 Bipolar I disorder, currently a depressive episode of unspecified severity
6A60.9双相I型障碍，目前为不伴精神病性症状的混合性发作6A60.9 Bipolar I disorder, currently mixed episodes without psychotic symptoms
6A60.A双相I型障碍，目前为伴精神病性症状的混合性发作6A60.A Bipolar I disorder, currently mixed episode with psychotic symptoms
6A60.B双相I型障碍，目前为部分缓解，最近为躁狂或轻躁狂发作6A60.B Bipolar I disorder, currently in partial remission, recent manic or hypomanic episode
6A60.C双相I型障碍，目前为部分缓解，最近为抑郁发作6A60.C Bipolar I disorder, currently in partial remission, recent depressive episode
6A60.D双相I型障碍，目前为部分缓解，最近为混合性发作6A60.D Bipolar I disorder, currently in partial remission, most recently in mixed episodes
6A60.E双相I型障碍，目前为部分缓解，最近为未特指的发作6A60.E Bipolar I disorder, currently in partial remission, recent unspecified episode
6A60.F双相I型障碍，目前为完全缓解6A60.F Bipolar I disorder, currently in complete remission
6A60.Y其他特指的双相障碍I型6A60.Y Other specified bipolar disorder type I
6A60.Z双相障碍I型，未特指的6A60.Z Bipolar disorder type I, unspecified
6A61双相障碍Ⅱ型6A61 Bipolar disorder type II
6A61.0双相Ⅱ型障碍，目前为轻躁狂发作6A61.0 Bipolar II disorder, current hypomanic episode
6A61.1双相Ⅱ型障碍，目前为轻度抑郁发作6A61.1 Bipolar II disorder, current mild depressive episode
6A61.2双相Ⅱ型障碍，目前为不伴精神病性症状的中度抑郁发作6A61.2 Bipolar II disorder, currently a moderate depressive episode without psychotic symptoms
6A61.3双相Ⅱ型障碍，目前为伴精神病性症状的中度抑郁发作6A61.3 Bipolar II disorder, currently a moderate depressive episode with psychotic symptoms
6A61.4双相Ⅱ型障碍，目前为不伴精神病性症状的重度抑郁发作6A61.4 Bipolar II disorder, currently a major depressive episode without psychotic symptoms
6A61.5双相Ⅱ型障碍，目前为伴精神病性症状的重度抑郁发作6A61.5 Bipolar II disorder, currently a major depressive episode with psychotic symptoms
6A61.6双相Ⅱ型障碍，目前为未特指严重程度的抑郁发作
6A61.6 Bipolar II disorder, currently a depressive episode of unspecified severity
6A61.7双相Ⅱ型障碍，目前为部分缓解，最近为轻躁狂发作6A61.7 Bipolar II disorder, currently in partial remission, recent hypomanic episode
6A61.8双相Ⅱ型障碍，目前为部分缓解，最近为抑郁发作6A61.8 Bipolar II disorder, currently in partial remission, recent depressive episode
6A61.9双相Ⅱ型障碍，目前为部分缓解，最近为未特指发作6A61.9 Bipolar II disorder, currently in partial remission, recent episode of unspecified
6A61.A双相Ⅱ型障碍，目前为完全缓解6A61.A Bipolar II disorder, currently in complete remission
6A61.Y其他特指的双相障碍Ⅱ型6A61.Y Other specified bipolar disorder type II
6A61.Z双相障碍Ⅱ型，未特指的6A61.Z Bipolar disorder type II, unspecified
6A62环性心境障碍6A62 cyclothymic disorder
6A6Y其他特指的双相及相关障碍6A6Y Other specified bipolar and related disorders
6A6Z双相及相关障碍，未特指的6A6Z Bipolar and related disorders, unspecified
L2-6A7抑郁障碍L2-6A7 depressive disorder
6A70单次发作的抑郁障碍6A70 Single episode depressive disorder
6A70.0单次发作的抑郁障碍，轻度6A70.0 Single episode of depressive disorder, mild
6A70.1单次发作的抑郁障碍，中度，不伴精神病性症状6A70.1 Single episode of depressive disorder, moderate, without psychotic symptoms
6A70.2单次发作的抑郁障碍，中度，伴精神病性症状6A70.2 Single episode of depressive disorder, moderate, with psychotic symptoms
6A70.3单次发作的抑郁障碍，重度，不伴精神病性症状6A70.3 Single episode of depressive disorder, severe, without psychotic symptoms
6A70.4单次发作的抑郁障碍，重度，伴精神病性症状6A70.4 Single episode of depressive disorder, severe, with psychotic symptoms
6A70.5单次发作抑郁障碍，未特指严重程度6A70.5 Single episode of depressive disorder, unspecified severity
6A70.6单次发作抑郁障碍，目前为部分缓解6A70.6 Single episode of depressive disorder, currently in partial remission
6A70.7单次发作抑郁障碍，目前为完全缓解6A70.7 Single episode of depressive disorder, currently in complete remission
6A70.Y其他特指的单次发作的抑郁障碍6A70.Y Other specified single-episode depressive disorders
6A70.Z单次发作的抑郁障碍，未特指的6A70.Z Single episode depressive disorder, unspecified
6A71复发性抑郁障碍6A71 Recurrent depressive disorder
6A71.0复发性抑郁障碍，目前为轻度发作6A71.0 Recurrent depressive disorder, currently mild episode
6A71.1复发性抑郁障碍，目前为中度发作，不伴精神病性症状6A71.1 Recurrent depressive disorder, currently of moderate severity, without psychotic symptoms
6A71.2复发性抑郁障碍，目前为中度发作，伴精神病性症状6A71.2 Recurrent depressive disorder, currently of moderate severity, with psychotic symptoms
6A71.3复发性抑郁障碍，目前为重度发作，不伴精神病性症状6A71.3 Recurrent depressive disorder, currently severe episode, without psychotic symptoms
6A71.4复发性抑郁障碍，目前为伴精神病性症状的重度发作6A71.4 Recurrent depressive disorder, currently severe episode with psychotic symptoms
6A71.5复发性抑郁障碍，目前发作，严重程度未特指6A71.5 Recurrent depressive disorder, current episode, severity unspecified
6A71.6复发性抑郁障碍，目前为部分缓解6A71.6 Recurrent depressive disorder, currently in partial remission
6A71.7复发性抑郁障碍，目前为完全缓解
6A71.7 Recurrent depressive disorder, currently in complete remission
6A71.Y其他特指的复发性抑郁障碍6A71.Y Other specified recurrent depressive disorders
6A71.Z复发性抑郁障碍，未特指的6A71.Z Recurrent depressive disorder, unspecified
6A72恶劣心境障碍6A72 Dysthymic Disorder
6A73混合性抑郁焦虑障碍6A73 Mixed depressive anxiety disorder
6A7Y其他特指的抑郁障碍6A7Y Other specified depressive disorders
6A7Z抑郁障碍，未特指的6A7Z Depressive disorder, unspecified
6A80心境障碍中，心境障碍发作的症状和病程表现6A80 Mood disorders, symptoms and course of mood disorder attacks
6A80.0心境障碍发作突出的焦虑症状6A80.0 Prominent anxiety symptoms in mood disorder attacks
6A80.1心境障碍中的惊恐发作6A80.1 Panic attacks in mood disorders
6A80.2目前抑郁发作持续6A80.2 Current depressive episode persists
6A80.3目前抑郁发作伴忧郁特征6A80.3 Current depressive episode with melancholic features
6A80.4心境障碍发作的季节特征6A80.4 Seasonal characteristics of mood disorder attacks
6A80.5快速循环6A80.5 fast cycle
6A8Y其他特指的心境障碍6A8Y Other specified mood disorders
6A8Z心境障碍，未特指的6A8Z Mood disorder, unspecified
L1-6B0焦虑或恐惧相关性障碍L1-6B0 Anxiety or fear-related disorder
6B00广泛性焦虑障碍6B00 Generalized Anxiety Disorder
6B01惊恐障碍6B01 Panic disorder
6B02广场恐怖6B02 Square Terror
6B03特定的恐怖6B03 specific horror
6B04社交性焦虑障碍6B04 Social anxiety disorder
6B05分离性焦虑障碍6B05 Separation anxiety disorder
6B06选择性缄默症6B06Selective Mutism
6B0Y其他特指的焦虑或恐惧相关性障碍6B0Y Other specified anxiety or fear-related disorders
6B0Z焦虑或恐惧相关性障碍，未特指的6B0Z Anxiety or fear-related disorder, unspecified
L1-6B4应激相关障碍L1-6B4 stress-related disorders
6B40创伤后应激障碍6B40 Post-traumatic stress disorder
6B41复杂性创伤后应激障碍6B41 Complex post-traumatic stress disorder
6B42延长哀伤障碍
6B42 Prolonged grief disorder
6B43适应障碍6B43 Adjustment disorder
6B44反应性依恋障碍6B44 Reactive Attachment Disorder
6B45脱抑制性社会参与障碍6B45 Disinhibitory social participation disorder
6B4Y其他特指的应激相关障碍6B4Y Other specified stress-related disorders
6B4Z应激相关障碍，未特指的。6B4Z Stress-related disorder, unspecified.
在本发明的其中一个方面，所述通过调节患者的运动皮层(特别是初级运动皮层)的兴奋性来治疗情绪障碍或应激障碍的方法的患者具有以上情绪或应激障碍中的一种或多种疾病或病征。例如，所述患者同时具有双相及相关障碍、抑郁障碍、焦虑或恐惧相关性障碍和应激相关障碍中的任意两种。又例如，所述患者同时具有双相及相关障碍、抑郁障碍、焦虑或恐惧相关性障碍和应激相关障碍中的任意三种或四种。In one aspect of the present invention, the method of treating a mood disorder or a stress disorder by modulating the excitability of the patient's motor cortex (especially the primary motor cortex) has a patient with one of the above mood or stress disorders or Various diseases or conditions. For example, the patient has any two of bipolar and related disorders, depressive disorders, anxiety or fear-related disorders, and stress-related disorders. For another example, the patient has any three or four of bipolar and related disorders, depressive disorders, anxiety or fear-related disorders, and stress-related disorders.
在本发明的其中一个方面，所述通过调节患者的运动皮层(特别是初级运动皮层)的兴奋性来治疗情绪障碍或应激障碍的方法特别适用于治疗应激障碍，尤其是治疗创伤后应激障碍和复杂性创伤后应激障碍。In one aspect of the present invention, the method of treating mood disorders or stress disorders by modulating the excitability of the patient's motor cortex (especially the primary motor cortex) is particularly suitable for the treatment of stress disorders, especially the treatment of post-traumatic stress. inflammatory disorder and complex post-traumatic stress disorder.
本发明中，“治疗”包括：改良、减轻、减少或预防与情绪障碍或应激障碍等精神疾病相关的症状的进行中的过程或结果；改善与所述精神疾病相关的症状的进行中的过程或结果；使处于导致特定机体功能损伤的疾病或病症中的机体功能正常化的进行中的过程或结果；或者引发疾病的一种或多种临床可测定的参数改善的进行中的过程或结果。在一个实施方案中，治疗目的是预防或减慢不希望的生理情况、病症或疾病，或获得有益的或期望的结果。该结果可以是，例如医学的、生理学的、临床的、物理治疗、职业治疗，面向保健人员或患者；或本领域理解为“生活品质”或日常生活活动的参数。本发明中，有益的或期望的临床结果包括但不限于，减轻症状；减小/缩小该情况、病症或疾病的程度；稳定该情况、病症或疾病的状态；延迟该情况、病症或疾病的开始或减慢其进展；改善或缓和该情况、病症或疾病。在一个实施方案中，治疗包括引发临床有效响应而没有过度水平的副作用。在一个实施方案中，治疗也包括与如果不接受治疗的预期的存活期相比延长存活期。本发明中，治疗可以是预防，治愈，或改良患者的临床情况，包括降低病程或疾病严重度，或主观改善患者的生活品质或延长患者的存活期等。In the present invention, "treatment" includes: the ongoing process or results of improving, alleviating, reducing or preventing symptoms related to mental disorders such as mood disorders or stress disorders; the ongoing process of improving symptoms related to the mental diseases. The process or result; the ongoing process or result of normalizing the functions of a body in a disease or condition that results in impairment of a specified body function; or the ongoing process or result of improving one or more clinically measurable parameters of the disease or result. In one embodiment, the purpose of treatment is to prevent or slow down an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired result. The outcome may be, for example, medical, physiological, clinical, physiotherapy, occupational therapy, for healthcare providers or patients; or a parameter understood in the art as "quality of life" or activities of daily living. In the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms; reducing/narrowing the extent of the condition, disorder, or disease; stabilizing the condition, disorder, or disease; delaying the onset of the condition, disorder, or disease To start or slow down the progress of a condition, disorder or disease; to improve or alleviate a condition, disorder or disease. In one embodiment, treatment involves eliciting a clinically effective response without undue levels of side effects. In one embodiment, treatment also includes prolonging survival compared to expected survival if not receiving treatment. In the present invention, treatment may be prevention, cure, or improvement of the patient's clinical condition, including reducing the course or severity of the disease, or subjectively improving the patient's quality of life or prolonging the patient's survival period.
本发明首次发现哺乳动物的运动皮层，特别是初级运动皮层参与双相
以及相关障碍，抑郁障碍，焦虑障碍和应激障碍等情绪障碍或应激障碍类精神疾病的发病过程。更重要的是，本发明发现了对哺乳动物的运动皮层，特别是初级运动皮层的兴奋性进行调节，可治疗具有双相以及相关障碍，抑郁障碍，焦虑障碍和应激障碍等情绪障碍或应激障碍类精神疾病或病征的动物。这是本领域已知的治疗情绪障碍或应激障碍类精神疾病的机制和药物未能针对的病理机制和进行治疗的脑部靶组织。因此，本发明提供的方法和药物或装置适合用于治疗情绪障碍或应激障碍类精神疾病。The present invention discovered for the first time that the motor cortex of mammals, especially the primary motor cortex, is involved in biphasic As well as related disorders, depressive disorders, anxiety disorders and stress disorders and other mood disorders or stress disorder-type mental illness pathogenesis. More importantly, the present invention has discovered that regulating the excitability of the motor cortex of mammals, especially the primary motor cortex, can treat mood disorders or stress disorders such as bipolar and related disorders, depressive disorders, anxiety disorders and stress disorders. Animals suffering from irritable mental illness or symptoms. This is a mechanism known in the art for treating mental disorders such as mood disorders or stress disorders, and drugs fail to target the pathological mechanisms and target brain tissues for treatment. Therefore, the methods, medicines or devices provided by the present invention are suitable for treating mental disorders such as mood disorders or stress disorders.
本发明还提供了用于治疗患者的情绪或应激障碍的药物组合物或试剂盒，或医疗设备，其含有调节运动皮层特别是初级运动皮层的兴奋性的试剂或装置，例如包括通过物理、化学或生物化学方法来调节运动皮层特别是初级运动皮层的兴奋性的试剂和装置。在本发明的其中一种实施方式中，提供了调节患者的运动皮层的兴奋性的设备，例如对患者运动皮层施加电刺激、磁刺激、光刺激、振动刺激、压力刺激、声刺激、超声刺激的一种或多种的设备。在本发明的其中一种实施方式中，提供了包含调节运动皮层兴奋性的试剂的药物组合物。The present invention also provides pharmaceutical compositions or kits, or medical devices for treating mood or stress disorders in patients, which contain agents or devices that modulate the excitability of the motor cortex, especially the primary motor cortex, for example, including through physical, Agents and devices for chemically or biochemically modulating the excitability of the motor cortex, especially the primary motor cortex. In one embodiment of the present invention, a device for regulating the excitability of the patient's motor cortex is provided, such as applying electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation, pressure stimulation, acoustic stimulation, and ultrasonic stimulation to the patient's motor cortex. one or more devices. In one embodiment of the invention, there is provided a pharmaceutical composition comprising an agent that modulates motor cortex excitability.
所述试剂或装置如前文中描述的调节运动皮层特别是初级运动皮层的兴奋性的方法中描述。例如为对患者运动皮层施加电刺激、磁刺激、光刺激、振动刺激、压力刺激、声刺激、超声刺激来调节运动皮层的兴奋性的试剂和装置。例如为光遗传(Optogenetics)制剂、化学遗传学(Chemogenetics)制剂或化学药物等。在本发明中，所述光遗传制剂为可在运动皮层的细胞中表达光感基因(例如ChR2，eBR，NaHR3.0，Arch或OptoXR)的试剂。在本发明中，所述化学遗传学制剂为可在运动皮层的细胞中表达调节细胞兴奋性蛋白的试剂，激活/拮抗调节细胞兴奋性蛋白的试剂，或为神经递质。在本发明中，能够增加运动皮层的细胞兴奋性的化学药物包括但不限于：a.神经递质；b.神经递质受体激动剂；c.神经递质再摄取抑制剂；或d.离子通道的激动剂。The agent or device is as described above in the method of modulating the excitability of the motor cortex, especially the primary motor cortex. For example, reagents and devices that apply electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation, pressure stimulation, acoustic stimulation, and ultrasonic stimulation to the patient's motor cortex to adjust the excitability of the motor cortex. For example, they are optogenetics preparations, chemogenetics preparations or chemical drugs. In the present invention, the optogenetic agent is an agent that can express light-sensing genes (eg, ChR2, eBR, NaHR3.0, Arch or OptoXR) in cells of the motor cortex. In the present invention, the chemical genetic agent is an agent that can express a protein that regulates cell excitability in cells of the motor cortex, an agent that activates/antagonizes a protein that regulates cell excitability, or is a neurotransmitter. In the present invention, chemical drugs that can increase the excitability of cells in the motor cortex include, but are not limited to: a. neurotransmitters; b. neurotransmitter receptor agonists; c. neurotransmitter reuptake inhibitors; or d. Ion channel agonist.
在本发明的其中一个方面，提供了含有调节运动皮层特别是初级运动皮层的兴奋性的试剂或装置在用于治疗患者的情绪或应激障碍的药物或设备中的用途。In one aspect of the present invention, there is provided the use of an agent or device containing an agent or device that modulates the excitability of the motor cortex, especially the primary motor cortex, in a medicament or device for treating mood or stress disorders in a patient.
在本发明的其中一个方面，本发明的通过调节患者的运动皮层(特别是初级运动皮层)的兴奋性来治疗情绪障碍或应激障碍的方法，以及所
述的治疗情绪障碍或应激障碍的药物或设备中，所述方法、药物或设备为在运动皮层特别是初级运动皮层中局部起效，即局部调节运动皮层的兴奋性的方法和药物或设备。用于治疗精神疾病的药物对于用于神经组织的方法和药物或设备，特别是脑部神经组织，例如运动皮层来说，将药物或刺激作用限定在目标组织是有益的。用于运动皮层的局部给药或刺激对治疗方法和制备药物或设备都是限制性的技术特征。用于运动皮层中的方法或药物需要考虑该方法或药物或刺激信号是否能够在运动皮层发挥有效性，包括药物或刺激信号是否能到达运动皮层，以及在运动皮层中是否能达到起效的浓度或刺激强度等。在本发明中，所述药物为在运动皮层或其附近组织中局部给药的剂型。可以通过局部给药的方式来达到将药物作用限定在目标组织，例如通过将药物制成可通过套管植入运动皮层中局部给药的剂型。又例如，将药物制成植入组织后缓释的剂型等。另外还可将上述药物制成组织特异性的靶向药物递送***的形式。例如可以通过将具有激活或加强运动皮层神经细胞兴奋性的制剂，包括小分子化合物或生物活性分子(核酸如蛋白编码DNA或mRNA分子、蛋白如抗体等)与能够特异性结合在运动皮层中特异性表达的蛋白结合的抗体或抗体片段连接形成能够识别和结合运动皮层的细胞的复合分子。在本发明中，所述医疗设备可为在运动皮层或其附近组织中局部作用的装置。例如通过外置于头皮的外部电圈，其可实现在运动皮层或其附近组织中局部作用的定向磁场的设置。In one aspect of the present invention, the method of the present invention for treating mood disorders or stress disorders by modulating the excitability of the patient's motor cortex (especially the primary motor cortex), and the Among the above-mentioned drugs or devices for treating mood disorders or stress disorders, the methods, drugs or devices are methods, drugs or devices that act locally in the motor cortex, especially the primary motor cortex, that is, locally regulate the excitability of the motor cortex. . Medications for Treating Psychiatric Disorders It may be advantageous for methods and medicaments or devices for use on neural tissue, particularly neural tissue of the brain, such as the motor cortex, to limit the action of the medication or stimulation to the target tissue. Local administration or stimulation of the motor cortex is a limiting technical feature of both the treatment method and the preparation of the drug or device. Methods or drugs used in the motor cortex need to consider whether the method, drug or stimulation signal can be effective in the motor cortex, including whether the drug or stimulation signal can reach the motor cortex, and whether the concentration in the motor cortex can be reached to be effective. or stimulus intensity, etc. In the present invention, the drug is a dosage form that is administered locally in the motor cortex or nearby tissues. The effect of the drug can be limited to the target tissue by local administration, for example, by formulating the drug into a dosage form that can be implanted into the motor cortex through a cannula for local administration. Another example is making the drug into a dosage form that can be sustained-released after being implanted into the tissue. In addition, the above-mentioned drugs can also be formulated into tissue-specific targeted drug delivery systems. For example, preparations that activate or enhance the excitability of motor cortex nerve cells, including small molecule compounds or bioactive molecules (nucleic acids such as protein-coding DNA or mRNA molecules, proteins such as antibodies, etc.), can be combined with those that can specifically bind to the motor cortex. The sexually expressed protein-binding antibodies or antibody fragments are linked to form complex molecules capable of recognizing and binding to cells of the motor cortex. In the present invention, the medical device may be a device that acts locally in the motor cortex or nearby tissue. For example, through an external electric coil externally placed on the scalp, it is possible to set a directional magnetic field that acts locally in the motor cortex or nearby tissues.
本发明提供的药物组合物中的活性成分可以以原料化合物的形式给药，另外也可将活性成分，任选地以生理上可接受的盐的形式，与一种或多种佐剂、赋形剂、载体、缓冲剂、稀释剂和/或其他常规的药物辅料一起引入药物组合物。The active ingredients in the pharmaceutical compositions provided by the present invention can be administered in the form of raw materials. In addition, the active ingredients can also be administered, optionally in the form of physiologically acceptable salts, with one or more adjuvants, excipients, etc. excipients, carriers, buffers, diluents and/or other conventional pharmaceutical excipients.
可以通过任意便利的适合于期望疗法的途径给予本发明的药物组合物。优选的给药途径包括口服给药，特别是以片剂、胶囊、锭剂、散剂和液体形式；和胃肠外给药，特别是皮肤、皮下、肌内和静脉内注射。本发明的药物组合物可以由本领域技术人员通过使用适合于期望制剂的标准方法和常规技术制备。如果需要，则可以使用适合于使活性成分缓释的组合物。The pharmaceutical compositions of the present invention may be administered by any convenient route suitable for the desired therapy. Preferred routes of administration include oral administration, especially in the form of tablets, capsules, lozenges, powders and liquids; and parenteral administration, especially cutaneous, subcutaneous, intramuscular and intravenous injection. Pharmaceutical compositions of the present invention may be prepared by those skilled in the art using standard methods and conventional techniques suitable for the desired formulation. If desired, compositions suitable for sustained release of the active ingredient may be used.
为从本发明药物组合物中的活性成分制备药物组合物，药学上可接受
的载体可以是固体或者液体。固体形式的制剂包括散剂、片剂、丸剂、胶囊、扁囊剂、栓剂以及可分散的颗粒剂。固体载体可以是一种或多种还能用作稀释剂、矫味剂、增溶剂、润滑剂、悬浮剂、粘合剂、防腐剂、片剂崩解剂或包囊材料的物质。In order to prepare pharmaceutical compositions from the active ingredients in the pharmaceutical compositions of the present invention, pharmaceutically acceptable The carrier can be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substances that may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or encapsulating materials.
需要时，可以应用适合提供活性成分缓释的组合物。If desired, compositions suitable to provide sustained release of the active ingredient may be used.
药物制剂优选为单位剂型。这类形式中，制剂被细分为含有适量活性组分的单位剂量。单位剂型可以是包装的制剂，该包装含有离散量的制剂，如包装的片剂、胶囊，以及小瓶或安瓿中的粉末。此外，单位剂型可以是胶囊、片剂、扁囊剂或锭剂本身，或者可以是适合数量的任何这些剂型的包装形式。Pharmaceutical preparations are preferably in unit dosage form. In such forms, the preparation is subdivided into unit doses containing appropriate quantities of the active ingredient. Unit dosage forms may be packaged preparations containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Additionally, the unit dosage form may be a capsule, tablet, cachet, or lozenge itself, or it may be the appropriate number of any of these in packaged form.
治疗有效剂量意指缓解症状或病况的活性成分的量。治疗功效和毒性，例如ED50和LD50，可以通过在细胞培养物或实验动物中的标准药理学程序而测定。治疗性和毒性效果之间的剂量比例是治疗指数，其可以通过LD50/ED50的比例而表达。A therapeutically effective dose means an amount of active ingredient that relieves a symptom or condition. Therapeutic efficacy and toxicity, such as ED50 and LD50, can be determined by standard pharmacological procedures in cell culture or experimental animals. The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed by the ratio LD50/ED50.
给予的剂量当然必须针对所治疗的个体的年龄、体重和病症，以及给药途径、剂型及给药方案，以及期望的结果而小心地调整，且确切的剂量当然应该由医师决定。The dosage administered must of course be carefully tailored to the age, weight and condition of the individual treated, as well as the route, dosage form and regimen, and desired results, and the exact dosage should of course be determined by the physician.
在本发明的另一个方面，还提供了通过对受试者的运动皮层的兴奋性进行检测来诊断情绪障碍或应激障碍的方法。本发明提供了检测运动皮层的兴奋性的试剂或医疗装置用于诊断情绪障碍或应激障碍的方法，特别是对运动皮层的兴奋性局部脑区检测的方法。本发明提供了用于检测运动皮层的兴奋性的试剂或装置在用于制备诊断情绪障碍或应激障碍的试剂盒或设备中的用途，特别是对运动皮层的兴奋性做局部脑区检测的试剂盒或设备中的用途。In another aspect of the present invention, a method for diagnosing mood disorders or stress disorders by detecting the excitability of the subject's motor cortex is also provided. The present invention provides reagents or medical devices for detecting the excitability of the motor cortex and methods for diagnosing mood disorders or stress disorders, especially methods for detecting the excitability of the motor cortex in local brain areas. The present invention provides the use of reagents or devices for detecting the excitability of the motor cortex in preparing kits or equipment for diagnosing mood disorders or stress disorders, especially for detecting the excitability of the motor cortex in local brain areas. Use in kits or devices.
在本发明的其中一个方面，在上述通过对受试者的运动皮层的兴奋性进行检测来诊断情绪障碍或应激障碍的方法中，其中当受试者的运动皮层的兴奋性下降时，判断所述受试者患有或具有发生所述情感障碍或应激障碍的风险。在本发明的其中又一个方面，所述诊断感障碍或应激障碍的方法包括比较受试者在不同时期(例如在不同情感障碍或应激障碍的各个发病阶段，或是在发病前或治疗后)所述运动皮层的兴奋性，观察其是否发生显著下调。
In one aspect of the present invention, in the above-mentioned method for diagnosing mood disorders or stress disorders by detecting the excitability of the subject's motor cortex, when the excitability of the subject's motor cortex decreases, it is determined that The subject suffers from or is at risk of developing the affective disorder or stress disorder. In yet another aspect of the present invention, the method for diagnosing sensory disorders or stress disorders includes comparing subjects at different stages (for example, at various stages of onset of different affective disorders or stress disorders, or before onset or treatment). (2) The excitability of the motor cortex was tested to see whether it was significantly down-regulated.
在本发明的其中一个方面，在上述通过对受试者的运动皮层的兴奋性进行检测来诊断情绪障碍或应激障碍的方法中，还包括对受试者进行刺激运动皮层的步骤。例如可先对受试者采用磁刺激、电刺激、光刺激或超声刺激等方式刺激运动皮层，然后检测运动皮层的兴奋性和其相对变化。In one aspect of the present invention, the method for diagnosing mood disorders or stress disorders by detecting the excitability of the subject's motor cortex further includes the step of stimulating the subject's motor cortex. For example, the subject can first stimulate the motor cortex using magnetic stimulation, electrical stimulation, light stimulation or ultrasonic stimulation, and then detect the excitability of the motor cortex and its relative changes.
在本发明的其中一个方面，在上述诊断情绪障碍或应激障碍的方法中，其中检测与运动皮层兴奋性相关的生物电信号，例如为神经元的静息膜电位、动作电位发放频率，基电流或电压阈值的检测等。In one aspect of the present invention, in the above-mentioned method for diagnosing mood disorders or stress disorders, bioelectric signals related to motor cortex excitability are detected, such as resting membrane potential and action potential firing frequency of neurons, based on Detection of current or voltage thresholds, etc.
在本发明的方法中，可以通过本领域已知的方法对生物电信号进行检测，包括单个或多个电极组成的检测装置，或是脑电图(EEG)等。可以对关注的脑区进行整体或局部的电信号检测和分析。In the method of the present invention, bioelectrical signals can be detected by methods known in the art, including detection devices composed of single or multiple electrodes, or electroencephalography (EEG), etc. Whole or partial electrical signal detection and analysis can be performed on the brain area of concern.
在本发明的其中一个方面，在上述诊断情绪障碍或应激障碍的方法中，其中检测与运动皮层兴奋性相关的生物标记物，例如为神经递质和信号通路相关蛋白或核酸。可举的例子包括但不限于检测谷氨酸AMPA或NMDA受体亚基，谷氨酸转运体的基因或蛋白表达等。In one aspect of the present invention, in the above method for diagnosing mood disorders or stress disorders, biomarkers related to motor cortex excitability, such as neurotransmitters and signaling pathway-related proteins or nucleic acids, are detected. Examples include, but are not limited to, detection of glutamate AMPA or NMDA receptor subunits, glutamate transporter gene or protein expression, etc.
在本发明的方法中，可以通过本领域已知的方法对运动皮层中神经细胞或信号传达途径进行检测，包括通过对神经细胞或信号传达途径中的神经递质，或对其受体蛋白、或神经递质的水解酶的量进行检测。可举的例子包括但不限于利用PCR，ELISA，Western Blot，电生理，微透析结合高效液相等技术检测相关基因及蛋白表达量。In the method of the present invention, the nerve cells or signal transmission pathways in the motor cortex can be detected by methods known in the art, including by detecting neurotransmitters in the nerve cells or signal transmission pathways, or their receptor proteins, Or detect the amount of neurotransmitter hydrolase. Examples include but are not limited to the use of PCR, ELISA, Western Blot, electrophysiology, microdialysis combined with high-performance liquid chromatography and other technologies to detect related gene and protein expression levels.
在本发明中可用的检测样品中蛋白质(表达)的方法包括免疫测定(immunoassay)。例如通过特异性识别相关蛋白的抗体进行ELISA或蛋白质印迹。抗体可以是单克隆或多克隆的。抗体可以是人源化或嵌合的。Methods of detecting protein (expression) in a sample useful in the present invention include immunoassays. For example, ELISA or Western blotting can be performed using antibodies that specifically recognize the relevant proteins. Antibodies can be monoclonal or polyclonal. Antibodies can be humanized or chimeric.
在本发明中可用的检测样品中蛋白质(表达)的方法还包括检测相关基因的mRNA的存在或其数量，例如通过RT-PCR检测样品中mRNA或其片段的量。Methods of detecting protein (expression) in a sample useful in the present invention also include detecting the presence or amount of mRNA of the relevant gene, for example by RT-PCR to detect the amount of mRNA or fragments thereof in the sample.
在本发明的其中一个方面，所述检测样品来自离体样品。In one aspect of the invention, the detection sample is from an ex vivo sample.
在本发明的其中一个方面，检测还可以对运动皮层进行活体观察和检测。In one aspect of the invention, the detection may also include in vivo observation and detection of the motor cortex.
在本发明的其中一个方面，通过对受试者的脑区成像来对受试者的运动皮层进行观察和检查。在本发明的其中又一个方面，是对受试者的运动皮层进行成像，例如为计算机断层成像(CT)或磁共振成像(MRI)
等。In one aspect of the invention, the subject's motor cortex is observed and examined by imaging the subject's brain region. In yet another aspect of the invention, the subject's motor cortex is imaged, such as computed tomography (CT) or magnetic resonance imaging (MRI). wait.
在本发明的其中又一个方面，所述成像为PET成像或SPECT。例如，可通过在静脉注射识别和显示结合正电子发射放射性核素示踪剂的信号，然后对相关脑区进行PET扫描。In yet another aspect of the invention, the imaging is PET imaging or SPECT. For example, the signal of a combined positron-emitting radionuclide tracer can be identified and visualized by injecting it intravenously and then performing a PET scan of the relevant brain region.
在本发明的其中又一个方面，所述成像结合磁共振成像(MRI)成像进行。例如在前述PET成像的结果基础上对运动皮层进行磁共振成像(MRI)成像以观察所述核团的活动。其中包括通过检测组织血流量，代谢状态等，观察相应功能脑区的局部脑组织生理及代谢改变，反映核团的活动变化。In yet another aspect of the invention, the imaging is performed in conjunction with magnetic resonance imaging (MRI) imaging. For example, based on the results of the aforementioned PET imaging, magnetic resonance imaging (MRI) imaging is performed on the motor cortex to observe the activity of the nuclei. This includes detecting tissue blood flow, metabolic status, etc., observing physiological and metabolic changes in local brain tissue in corresponding functional brain areas, and reflecting changes in nuclear activity.
本发明还提供了通过上述本发明的方法来诊断情绪障碍或应激障碍的试剂盒。The present invention also provides a kit for diagnosing mood disorders or stress disorders through the above-mentioned method of the present invention.
本发明还提供了通过上述本发明的方法来诊断情绪障碍或应激障碍的医疗装置。The present invention also provides a medical device for diagnosing mood disorders or stress disorders through the method of the present invention.
图1显示了通过恐惧增强学***进行检测。B-E.对每一只造模动物的四项行为学指标进行记录，并与对照组(fear conditioning,FC)进行对比，每种行为学都可以获得行为异常的动物，已用虚线框标注出来。其中B为高架十字迷宫(elevated-plus maze，EPM),用来检测焦虑水平；C为声音惊吓反射(acoustic startle response，ASR)用来检测惊吓水平；D为强迫游泳(forced-swim test，FST)用来检测抑郁水平；E为恐惧回忆(fear recall或fear memory recall)用来检测恐惧记忆水平。F-I，综合评估动物的焦虑、惊吓、抑郁和恐惧记忆水平之后，将具备三种或以上行为异常的动物定义为diseased，将至多表现出一项行为学异常的动物定义为resistant。通过对比发现diseased动物整体相对于对照(Con)都表现出了异常的焦虑、惊吓、抑郁和恐惧，而resistant的动物则这四项表现和对照没有显著差别。*P<0.5,**P<0.01,***P<0.001,****P<0.0001，Control组，疾病易感组和耐受组中n分别为20,11and 17。单向方差分析，然后进行Tukey多重比较
(Tukey’s multiple comparisons test)。所有数据均以平均值±SEM表示。Figure 1 shows the preparation of disease or disease-susceptible (diseased) and disease-resistant (resistant) mice through the fear-enhanced feel learning model (Stress-enhanced feel learning, SEFL). A. Use restraint stress combined with plantar electrical stimulation to create a model in mice, and detect the mice's anxiety, depression, fright and fear levels after modeling. BE. Record the four behavioral indicators of each modeled animal and compare them with the control group (fear conditioning, FC). Animals with abnormal behavior can be obtained for each behavioral method, which are marked with dotted boxes. Among them, B is the elevated-plus maze (EPM), used to detect anxiety levels; C is the acoustic startle response (ASR), used to detect startle levels; D is the forced-swim test (FST) ) is used to detect the level of depression; E is fear recall (fear recall or fear memory recall), which is used to detect the level of fear memory. FI, after comprehensively assessing the animal's anxiety, startle, depression and fear memory levels, animals with three or more behavioral abnormalities are defined as diseased, and animals showing at most one behavioral abnormality are defined as resistant. Through comparison, it was found that the diseased animals as a whole showed abnormal anxiety, fright, depression and fear compared to the control (Con), while the resistant animals had no significant difference in these four performances from the control. *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001, n in Control group, disease susceptible group and tolerant group were 20, 11 and 17 respectively. One-way ANOVA followed by Tukey's multiple comparisons (Tukey's multiple comparisons test). All data are expressed as mean ± SEM.
图2显示疾病小鼠、耐受小鼠与对照组小鼠的脑区的兴奋性。A.VS120全脑扫描获取的大脑c-fos染色图谱。具有c-fos标记的为兴奋的神经元。比例尺：1mm。图中左上角标记为脑片所在的坐标(相对于bregma)。B-D疾病(diseased)与耐受(resistant)小鼠相对于Control组小鼠在皮质(Cortex)、杏仁核(Amygdala)和海马(hippocampus)区域表现出的神经元激活程度的差异。B.对照与疾病/耐受小鼠在皮质所包含的扣带回皮层(Cg)，边缘下皮层(IL)和前边缘皮层(PrL)均表现为疾病小鼠c-fos显著下降,而耐受小鼠有所回升的趋势。C.对照与疾病/耐受小鼠在杏仁核各个亚区表现出的c-fos神经元数目差异。在耐受小鼠中，c-fos神经元相比疾病小鼠有相对明显下降的趋势。而疾病组相比对照组，则在外侧杏仁核(LA)区域有明显上升的趋势。D.对照与疾病/耐受小鼠在腹侧海马的CA1,CA3和DG区域表现出的c-fos数目差异。其中DG组表现为疾病动物中显著减少，耐受组则回升到与对照组无差异。*P<0.5,**P<0.01,***P<0.001,****P<0.0001,单向方差分析，然后进行Tukey多重比较；Control组，疾病易感组和耐受组n＝11,8and 9。所有数据均以平均值±SEM表示。Figure 2 shows the excitability of brain regions in diseased mice, tolerant mice and control mice. A. Brain c-fos staining pattern obtained by VS120 whole-brain scan. Neurons labeled with c-fos are excitable neurons. Scale bar: 1mm. The upper left corner of the figure is marked as the coordinates of the brain slice (relative to bregma). B-D Differences in the degree of neuronal activation in the cortex, amygdala, and hippocampus regions between mice in the diseased and resistant groups compared to mice in the Control group. B. Control and disease/tolerant mice showed a significant decrease in c-fos in disease mice in the cingulate cortex (Cg), infralimbic cortex (IL) and prelimbic cortex (PrL) contained in the cortex, while the resistant mice showed a significant decrease in c-fos. Affected mice have a rebound trend. C. Differences in the number of c-fos neurons in various subregions of the amygdala between control and disease/tolerant mice. In tolerant mice, c-fos neurons showed a relatively significant decrease compared with disease mice. Compared with the control group, the disease group showed a significant upward trend in the lateral amygdala (LA) area. D. Differences in c-fos numbers between control and disease/tolerant mice in the CA1, CA3 and DG regions of the ventral hippocampus. Among them, the DG group showed a significant decrease in diseased animals, while the tolerance group increased to no difference from the control group. *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001, one-way analysis of variance, followed by Tukey multiple comparisons; Control group, disease susceptible group and tolerant group n= 11,8and 9. All data are expressed as mean ± SEM.
图3显示初级运动皮层(Primary motor cortex)和次级运动皮层(Secondary motor cortex)在疾病易感与耐受小鼠中的活性调控。A.初级运动皮层的活性在疾病小鼠中相比于对照显著下调，而在耐受小鼠中活性恢复。B.次级运动皮层活性在疾病小鼠中相比于对照有下调趋势且显著低于耐受小鼠，而耐受小鼠中活性则与对照无殊。*P<0.5,单向方差分析，然后进行Tukey多重比较；Control组，疾病易感组和耐受组n＝11,8and 9。所有数据均以平均值±SEM表示。Figure 3 shows the regulation of activity of primary motor cortex (Primary motor cortex) and secondary motor cortex (Secondary motor cortex) in disease-susceptible and tolerant mice. A. The activity of the primary motor cortex was significantly down-regulated in diseased mice compared with controls, while the activity was restored in tolerant mice. B. The activity of the secondary motor cortex in diseased mice has a downward trend compared with the control and is significantly lower than that in the tolerant mice, while the activity in the tolerant mice is no different from the control. *P<0.5, one-way ANOVA followed by Tukey multiple comparisons; Control group, disease susceptible group and tolerant group n=11,8and 9. All data are expressed as mean ± SEM.
图4显示通过RNA测序分析显示疾病小鼠的突触传递功能异常。图4A显示疾病小鼠的M1区域在关联性学习(associative learning)、生物钟节律(circadian sleep/wake cycle)、睡眠(sleep)等方面主要表现为核心基因下调的趋势。图4B显示通过GSEA基因集富集分析法发现疾病小鼠在多巴胺合成与转运(dopamine transport)、G蛋白信号通路(G protein-coupled receptor signalling)以及突触传递(dopaminergic synaptic transmission)方面，都呈现了核心基因的下调，指示M1区域突触信号传递受损，关键信号通路被抑制。
Figure 4 shows functional abnormalities in synaptic transmission in diseased mice revealed by RNA sequencing analysis. Figure 4A shows that the M1 region of diseased mice mainly shows a trend of down-regulation of core genes in aspects such as associative learning, circadian sleep/wake cycle, and sleep. Figure 4B shows that through GSEA gene set enrichment analysis, it was found that diseased mice showed poor performance in dopamine synthesis and transport (dopamine transport), G protein-coupled receptor signaling pathway (G protein-coupled receptor signaling) and synaptic transmission (dopaminergic synaptic transmission). The downregulation of core genes indicates that synaptic signaling in the M1 region is impaired and key signaling pathways are inhibited.
图5显示激活疾病小鼠的初级运动皮层显著改善其焦虑、抑郁、惊吓和恐惧水平。图5A显示在对照Con和疾病(diseased)小鼠的双侧M1中注射rAAV-CaMKIIa-hM3D(Gq)-mCherry兴奋性病毒。图5B显示行为学检测前给小鼠腹腔注射CNO(3mg/kg)，特异性激活Con和diseased小鼠的初级运动皮层。图5C-F显示对比发现，激活初级运动皮层后，diseased小鼠的焦虑水平(C)、抑郁水平(D)、惊吓水平(E)和恐惧水平(F)均有相比于激活前明显好转的趋势；而在对照小鼠中，激活初级运动皮层对焦虑(C)、抑郁(D)、惊吓(E)和恐惧(F)则没有有益的影响。说明激活初级运动皮层特异性地可以综合改善疾病小鼠的焦虑状态、抑郁状态、惊吓水平和恐惧水平。Control组和疾病组n＝11每组。所有数据均以平均值±SEM表示。Figure 5 shows that activating the primary motor cortex of diseased mice significantly improved their levels of anxiety, depression, startle, and fear. Figure 5A shows injection of rAAV-CaMKIIa-hM3D(Gq)-mCherry excitovirus in bilateral M1 of control Con and diseased mice. Figure 5B shows that intraperitoneal injection of CNO (3 mg/kg) into mice before behavioral testing specifically activated the primary motor cortex of Con and diseased mice. Figure 5C-F shows the comparison and found that after activating the primary motor cortex, the anxiety level (C), depression level (D), startle level (E) and fear level (F) of the diseased mice were significantly improved compared with before activation. trend; whereas in control mice, activating the primary motor cortex had no beneficial effects on anxiety (C), depression (D), startle (E), and fear (F). This shows that activating the primary motor cortex can specifically and comprehensively improve the anxiety state, depression state, startle level and fear level of diseased mice. Control group and disease group n=11 in each group. All data are expressed as mean ± SEM.
图6显示临床上通过经颅磁刺激患者初级运动皮层对PTSD核心症状具有显著的疗效，在对其伴随的焦虑、抑郁、睡眠异常、社交异常等方面也具有良好的治疗效果。图6A和B显示患者在间歇性theta爆发刺激(Intermittent theta burst stimulation,iTBS)的方式进行运动皮层治疗方案。处理后，PTSD患者的疾病严重指数(CAPS得分)显著下降(图6C)。而其伴随的焦虑水平(图6D)、抑郁水平(图6E)、睡眠质量(图6F)和社交表现(图6G)都在不同的治疗阶段反映出显著的好转。Figure 6 shows that clinically, transcranial magnetic stimulation of the primary motor cortex of patients has a significant effect on the core symptoms of PTSD, and also has a good therapeutic effect on the accompanying anxiety, depression, sleep abnormalities, social abnormalities, etc. Figure 6A and B shows a patient undergoing a motor cortex treatment protocol in the form of intermittent theta burst stimulation (iTBS). After treatment, the disease severity index (CAPS score) of PTSD patients decreased significantly (Figure 6C). The accompanying anxiety levels (Figure 6D), depression levels (Figure 6E), sleep quality (Figure 6F) and social performance (Figure 6G) all reflected significant improvements at different treatment stages.
下面将结合实施例进一步说明本发明的实质内容和有益效果，该实施例仅用于说明本发明而非对本发明的限制。The essence and beneficial effects of the present invention will be further described below with reference to examples. The examples are only used to illustrate the present invention but not to limit the present invention.
实施例1材料和方法Example 1 Materials and Methods
一.实验材料1. Experimental materials
1.实验动物：6-8周龄雄性C57小鼠，购买自江苏集萃药康生物科技股份有限公司，用于构建小鼠受到负性刺激后产生应激障碍的动物模型。1. Experimental animals: 6-8 week old male C57 mice, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., are used to construct an animal model of stress disorder in mice after being exposed to negative stimulation.
2.抗体、病毒和试剂：2. Antibodies, viruses and reagents:
用于标记神经元活性的c-fos抗体购自Synaptic Systems(#226 008)；The c-fos antibody used to label neuronal activity was purchased from Synaptic Systems (#226 008);
用于鼠脑的固定和包埋以制备鼠脑切片样本的多聚甲醛(PFA)溶液(上海阿拉丁生化科技股份有限公司)和OCT(SAKURA，美国)。
Paraformaldehyde (PFA) solution (Shanghai Aladdin Biochemical Technology Co., Ltd.) and OCT (SAKURA, USA) used for fixation and embedding of mouse brain to prepare mouse brain slice samples.
用于初级运动皮层激活的病毒rAAV-CaMKIIa-hM3D(Gq)-mCherry-WPREs-pA和抑制的病毒rAAV-hSyn-hM4D(Gi)-EGFP-WPRE-hGH pA购于武汉枢密脑科学技术有限公司。用于激活Gq的试剂Clozapine N-oxide(CNO)购于Sigma-Aldrich(34233-69-7)。The virus rAAV-CaMKIIa-hM3D(Gq)-mCherry-WPREs-pA for primary motor cortex activation and the virus rAAV-hSyn-hM4D(Gi)-EGFP-WPRE-hGH pA for inhibition were purchased from Wuhan Primus Brain Science and Technology Co., Ltd. . The reagent Clozapine N-oxide (CNO) used to activate Gq was purchased from Sigma-Aldrich (34233-69-7).
3.仪器：3. Instruments:
检测动物焦虑、抑郁、惊吓和恐惧的行为学仪器分别为Any-maze***(stoelting Any-maze北京京冠卓艺商贸中心)、强迫游泳(上海移数)、惊跳反射***(San Diego)和条件恐惧***(Coulbourn Instruments)。拍摄鼠脑样本荧光的显微镜为虚拟数字切片扫描***VS120(Olympus)。鼠脑立体定位仪(瑞沃德，深圳，型号：68030)和微量注射器(Stoelting)用于小鼠脑内病毒注射。Behavioral instruments for detecting animal anxiety, depression, startle and fear are Any-maze system (stoelting Any-maze Beijing Jingguan Zhuoyi Business Center), forced swimming (Shanghai Shishu), startle reflex system (San Diego) and Conditioned Fear System (Coulbourn Instruments). The microscope used to capture the fluorescence of mouse brain samples was the Virtual Digital Slice Scanning System VS120 (Olympus). A mouse brain stereotaxic instrument (Reward, Shenzhen, model: 68030) and a microsyringe (Stoelting) were used for virus injection into the mouse brain.
二、动物造模及行为学检验方法2. Animal modeling and behavioral testing methods
1.负性刺激造模方法：结合压力应激和条件性恐惧，实现恐惧的过度增强。用50ml的离心管对小鼠进行2小时的束缚应激。七天后将对包括对照组在内的所有动物进行条件性恐惧实验，分为两个阶段分别为训练阶段和恐惧消退阶段。训练阶段给予声音信号配合共终止的足底电击训练结束后，让实验鼠在环境中停留1分钟后放入小鼠饲养笼。24小时后进行恐惧消退实验。声音消退实验时，将小鼠置于相同声音信号的不同环境中进行。最后同样让小鼠在环境中停留1分钟，然后放回原有鼠笼。1. Negative stimulus modeling method: combine stress and conditioned fear to achieve excessive fear enhancement. Mice were subjected to restraint stress in 50 ml centrifuge tubes for 2 hours. Seven days later, all animals, including the control group, will be subjected to a conditioned fear experiment, which is divided into two stages: the training stage and the fear extinction stage. During the training phase, a sound signal was given in conjunction with the co-terminated foot shock training. After the training, the experimental mice were allowed to stay in the environment for 1 minute and then placed in the mouse cage. A fear extinction experiment was performed 24 hours later. During the sound extinction experiment, mice were placed in different environments with the same sound signal. Finally, the mice were also allowed to stay in the environment for 1 minute, and then returned to the original cage.
2.行为学检测方法：2. Behavioral detection methods:
高架十字迷宫(EPM)即将小鼠放置在一个被架高离地面约0.5m的十字迷宫里。十字迷宫有两个开臂(30x5cm2)，两个闭臂(30x5x20cm3)。让小鼠在迷宫里自由移动5分钟，最后记录小鼠在开臂和闭臂里停留的时间。小鼠在开臂停留时间越长，表明小鼠的焦虑程度越低。The elevated plus maze (EPM) places mice in a plus maze that is elevated approximately 0.5 m above the ground. The plus maze has two open arms (30x5cm 2 ) and two closed arms (30x5x20cm 3 ). Let the mice move freely in the maze for 5 minutes, and finally record the time the mice stay in the open and closed arms. The longer the mouse stayed in the open arm, the less anxious the mouse was.
强迫游泳(FST)即将小鼠投入盛有约高约30cm、2L水的透明玻璃量筒中。小鼠将在水中持续挣扎保持游泳状态，也会不时出现小鼠完全不动的放弃状态。6分钟后，将小鼠从水中取出，根据拍摄的视频计数小鼠在后5分钟内保持身体完全不动的漂浮状态的时间。小鼠保持完全不动的时间越久，反映出的小鼠的抑郁程度越高。Forced swimming (FST) is to put the mouse into a transparent glass measuring cylinder containing 2L of water with a height of about 30cm. The mouse will continue to struggle in the water to keep swimming, and from time to time there will be a state of giving up where the mouse is completely motionless. After 6 minutes, the mice were taken out of the water, and the time the mice remained in a completely motionless floating state for the next 5 minutes was counted based on the captured video. The longer the mice remained completely still, the more depressed the mice were.
声音惊吓反射(ASR)即将小鼠放置在惊跳反射装置内，首先给与一个
背景噪音(70dB)，记录小鼠对背景噪音的反应程度。后面以随机的间隔，分别给与十个强度为100dB的强噪音刺激(40ms)，并依次记录噪音时小鼠的反应。最后分析每一只小鼠十次强刺激时的平均反应。反应越大则表明小鼠对于外界刺激的惊吓程度越高。Acoustic startle reflex (ASR) is to place the mouse in the startle reflex device and first give it a Background noise (70dB), record the response of mice to background noise. Later, ten strong noise stimuli (40 ms) with an intensity of 100 dB were given at random intervals, and the mice's responses to the noise were recorded sequentially. Finally, the average response of each mouse to ten strong stimulations was analyzed. The greater the response, the higher the degree of fright the mouse is to external stimuli.
恐惧回忆(Fear memory recall)将小鼠放置于与恐惧消退时同样的环境当中，并接受与足底刺激偶联的声音，总共接受四次，每次持续30s。每次声音之间的间隙随机，大致在60s-90s之间。最后，利用软件统计小鼠四次声音期间的平均freezing的时间。freezing的时间越久，则表明小鼠恐惧记忆越强。For fear memory recall, mice were placed in the same environment as when fear was extinguished and received sounds coupled with plantar stimulation four times in total, each lasting 30 seconds. The gap between each sound is random, roughly between 60s-90s. Finally, use software to count the average freezing time of the mouse during the four sounds. The longer the freezing time, the stronger the fear memory of the mice.
三、样本处理3. Sample processing
用溶解有4％多聚甲醛的0.1M PBS对小鼠进行心脏灌流以固定组织。脑组织取出后依次浸泡在4℃的多聚甲醛与蔗糖溶液中24小时，固定后用振动切片机(VT 1000S)切出50μm的冠状切片。接着利用免疫组化染色技术添加抗体识别立早基因(immediate early gene)c-fos以标记全脑被激活的神经元。最后用DAPI染细胞核5分钟后进行封片。利用Olympus的VS120数字切片工作站拍摄染色的脑片。拍摄好的图像利用imageJ进行处理，并和对应的脑图谱拟合之后，进行全脑各个脑区的c-fos神经元计数。Mice were cardiac perfused with 4% paraformaldehyde dissolved in 0.1M PBS to fix the tissue. After the brain tissue was taken out, it was soaked in paraformaldehyde and sucrose solutions at 4°C for 24 hours. After fixation, 50 μm coronal sections were cut out with a vibrating microtome (VT 1000S). Immunohistochemical staining was then used to add antibodies identifying the immediate early gene c-fos to label activated neurons throughout the brain. Finally, the nuclei were stained with DAPI for 5 minutes and then mounted. Stained brain slices were captured using Olympus's VS120 digital sectioning workstation. The captured images were processed using imageJ and fitted to the corresponding brain atlas, and c-fos neurons were counted in each brain region of the whole brain.
四、手术方案4. Surgical plan
9-10周龄雄性小鼠经过1％戊巴比妥钠以50mg/kg麻醉后，剃毛暴露头皮并固定在立体定位仪上。用眼科剪沿小鼠头部正中线从两眼连线后缘到两耳连线前缘轻轻切开皮肤，暴露出小鼠颅骨顶部。参照小鼠的颅骨上正中矢状缝与冠状缝的焦点前囟(Bregma)和正中矢状缝与人字形缝的交点后囟(lambda)进行立体定位调平小鼠颅骨。实施开颅手术，将目标脑区正上方的颅骨用直径0.5mm的颅钻垂直打通，并避免戳破脑膜引起脑出血。沿着Z轴将玻璃针尖慢慢***脑组织，直至到达初级运动皮层的位置(AP：+1.54mm；ML：±1.30mm；DV：-1.60mm)以20nl/min的速率双边各注射病毒(rAAV-CaMKIIa-hM3D(Gq)-mCherry或rAAV-hSyn-hM4D(Gi)-EGFP)200nl，注射结束后玻璃针停留7min再缓慢提升直至移出颅骨。病毒注射完后对小鼠进行缝合，在缝合处涂抹林可霉素软膏，减少术后感染的风险。操作结束后需将小鼠放置在37℃的加热垫上
待其慢慢恢复意识，并在术后3天内每天观察动物的健康状态。Male mice aged 9-10 weeks were anesthetized with 1% sodium pentobarbital at 50 mg/kg, shaved to expose the scalp, and fixed on a stereotaxic apparatus. Use ophthalmic scissors to gently cut the skin along the midline of the mouse's head from the posterior edge of the line connecting the eyes to the front edge of the line connecting the ears to expose the top of the mouse skull. The mouse skull was leveled stereotactically with reference to the bregma (Bregma), the focal point of the median sagittal suture and coronal suture, and the posterior fontanel (lambda), the intersection point of the median sagittal suture and herringbone suture, on the mouse skull. A craniotomy is performed, and the skull directly above the target brain area is opened vertically with a cranial drill with a diameter of 0.5 mm to avoid puncturing the meninges and causing cerebral hemorrhage. Slowly insert the glass needle tip into the brain tissue along the Z-axis until it reaches the position of the primary motor cortex (AP: +1.54mm; ML: ±1.30mm; DV: -1.60mm). Inject the virus bilaterally at a rate of 20nl/min ( rAAV-CaMKIIa-hM3D(Gq)-mCherry or rAAV-hSyn-hM4D(Gi)-EGFP) 200nl. After the injection, the glass needle stayed for 7 minutes and then slowly lifted until it moved out of the skull. After the virus injection, the mice were sutured, and lincomycin ointment was applied to the sutures to reduce the risk of postoperative infection. After the operation, the mice need to be placed on a heating pad at 37°C. Wait for the animal to slowly regain consciousness, and observe the animal's health every day for 3 days after the operation.
对行为学检测综合筛选出的同时表现出焦虑、抑郁、惊吓和恐惧的疾病小鼠，在其双侧初级运动皮层进行Gq兴奋性病毒的注射。对照组注射同样的病毒。病毒表达2-3周后，对小鼠依次进行了焦虑、抑郁、惊吓和恐惧水平的检测。在每项行为学检测前，给小鼠腹腔注射了CNO(3mg/kg)激活运动皮层，并于半小时之后检测行为学指标。最后结合行为学检测技术，评估易感小鼠的焦虑、抑郁、惊吓及恐惧指标是否发生显著改善。Mice with symptoms of anxiety, depression, fright, and fear that were comprehensively screened through behavioral tests were injected with Gq excitatory virus in their bilateral primary motor cortex. The control group was injected with the same virus. After 2-3 weeks of virus expression, the mice were tested for levels of anxiety, depression, fright and fear. Before each behavioral test, mice were intraperitoneally injected with CNO (3 mg/kg) to activate the motor cortex, and behavioral indicators were measured half an hour later. Finally, behavioral testing technology was combined to evaluate whether the anxiety, depression, fright, and fear indicators of susceptible mice were significantly improved.
五、数据分析5. Data analysis
本研究中的所有实验和数据分析均为盲法，每个图例中都标明了重复次数(n)，除非另有说明，否则所有实验至少独立进行了三次。数据以平均值±SEM表示。使用非配对、双侧Student t检验或单向方差分析(ANOVA)，然后进行Tukey多重比较，分别评估两个实验条件和三个或三个以上实验条件之间差异的显著性。显著性水平设置为*P<0.05，**P<0.01，***P<0.001，****P<0.0001。All experiments and data analyzes in this study were blinded, the number of replicates (n) is indicated in each figure legend, and all experiments were performed at least three times independently unless otherwise stated. Data are expressed as mean ± SEM. The significance of differences between two experimental conditions and three or more experimental conditions was assessed using unpaired, two-sided Student's t test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons. The significance level is set as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
实施例2模型动物制备和筛选，检测其精神疾病指征Example 2 Preparation and screening of model animals to detect signs of mental illness
根据图1A描述的恐惧增强学***；acoustic startle response(ASR)来检测惊吓水平；forced-swim test(FST)来检测抑郁水平；以及fear recall用来检测恐惧记忆水平，由此筛选出对负性刺激易感的疾病小鼠和耐受小鼠。FC对照组和Stress+FC造模组在后续行为学检测中的表现如图1.B-E所示。参照对照组的mean±SD，在Stress+FC中挑选出了精神异常的小鼠，称为疾病(diseased)小鼠，即结合B-E四个行为学检测结果，有3-4个指标呈现异常。该界定标准参照Ritov G et al,Mol Psychiatry.2016。Posttraumatic stress disorder (PTSD) model mice were prepared according to the method of fear-enhanced learning (SEFL) described in Figure 1A. This model is described in Sillivan SE et al, Biol Psychiatry. 2017 and is widely recognized in reviews of animal models of PTSD (e.g. Richter-Levin G et al, Mol Psychiatry. 2019). After modeling, mice were subjected to elevated-plus maze (EPM) to detect anxiety levels; acoustic startle response (ASR) to detect startle levels; forced-swim test (FST) to detect depression levels; and fear recall to detect fear memory. levels, thereby screening disease mice that are susceptible to negative stimuli and those that are resistant. The performance of the FC control group and the Stress+FC modeling group in subsequent behavioral tests is shown in Figure 1.B-E. Referring to the mean±SD of the control group, mice with mental disorders were selected in Stress+FC and were called diseased mice. That is, combined with the four behavioral test results of B-E, 3-4 indicators were abnormal. This definition standard refers to Ritov G et al, Mol Psychiatry.2016.
将筛选出的疾病小鼠和对照组以及疾病耐受小鼠进行对比，确认了疾病小鼠在焦虑(EPM)、抑郁(FST)、惊吓(ASR)和恐惧(Recall)四个方面都有明显异常(图1.F-I)。
Comparing the selected disease mice with the control group and disease-tolerant mice, it was confirmed that the disease mice had significant symptoms in four aspects: anxiety (EPM), depression (FST), startle (ASR) and fear (Recall). Abnormal (Fig. 1.FI).
实施例3动物脑内神经元兴奋性研究显示脑内多个重要核团活性发生显著改变Example 3: Research on neuronal excitability in animal brains shows significant changes in the activity of multiple important nuclei in the brain
对实施例2获得的疾病发生小鼠和耐受小鼠进行了全脑c-fos染色，探索疾病与耐受小鼠相比对照组发生的脑区活性的改变。结果如图2A所示。对鼠脑的后内侧前额叶皮层(mPFC)、海马以及杏仁核区域的c-fos染色结果进行统计分析。结果表明，疾病小鼠具有mPFC的c-fos神经元显著减少(图2B)，杏仁核则在LA区域相比对照组有增多的趋势(图2C)，而海马的CA1,CA3以及DG区域均呈现c-fos神经元显著减少的趋势(图2D)。而这些变化在耐受小鼠中都有了显著的改善。Whole-brain c-fos staining was performed on the disease-producing mice and tolerant mice obtained in Example 2 to explore the changes in brain area activity in the disease- and tolerant mice compared with the control group. The results are shown in Figure 2A. Statistical analysis was performed on the c-fos staining results of the posterior medial prefrontal cortex (mPFC), hippocampus and amygdala areas of the mouse brain. The results showed that the c-fos neurons in the mPFC of diseased mice were significantly reduced (Figure 2B), the amygdala tended to increase in the LA area compared with the control group (Figure 2C), and the CA1, CA3 and DG areas of the hippocampus were all There was a trend of significant decrease in c-fos neurons (Figure 2D). These changes were significantly improved in tolerant mice.
实施例4疾病发生动物的初级运动皮层的活性显著下调Example 4 The activity of the primary motor cortex of animals with disease is significantly down-regulated.
对脑区活性的观察发现，M1脑区的活性在疾病小鼠中显著下调，而对照组和耐受组的小鼠M1脑区活性没有显著差异(图3A)。在M2脑区的活性同样呈现在疾病组下降，而在耐受组中活性则显著高于疾病组(图3B)。鉴于此，发明人推测运动皮层活性的变化影响小鼠的精神疾病。Observation of brain area activity found that the activity of the M1 brain area was significantly down-regulated in diseased mice, while there was no significant difference in the activity of the M1 brain area between mice in the control group and the tolerance group (Figure 3A). The activity in the M2 brain region also showed a decrease in the disease group, while the activity in the tolerance group was significantly higher than that in the disease group (Figure 3B). In view of this, the inventors hypothesized that changes in motor cortex activity affect psychiatric disorders in mice.
实施例5疾病发生动物的初级运动皮层RNA测序分析，指示突触传递功能异常Example 5 RNA sequencing analysis of primary motor cortex of animals with disease, indicating abnormal synaptic transmission function
对实施例2获得的疾病发生小鼠和对照小鼠进行初级运动皮层的解剖，将组织裂解提取RNA并进行转录组测序分析。转录组测序分析委托开泰生物技术公司通过RNA-seq进行，实验部分流程主要包括：样品的质量检测,双链cDNA的合成,转录组文库的构建与质检,样本的上机测序，测序采用Illumina Novaseq 6000进行。测序获得原始测序序列(Raw data)后进入信息分析流程，流程分为两个阶段：1)测序数据质量评估：通过对测序错误率、数据量、比对率等进行统计，符合标准则进行后续的分析；2)信息挖掘及分析：通过质控、比对、定量、差异显著性分析和功能富集等环节，以及可变剪切分析，将测序获得的疾病小鼠中RNA与对照组进行对比和差异排序，最后利用GSEA，即基因集富集分析法解析疾病小鼠中发生的异常基因的功能聚类。The disease-producing mice and control mice obtained in Example 2 were dissected from the primary motor cortex, and the tissues were lysed to extract RNA and conduct transcriptome sequencing analysis. Transcriptome sequencing analysis was entrusted to Kaitai Biotechnology Company to conduct RNA-seq. The experimental process mainly includes: sample quality inspection, double-stranded cDNA synthesis, transcriptome library construction and quality inspection, sample sequencing, and sequencing using Performed on Illumina Novaseq 6000. After sequencing obtains the raw sequencing sequence (Raw data), it enters the information analysis process. The process is divided into two stages: 1) Sequencing data quality assessment: By counting the sequencing error rate, data volume, comparison rate, etc., follow-up will be carried out if the standards are met. Analysis; 2) Information mining and analysis: Through quality control, comparison, quantification, difference significance analysis and functional enrichment, as well as alternative splicing analysis, the RNA in the disease mice obtained by sequencing was compared with the control group. Comparison and differential ranking were performed, and finally GSEA, a gene set enrichment analysis method, was used to analyze the functional clustering of abnormal genes occurring in diseased mice.
结果显示，疾病小鼠在学习、生物钟节律、睡眠等方面主要表现为核
心基因的下调的趋势(图4A)，而这也符合临床上PTSD患者认知学习能力受损，睡眠异常等现象。另一方面，GSEA结果显示，疾病小鼠在多巴胺合成与转运、G蛋白信号通路以及突触传递方面，都呈现了核心基因的下调(图4B)，指示M1区域突触信号传递受损，关键信号通路被抑制。鉴于此，发明人认为运动皮层活性的降低是源于突触传递的减弱，而激活M1神经元的兴奋性可修复部分脑区的功能。The results showed that the diseased mice mainly showed nuclear abnormalities in learning, biological clock rhythm, sleep and other aspects. There is a downward trend in cardiac genes (Figure 4A), which is also consistent with the clinical phenomena of impaired cognitive learning ability and abnormal sleep in PTSD patients. On the other hand, GSEA results showed that disease mice showed downregulation of core genes in dopamine synthesis and transport, G protein signaling pathways, and synaptic transmission (Figure 4B), indicating that synaptic signaling in the M1 region is impaired, which is key. Signaling pathways are inhibited. In view of this, the inventor believes that the reduction in motor cortex activity is due to the weakening of synaptic transmission, and activating the excitability of M1 neurons can restore the function of some brain areas.
实施例6激活疾病小鼠的初级运动皮层的活性改善其疾病指症Example 6 Activating the activity of the primary motor cortex of diseased mice improves their disease symptoms
激活疾病小鼠M1区域并检测焦虑、抑郁、惊吓以及恐惧水平是否发生改善。Activate the M1 region of diseased mice and detect improvements in anxiety, depression, startle, and fear levels.
首先向对照和疾病小鼠的双侧M1均注射了激活病毒rAAV-CaMKIIa-hM3D(Gq)-mCherry，该病毒主要表达于M1的谷氨酸能神经元内，依赖于体外CNO的注射从而激活神经元(图5A)。病毒稳定表达2-3周后检测疾病小鼠的各项指标，并于检测前半个小时给小鼠进行腹腔注射CNO(3mg/kg)。染色结果表明，CNO给药后，动物的M1区域激活神经元的数目显著上升，提示该区域确实被激活(图5B)。First, the activating virus rAAV-CaMKIIa-hM3D(Gq)-mCherry was injected into the bilateral M1 of both control and disease mice. The virus is mainly expressed in the glutamatergic neurons of M1 and is activated by the injection of CNO in vitro. neurons (Fig. 5A). After the virus was stably expressed for 2-3 weeks, various indicators of the diseased mice were detected, and the mice were intraperitoneally injected with CNO (3 mg/kg) half an hour before the test. The staining results showed that after CNO administration, the number of activated neurons in the M1 area of the animals increased significantly, suggesting that this area was indeed activated (Figure 5B).
行为学实验结果表明，M1被激活之后，疾病发生小鼠的抗焦虑(图5C)水平上升，以及抑郁(图5D)、惊吓(图5E)以及恐惧(图5F)水平均比激活前有了明显的好转。Behavioral experimental results showed that after M1 was activated, the anxiolytic (Figure 5C) levels of disease-prone mice increased, and the levels of depression (Figure 5D), fright (Figure 5E), and fear (Figure 5F) were all higher than before activation. Significant improvement.
对对照小鼠做同样的M1的激活，则表现出了焦虑水平的上升(伴随适应环境之后探索欲的下降)，且没有表现出抑郁、惊吓以及恐惧的改变。The same M1 activation was performed on control mice, which showed an increase in anxiety levels (accompanied by a decrease in the desire to explore after adapting to the environment), and did not show changes in depression, fright, and fear.
实验证明了M1的活性对于疾病小鼠病症改善的特异性和有效性。Experiments have demonstrated the specificity and effectiveness of M1 activity in improving symptoms in diseased mice.
实施例7对人类患者进行临床治疗改善疾病指症Example 7 Clinical treatment of human patients to improve disease symptoms
本申请的研究获得杭州市第七人民医院伦理审查委员会审批，并已在中国临床试验注册中心注册。每名受试者均签署了正式的知情同意书。按照纳排标准招募创伤后应激障碍(PTSD)患者和正常人对照，按照随机数字对照表法将PTSD被试和正常对照随机分配至M1真刺激和伪刺激组。纳排表准如下：The research applied for this application was approved by the Ethics Review Committee of Hangzhou Seventh People's Hospital and has been registered with the China Clinical Trial Registration Center. Each subject signed a formal informed consent form. Posttraumatic stress disorder (PTSD) patients and normal controls were recruited according to inclusion and exclusion criteria, and PTSD subjects and normal controls were randomly assigned to M1 real stimulation and sham stimulation groups according to the random number comparison table method. The inclusion schedule is as follows:
1.PTSD患者纳入标准：1. Inclusion criteria for PTSD patients:
1)年龄：16-60岁；
1)Age: 16-60 years old;
2)文化：小学毕业以上文化；2) Culture: primary school graduate or above;
3)种族：汉族；3) Ethnicity: Han;
4)利手：右利手；4) Handy: right-handed;
5)符合美国精神病学会《精神障碍诊断与统计手册第5版》(DSM-5)创伤后应激障碍诊断标准；5) Meet the diagnostic criteria for post-traumatic stress disorder in the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5);
6)PTSD诊断量表(CAPS-5)>45分；6)PTSD Diagnostic Scale (CAPS-5)>45 points;
7)HAMD-17量表***项(项目3)≤1分；7) HAMD-17 scale suicide item (item 3) ≤ 1 point;
8)合并用药剂量稳定维持四周以上；8) The combined dosage should be maintained stably for more than four weeks;
9)自愿参加本研究，并签署知情同意书。9) Voluntarily participate in this study and sign the informed consent form.
2.正常人纳入标准：2. Inclusion criteria for normal people:
1)年龄：16-60岁；1)Age: 16-60 years old;
2)文化：小学毕业以上文化；2) Culture: primary school graduate or above;
3)种族：汉族；3) Ethnicity: Han;
4)利手：右利手4) Handy: right-handed
5)健康受试者CAPS-5<30；5) CAPS-5<30 in healthy subjects;
6)HAMD-17量表评分≤3分；6) HAMD-17 scale score ≤3 points;
7)自愿参加本研究，并签署知情同意书。7) Voluntarily participate in this study and sign the informed consent form.
3.PTSD被试排除标准：3.Exclusion criteria for PTSD subjects:
1)有神经***疾病及其它严重躯体疾病者；1) Those with neurological diseases and other serious physical diseases;
2)符合DSM-5其他精神障碍诊断标准(精神***症谱系及其他精神病性障碍、双相及相关障碍、抑郁障碍、焦虑障碍、强迫及相关障碍、躯体症状及相关障碍等精神障碍)的患者；2) Patients who meet the DSM-5 diagnostic criteria for other mental disorders (schizophrenia spectrum and other psychotic disorders, bipolar and related disorders, depressive disorders, anxiety disorders, obsessive-compulsive and related disorders, somatic symptoms and related disorders and other mental disorders) ;
3)目前或过去12个月内有明显***倾向者；3) Those who have obvious suicidal tendencies currently or within the past 12 months;
4)目前(或在过去4周内)服用可能限制TMS有效性的足量苯二氮卓类药物超过2周或任何剂量的抗癫痫、抗惊厥药物者；4) Those who are currently (or within the past 4 weeks) taking a sufficient amount of benzodiazepines for more than 2 weeks or any dose of anti-epileptic or anticonvulsant drugs that may limit the effectiveness of TMS;
5)有金属植入物或不能安全移除的任何其他金属植入物者以至于不能完成磁共振扫描者或存在明显TMS禁忌症者；5) Those who have metal implants or any other metal implants that cannot be safely removed so that they cannot complete an MRI scan or have obvious contraindications to TMS;
6)既往已经接受过rTMS的治疗，可能会破坏受试者盲态者。6) Those who have received rTMS treatment in the past may damage the subject's blindness.
4.正常对照排除标准：4. Normal control exclusion criteria:
1)疾病史:患有脑器质性疾病、神经***疾病及严重的内分泌或代谢性疾病者；患有
1) Disease history: Those suffering from organic brain diseases, neurological diseases and severe endocrine or metabolic diseases;
2)DSM-5轴I、II任何疾病，头颅外伤者；曾有意识丧失史；2) Any disease of DSM-5 Axis I and II, head trauma; history of loss of consciousness;
3)家族史:两系三代精神病家族史阳性者；3) Family history: Those with positive family history of mental illness in two or three generations;
4)治疗史:曾服用过抗精神病药、抗抑郁药；MRI检查前一个月使用过苯二氮卓类或其他影响中枢神经***的药物；六个月接受连续药物治疗；4) Treatment history: Have taken antipsychotics and antidepressants; have used benzodiazepines or other drugs that affect the central nervous system one month before the MRI examination; have received continuous drug treatment for six months;
5)对测试不合作者或不能有效完成者；5) Those who do not cooperate with the test or cannot complete it effectively;
6)安装有固定金属假牙、心脏起搏器或金属假体植入物者；6) Those with fixed metal dentures, pacemakers or metal prosthetic implants;
7)色盲；7) Color blindness;
8)1年内处于孕产期8) In the pregnancy and childbirth period within 1 year
9)有输血史9) Have a history of blood transfusion
5.剔除标准5. Elimination criteria
1)不执行试验要求的受试者；1) Subjects who do not perform the test requirements;
2)严重违背方案，研究者认为有必要停止研究；2) The protocol is seriously violated and the researcher believes it is necessary to stop the study;
6.中止标准6. Termination criteria
1)研究中出现严重不良反应，不宜继续参加研究者。1) Those who have serious adverse reactions during the study and are not suitable to continue to participate in the study.
2)研究期间受试者出现其他急重症，需采取紧急措施者。2) During the study period, the subject develops other acute or serious illness and requires emergency measures.
3)受试者不合作、不服从治疗，经研究者反复解释无效者。3) The subject is uncooperative and disobedient to treatment, and the treatment is ineffective after repeated explanations by the researcher.
在治疗前收集所有受试的一般情况问卷，并进行临床量表调查。分别于治疗前、rTMS治疗开始后每周以及整个治疗周期完成后的1、2、4周后进行临床量表评估，具体评估项目如下：Before treatment, a general situation questionnaire was collected from all subjects and a clinical scale survey was conducted. Clinical scale assessments were conducted before treatment, every week after the start of rTMS treatment, and 1, 2, and 4 weeks after the completion of the entire treatment cycle. The specific assessment items are as follows:
1)临床用PTSD量表Clinician-Administered PTSD Scale(CAPS-5)1) Clinical-Administered PTSD Scale (CAPS-5)
2)汉密尔顿焦虑量表Hamilton Anxiety Scale(HAMA)2) Hamilton Anxiety Scale (HAMA)
3)汉密尔顿抑郁量表Hamilton Depression Scale(HAMD)3) Hamilton Depression Scale (HAMD)
4)个人和社会功能量表Personal and Social Performance Scale(PSP)4) Personal and Social Performance Scale (PSP)
5)匹兹堡睡眠质量指数量表Pittsburgh Sleep Quality Index(PSQI)5) Pittsburgh Sleep Quality Index (PSQI)
6)TMS不良反应问卷(TASS)6)TMS Adverse Effect Questionnaire (TASS)
7)状态-特质焦虑量表(STAI)量表7) State-Trait Anxiety Inventory (STAI) scale
开始治疗之后，针对M1区试验组，对PTSD患者(7名)的左侧M1区域进行rTMS干预，其方案为iTBS范式，总的脉冲数为1800，刺激强度保持在70-120％RMT(图6B)。每天进行两次，间隔两小时以上；每周连续5次，休息2天后再连续5次治疗，共计两周治疗20次。
After starting treatment, for the M1 area experimental group, rTMS intervention was performed on the left M1 area of PTSD patients (7 patients). The protocol was iTBS paradigm, the total number of pulses was 1800, and the stimulation intensity was maintained at 70-120% RMT (Figure 6B). Carry out twice a day, with an interval of more than two hours; 5 consecutive times a week, and then 5 consecutive treatments after 2 days of rest, for a total of 20 treatments in two weeks.
本次研究脑电10-20***定位M1区(图6A)。静息运动阈值(RMT)是患者放松状态下连续进行10次刺激，其中至少5次，能引出大于50μV运动诱发电位的最小刺激强度。由经过TMS培训的专业治疗师操作，保证治疗部位及治疗参数的稳定。In this study, the EEG 10-20 system located the M1 area (Figure 6A). The resting motor threshold (RMT) is the minimum stimulation intensity that can elicit a motor evoked potential greater than 50 μV when the patient is in a relaxed state for 10 consecutive stimulations, of which at least 5 are. It is operated by professional therapists who have received TMS training to ensure the stability of the treatment area and treatment parameters.
治疗前及每周结束治疗后，以及整个治疗周期完成一周后、两周后、四周后，对iTBS刺激的患者进行量表评估。通过对比治疗前后各项量表的差异，明确iTBS对M1区治疗对患者PTSD症状，以及焦虑、抑郁、社交障碍、幸福感缺乏和睡眠障碍等伴随症状的治疗效果及效果的持续性。Patients stimulated with iTBS were evaluated on the scale before treatment and after each week of treatment, as well as one week, two weeks, and four weeks after the completion of the entire treatment cycle. By comparing the differences in various scales before and after treatment, we can clarify the therapeutic effect and persistence of iTBS treatment in the M1 area on patients' PTSD symptoms, as well as accompanying symptoms such as anxiety, depression, social disorder, lack of happiness, and sleep disorders.
量表统计结果显示，PTSD患者在iTBS刺激运动皮层治疗之后，其PTSD症状的严重程度指标(CAPS量表)呈现显著下降的趋势(图6C)。另外，伴随症状如焦虑水平(图6D)、抑郁水平(图6E)、睡眠质量(图6F)和社交表现(图6G)都在不同的治疗阶段反映出显著的好转。分别具体表现为汉密尔顿焦虑指数(hamilton anxiety scale)的下降，汉密尔顿抑郁指数(hamilton depression scale)的下降，睡眠治疗问题指数(sleep quality index)下降，社交表现分数(social performance scale)提升。The statistical results of the scale showed that after iTBS stimulation of the motor cortex, the PTSD symptom severity index (CAPS scale) of PTSD patients showed a significant downward trend (Figure 6C). In addition, accompanying symptoms such as anxiety level (Figure 6D), depression level (Figure 6E), sleep quality (Figure 6F), and social performance (Figure 6G) all reflected significant improvements at different treatment stages. The specific manifestations include a decrease in the Hamilton Anxiety Scale, a decrease in the Hamilton Depression Scale, a decrease in the Sleep Quality Index, and an increase in the Social Performance Scale.
鉴于此，发明人在临床上验证了经颅磁刺激初级运动皮层对PTSD核心症状具有显著的疗效，在对其伴随的焦虑、抑郁、睡眠异常、社交异常等方面也具有良好的治疗效果。In view of this, the inventor has clinically verified that transcranial magnetic stimulation of the primary motor cortex has a significant effect on the core symptoms of PTSD, and also has a good therapeutic effect on its accompanying anxiety, depression, sleep abnormalities, social abnormalities, etc.
上面是对本发明进行的说明，不能将其看成是对本发明进行的限制。除非另外指出，本发明的实践将使用有机化学、聚合物化学、生物技术等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。The above is a description of the present invention, and it cannot be regarded as a limitation of the present invention. Unless otherwise indicated, the practice of the present invention will employ conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc. It will be apparent that the present invention may be carried out in other ways than those specifically described in the above illustration and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art to which this invention belongs. Many modifications and variations are possible in light of the teachings of this invention and are therefore within the scope of this invention.
如无特别表示，本文中出现的温度的单位“度”是指摄氏度，即℃。
Unless otherwise stated, the unit "degree" for temperature appearing in this article refers to degrees Celsius, that is, °C.
Claims (20)
- 一种治疗患者的情绪或应激障碍的方法，所述方法包括调节患者的运动皮层的兴奋性，其中所述情绪或应激障碍例如为双相及相关障碍、抑郁障碍、焦虑或恐惧相关性障碍和应激相关障碍中的一种或多种。A method of treating a mood or stress disorder in a patient, the method comprising modulating the excitability of the patient's motor cortex, wherein the mood or stress disorder is, for example, bipolar and related disorders, depressive disorders, anxiety or fear-related disorders. One or more of the following disorders and stress-related disorders.
- 根据权利要求1所述的方法，其中所述方法包括增加患者的运动皮层的兴奋性。The method of claim 1, wherein the method includes increasing the excitability of the patient's motor cortex.
- 根据权利要求1所述的方法，其中所述运动皮层选自初级运动皮层、前运动皮层和辅助运动区，优选为初级运动皮层。The method according to claim 1, wherein the motor cortex is selected from the group consisting of primary motor cortex, premotor cortex and supplementary motor area, preferably primary motor cortex.
- 根据权利要求1所述的方法，所述方法包括通过物理方法调节患者的运动皮层的兴奋性，例如对患者运动皮层施加电刺激、磁刺激、光刺激、振动刺激、压力刺激、声刺激、超声刺激中的一种或多种。The method according to claim 1, which method includes regulating the excitability of the patient's motor cortex through physical methods, such as applying electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation, pressure stimulation, acoustic stimulation, ultrasound to the patient's motor cortex. one or more of the stimuli.
- 根据权利要求1所述的方法，所述方法包括给予患者调节运动皮层兴奋性的制剂，例如通过给予患者光遗传学(Optogenetics)制剂、化学遗传学(Chemogenetics)制剂或化学制剂。The method of claim 1, comprising administering to the patient a preparation that modulates motor cortex excitability, for example by administering to the patient an optogenetics preparation, a chemogenetics preparation or a chemical preparation.
- 根据权利要求1-5中任一项所述的方法，其中所述方法为通过全身或局部调节运动皮层兴奋性的方法，优选的，其中所述方法为局部调节运动皮层兴奋性的方法，The method according to any one of claims 1 to 5, wherein the method is a method of regulating the excitability of the motor cortex systemically or locally, preferably, wherein the method is a method of regulating the excitability of the motor cortex locally,例如，其中通过对运动皮层进行局部刺激的物理方法或局部给药来调节运动皮层兴奋性。For example, in which motor cortex excitability is modulated by physical means of local stimulation of the motor cortex or local administration.
- 根据权利要求1-6中任一项所述的方法，其中所述患者具有应激障碍，例如为创伤后应激障碍或复杂性创伤后应激障碍。 The method according to any one of claims 1 to 6, wherein the patient has a stress disorder, such as post-traumatic stress disorder or complex post-traumatic stress disorder.
- 一种诊断受试者的情感或应激障碍的方法，所述方法包括检测运动皮层的兴奋性，其中所述情绪或应激障碍例如为双相及相关障碍、抑郁障碍、焦虑或恐惧相关性障碍和应激相关障碍中的一种或多种。A method of diagnosing an affective or stress disorder in a subject, the method comprising detecting excitability of the motor cortex, wherein the affective or stress disorder is, for example, bipolar and related disorders, depressive disorders, anxiety or fear-related disorders One or more of the following disorders and stress-related disorders.
- 根据权利要求8所述的方法，其中当受试者的运动皮层的兴奋性下降时，判断所述受试者患有所述的或具有发生所述情感或应激障碍的风险。The method of claim 8, wherein the subject is determined to be suffering from or at risk of developing the affective or stress disorder when the excitability of the subject's motor cortex decreases.
- 根据权利要求8所述的方法，其中所述运动皮层选自初级运动皮层、前运动皮层和辅助运动区，优选为初级运动皮层。The method according to claim 8, wherein the motor cortex is selected from the group consisting of primary motor cortex, premotor cortex and supplementary motor area, preferably primary motor cortex.
- 根据权利要求8-10中任一项所述的方法，其中检测与运动皮层相关的生物电信号，例如为神经元的静息膜电位、动作电位发放频率，基电流或电压阈值的检测等。The method according to any one of claims 8-10, wherein bioelectrical signals related to the motor cortex are detected, such as resting membrane potential of neurons, action potential firing frequency, detection of base current or voltage threshold, etc.
- 根据权利要求8-10中任一项的方法，其中检测与运动皮层相关的生物标记物，例如为神经递质和信号通路相关蛋白或核酸。The method according to any one of claims 8-10, wherein biomarkers related to the motor cortex, such as neurotransmitters and signaling pathway-related proteins or nucleic acids, are detected.
- 根据权利要求8所述的方法，其为局部检测脑区即运动皮层的兴奋性的方法。The method according to claim 8, which is a method of locally detecting the excitability of a brain area, namely the motor cortex.
- 根据权利要求8所述的方法，其中所述患者具有应激障碍，例如为创伤后应激障碍或复杂性创伤后应激障碍。The method of claim 8, wherein the patient has a stress disorder, such as post-traumatic stress disorder or complex post-traumatic stress disorder.
- 用于治疗患者的情绪或应激障碍的药物组合物或设备，所述药物组合物或设备可调节患者的运动皮层的兴奋性，其中所述情绪或应激障碍例如为双相及相关障碍、抑郁障碍、焦虑或恐惧相关性障碍和应激相关障碍中的一种或多种。A pharmaceutical composition or device for treating a mood or stress disorder in a patient, such as bipolar and related disorders, which modulates the excitability of the motor cortex of the patient. One or more of a depressive disorder, an anxiety- or fear-related disorder, and a stress-related disorder.
- 根据权利要求15所述的药物组合物或设备，其中所述药物组合 物或设备可增加患者的运动皮层的兴奋性。A pharmaceutical composition or device according to claim 15, wherein said pharmaceutical combination Objects or devices can increase the excitability of the patient's motor cortex.
- 根据权利要求15所述的药物组合物或设备，其中所述运动皮层选自初级运动皮层、前运动皮层和辅助运动区，优选为初级运动皮层。The pharmaceutical composition or device according to claim 15, wherein the motor cortex is selected from the group consisting of primary motor cortex, premotor cortex and supplementary motor area, preferably primary motor cortex.
- 根据权利要求15-17中任一项所述的药物组合物或设备，其为调节患者的运动皮层的兴奋性的设备，例如对患者运动皮层施加电刺激、磁刺激、光刺激、振动刺激、压力刺激、声刺激、超声刺激的一种或多种的设备。The pharmaceutical composition or device according to any one of claims 15 to 17, which is a device for regulating the excitability of the patient's motor cortex, such as applying electrical stimulation, magnetic stimulation, light stimulation, vibration stimulation to the patient's motor cortex, One or more devices for pressure stimulation, sound stimulation, and ultrasonic stimulation.
- 根据权利要求15-17中任一项所述的药物组合物或设备，其为包含调节运动皮层兴奋性的制剂的药物组合物，例如为光遗传学(Optogenetics)制剂、化学遗传学(Chemogenetics)制剂或化学制剂。The pharmaceutical composition or device according to any one of claims 15 to 17, which is a pharmaceutical composition containing a preparation that modulates motor cortex excitability, such as an optogenetics preparation or a chemogenetics preparation. Preparations or chemical preparations.
- 根据权利要求15-19中任一项所述的药物组合物或设备，其为通过全身或局部调节运动皮层兴奋性的药物组合物或设备，优选的，其中所述方法为局部调节运动皮层兴奋性的药物组合物或设备，The pharmaceutical composition or device according to any one of claims 15 to 19, which is a pharmaceutical composition or device for regulating motor cortex excitability systemically or locally, preferably, wherein the method is to locally regulate motor cortex excitability. Sexual pharmaceutical compositions or devices,例如，其为在运动皮层局部给药的剂型的药物组合物，或为具有在运动皮层局部作用的装置的设备。 For example, it is a pharmaceutical composition in a dosage form for local administration in the motor cortex, or a device having a device that acts locally on the motor cortex.
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