WO2018205935A1 - 治疗抑郁症的方法和药物组合物 - Google Patents

治疗抑郁症的方法和药物组合物 Download PDF

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WO2018205935A1
WO2018205935A1 PCT/CN2018/086043 CN2018086043W WO2018205935A1 WO 2018205935 A1 WO2018205935 A1 WO 2018205935A1 CN 2018086043 W CN2018086043 W CN 2018086043W WO 2018205935 A1 WO2018205935 A1 WO 2018205935A1
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discharge
component
depression
cluster
nucleus
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PCT/CN2018/086043
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English (en)
French (fr)
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胡海岚
杨艳
崔一卉
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浙江大学
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Priority claimed from CN201710322647.XA external-priority patent/CN108853502B/zh
Application filed by 浙江大学 filed Critical 浙江大学
Priority to EP18798176.6A priority Critical patent/EP3636281B1/en
Publication of WO2018205935A1 publication Critical patent/WO2018205935A1/zh
Priority to US16/679,195 priority patent/US11471428B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4015—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having oxo groups directly attached to the heterocyclic ring, e.g. piracetam, ethosuximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry
    • G01N2800/304—Mood disorders, e.g. bipolar, depression

Definitions

  • the present invention relates to the field of disease treatment and medicine.
  • the present invention relates to the treatment of depression and pharmaceutical compositions for the treatment of depression and methods for their preparation.
  • depression has a high incidence in the population. People with depression can be significantly affected both emotionally and physically without proper treatment. According to the definition and description of the US National Institute of Mental Health (NIMH), depression includes the following symptoms: "continuous sadness, anxiety, or emptiness”; despair, pessimism; guilt, no Sense of value, helplessness; loss of interest and happiness in the enjoyment of hobbies and activities; reduction of energy, fatigue; difficulty in concentration, difficulty in memory, difficulty in making decisions; hyperactivity, irritability, etc.
  • NIMH National Institute of Mental Health
  • Lateral habenula is a component of the nucleus and is located in the upper thalamus.
  • the lateral nucleus is the main organization that transmits information between the marginal forebrain and the midbrain.
  • the lateral habenular nucleus is linked and regulated by dopaminergic and serotoninergic nerve fibers, which makes the lateral habenular nucleus involved in various physiological activities, affecting body functions, and drug addiction, reward-disgust, pain, sleep, etc.
  • Mental state is related to illness.
  • Lateral habenular nucleus has been found to be associated with the development of depression. Studies have shown that in depressed rats, the frequency of microexcitatory postsynaptic current mEPSC in the lateral habenular nucleus to VTA projection neurons is significantly higher than that in normal rats, suggesting that this depression-related hyperexcitability in the nucleus The state may be mediated by synaptic plasticity mechanisms (Li, B. et al. Nature 470, 535-539, 2011). Under normal conditions, the lateral nucleus has a low level of inhibition of VTA and DRN.
  • the present invention for the first time and unexpectedly found that clustered discharge of neurons of the lateral nucleus has an important role in the production of depression, and found a key factor affecting the cluster discharge of the lateral nucleus, thereby providing inhibition by Methods and drugs for treating (suppressing) depression by cluster discharge of the lateral nucleus, especially methods and drugs for rapidly treating (suppressing) depression.
  • the present invention provides a method of treating depression in a subject by inhibiting cluster discharge of the lateral nucleus.
  • the present invention provides the use of an agent for inhibiting cluster discharge in the lateral nucleus nucleus for the treatment of depression, in particular for administration of a drug for the treatment of depression in the lateral nucleus.
  • the present invention also provides a pharmaceutical composition for treating depression, particularly a pharmaceutical composition for treating depression in the lateral nucleus, which comprises an agent for inhibiting cluster discharge in the lateral nucleus.
  • Subjects in need of the methods and medicaments (pharmaceutical compositions) described herein include subjects diagnosed with depression.
  • the subject to be treated may be a mammal, including a human or a non-human primate such as a monkey.
  • the mammal can be other animals such as rats, mice, rabbits, pigs, dogs, and the like.
  • the mammal can be a domestic animal such as a cat or a dog.
  • cluster discharge refers to a discharge pattern in which two or more spikes are simultaneously generated by a neuron during discharge.
  • Suppressing cluster discharge refers to suppressing the degree of distribution of cluster discharges, including reducing the frequency of cluster discharges or the number of peak potentials in clusters during cluster discharge, reducing the intensity of cluster discharges, and even eliminating the occurrence of cluster discharges. .
  • single dispense or “single discharge” is a discharge pattern in which a neuron issues a spike each time during discharge.
  • the reagent for suppressing the cluster discharge includes a compound, a complex or a mixture capable of suppressing the cluster discharge, a preparation used in a method for suppressing the cluster discharge (including a surgical method), and the like.
  • the reagent includes a small molecule compound or complex, or a macromolecular active component such as a protein or a nucleic acid, for example, an antagonist such as an antibody that binds to a protein on a cluster discharge physiological pathway, or a nucleic acid that affects the expression level of these proteins. .
  • treatment includes: an ongoing process or result of ameliorating, alleviating, reducing or preventing symptoms associated with depression; an ongoing process or result of ameliorating symptoms associated with depression; causing a particular organism to be caused An ongoing process or result of normalization of the body's function in a disease or condition that is impaired; or an ongoing process or result that results in the improvement of one or more clinically measurable parameters of the disease.
  • the therapeutic goal is to prevent or slow (reduce) undesired physiological conditions, conditions or diseases, or to obtain beneficial or desired results.
  • the result may be, for example, medical, physiological, clinical, physical therapy, occupational therapy, to a health care professional or patient; or as a parameter in the art as "quality of life" or activities of daily living.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms; reducing/reducing the extent of the condition, disorder or disease; stabilizing (ie, not worsening) the condition, condition or state of the disease; delaying the condition , the onset or slowing of the progression of the condition or disease; the improvement or alleviation of the condition, disorder or disease; and the reduction (whether partial or general), whether detectable or undetectable; or the enhancement or amelioration of the condition, condition Or disease.
  • the treatment comprises eliciting a clinically effective response without excessive levels of side effects.
  • the treatment also includes prolonging the survival period as compared to the expected survival if not receiving treatment.
  • treatment refers to administering a drug or performing a medical procedure on a patient.
  • the treatment may be prevention (prevention), cure of weakness or disease, or improvement of the clinical condition of the patient, including lowering the course of the disease or the severity of the disease, or subjectively improving the quality of life of the patient or prolonging the survival of the patient.
  • the method for treating depression by inhibiting cluster discharge of the lateral nucleus of the present invention the use of the agent for inhibiting cluster discharge in the lateral nucleus of the present invention for treating a drug for depression, And in the pharmaceutical composition for treating depression of the present invention, the agent for inhibiting cluster discharge is an N-methyl-D-aspartate receptor inhibitor.
  • N-methyl-D-aspartate is an excitatory amino acid (EAA), an excitatory neurotransmitter of the central nervous system.
  • EAA excitatory amino acid
  • NMDA receptor or NMDAR N-methyl-D-aspartate receptor
  • NMDA receptor or NMDAR is an ionic receptor involved in excitatory synaptic transmission. Modulation of NMDA receptors modulates glutamatergic neurotransmitter-mediated neurological effects.
  • N-methyl-D-aspartate receptor inhibitors useful in the present invention include, but are not limited to:
  • NMDA receptors (inhibitors that compete with glutamate binding sites): AP5, AP7, CPPene, Selfotel;
  • Non-competitive inhibitors of NMDA receptors inhibitors that block allosteric binding sites: Aptiganel, ketamine, memantine, Huperzine A, Ibogaine, HU-211, Gabapentin, PD-137889, etc.;
  • NMDA receptor anti-competitive channel blockers (channel blockers): Amantadine, Atomoxetine, AZD6765, Dextromethorphan, Magnesium Ammonium Hydrochloride, MK801 ( Dizocilpine);
  • Glycine binding site inhibitor TK-40, Kynurenic acid and the like.
  • the method for treating depression by inhibiting cluster discharge of the lateral nucleus of the present invention the use of the agent for inhibiting cluster discharge in the lateral nucleus of the present invention for treating a drug for depression, And in the pharmaceutical composition for treating depression of the present invention, the agent for inhibiting cluster discharge is a T-type calcium ion channel inhibitor.
  • the T-type calcium channel or the T-type calcium channel is also known as the low voltage activated calcium channel (T-type calcium channel).
  • T-type calcium channels play an important role in the regulation of excitability in the central and peripheral nervous systems.
  • the T-type calcium channel family includes three different ⁇ 1 subunit genes: CACNA1G, CACNA1H, CACAN1I, encoding ⁇ 1G, ⁇ 1H, and ⁇ 1I, respectively, thus constituting Cav3.1, Cav3.2, and Cav3.3, 3
  • the T-type calcium channel protein is a structure composed of tetramers, and each monomer, the ⁇ 1 subunit, contains four homologous regions.
  • the channel protein pores are composed of the above four homologous regions.
  • the pore helix is connected to the end of the extracellular S6 fragment to form a calcium ion selective passage filter.
  • the S4 fragment of each homologous domain has a positively charged amino acid residue every three amino acids, forming a channel voltage receptor. Based on this structure, the opening and closing of the channel can be controlled when the membrane potential changes.
  • T-type calcium ion channel inhibitors useful in the present invention include, but are not limited to:
  • Succinimides such as ethosuximide, methsuximide; hydantoins; zonisamide; valproate sodium; Pheytoin; Mibefradil; Phenytoin; sipatrigine; piperazine analogs such as Flunarikine, Z941; piperidine analogs such as Z944 and Fluoropiperidine; TTA-P1; TTA-P2; quinazolinone; pimozide Pimozide); Trimethadione and metformin; TTA-Q4; ML218 and the like.
  • the method for treating depression by inhibiting cluster discharge of the lateral nucleus of the present invention the use of the agent for inhibiting cluster discharge in the lateral nucleus of the present invention for treating a drug for depression, And in the pharmaceutical composition for treating depression of the present invention, the agent for inhibiting cluster discharge is a combination of an N-methyl-D-aspartate receptor inhibitor and a T-type calcium ion channel inhibitor.
  • the present invention provides a method of treating depression in a subject, comprising administering to the subject a component (a) an N-methyl-D-aspartate receptor (NMDAR) inhibitor; and a component (b) A combination of T-type calcium channel or T-VSCC inhibitors.
  • a component a) an N-methyl-D-aspartate receptor (NMDAR) inhibitor
  • a component b) A combination of T-type calcium channel or T-VSCC inhibitors.
  • the present invention also provides a pharmaceutical composition for treating depression comprising: a component (a) an N-methyl-D-aspartate receptor (NMDAR) inhibitor; and a component (b) a T-type calcium ion Channel (T-type calcium channel or T-VSCC) inhibitor.
  • NMDAR N-methyl-D-aspartate receptor
  • T-type calcium ion Channel T-type calcium channel or T-VSCC
  • the method and pharmaceutical composition for treating depression provided by the present invention the therapeutically effective amount of at least one of component (a) and component (b) administered to the subject is low A therapeutically effective amount of the component administered in the absence of another component. In yet another aspect of the invention, the therapeutically effective amount of component (a) and component (b) administered to the subject is lower than the therapeutically effective amount of the component administered in the absence of the other component. .
  • At least one of component (a) and component (b) administered to the subject particularly two
  • the therapeutically effective amount of the species is at least 5% lower, at least 10% lower, at least 25% lower, at least 50% lower, at least 60% lower, and at least lower than the therapeutically effective amount of the component administered in the absence of the other component. 70%, at least 80% lower, or at least 90% lower.
  • the therapeutically effective amount ratio of at least one of component (a) and component (b) administered to the subject in a method and pharmaceutical composition for treating depression provided by the present invention
  • the therapeutically effective amount of the component administered in the absence of another component is 5% to 90% lower, 10% to 90% lower, 25% to 90% lower, or 50% to 90% lower.
  • the therapeutically effective amount of the NMDAR inhibitor is lower than the therapeutically effective amount of the antidepressant drug administered in the absence of the T-VSCC inhibitor. At least 5%, at least 10% lower, at least 25% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, or at least 90% lower. That is, in the pharmaceutical composition of the present invention, the dose of the NMDAR inhibitor is at least 5% lower than the normal dose (recommended dose) of the NMDAR inhibitor alone, at least 10% lower, at least 25% lower, and at least 50% lower. , at least 60% lower, at least 70% lower, at least 80% lower, or at least 90% lower.
  • the therapeutically effective amount of the NMDAR inhibitor is greater than the antidepressant administered in the absence of the T-VSCC inhibitor.
  • the therapeutically effective amount of the drug is 5% to 90% lower, 10% to 90% lower, 25% to 90% lower, or 50% to 90% lower. That is, in the pharmaceutical composition of the present invention, the dose of the NMDAR inhibitor agent is 5% to 90% lower, 10% to 90% lower, and 25% lower than the normal dose (recommended dose) of the NMDAR inhibitor alone. 90%, or 50% to 90% lower.
  • the NMDAR inhibitor and the T-VSCC inhibitor are formulated in the same pharmaceutical composition, or the NMDAR inhibitor is formulated in the first pharmaceutical combination And the T-VSCC inhibitor is formulated in a second pharmaceutical composition.
  • the NMDAR inhibitor and the T-VSCC inhibitor are administered simultaneously.
  • the NMDAR inhibitor and the T-VSCC inhibitor are administered separately.
  • depression may specifically refer to "lateral lateral nucleus-mediated depression", especially "outside nucleus cluster discharge-mediated depression”.
  • the inventors of the present application have found and demonstrated abnormal distribution of neurons of the lateral nucleus, especially the abnormal distribution of cluster discharges, which plays an important role in the generation of depression.
  • the inventors of the present application have also discovered key factors affecting the cluster discharge of the lateral nucleus, and by adjusting these key factors, depression can be suppressed or eliminated.
  • the method for treating depression by inhibiting cluster discharge of the lateral nucleus of the present invention the use of the agent for inhibiting cluster discharge in the lateral nucleus of the present invention for treating a drug for depression,
  • the method or medicament is suitable for use in other depression patients and drug-ineffective depression patients.
  • Antidepressants that have been employed in the art, classified according to their mechanisms for inhibiting depression, may include:
  • SSRIs Selective serotonin reuptake inhibitors
  • SNRIs norepinephrine reuptake dual inhibitors
  • MAOIs monoamine oxidase inhibitors
  • TCAs tricyclic depressors
  • mGluRs Metabolic glutamate receptors
  • the inventors of the present application have first discovered and demonstrated abnormal distribution of neurons of the lateral nucleus, especially the abnormal distribution of cluster discharges, which plays an important role in the production of depression, thus providing neurons that inhibit lateral nucleus Abnormal release, especially the abnormal release of cluster discharges to treat (suppress) depression methods and drugs.
  • This is a mechanism known in the art for treating depression and a pathological mechanism against which a drug fails to target and a target target for brain tissue or its molecular level for treatment.
  • the methods and medicaments or pharmaceutical compositions provided by the present invention are particularly suitable for use in the above-described antidepressant methods and drug-ineffective depression patients.
  • NMDA receptor antagonists are known in the art for use in the treatment of depression. However, in these reports, the discovered or presumed antidepressant mechanism is completely different from the mechanism discovered by the present invention, that is, by inhibiting the abnormal release of lateral nucleus neurons, particularly the abnormal release of cluster discharges.
  • the method for treating depression by inhibiting the cluster discharge of the lateral nucleus of the present invention inhibits the cluster discharge in the lateral nucleus
  • the agent for inhibiting cluster discharge does not include these NMDA receptor antagonists, such as AP5, CPPene, MK801, Memantine, ketamine, felbamate, glycine, D-serine, D-cycloserine, L-glutamic acid, efendil, and the like.
  • the method for treating depression by inhibiting the cluster discharge of the lateral nucleus of the present invention inhibits the cluster discharge in the lateral nucleus
  • the agent for treating a drug for depression, and the pharmaceutical composition for treating depression of the present invention does not include fluoxetine, trazodone, ethosylamine, trimethyl ketone , sodium valproate, pimozide and zonisamide.
  • a method of treating depression by inhibiting cluster discharge of the lateral nucleus of the present invention and an agent for inhibiting cluster discharge in the lateral nucleus of the present invention are used for the medicament for treating depression
  • the agent for suppressing cluster discharge does not inhibit a tonic pulse.
  • the method for treating depression by inhibiting cluster discharge of the lateral nucleus of the present invention the use of the agent for inhibiting cluster discharge in the lateral nucleus of the present invention for treating a drug for depression
  • the method, medicament or pharmaceutical composition is a method and a pharmaceutical or pharmaceutical composition which is locally activated in the lateral nucleus, that is, administered to the lateral nucleus.
  • nerve tissue particularly brain nerve tissue, such as the lateral nucleus
  • Administration for administration in the lateral nucleus is a technical feature that is both restrictive to the method of treatment and preparation of the drug.
  • the pharmaceutical or pharmaceutical composition is a dosage form for topical administration to the lateral nucleus.
  • the action of the drug can be limited to the target tissue by topical administration, for example by making the drug into a dosage form that can be administered topically to the lateral nucleus by cannulation.
  • the drug is prepared into a dosage form or the like which is sustained release after being implanted into a tissue.
  • the above drugs can also be formulated in the form of tissue-specific targeted drug delivery systems.
  • an antibody which binds to a small molecule compound or a biologically active molecule capable of specifically binding to a protein expressed in the lateral nucleus can be used.
  • a small molecule compound or a biologically active molecule nucleic acid such as a protein-encoding DNA or mRNA molecule, a protein such as an antibody, etc.
  • the antibody fragments are ligated to form a composite molecule capable of recognizing and binding to cells of the lateral nucleus.
  • the above-described method for treating depression by locally inhibiting cluster discharge in the lateral nucleus of the present invention including administering NMDAR inhibitor or T-VSCC inhibitor alone, or administering T in combination) - VSCC inhibitors and NMDAR inhibitors) and drugs (including pharmaceutical compositions or combination pharmaceutical compositions)
  • the T-type calcium channel inhibitors may also be fluoxetine, trazodone, ethosuxamine, trimethyl shuang Ketone, sodium valproate, pimozide, zonisamide, etc.
  • the NMDA receptor antagonist may also be AP5, CPPene, MK801, memantine, ketamine, felfalide, glycine, D-serine , D-cycloserine, L-glutamic acid, Effendil and the like.
  • the method for treating depression by inhibiting cluster discharge of the lateral nucleus of the present invention the use of the agent for inhibiting cluster discharge in the lateral nucleus of the present invention for treating a drug for depression,
  • the pharmaceutical composition for treating depression of the present invention is particularly suitable for rapid treatment (inhibition) of depression.
  • the medicament provided by the present invention is suitable as a rapid onset of treatment (inhibition) of depression.
  • Most antidepressants in the field generally take a week to several weeks to exert antidepressant effects.
  • the commonly used 5-HT reuptake inhibitor (SSRI) is usually effective in 2-3 weeks
  • 5-HT and norepinephrine Reuptake of dual inhibitors is usually only effective in 1 week.
  • the antidepressant method and the pharmaceutical or pharmaceutical composition provided by the present invention have an onset time of less than one week, preferably less than three days, more preferably less than one day, such as less than 12 hours.
  • the medicament provided by the present invention is also suitable as a fast-acting and intermediate-effect or long-acting therapeutic (suppressive) depression, wherein the single-dose antidepressant effect can last for more than one day, preferably for more than three days, more preferably for more than one week.
  • the present invention provides a pharmaceutical composition for treating depression comprising a therapeutically effective amount of an agent that inhibits cluster discharge in the lateral nucleus.
  • the active ingredient in the pharmaceutical composition provided by the present invention is an agent which inhibits cluster discharge in the lateral nucleus.
  • the active ingredient in the pharmaceutical composition of the present invention which is suitable for use in the treatment may be administered in the form of a raw material compound, it is preferred to administer the active ingredient, optionally in the form of a physiologically acceptable salt, with one or more
  • the pharmaceutical composition is introduced together with the agent, excipient, carrier, buffer, diluent, and/or other conventional pharmaceutical excipients.
  • compositions of the 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 dermal, subcutaneous, intramuscular and intravenous injection.
  • the pharmaceutical compositions of the present invention can be prepared by those skilled in the art by using standard methods and conventional techniques appropriate to the desired formulation. If desired, a composition suitable for sustained release of the active ingredient can be used.
  • compositions of the invention may be those suitable for oral, rectal, bronchial, nasal, pulmonary, topical (including buccal and sublingual), transdermal, vaginal or parenteral (including skin, subcutaneous, intramuscular, intraperitoneal, intravenous)
  • Pharmaceutical compositions for administration by internal, intraarterial, intracerebral, intraocular injection or infusion, or those suitable for administration by inhalation or insufflation (including powder and liquid aerosol administration) or for sustained release A pharmaceutical composition in the form of a systemic administration.
  • suitable sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compounds of the invention, which matrices may be in the form of shaped articles, such as films or microcapsules.
  • the active ingredient of the pharmaceutical compositions of the present invention can thus be formulated into a pharmaceutical composition and unit dosage form together with conventional adjuvants, carriers or diluents.
  • Such forms include solids, and especially in the form of tablets, filled capsules, powders and pellets, as well as liquids, especially aqueous or nonaqueous solutions, suspensions, emulsions, elixirs, and capsules filled in the form described above, all of these forms Both are used orally, suppositories for rectal administration, and sterile injectable solutions for parenteral administration.
  • Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or ingredients, and such unit dosage forms may contain any suitable effective amount equivalent to the desired daily application dosage range. Active ingredient.
  • the pharmaceutically acceptable carrier can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • the solid carrier can be one or more materials which are also useful as diluents, flavors, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.
  • Aqueous suspensions suitable for oral use can be prepared by dispersing the finely divided active ingredient in a viscous substance such as a natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose, or other well known suspensions Prepared in water.
  • a viscous substance such as a natural or synthetic gum, resin, methylcellulose, sodium carboxymethylcellulose, or other well known suspensions Prepared in water.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • Such preparations may contain, in addition to the active ingredient, coloring agents, flavoring agents, stabilizers, buffers, artificial and natural sweeteners, dispersing agents, thickening agents, solubilizing agents and the like.
  • the compounds of the invention may be formulated as ointments, creams, or lotions, or as a transdermal patch.
  • ointments and creams may be formulated with aqueous or oily bases, together with suitable thickening and/or gelling agents.
  • Lotions may be formulated with aqueous or oily bases and usually contain one or more emulsifying, stabilizing, dispersing, suspending, thickening or coloring agents.
  • Respiratory administration can also be achieved by means of an aerosol wherein the active ingredient is provided in a pressurized pack together with a suitable propellant such as a chlorofluorocarbon (CFC) such as dichlorodifluoromethane or trichlorofluoride. Methane or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. Aerosols may also suitably contain a surfactant such as lecithin. The dose of the drug can be controlled by a metering valve.
  • a suitable propellant such as a chlorofluorocarbon (CFC) such as dichlorodifluoromethane or trichlorofluoride. Methane or dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • Aerosols may also suitably contain a surfactant such as lecithin.
  • the dose of the drug can be controlled by a metering valve.
  • the active ingredient in the pharmaceutical compositions of the invention may be presented in the form of a dry powder such as a compound in a suitable powder base such as lactose, starch, starch derivatives (such as hydroxypropylmethylcellulose) and polyvinylpyrrolidone (PVP). a mixture of powders.
  • a suitable powder base such as lactose, starch, starch derivatives (such as hydroxypropylmethylcellulose) and polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • the powder carrier will form a gel within the nasal cavity.
  • the powder composition may be presented in unit dosage form, for example in the form of a capsule or cartridge (such as a capsule or cartridge of gelatin) or in the form of a blister pack from which the powder can be administered by means of an inhaler.
  • a composition suitable for providing sustained release of the active ingredient can be applied as needed.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active ingredient.
  • the unit dosage form can be a packaged preparation containing discrete quantities of preparation such as a packaged tablet, capsule, and powder in a vial or ampule.
  • the unit dosage form can be a capsule, tablet, cachet or lozenge itself, or can be in the form of a suitable quantity of any of these dosage forms.
  • Tablets or capsules for oral administration and liquids for intravenous administration and continuous infusion are preferred compositions.
  • a therapeutically effective dose means the amount of active ingredient that relieves symptoms or conditions.
  • Therapeutic efficacy and toxicity such as ED50 and LD50, can be determined by standard pharmacological procedures in cell culture or laboratory animals. The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed by the ratio of LD50/ED50.
  • the dosage administered will of course have to be carefully adjusted for the age, weight and condition of the individual being treated, as well as the route of administration, the dosage form and dosage regimen, and the desired result, and the exact dosage should of course be determined by the physician.
  • a pharmaceutical composition comprising from about 0.1 to about 1000 mg, preferably from about 1 to about 750 mg, of the active ingredient per single dose is suitable for therapeutic treatment.
  • the invention also provides an animal model of depression, preferably a rat or a mouse.
  • the animal model of depression according to the present invention is characterized by depression, which has abnormal distribution of cluster discharge of lateral nucleus neurons.
  • the present invention also provides a method for screening potential substances for treating depression using the above animal model, comprising the steps of:
  • test substance is a potential substance that can be used to treat depression.
  • the method of screening for a potential substance for treating depression further comprises one or more of the following steps:
  • Test substances are potential substances for the treatment of depression.
  • FIG. 1 Schematic diagram of bilateral cannula implantation of cLH rat lateral nucleus. The white dashed line indicates the position of the helium core.
  • BG topical bilateral application of ketamine (25 ⁇ g per side, BD) and AP5 (40nmol per side, EG) to LHb, can effectively reverse the depression phenotype of cLH rats in a short time (0.5 or 1 hour): significantly reduced Immobility time (C and F) in forced swimming and significantly increased preference for syrup (D and G) in depressed animals.
  • FIG. 1 The clustering of lateral habenular nucleus neurons in rat and mouse depressive animal models is enhanced and can be reversed by ketamine.
  • A indicates the recording sites recorded by whole-cell patch clamps, which are distributed in different sub-regions of the lateral habenula.
  • B-D Typical diagrams of three self-issuing modes of neurons in the lateral nucleus, namely, silent, single tonic and burst. In the middle is the reaction of the same neuron to TTX, which blocks the peak potential of single and cluster discharges. The discharge trajectory on the right is an enlarged view of the shaded area on the left.
  • the (E-F) scatter plot (E) and the cumulative plot (F) show the mean and distribution of resting membrane potentials (RMPs).
  • the intra-cluster release frequency is positively correlated with the degree of superposition of resting membrane potential.
  • I-N In the congenital depression (cLH) rats and the chronic restraint stress-induced mouse depression model, the proportion of neurons in the cluster discharge was significantly increased.
  • the (I, L) pie chart shows an increase in the number of clustered discharge neurons in the rat and mouse depression models.
  • the (J, M) bar graph shows the proportion of cells in each of the released cells with a single discharge and a cluster discharge.
  • the (K, N) column chart shows the distribution of neuronal peak potential intervals in the nucleus of the immobile animals.
  • Figure 3 shows in vivo electrophysiological recordings that ketamine inhibits cluster-like discharge activity and ⁇ -band synchronization activity in the nucleus of neurons in chronic restraint stress mice.
  • A Recording sites in the body recording electrode in control and CRS mouse LHb.
  • B Representative examples of the LHb neuron firing in mice, CRS and CRS + ketamine, and the mean firing waveform (right) were isolated, and the cluster discharge was separated by analysis of the peak potential interval (ISI).
  • C-D CRS mice LHb neurons cluster discharge ratio and the number of cluster discharges per minute were significantly higher than the control mice, and can be reversed by ketamine.
  • FIG. 4LHb The clustered discharge of Figure 4LHb requires activation of the NMDA receptor.
  • NMDA receptor-mediated excitatory postsynaptic currents (NMDAR-EPSCS) were isolated by the addition of GABA receptor inhibitors (picrotoxin) and AMPA receptor blockers (NBQX) to artificial blood cerebrospinal fluid (ACSF) without Mg2+, The current was confirmed by the NMDA receptor blocker AP5.
  • B LHb neurons clamped NMDAR-EPSCs recorded at different voltages, which were completely blocked by AP5.
  • C-H Effect of ketamine (C-D), AP5 (E-F) and NBQX (G-H) on spontaneous cluster discharge in LHb.
  • the left side is a typical picture and the right side is a statistical chart.
  • I-J NMDA perfusion can cause cluster discharges in cells that are not released, and this induced cluster discharge can be inhibited by ketamine.
  • NMDA induces large excitatory postsynaptic potentials and cluster discharges. All data are expressed as mean ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001 compared to the control group. N.S. indicates that the difference is not significant. The other icons are the same.
  • LHb cluster discharge requires the involvement of neuronal membrane super- and T-type voltage-sensitive dry channels.
  • A Ramp current injection induces a typical map of LHb neurons from cluster discharge to single discharge, with neurons prone to cluster discharges in a relatively super-state and a single discharge in a relatively depolarized state.
  • B The statistical graph shows the proportion of neurons that can induce cluster discharge after LHb neurons are injected into the super-current in large mice.
  • C-E Current clamp plus recorded cluster discharge frequency (C), cluster discharge duration (D) and number of cluster discharges (E) correlated with neuron resting membrane potential.
  • F A typical example plot of spontaneous single-discharge neuron transition to clustered discharge under superfluidization.
  • FIG. 1 A typical example plot of spontaneous clustered discharge to single discharge under depolarization. Effect of (H, I) T-VSCC blocker Mibefradil (H) and HCN channel blocker ZD7288 (I) on spontaneous cluster discharge of LHb neurons. The left side is a typical picture and the right side is a statistical chart.
  • Activated T-VSCC causes the magnesium ion blocking the NMDA receptor to be removed, and the opening of the T-VSCC and NMDA receptor channels drives the neuron membrane potential to super-clustered discharge threshold change.
  • T-VSCC antagonists exhibit a rapid antidepressant effect.
  • A-C Local injection of mibefradil to bilateral LHb showed rapid antidepressant effects in both FST (B) and SPT (C) behaviors.
  • A The injection site map of the cannula was determined by injecting CTB into LHb. All data are expressed as mean ⁇ SEM. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, ****P ⁇ 0.0001 compared to the control group. N.S. indicates that the difference is not significant. The other icons are the same.
  • Figure 7e NpHR light activation-induced rebound cluster discharges show animals aversive and depressive phenotypes that can be reversed by ketamine.
  • A Schematic diagram of eNpHR virus expression vector construction (top panel), photoelectric and recording schematic (below).
  • B, C Representative images of yellow-activated brain slice neurons (B) and in vivo recorded neurons (C) rebound cluster discharges in mouse LHb expressed by AAV2/9-eNpHR virus. The percentage of cells that successfully induced cluster discharges is shown in the right graph.
  • D The dot plot and the post-stimulation time bar plot show the response of a representative LHb neuron to 100 ms yellow light stimulation in the bulk photoelectrode recording.
  • Figure 8 shows that a single discharge at the same frequency as the cluster discharge does not cause a depressive phenotype.
  • A Ex vivo electrophysiological recordings show that a 5 Hz light activated oChIEF optical channel produces a single discharge of 5 Hz.
  • B Photoactivation did not alter the animal's ability to exercise compared to eGFP mice expressing control non-light channels.
  • C A single discharge from 5 Hz photoactivation does not induce a depressive phenotype.
  • Figure 9 co-administered a low dose of NMDA receptor inhibitor and a T-type calcium channel inhibitor to produce a rapid antidepressant effect and reduce the side effects of both drugs.
  • Figure 10 Effect of NMDAR inhibitor in combination with T-VSCC inhibitor on induction of cluster discharge.
  • the lateral nucleus neurons were injected with a super-charging current of -100 pA for 100 ms, which made the neurons hyperpolarized, and the super-state induced the cluster discharge of the lateral nucleus neurons.
  • the effect of lateral sputum nucleus slices on the probability of neuron super-induced cluster discharge when perfused with ketamine (100 ⁇ M, A), AP5 (100 ⁇ M, B) or Mibefradil (10 ⁇ M, C) the left graph is cluster discharge
  • the waveform example is on the right side for statistical analysis.
  • cLH rats Male cLH rats (4-12 weeks old), Sprague Dawley rats (4-12 weeks old).
  • cLH rats are a selectively cultured animal model of depression with a congenital acquired helpless depression phenotype (D. Schulz, M. M. Mirrione, F. A. Henn, Neurobiol Learn Mem 93, 291, Feb, 2010).
  • the cLH rats of this experiment were introduced from the Malinow Laboratory in Cold Spring Harbor, USA. CLH rat feeding and propagation is described in the aforementioned D. Schulz, et al, Feb, 2010. Rats 4/cage, 12-hour light-dark cycle (light at 7am-7pm).
  • One cLLH rat/cage was used for cannulation experiments.
  • AAV9-CaMKII-eNpHR3.0-eYFP the plasmid was purchased from Addgene, Cat#26971, the virus was coated by Shanghai Taiting Biotechnology Co., Ltd.; AAV9-Ubi-eGFP was donated by Gao Guangping Laboratory of UMass; AAV9-hSyn- oChIEF-tdTomato, the plasmid was purchased from Addgene, Cat#50977, and the virus was coated by Shanghai Taiting Biotechnology Co., Ltd.
  • mice were anesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (8 mg/kg) and fixed on a stereotaxic instrument (Stoelting instruments). Each mouse was injected with 0.1-0.2 ul of purified AAV virus ( ⁇ 10 13 infectious units/ml) per LHb, and the LHb stereotactic coordinates (front and back distance Bregma: -1.7 mm (AP), ⁇ 0.46 mm left and right) ML), the cortical surface is down -2.56 mm (DV)). Slowly inject ( ⁇ 100-150nl/min) using a self-drawn glass microelectrode, leave the needle at the end of the injection for 5 min, and then slowly remove the injection electrode within 5 min.
  • AAV virus ⁇ 10 13 infectious units/ml
  • LHb stereotactic coordinates front and back distance Bregma: -1.7 mm (AP), ⁇ 0.46 mm left and right) ML
  • ML cor
  • Behavioral experiments or electrophysiological experiments were performed at least 14 days after surgery. At the end of the behavioral experiment, the injection site was examined and only the animal data for the correct injection was used.
  • Brain sections injected with AAV virus were examined under a fluorescence microscope, or other viruses labeled with GFP were examined for antibodies with antibodies prior to microscopic examination.
  • the nucleus of each brain was cut into 6 consecutive sections (30 um sections of mice, 6 per group; 40 um sections of rats, 8-9 per group). All sections were counterstained with Hoechst before being mounted to the fixed piece.
  • Rat bilateral LHb embedded cannula Rats were anesthetized by intraperitoneal injection of 4% pentobarbital (60 mg/kg body weight) and fixed on a rat stereotaxic instrument. LHb stereotactic coordinates for catheter placement (front and back distance Bregma: -3.7 mm (AP), left and right side ⁇ 0.7 mm (ML), cortical surface down -4.1 mm (DV)). Above the skull corresponding to LHb, the cranial drill is drilled, and then three screws are fixed on the skull.
  • AP front and back distance Bregma
  • ML left and right side ⁇ 0.7 mm
  • DV cortical surface down -4.1 mm
  • the double-sided sleeve (purchased from Plastics One Company of the United States) enters the top of the nucleus, and the sleeve is fixed with the cement. After the cement is completely solidified, the sleeve is inserted into the sleeve, etc. The long flat end plugs the core and the nut is screwed to prevent the plug from falling out. Seven days after surgery, the rats can be used to detect the behavioral effects of the drug after recovery from surgical trauma.
  • Rats 40-50 days after birth or mice 8 weeks old were anesthetized with isoflurane and perfused with 20 ml of ice-cold oxygenated section.
  • the brain is quickly decapitated and placed in an oxygenated section.
  • a 350 um tubular section was then sectioned in an oxygenated, ice-cold slice using a Leica vibratome.
  • the nucleus slices were enriched in ACSF at 34 ° C (118 mM NaCl, 2.5 mM KCl, 26 mM NaHCO 3 , 1 mM NaH 2 PO 4 , 10 mM glucose, 1.3 mM MgCl 2 and 2.5 mM CaCl 2 , gassed with 95% O 2 and In 5% CO 2 ), recovery was carried out for at least 1 hour and then transferred to room temperature for recording. Rats and mice injected with the ketamine group were performed 1 h before the animals were taken.
  • the patch clamp recording of the lateral habenular nucleus was recorded using an Axon Multiclamp 700B amplifier under an Olympus microscope equipped with an infrared differential interference phase contrast optical lens at 32 ⁇ 1 °C. All cells were recorded in whole cell mode.
  • the neuron recording electrode impedance was 4-6 M ⁇ , and the electrode internal liquid composition was (mM): 105K-Gluconate, 30KCl, 4Mg-ATP, 0.3Na-GTP, 0.3EGTA, 10HEPES and 10Na-phosphocreatine, and the pH was 7.35.
  • the artificial cerebrospinal fluid (ACSF) components used to record the external fluid were (mM): 125 NaCl, 2.5 KCl, 25 NaHCO 3 , 1.25 NaH 2 PO 4 , 1 MgCl 2 and 25 glucose.
  • the data was filtered at 2 kHz and recorded using a Digidata 1322A at 10 kHz. Data were analyzed using the pClamp 10 software.
  • the different discharge modes produced in LHb are defined as: cells that are not released, refer to cells that have no action potential release during the entire recording process; cells that are single-discharged have a discharge frequency of 0.1-10 Hz, and rarely 10-20 Hz; Clustered discharge cells refer to the generation of clusters, and the frequency of intra-cluster emission is extremely high, but it shows a gradual decreasing trend, and the frequency between clusters is as high as 200 Hz.
  • the induced NMDA receptor-mediated excitatory postsynaptic current was recorded at -50 mV to -80 mV at zero-magnesium ACSF.
  • the induced T-type voltage-sensitive calcium channel current was clamped at -50 mV and then clamped to -100 mV for 1 second. Stimulation is given at a normal frequency of 0.1 Hz. Calcium current is obtained by linear leakage subtraction.
  • mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (8 mg/kg), and then fixed on a stereotaxic instrument (Stoelting instruments).
  • the stainless steel wire is wound onto two screws attached to the skull for grounding.
  • the electrodes are fixed to the surface of the skull with cement.
  • the 64-channel OmniPlex-D neural signal acquisition system (Plexon Inc., Dallas, TX) was recorded and the spontaneous discharge activity of the animal LHb (sampling frequency 40 kHz, 300-6000 Hz bandpass filtering) and field were recorded in a cage for 30 minutes. Potential (LFP, 1 kHz sampling rate, 250 Hz low pass filtering) with a gain of 5000. A channel having no discernible neuronal firing signal is used as a reference electrode.
  • LFP 1 kHz sampling rate, 250 Hz low pass filtering
  • a channel having no discernible neuronal firing signal is used as a reference electrode.
  • the tetrode was stepped down at a depth of 70 ⁇ m and recovered for 2 days to start the next recording. For chronic restraint stress mice, discharge activity was recorded 30 min before ketamine administration and 1 h after administration. The recorded animals were used to determine the electrode sites in the form of electrical damage.
  • Action potential sorting All recorded electrical signals were imported into Offline Sorter V3 (Plexon Inc.), and then individual neuron discharges were manually sorted using threshold method and principal component analysis (PCA). The release of the peak potential interval less than the refractory period (1.4 ms) was excluded, and cross-correlation analysis was performed to ensure that no sorted neurons were not duplicated. Signals that are inseparable from background noise are excluded.
  • Data Analysis The software used in this part of the data analysis was Neuroexplorer 4 (Plexon Inc.) and MATLAB.
  • Data Analysis The software used in this part of the data analysis was Neuroexplorer 4 (Plexon Inc.) and MATLAB.
  • the experiment was carried out under normal fluorescent lighting.
  • the mouse forced swimming cylindrical container has a diameter of 12 cm and a height of 25 cm.
  • the test water depth is 14 cm and the water temperature is 23-24 °C.
  • the camera recorded the swimming of the mice within 6 min from the side.
  • the double-blind method was used to count the immobility time (the floating position of the animal or the time when the limbs were completely inactive) after 4 minutes of swimming in the mouse.
  • mice were housed separately for 1 week, and then the mice were given two bottles of normal water for 2 consecutive days, and then the water was exchanged for two bottles of 2% sucrose water for training for two days. After the training, give the animal a bottle of normal water and a bottle of 2% sucrose water for testing, exchange the position of the water bottle every 12 hours, record the consumption of water and sugar water every 24 hours (weigh the water bottle), record a total 48 hours.
  • Animals were randomly divided into two groups. One group was placed in a 50 ml centrifuge tube for 2 h every day from 11:00 to 14:00, and was continuously restrained for 14 days. In order to facilitate animal breathing, a 50 ml centrifuge tube was used to drill several small holes of 2 mm diameter. Another group of control animals did not receive restraint stress. After the end of the daily restraint, the animals were returned to their cages and housed in the same breeding room as the control animals. Forced swimming and syrup preference tests were performed on day 15 to assess the depressive phenotype of the animals.
  • the implanted fiber was connected to the patch cable via a ceramic sleeve (ceramic sleeve was purchased from NEWDOON, Hangzhou, China), and the patch cable was connected to a rotary joint via an FC/PC adapter (purchased from Doric, Québec, Canada) ) allows animals to move freely without restriction.
  • the other patch cable is connected to the computer via a FC/PC adapter and a 473 nm DPSS laser (Aurora-220-473) or a 589 nm DPSS laser (Aurora-220-589).
  • the lasers are all purchased from NEWDOON, Hangzhou, China.
  • RTPA Real-time place aversion
  • an intermediately connected 52x26x23 cm open box was divided into two boxes of the same size (26 x 26 x 23 cm) for behavioral testing. Mice were placed in the box for free movement for 20 minutes to assess the basic preference level of the mice for the boxes on both sides. In the next 20-minute test period, the mice were evenly distributed to the left and right side boxes, with this side as the stimulation box and the other side the corresponding non-stimulation box for each mouse. The mice were placed in a non-stimulation box and the experiment was started.
  • the mouse can activate yellow light stimulation (eNpHR 3.0: 589 nm, 1 Hz, 16 mW, 100 ms interval) until the mouse returns to the non-stimulated side.
  • a camera is mounted directly above the avoidance box to record the activity of each experimental animal.
  • the animal-related behavioral indicators were analyzed using Any-maze software (Stoelting, USA).
  • Avoidance score difference between the stimulation side and the non-stimulus side after 20 minutes - the difference between the stimulation side and the non-stimulus side of the first 20 minutes.
  • Open field test Based on the experimental method of previous research (Matthews et al., 2016), the open field test box size is 45x45x45 cm, and the four walls and the bottom are white resin materials. Animals were free to explore for 9 minutes in the open field and laser stimulation (eNpHR 3.0: 589 nm, 1 Hz, 16 mW, 100 ms interval) for 3 minutes in the middle. The camera directly above the market recorded the movement of each experimental animal, and the behavioral indicators of the animals were analyzed using Any-maze software.
  • the experiment was carried out under normal illumination (about 100 lux).
  • the mouse forced swimming cylindrical transparent container has a diameter of 12 cm and a height of 25 cm.
  • the test water depth is 14 cm and the water temperature is 22.5-23 °C.
  • laser stimulation was initiated for 6 minutes (eNpHR 3.0: 589 nm, 1 Hz, 16 mW, 100 ms interval).
  • the camera recorded the swimming of the mice within 6 min from the side.
  • the double-blind method was used to measure the immobility time (the floating position of the animal or the time when the limbs were completely inactive) in the mouse for 6 minutes after swimming for 6 minutes.
  • Figure 1A is a schematic diagram of bilateral cannula implantation of the lateral nucleus of cLH rats, with white dotted lines indicating the position of the nucleus.
  • BG of Figure 1 gives the results of the experiment: localized bilateral application of different NMDAR inhibitors ketamine (25 ⁇ g per side, BD of Figure 1) and AP5 (40 nmol per side, EG of Figure 1) to LHb, in a short time ( 0.5 or 1 hour) can effectively reverse the depressive phenotype of cLH rats, including significantly reducing the immobility time in forced swimming (C and F in Figure 1), significantly increasing the preference for syrup in depressed animals (Figure 1 D and G).
  • NMDA receptor inhibitors such as ketamine
  • Example 3 Spontaneous discharge patterns of three neurons in the lateral habenular nucleus (non-discharge, single discharge and cluster discharge) in the release characteristics of depressed animals
  • a of Figure 2 shows the recording sites recorded by whole-cell patch clamps, which are distributed in different sub-regions of the lateral nucleus. Neurons in the lateral habenular nucleus were found to have three typical patterns of self-issued patterns, namely, silent (shown as B in Figure 2), single tonic (shown as C in Figure 2), and clustered discharge ( Burst) (shown in D of Figure 2).
  • E (scatter plot) and F (accumulation plot) of Figure 2 show the mean and distribution of resting membrane potentials (RMPs). The results showed that the resting membrane potential of a single discharge cell showed polarization and the cluster discharge cells showed super-sensitivity compared to cells that were not released.
  • Example 4 In vivo experiment to observe the discharge pattern of lateral nucleus neurons in depressive animals
  • a multi-channel electrophysiological recording method a better recording method for simulating animal physiological state, was used to insert more nucleus in the lateral nucleus of awake mice.
  • the channel electrophysiological recording electrode records the release of lateral nucleus neurons, including the cluster discharge activity of the mouse and the synchronization activity of the ⁇ band. The result is shown in Figure 3.
  • a of Fig. 3 shows the recording sites in the LHb of the body recording electrode in the control and CRS (chronic restraint stress).
  • B of Fig. 3 is a representative example (left) and average release waveform (right) of the LHb neuron firing in the body recording control, CRS and CRS + ketamine, and the cluster discharge was separated by analyzing the peak potential interval (ISI).
  • the C-D of Figure 3 shows that the CRS mouse LHb neuron cluster discharge ratio and the number of cluster discharges per minute are significantly higher than the control mice, and can be reversed by ketamine.
  • control group 143 ms
  • CRS group 33 ms
  • CRS + ketamine group 121 ms
  • the dotted line indicates the point at which the peak potential changes by 50%.
  • Figure 3G further analyzes the correlation between neuronal discharge and field potential (SFC, left) (), average SFC (middle) and ⁇ band (4-10 Hz) SFC percentage (right) for each discharge unit, further It was confirmed that the network of the lateral habenular nucleus neurons of CRS-depressed mice exhibited a ⁇ -band (4-10 Hz) rhythm that could be blocked by ketamine.
  • Example 5 In vitro experiments demonstrated that activation of NMDA receptors is a necessary and sufficient condition for LHb neurons to produce cluster discharges.
  • a of Figure 4 is a typical graph of excitatory postsynaptic currents generated when a neuron is clamped at -80 mV.
  • NMD-receptor-mediated excitatory postsynaptic currents by the addition of GABA receptor inhibitors (picrotoxin) and AMPA receptor blockers (NBQX) in artificial cerebral spinal fluid (ACSF) without Mg 2+ ), and confirm the current with the NMDA receptor blocker AP5.
  • C-H of Figure 4 shows the effect of ketamine (C-D), AP5 (E-F) and NBQX (G-H) on spontaneous tuft discharge in the lateral nucleus.
  • the left side is a typical picture and the right side is a statistical chart.
  • ketamine does not affect resting membrane potentials (RMPs) of neurons, but almost completely blocks spontaneous cluster discharges.
  • RMPs resting membrane potentials
  • the cluster discharge of LHb was converted to a single discharge 10 seconds after treatment with ketamine.
  • NMDA perfusion produces a cluster discharge of cells that are not released, and this induced cluster discharge can be inhibited by ketamine.
  • NMDA induces large excitatory postsynaptic potentials and cluster discharges.
  • Example 6 LHb cluster discharge requires the involvement of neuronal membrane super-type and T-type voltage-sensitive dry channel
  • Example 3 The phenomenon found in Example 3, that is, neurons of different release modes in LHb have different resting membrane potentials (RMP), and neurons with spontaneous cluster discharges exhibit superimposed resting membrane potential.
  • RMP resting membrane potentials
  • the inventors further confirmed the relationship between the resting membrane potential and the neuron firing pattern.
  • the recorded neurons were injected with an increasing ramp current to vary the resting membrane potential of the cells from -80 to -40 mV.
  • Super current injection causes the cells to produce cluster discharges.
  • the peak number of clusters in the cluster discharge is between -56 and -60 mV, which is similar to the resting membrane potential of spontaneous cluster-shaped discharge cells.
  • super- or depolarized current injection of cells from self-discharge can also cause cells to transform between individual and cluster-like dispensing.
  • a in Figure 5 is a typical plot of ramp current injection induced LHb neurons from cluster discharge to single discharge. As shown, the neurons are prone to clustered discharges in a relatively super-densified state and a single discharge in a relatively depolarized state.
  • B in Fig. 5 is a statistical graph showing the proportion of neurons capable of inducing a cluster discharge after injecting a super-current into LHb neurons in a large mouse.
  • C-E in Fig. 5 is the correlation between the clustered discharge frequency (C) recorded by the current clamp, the cluster discharge duration (D) and the number of discharges in the cluster (E) and the resting membrane potential of the neurons.
  • the inventors of the present application further studied how the NMDA receptor is under the super-condition. Activate to participate in the cluster discharge.
  • T-type voltage-sensitive calcium ion channels that are activated and depolarize neurons when the neurons are super-sized: T-type voltage-sensitive calcium ion channels.
  • the T-type voltage-sensitive calcium channel is a type of super-activated calcium channel. After channel activation, calcium ions are influxed and the neurons are depolarized.
  • the channel has three subtypes, Cav3.1, Cav3.2 and Cav3.3, and all three subtypes are expressed in LHb.
  • F in Fig. 5 is a typical example diagram of the conversion of spontaneous single discharge neurons to cluster discharges under superization.
  • G in Fig. 5 is a typical example of the conversion of spontaneous cluster discharge to single discharge under depolarization.
  • the effect of a hyperpolarization-activated cyclic nucleotide-gated channel on the spontaneous or induced cluster discharge of LHb was tested.
  • H, I in Figure 5 shows the effect of T-VSCC blocker Mibefradil (H) and HCN channel blocker ZD7288 (I) on spontaneous cluster discharge of LHb neurons.
  • the left side is a typical picture and the right side is a statistical chart.
  • the effect of HCN channel blockers on spontaneous or induced cluster discharge of LHb is much smaller than that of T-VSCC blocker.
  • the inventors first discovered that the NMDA receptor and T-VSCCs synergistically cause spontaneous cluster discharge in the lateral nucleus.
  • the cluster discharge physiological process is shown as J in Figure 5: activated T-VSCC causes magnesium ions blocking the NMDA receptor to be removed, and T-VSCC and NMDA receptor channels open to drive neuronal membrane potential superclusters. The direction of the discharge threshold changes. When the T-VSCC and NMDA receptor channels were rapidly inactivated, the resting membrane potential of the neurons recovered to -55 mV and another cluster-like discharge cycle was initiated.
  • Example 7 Topical administration of the nucleus of the lateral nucleus of the animal model, blocking the T-VSCC of the lateral habenular nucleus, eliminating the symptoms of depression
  • FIG. A of Figure 6 is an injection site map of the LHb injection CTB to determine the cannula. It was observed that Mibefradil had a rapid antidepressant effect after 1 h of action: it showed rapid antidepressant effects in both FST (Fig. 6 B) and SPT (Fig. 6 C) behavior. It was demonstrated that local blockade of LHb T-VSCC is rapid antidepressant.
  • the inventors first discovered that the increase in the cluster discharge pattern rather than the entire firing frequency contributes to the development of depression.
  • the inventors were able to detect rebounded cluster discharges on isolated brain slices by activating the inhibitory light-sensitive channel eNpHR3.0. At the same time, the inventors through light experiments in the animal to activate the eNpHR3.0 light-sensitive channel expressed by the lateral nucleus of the nucleus, and found that it can also quickly mediate the generation of aversion and depression phenotype.
  • Figure 7A shows a schematic diagram of the construction of the eNpHR virus expression vector (top panel), photoelectron and recording schematic (bottom panel).
  • Figure B, B, C is a typical diagram of the reverberative cluster discharge of yellow-activated brain slice neurons (B) and in vivo recorded neurons (C) in mouse LHb expressed by AAV2/9-eNpHR virus. .
  • the percentage of cells that successfully induced cluster discharges is shown in the right panel of Figure C.
  • D The dot plot and the post-stimulation time bar plot show the response of a representative LHb neuron to 100 ms yellow light stimulation in the bulk photoelectrode recording. After the end of the yellow light, the neurons have a rebounding discharge frequency that increases.
  • Example 9 In vitro experiments demonstrated that partial blocking of the NMDA receptor inhibitor at the concentration of the clustered discharge was combined with the T-VSCC receptor inhibitor to completely block the induction of cluster discharge.
  • the lateral nucleus neurons were injected with a super-charging current of -100 pA for 100 ms, which made the neurons hyperpolarized, and the super-state induced the cluster discharge of the lateral nucleus neurons.
  • the lateral nucleus slices were perfused with ketamine (100 ⁇ M, Figure 10A), AP5 (100 ⁇ M, Figure 10B) or Mibefradil (10 ⁇ M, Figure 10C)
  • the probability of neuron super-induced cluster discharge was partially reduced to 0.19, 0.12 and 0.05.
  • Example 10 The in vivo experiment of Example 10 demonstrates that an NMDA receptor inhibitor administered in combination with an effective dose lower than that administered alone, in combination with an effective dose of a T-VSCC receptor inhibitor administered alone, can effectively produce an antidepressant effect, and Reduce the side effects of both drugs
  • the forced swimming behavior paradigm can reduce the time for mice to give up.
  • the present invention for the first time and accidentally found that the cluster discharge of neurons of the lateral nucleus has an important role in the production of depression, and found that the key factors affecting the cluster discharge of the lateral nucleus, including the activation of the NMDA receptor is the LHb nerve.
  • the accumulation of clustered discharges rather than the entire frequency of distribution contributes to the development of depression.
  • the inventors have provided methods and medicaments for treating (suppressing) depression by inhibiting cluster discharge of the lateral nucleus, particularly methods and medicaments for rapidly treating (suppressing) depression.
  • the unit "degree” of temperature appearing in this document refers to degrees Celsius, or °C.

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Abstract

抑制外侧缰核中簇状放电的试剂在制备治疗抑郁症的药物中的用途及包含其的药物组合物，其中所述抑制外侧缰核中簇状放电的试剂为N-甲基-D-天冬氨酸受体抑制剂和/或T型钙离子通道抑制剂。

Description

治疗抑郁症的方法和药物组合物
本申请要求以下中国专利申请的优先权：2017年5月9日提交的、申请号为201710322647.X、发明名称为“治疗抑郁症的方法和药物组合物”；2017年5月9日提交的、申请号为201710322266.1、发明名称为“治疗抑郁症的联合用药”；和2017年5月9日提交的、申请号为201710322646.5、发明名称为“抑郁症的调节因子及其应用”，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及疾病治疗和药物领域。具体的，本发明涉及抑郁症的治疗和用于治疗抑郁症的药物组合物和其制备方法。
背景技术
抑郁症在人群中具有很高的发生比例。抑郁症患者如果不进行合适的治疗，在情绪和身体上都会受到重大影响。根据美国国家精神健康研究所(US National Institute of Mental Health,NIMH)的定义和描述，抑郁症包括以下症状：“持续性悲伤、焦虑或空虚”的情绪；绝望感、悲观感；罪恶感、无价值感、无助感；失去对享受过的爱好和活动的兴趣和快乐；精力减少、疲劳；难于集中精神、记忆困难、难于做决定；多动、易怒等。
外侧缰核(lateral habenula，LHb)是缰核的组成部分，位于上丘脑。外侧缰核是边缘前脑和中脑间传递信息的主要组织。近年来，发现外侧缰核与多巴胺能和5-羟色胺能的神经纤维联接和调控，使得外侧缰核参与了多种生理活动，影响机体功能，与药物成瘾、奖赏-厌恶、疼痛、睡眠等精神状态和病症相关。
已经发现外侧缰核与抑郁症的发生具有关联。研究表明，在抑郁大鼠中，外侧缰核至VTA投射神经元的微小兴奋性突触后电流mEPSC的频率相对于正常大鼠显著增高，这提示在缰核中，这种抑郁相关的过度兴奋状态有可能是由突触可塑性机制所介导的(Li,B.et al.Nature 470,535–539，2011)。在正常情况下，外侧缰核对VTA与DRN有低水平的 抑制作用。在抑郁症中，精神压力导致βCaMKII表达量显著上升，导致外侧缰核神经元的GluR1上膜，神经元兴奋性和突触传递效率均显著增强。外侧缰核的高度兴奋增强对VTA与DRN抑制，导致快感缺失和行为上的绝望表现(Li et al.,Science 341,1016–1020，2013)。.
本领域已经具有一些常用的抗抑郁药物，但这些药物通常在比较长的一段时间后才能见效。而且导致抑郁症的病理机制还未完全被认识。本领域还需要新的，或是起效更快速、使用剂量更安全的治疗抑郁症的方法和药物。
发明内容
本发明首次和意外发现外侧缰核的神经元的簇状放电(burst)在抑郁症的产生中具有重要作用，并发现了影响外侧缰核的簇状放电的关键因素，由此提供了通过抑制外侧缰核的簇状放电来治疗(抑制)抑郁症的方法和药物，特别是快速治疗(抑制)抑郁症的方法和药物。
具体的，本发明提供了通过抑制外侧缰核的簇状放电来治疗对象中的抑郁症的方法。本发明提供了抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，特别是施用在外侧缰核的用于治疗抑郁症的药物。本发明还提供了治疗抑郁症的药物组合物，特别是施用在外侧缰核的用于治疗抑郁症的药物组合物，其含有抑制外侧缰核中簇状放电的试剂。
需要本文所述的方法和药物(药物组合物)的对象包括被诊断患有抑郁症的对象。将要治疗的对象可以是哺乳动物，包括人或者非人灵长类如猴。哺乳动物可以是其它动物，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。
术语“簇状发放”，或“簇状放电”，是指神经元在放电过程中同时产生两个或两个以上锋电位的放电模式。
抑制簇状放电是指抑制簇状放电的发放程度，包括减少簇状放电的频率或簇状放电过程中簇内峰电位的个数，降低簇状放电的强度，甚至是消除簇状放电的发生。
术语“单个发放”，或“单个放电”，是神经元在放电过程中每次发放一个锋电位的放电模式。
抑制簇状放电的试剂包括能够在对簇状放电起抑制作用的化合物、复合物或混合物，以及在抑制簇状放电的方法(含外科手术方法)中使用的制剂等。所述试剂包括小分子化合物或复合物，或是蛋白、核酸等大分子活性成分，例如与簇状放电生理途径上的蛋白结合的拮抗剂如抗体，或是影响这些蛋白的表达水平的核酸等。
本发明中，“治疗”包括：改良、减轻、减少或预防与抑郁症相关的症状的进行中的过程或结果；改善与抑郁症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(减轻)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(即非恶化)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；和减轻(无论部分或总体)、无论可检测出的或不可检测出的；或增强或改善该情况、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。在一个实施方案中，治疗指给药药物或对患者执行医疗程序。本发明中，治疗可以是预防(防止)，治愈虚弱或病，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。
在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法，本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，以及本发明的治疗抑郁症的药物组合物中，所述抑制簇状放电的试剂为N-甲基-D-天冬氨酸受体抑制剂。
N-甲基-D-天冬氨酸是一种兴奋性氨基酸(excitatory amino acids，EAA)，是中枢神经系统的兴奋性神经递质。N-甲基-D-天冬氨酸受体(NMDA受体或NMDAR)是一种离子型受体，参与兴奋性突触传递。对 NMDA受体的调节可调节谷氨酸能神经递质介导的神经作用。
可用于本发明的N-甲基-D-天冬氨酸受体抑制剂包括但不限于：
1)NMDA受体的竞争性抑制剂(与谷氨酸结合位点竞争的抑制剂)：AP5，AP7，CPPene，塞福太(Selfotel)；
2)NMDA受体的非竞争性抑制剂(阻断变构结合位点的抑制剂)：阿替加奈(Aptiganel)，氯胺酮，美金刚(memantine)、Huperzine A，伊博格碱(Ibogaine)，HU-211，加巴喷丁(Gabapentin)，PD-137889等；
3)NMDA受体反竞争性的通道阻断剂(通道阻断剂)：金刚胺(Amantadine)，阿托西汀(Atomoxetine)，AZD6765，右美沙芬(Dextromethorphan)，盐酸镁金刚胺，MK801(Dizocilpine)；
4)甘氨酸结合位点抑制剂：TK-40，犬尿酸(Kynurenic acid)等。
在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法，本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，以及本发明的治疗抑郁症的药物组合物中，所述抑制簇状放电的试剂为T型钙离子通道抑制剂。
T型钙离子通道或T型钙通道(transient calcium channel)又名低电压激活钙通道(Low voltage activate calcium channel,T-type calcium channel)。T型钙通道在中枢与外周神经系统的兴奋性调节中均有着重要的作用。脊椎动物中，T型钙通道家族包括3个不同的α1亚基基因：CACNA1G、CACNA1H、CACAN1I，分别编码α1G、α1H、和α1I，从而构成了Cav3.1，Cav3.2和Cav3.3，3种T型钙通道亚型。T型钙通道蛋白是四聚体组成的结构，每个单体即α1亚单位含四个同源区域。通道蛋白孔道由上述四个同源区域构成。孔道螺旋与细胞外S6片段的末端连接够成了钙离子选择性通过滤器。每个同源结构域的S4片段每隔三个氨基酸都有带正电的氨基酸残基，形成了通道的电压感受器，以此结构为基础在膜电位发生变化时可以控制通道的开放和关闭。
可用于本发明的T型钙离子通道抑制剂包括但不限于：
琥珀酰亚胺类(Succinimides)，例如乙琥胺(ethosuximide)、甲琥胺(methsuximide)；乙内酰脲类(hydantoins)；唑尼沙胺(zonisamide)；丙戊酸钠(valproate sodium)；phenytoin；Mibefradil；苯妥英(Phenytoin)； sipatrigine；哌嗪类似物如Flunarizine、Z941；哌啶类似物如Z944和Fluoropiperidine；TTA-P1；TTA-P2；喹唑啉酮(quinazolinone)；匹莫齐特(Pimozide)；三甲双酮(Trimethadione)和二甲双酮；TTA-Q4；ML218等。
在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法，本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，以及本发明的治疗抑郁症的药物组合物中，所述抑制簇状放电的试剂为N-甲基-D-天冬氨酸受体抑制剂和T型钙离子通道抑制剂的组合。
本发明提供了治疗对象中的抑郁症的方法，其包括向所述对象施用组分(a)N-甲基-D-天冬氨酸受体(NMDAR)抑制剂；和组分(b)T型钙离子通道(T-type calcium channel或T-VSCC)抑制剂的组合。
本发明还提供了治疗抑郁症的药物组合物，其包含：组分(a)N-甲基-D-天冬氨酸受体(NMDAR)抑制剂；和组分(b)T型钙离子通道(T-type calcium channel或T-VSCC)抑制剂。
在本发明的其中一个方面，本发明提供的治疗抑郁症的方法和药物组合物中，施用于所述对象的组分(a)和组分(b)的其中至少一种的治疗有效量低于不存在另一组分时施用的所述组分的治疗有效量。在本发明的其中又一个方面，施用于所述对象的组分(a)和组分(b)的治疗有效量都低于不存在另一组分时施用的所述组分的治疗有效量。
在本发明的其中又一个方面，本发明提供的治疗抑郁症的方法和药物组合物中，施用于所述对象的组分(a)和组分(b)的其中至少一种，特别是两种的治疗有效量比不存在另一组分时施用的所述组分的治疗有效量低至少5％，低至少10％，低至少25％，低至少50％，低至少60％，低至少70％，低至少80％，或低至少90％。在本发明的其中一个方面，本发明提供的治疗抑郁症的方法和药物组合物中，施用于所述对象的组分(a)和组分(b)的其中至少一种的治疗有效量比不存在另一组分时施用的所述组分的治疗有效量低5％至90％，低10％至90％，低25％至90％，或低50％至90％。
例如，本发明提供的治疗抑郁症的方法和药物组合物中，所述NMDAR抑制剂的治疗有效量比不存在所述T-VSCC抑制剂时施用的所述抗抑郁症 药物的治疗有效量低至少5％，低至少10％，低至少25％，低至少50％，低至少60％，低至少70％，低至少80％，或低至少90％。即在本发明的药物组合物中，所述NMDAR抑制剂的剂量比单独使用该NMDAR抑制剂的正常剂量(建议剂量)低至少5％，低至少10％，低至少25％，低至少50％，低至少60％，低至少70％，低至少80％，或低至少90％。在本发明的其中一个方面，本发明提供的治疗抑郁症的方法和药物组合物中，所述NMDAR抑制剂的治疗有效量比不存在所述T-VSCC抑制剂时施用的所述抗抑郁症药物的治疗有效量低5％至90％，低10％至90％，低25％至90％，或低50％至90％。即在本发明的药物组合物中，所述NMDAR抑制剂剂的剂量比单独使用该NMDAR抑制剂的正常剂量(建议剂量)低5％至90％，低10％至90％，低25％至90％，或低50％至90％。
本发明提供的治疗抑郁症的方法和药物组合物中，所述NMDAR抑制剂和所述T-VSCC抑制剂被配制在同一药物组合物中，或所述NMDAR抑制剂被配制在第一药物组合物中并且所述T-VSCC抑制剂被配制在第二药物组合物中。
本发明提供的治疗抑郁症的方法和药物组合物中，将所述NMDAR抑制剂和所述T-VSCC抑制剂同时施用。在本发明的另一个方面，将所述NMDAR抑制剂和所述T-VSCC抑制剂分开施用。
在本发明中，抑郁症可以特别指“外侧缰核介导的抑郁症”，尤其是指“外侧缰核簇状放电介导的抑郁症”。本申请的发明人发现并证明了外侧缰核的神经元的异常发放，特别是簇状放电的异常发放在抑郁症的产生中具有重要作用。本申请的发明人还发现了影响外侧缰核的簇状放电的关键因素，通过对这些关键因素的调节，能够抑制或消除抑郁症。
在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法，本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，以及本发明的治疗抑郁症的药物组合物中，该方法或药物适合用于在其它抗抑郁方法和药物不起效的抑郁症患者中使用。
本领域已经采用的抗抑郁药，根据其抑制抑郁症的机制分类，可包括：
●褪黑激素激动剂；
●选择性5-羟色胺重摄取抑制剂(SSRIs)；
●5-HT和去甲肾上腺素再摄取双重抑制剂(SNRIs)；
●单胺氧化酶抑制剂(MAOIs)；
●三环类抑郁药(TCAs)；
●三重单胺摄取阻断剂；
●代谢型谷氨酸受体(mGluRs)；
●GABA拮抗剂；
●NK1拮抗剂；
●NK2拮抗剂；
●CRF1拮抗剂；
●精氨加压素V1b拮抗剂；
●MCH受体拮抗剂；
●NT-3、NT-4拮抗剂；
●CREB拮抗剂等。
以上种类的抗抑郁药和其具体药物在WO2007/137247中列出。在此通过全文引入。
本申请的发明人首次发现并证明了外侧缰核的神经元的异常发放，特别是簇状放电的异常发放在抑郁症的产生中具有重要作用，因此提供了通过抑制外侧缰核的神经元的异常发放，特别是簇状放电的异常发放来治疗(抑制)抑郁症的方法和药物。这是本领域已知的治疗抑郁症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或其分子水平上的靶目标。因此，本发明提供的方法和药物或药物组合物特别适合用于在上述抗抑郁方法和药物不起效的抑郁症患者中使用。
本领域已知某些NMDA受体拮抗剂可用于治疗抑郁症。但在这些报道中，其发现的或推测的抗抑郁机制与本发明发现的机制，即通过抑制外侧缰核神经元的异常发放，特别是簇状放电的异常发放来抑制抑郁症，完全不同。在不破坏本发明的创新性的情况下，在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法，本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，以及本发明的治疗抑郁症的药物组合物中，所述抑制簇状放电的试剂不包括这些NMDA受体拮抗剂，例如AP5，CPPene，MK801、美金刚(memantine)、氯胺酮、 非尔氨脂(felbamate)、甘氨酸、D-丝氨酸、D-环丝氨酸、L-谷氨酸艾芬地尔等。
本领域存在个别的报道发现某些化合物可抑制T型钙离子通道，也具有抗抑郁症的现象。例如，氟西汀、曲唑酮、乙琥胺、三甲双酮、丙戊酸钠、匹莫齐特和唑尼沙胺。但在这些报道中，其提出或假设的抗抑郁机制与本发明发现的机制，即通过抑制外侧缰核神经元中簇状放电的异常发放来抑制抑郁症，完全不同。在不破坏本发明的创新性的情况下，在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法，本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，以及本发明的治疗抑郁症的药物组合物中，所述抑制簇状放电的试剂不包括氟西汀、曲唑酮、乙琥胺、三甲双酮、丙戊酸钠、匹莫齐特和唑尼沙胺。
在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法以及本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途中，所述的抑制簇状放电的试剂不抑制单个放电(tonic pulse)。
在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法，本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，以及本发明的治疗抑郁症的药物组合物中，所述方法、药物或药物组合物为在外侧缰核中局部起效，即施用在外侧缰核的方法和药物或药物组合物。对于用于神经组织的方法和药物，特别是脑部神经组织，例如外侧缰核来说，将药物的作用限定在目标组织是有益的。用于在外侧缰核中给药对治疗方法和制备药物都是限制性的技术特征。用于LHb的方法或药物需要考虑该方法或药物是否能够在LHb发挥有效性，包括药物是否能到达LHb，以及在LHb中是否能达到起效的浓度等。在本发明中，所述药物或药物组合物为在外侧缰核局部给药的剂型。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外侧缰核局部给药的剂型。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系统的形式。例如可以通过将具有抑制簇状放电功能的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外侧缰核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外侧缰核的细胞的复合分子。
在本发明的其中一个方面，在本发明提供的上述在通过在外侧缰核中局部抑制簇状放电来治疗抑郁症的方法(包括单独给予NMDAR抑制剂或T-VSCC抑制剂，或联合给予T-VSCC抑制剂和NMDAR抑制剂)和药物(包括药物组合物或联合药物组合物)中，所述T型钙离子通道抑制剂也可为氟西汀、曲唑酮、乙琥胺、三甲双酮、丙戊酸钠、匹莫齐特和唑尼沙胺等；以及所述NMDA受体拮抗剂也可为AP5，CPPene，MK801、美金刚、氯胺酮、非尔氨脂、甘氨酸、D-丝氨酸、D-环丝氨酸、L-谷氨酸艾芬地尔等。
在本发明的其中一个方面，本发明的通过抑制外侧缰核的簇状放电来治疗抑郁症的方法，本发明的抑制外侧缰核中簇状放电的试剂用于治疗抑郁症的药物的用途，以及本发明的治疗抑郁症的药物组合物，特别适合用于快速治疗(抑制)抑郁症。本发明提供的药物适合作为快速起效的治疗(抑制)抑郁症。本领域大多数抗抑郁药一般需要一周到几周时间才能发挥抗抑郁的作用，例如常用的5-HT再摄取抑制剂(SSRI)通常在2-3周才显效，5-HT和去甲肾上腺素再摄取双重抑制剂通常在1周才显效。本发明提供的抗抑郁方法和药物或药物组合物的起效时间低于一周，优选低于三天，更优选低于一天，例如为低于12小时。本发明提供的药物还适合作为快速起效以及具有中效或长效的治疗(抑制)抑郁症，其单剂量的抗抑郁效果能够持续一天以上，优选持续三天以上，更优选持续一周以上。
本发明提供的治疗抑郁症的药物组合物，其包含治疗有效量的抑制外侧缰核中簇状放电的试剂。
本发明提供的药物组合物中的活性成分为抑制外侧缰核中簇状放电的试剂。尽管适用于治疗的本发明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，但优选将活性成分，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。
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。
本发明的药物组合物可以是那些适合于口服、直肠、支气管、鼻、肺、局部(包括颊和舌下)、透皮、阴道或肠胃外(包括皮肤、皮下、肌内、腹膜内、静脉内、动脉内、脑内、眼内注射或输注)给药的药物组合物，或那些适合于通过吸入或吹入给药(包括粉末和液体气雾剂给药)或适合于通过缓释系统给药的形式的药物组合物。适合的缓释系统的实例包括含有本发明化合物的固体疏水性聚合物的半渗透基质，该基质可以是成形的制品形式，例如薄膜或微囊。
因此可将本发明的药物组合物中的活性成分与常规的佐剂、载体或稀释剂一起制成药物组合物及其单位剂量的形式。这样的形式包括固体、并尤其是片剂、填充胶囊、散剂和微丸的形式，以及液体、尤其是水溶液或非水溶液、混悬剂、乳剂、酏剂和填充上述形式的胶囊，所有这些形式均用于口服，用于直肠给药的栓剂、以及用于胃肠外的无菌可注射溶液。这样的药物组合物及其单位剂型可包括常规比例的常规成分，含有或不含另外的活性化合物或成分，并且这样的单位剂型可含有与所需每日应用剂量范围相当的任何适合的有效量的活性成分。
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。
适合于口服使用的含水混悬剂可通过将细粉碎的活性成分分散在含黏性物质、如天然或合成的树胶、树脂、甲基纤维素、羧甲基纤维素钠、或其他众所周知的悬浮剂的水中而制备。
还包括欲在临用之前转化为用于口服给药的液体形式制剂的固体形式制剂。这样的液体形式包括溶液、混悬剂和乳剂。除活性成分之外，这样 的制剂还可包含着色剂、矫味剂、稳定剂、缓冲剂、人造和天然的甜味剂、分散剂、增稠剂、增溶剂等。
为了局部施用到表皮，可将本发明化合物配制成软膏剂、霜剂，或洗剂，或透皮贴剂。例如，软膏剂和霜剂可用水性或油性基质外加适合的增稠剂和/或胶凝剂配制而成。洗剂可用水性或油性基质配制而成，且通常还含一种或多种乳化剂、稳定剂、分散剂、悬浮剂、增稠剂或着色剂。
呼吸道给药也可以借助气雾剂实现，其中活性成分与适合的抛射剂一起在加压包装中提供，适合的抛射剂如含氯氟烃(CFC)，例如二氯二氟甲烷、三氯氟甲烷或二氯四氟乙烷，二氧化碳或其他适合的气体。气雾剂还可适当地含有表面活性剂，如卵磷脂。药物的剂量可通过配备计量阀控制。
或者，本发明药物组合物中的活性成分可以干粉形式提供，例如化合物在适合的粉末基质(如乳糖、淀粉、淀粉衍生物(如羟丙基甲基纤维素)和聚乙烯吡咯烷酮(PVP))中的粉末混合物。适宜地，粉末载体将在鼻腔内形成凝胶。粉末组合物可以单位剂型呈现，例如以胶囊或药筒(如明胶的胶囊或药筒)形式，或以粉末可借助吸入器从中给药的泡罩包装形式。
需要时，可以应用适合提供活性成分缓释的组合物。
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。
用于口服给药的片剂或胶囊和用于静脉内给药和连续输注的液体是优选的组合物。
在一个实施方案中，当意欲使用本发明的药物组合物治疗具有滥用倾向和因烟碱成瘾导致的脱瘾症状时，关注例如树胶、贴剂、喷雾剂、吸入剂、气雾剂等这样的制剂。
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。
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。
实际的剂量取决于所治疗疾病的性质及严重程度、确切的给药方式和给药剂型，且在医师的判断范围之内，可以根据本发明具体情况通过递增剂量而改变，以产生期望的治疗效果。然而，目前认为含有约0.1-约1000mg、优选约1-约750mg的活性成分/单个剂量的药物组合物对于治疗性处理是适合的。
本发明还提供了一种抑郁症动物模型，优选为大鼠或小鼠。本发明所述抑郁症动物模型具有抑郁症特征，其存在外侧缰核神经元簇状放电的异常发放。
本发明还提供了采用上述动物模型筛选用于治疗抑郁症的潜在物质的方法，包括步骤：
(1)给抑郁症动物模型施用待筛选的测试物；和
(2)观察所述抑郁症动物模型中的抑郁症的相关症状和/或指标，并与对照组进行比较。
其中，如果所述抑郁症动物模型中抑郁症的相关症状有显著改善，则表示该测试物是可用于治疗抑郁症潜在物质。
在本发明的其中又一个方面，所述筛选用于治疗抑郁症的潜在物质的方法还包括以下一个或多个步骤：
对上一步骤筛选出的潜在物质，进一步测试其对神经元簇状发放的影响；
对上一步骤筛选出的潜在物质，施用于动物模型，观察其对抑郁症症状的影响；
在测试其对神经元簇状发放的影响时，如果与阴性对照组(或空白对照组)相比，加入或施用所述测试物的测试组中神经元簇状发放比例显著降低，则表示该测试物是治疗抑郁症的潜在物质。
附图说明
图1外侧缰核局部阻断NMDA受体足以产生快速抗抑郁效果。(A)cLH大鼠外侧缰核双侧套管植入示意图。白色虚线指示缰核位置。(B-G)局部双侧施加氯胺酮(25μg每侧，B-D)和AP5(40nmol每侧，E-G)到LHb，在短时间内(0.5或1小时)能有效逆转cLH大鼠的抑郁表型：显著降低强迫游泳中的不动时间(C和F)和显著增高抑郁动物对糖水的偏好性(D和G)。(H-I)LHb双侧施加氯胺酮的抗抑郁效果可以持续到给药后第14天。所有数据均表示为平均值±SEM。*P<0.05，**P<0.01，***P<0.001，****P<0.0001与对照组相比。N.S.表示差异不显著。其他的图标相同。
图2大鼠和小鼠抑郁动物模型中外侧缰核神经元簇状发放增强并可被氯胺酮所反转。(A)指示了全细胞膜片钳记录的记录位点，记录位点分布于外侧缰核的不同亚区。(B-D)外侧缰核中神经元的三种自发放模式典型图，分别是不放电(silent)，单个放电(tonic)和簇状放电(burst)。中间是同一个神经元对TTX的反应，TTX可阻断单个放电和簇状放电的峰电位。右边放电轨迹图是左边阴影区的放大图。(E-F)散点图(E)和累积曲线(F)显示静息膜电位(RMPs)的均值和分布。(G-H)簇内发放频率而非簇间发放频率与静息膜电位超级化程度正相关。(I-N)在先天抑郁(cLH)大鼠和慢性束缚应激诱导的小鼠抑郁模型中，簇状放电的神经元比例显著增高。(I，L)饼状统计图显示在大鼠和小鼠抑郁模型中，簇状放电神经元数目增加。(J，M)柱图显示所有发放细胞中单个放电和簇状放电的细胞比例。(K，N)柱形统计图显示不动抑郁动物缰核中神经元峰电位间隔的分布。
图3在体电生理记录显示氯胺酮抑制慢性束缚应激小鼠缰核神经元簇状放电活性和θ波段的同步化活动。(A)在体记录电极在对照和CRS小鼠LHb中的记录位点。(B)在体记录对照，CRS和CRS+氯胺酮的小鼠LHb神经元放电的代表性示例(左)和平均发放波形(右)，通过分析峰电位间隔(ISI)来分离簇状放电。(C-D)CRS小鼠LHb神经元簇状放电比例和每分钟簇状放电的个数都显著高于对照小鼠，且可以被氯胺酮所反转。(E)对照小鼠和CRS小鼠在氯胺酮注射前后峰电位间隔的累积分布曲线(对照组：143ms，CRS组：33ms，CRS+氯胺酮组：121ms)。虚线 指示峰电位50％变化的点。(F)对照组和CRS组小鼠在给氯胺酮前后神经元发放相关场电位，CRS组相邻波谷之间的时间间隔为140ms左右(周期约为7hz)(G)对照小鼠和CRS小鼠在氯胺酮注射前后单个神经元放电与场电位的相关性(SFC)。每个放电单位的SFC(左)，平均的SFC(中)，θ波段(4-10Hz)中SFC百分比。所有数据均表示为平均值±SEM。*P<0.05，**P<0.01，***P<0.001，****P<0.0001与对照组相比。N.S.表示差异不显著。其他的图标相同。
图4LHb的簇状放电需要激活NMDA受体。(A)将神经元钳制在-80mV时产生的兴奋性突触后电流典型图。通过在无Mg2+的人工脑脊液(ACSF)中加入GABA受体抑制剂(picrotoxin)和AMPA受体阻断剂(NBQX)来分离NMDA受体介导的兴奋性突触后电流(NMDAR-EPSCS)，并用NMDA受体阻断剂AP5来确认电流。(B)LHb神经元钳制在不同电压下记录到的NMDAR-EPSCs，该电流可被AP5完全阻断。(C-H)LHb中氯胺酮(C-D)，AP5(E-F)和NBQX(G-H)对自发簇状放电的影响。左侧为典型图，右侧为统计图。(I-J)NMDA灌流可使不发放的细胞产生簇状放电，这种诱发的簇状放电可被氯胺酮所抑制。NMDA可诱发大的兴奋性突触后电位和簇状放电。所有数据均表示为平均值±SEM。*P<0.05，**P<0.01，***P<0.001，****P<0.0001与对照组相比。N.S.表示差异不显著。其他的图标相同。
图5LHb簇状放电需要神经元膜超级化和T型电压敏感型干通道的参与。(A)斜坡电流注射诱导LHb神经元从簇状放电向单个放电转化的典型图，神经元在相对超级化的状态下容易产生簇状放电，而在相对去极化的状态下产生单个放电。(B)统计图显示大小鼠中LHb神经元注入超级化电流后能诱导产生簇状放电的神经元比例。(C-E)电流钳加记录到的簇状放电频率(C)，簇状放电持续时间(D)和簇内放电个数(E)与神经元静息膜电位的相关性。(F)超级化下自发单个放电神经元向簇状放电转化的典型示例图。(G)去极化下自发簇状放电向单个放电转化的典型示例图。(H，I)T-VSCC阻断剂Mibefradil(H)和HCN通道阻断剂ZD7288(I)对LHb神经元自发簇状放电的影响。左侧为典型图，右侧为统计图。(J)一个发放示例图总结了LHb神经元簇状放电中所需要的各种离子和 通道。激活的T-VSCC使得阻断NMDA受体的镁离子被移开，T-VSCC和NMDA受体通道的打开驱动神经元膜电位超簇状放电阈值方向变化。当快速失活T-VSCC和NMDA受体通道时，神经元静息膜电位恢复到-55mV一下，起始另一个簇状放电周期。所有数据均表示为平均值±SEM。*P<0.05，**P<0.01，***P<0.001，****P<0.0001与对照组相比。N.S.表示差异不显著。其他的图标相同。
图6T-VSCC拮抗剂表现出快速抗抑郁的作用。(A-C)局部注射mibefradil到双侧LHb，在FST(B)和SPT(C)行为中均表现出快速的抗抑郁效果。(A)为LHb注射CTB确定套管的注射位点图。所有数据均表示为平均值±SEM。*P<0.05，**P<0.01，***P<0.001，****P<0.0001与对照组相比。N.S.表示差异不显著。其他的图标相同。
图7eNpHR光激活诱导的反弹式簇状放电使动物表现出可被氯胺酮反转的厌恶和抑郁表型。(A)eNpHR病毒表达载体构建示意图(上图)，光电及记录示意图(下图)。(B，C)在AAV2/9-eNpHR病毒表达的小鼠LHb中，黄光激活的脑片神经元(B)和在体记录到的神经元(C)反弹簇状放电的典型图。成功诱导出簇状放电的细胞百分比显示于右侧统计图。(D)点阵图和刺激后时间柱图显示在体光电极记录中一个代表性的LHb神经元对100ms黄光刺激的反应。(E)eNpHR光激活引起的反弹式簇状放电的簇内发放频率和簇内发放个数的分布与CRS小鼠缰核中记录到的相当。中央的十字交叉代表平均值。(F)eNpHR光激活引起的反弹式簇状放电诱发的实时位置厌恶(RTPA)。左侧显示RTPA的代表性热图，右侧显示定量分析的厌恶回避参数。(G)eNpHR光激活引起的反弹式簇状放电诱发的FST抑郁表型。(H)eNpHR光激活引起的反弹式簇状放电诱发的SPT抑郁表型。所有数据均表示为平均值±SEM。*P<0.05，**P<0.01，***P<0.001，****P<0.0001与对照组相比。N.S.表示差异不显著。其他的图标相同。
图8与簇状放电相同频率的单个放电不能引起抑郁表型。(A)离体电生理记录显示5Hz的光激活oChIEF光通道产生5Hz的单个放电。(B)与表达对照非光通道的eGFP小鼠相比，光激活并不改变动物的运动能力。(C)5Hz的光激活产生的单个放电不能诱导出抑郁表型。
图9联合施用低剂量NMDA受体抑制剂和T型钙离子通道抑制剂能够产生快速抗抑郁效果，并且降低两种药物的副作用。
图10NMDAR抑制剂与T-VSCC抑制剂联合用药对诱发簇状放电的影响。给予外侧缰核神经元注射-100pA的超级化电流100ms，使神经元处于超极化状态，超级化状态诱发外侧缰核神经元簇状放电。外侧缰核脑片单独灌流氯胺酮(100μM，A)，AP5(100μM，B)或Mibefradil(10μM,C)时，对神经元超级化诱发簇状放电的概率的影响，左侧图形为簇状放电的波形示例，右侧为统计分析。当氯胺酮(100μM)与Mibefradil(10μM)联合给药(D)或AP5(100μM)与Mibefradil(10μM)联合给药(E)，对神经元超级化诱发簇状放电的概率的影响，左侧图形为簇状放电的波形示例，右侧为统计分析。所有数据均表示为平均值±SEM。****P<0.0001与对照组相比。其他的图标相同。
具体实施方式
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。
实施例1 材料和方法
动物材料
雄性cLH大鼠(4-12周龄)，Sprague Dawley大鼠(4-12周龄)。cLH大鼠是一个选择性培育的具有先天习得性无助抑郁表型的抑郁症动物模型(D.Schulz,M.M.Mirrione,F.A.Henn,Neurobiol Learn Mem 93,291，Feb,2010)。本实验的cLH大鼠从美国冷泉港Malinow实验室引进。cLH大鼠饲养和繁殖如前述D.Schulz,et al，Feb,2010中描述。大鼠4只/笼，12小时的明暗周期(7am-7pm有光)。用于套管实验的cLH大鼠1只/笼饲养。成年(8-12周龄)C57BL/6小鼠被用于行为测试：4只/笼，12小时明暗周期(5am-5pm有光)。大鼠和小鼠都能够自由摄取稳定的水和食物，所有的动物实验经过浙江大学动物保护和使用委员会的批准。
病毒构建
AAV9-CaMKII-eNpHR3.0-eYFP，质粒购自于Addgene，Cat#26971， 病毒由上海泰廷生物科技有限公司包被；AAV9-Ubi-eGFP由UMass的Gao Guangping实验室馈赠；AAV9-hSyn-oChIEF-tdTomato，质粒购自于Addgene，Cat#50977，病毒由上海泰廷生物科技有限公司包被。
立体定位注射和组织学
小鼠注射病毒:小鼠腹腔注射氯胺酮(100mg/kg体重)和赛拉嗪(8mg/kg)混合液麻醉后，固定于立体定位仪上(Stoelting instruments)。每只小鼠每侧LHb注入0.1-0.2ul纯化浓缩的AAV病毒(～10 13感染单位/ml)，LHb立体定位坐标(前后距离Bregma：-1.7mm(AP)，左右旁开±0.46mm(ML)，皮层表面往下-2.56mm(DV))。使用自行拉制的玻璃微电极缓慢注入(～100-150nl/min)，注射结束留针5min，然后再5min内缓慢移出注射电极。
术后至少14天，开展行为实验或者电生理实验。行为实验结束后检查注射位置，只使用正确注射的那些动物数据。
注射了AAV病毒的脑切片在荧光显微镜下检查，或其它标记了GFP的病毒在显微检查之前用抗体检查GFP蛋白。每个大脑的缰核区切成6组连续的切片(小鼠30um的切片，每组6片；大鼠40um切片，每组8-9片)。所有的切片在安装到固定片之前用Hoechst复染色。
大鼠双侧LHb埋置套管：大鼠腹腔注射4％戊巴比妥那(60mg/kg体重)麻醉后，固定于大鼠立体定位仪上。置管用LHb立体定位坐标(前后距离Bregma：-3.7mm(AP)，左右旁开±0.7mm(ML)，皮层表面往下-4.1mm(DV))。在LHb对应的颅骨上方，颅钻钻孔，之后在颅骨上固定三颗螺丝。按置管LHb坐标使双侧套管(购自于美国plastics one公司)进入缰核的上方，并用牙托水泥固定套管，待牙托水泥完全凝固后，在套管中插入与套管等长的平端堵芯，并拧上螺帽以防止堵芯脱落。术后7天，待大鼠从手术创伤中恢复后，可用于检测药物的行为学效果。大鼠使用气体(异氟烷)麻醉机麻醉后，用套管配套的注射针，自套管中缓慢注入1ul待测试药物(约100nl/min)，注射结束留针10min，然后移出注射内芯。根据药物的有效时程进行行为学测试。行为实验结束后，通过套管注射CTB-488or 555检查套管位置，LHb注射位点准确的动物行为学数据才用于统计分析。
离体电生理记录
出生后40-50天的大鼠或出生8周的小鼠经异氟烷麻醉后，用20ml冰 冷充氧的切片液进行灌流。快速断头取出大脑，放进充氧的切片液中。随后利用Leica振动切片机在充氧的冰冷的切片液中，进行350um的管状切面切片。缰核脑片在充氧的34℃的ACSF(118mM NaCl,2.5mM KCl,26mM NaHCO 3,1mM NaH 2PO 4,10mM glucose,1.3mM MgCl 2and 2.5mM CaCl 2,gassed with 95％O 2and 5％CO 2)中，恢复至少1小时后转移到室温进行记录。注射氯胺酮组的大鼠和小鼠均在动物取脑前1h进行。
外侧缰核脑片的膜片钳记录采用Axon Multiclamp 700B放大器，在32±1℃环境下，在装配红外微分干涉相差光学镜头的Olympus显微镜下进行记录。所有细胞均在全细胞模式下记录。神经元记录电极阻抗为4-6MΩ，电极内液成分为(mM)：105K-Gluconate,30KCl,4Mg-ATP,0.3Na-GTP,0.3EGTA,10HEPES and 10Na-phosphocreatine,pH为7.35。记录外液所用人工脑脊液(ACSF)成分为(mM)：125NaCl,2.5KCl,25NaHCO 3,1.25NaH 2PO 4,1MgCl 2and 25葡萄糖。数据经过2kHz过滤后使用Digidata1322A在10kHz下采样记录。数据使用pClamp 10软件进行分析。
大小鼠LHb神经元中动作电位自发放频率，是在I＝0电流钳记录模式下进行，持续60秒，平均发放频率由这60s发放来统计。对LHb中产生的不同的放电模式的定义为：不发放细胞，指在整个记录过程中没有动作电位发放的细胞；单个放电的细胞，指放电频率在0.1-10Hz，极少有10-20Hz；簇状放电细胞，是指能产生成簇的发放，且簇内发放频率极高，但表现出逐渐递减趋势，簇间发刚频率高达200Hz。
诱发的NMDA受体介导的兴奋性突触后电流是在零镁的ACSF下，将细胞牵制在-50mV到-80mV下所记录到的。诱发的T型电压敏感型钙通道电流，是将细胞钳制在-50mV下，然后再将细胞钳制到-100mV，持续1秒。刺激按照常规频率0.1Hz给出。钙电流通过线性渗漏相减得到。
在体电生理记录
成年雄性小鼠腹腔注射氯胺酮(100mg/kg体重)和赛拉嗪(8mg/kg)混合液麻醉后，固定于立体定位仪上(Stoelting instruments)。将由8个tetrode(由四根电极丝组成的)(电阻为250-500KΩ,California fine wire)组成的可移动电极阵列植入到LHb(AP：-1.72mm；ML：±0.46mm；DV：-2.44mm)。不锈钢丝缠绕到固定在颅骨上的两个螺钉上用于接地。电极用 牙托水泥固定在颅骨表面。动物恢复5-7天后，开始适应记录所用的转接头，一天10分钟，共适应2-3天。记录采用64通道的OmniPlex-D神经信号采集系统(Plexon Inc.,Dallas,TX)，在饲养笼中记录30分钟动物LHb的自发放电活动(采样频率40kHz,300-6000Hz带通滤波))和场电位(LFP，采样率为1kHz,250Hz低通滤波)，增益为5000。将没有可辨别的神经元放电信号的通道作为参考电极。每次记录后tetrode以70μm深度往下步进，并恢复2天以开始下一次记录。对于慢性束缚应激小鼠，记录氯胺酮给药前30min和给药1h后的放电活动。所用记录的动物最后都用电损毁的方式确定电极位点。
动作电位分选：将所有记录到的电信号导入到Offline Sorter V3(Plexon Inc.)，然后使用阈值法和主成分分析(PCA)对单个神经元放电进行手动分选。峰电位间隔小于不应期(1.4ms)的发放被排除在外，并通过互相关分析确保没有分选出的神经元不发生重复。与背景噪音无法分离的信号被排除。数据分析：该部分数据分析所用软件为Neuroexplorer4(Plexon Inc.)和MATLAB。数据分析：该部分数据分析所用软件为Neuroexplorer4(Plexon Inc.)和MATLAB。
行为学实验
强迫游泳测试(forced swim test，FST)
实验在正常日光灯下进行。小鼠强迫游泳圆柱形容器的直径为12cm，高25cm。测试水深为14cm，水温23-24℃。摄像头从侧边记录小鼠在6min内的游泳情况。采用双盲方式统计小鼠游泳6min内后4min的不动时间(动物的漂浮姿势或者四肢完全没有活动的时间)。
糖水偏好测试(sucrose reference test，SPT)
实验小鼠单独饲养1周，然后连续2天给予小鼠两瓶普通水，之后两天将水换为两瓶2％的蔗糖水进行训练。训练结束后，给予动物一瓶普通水和一瓶2％的蔗糖水进行测试，每12小时交换一次水瓶的位置，每24小时记录一次水和糖水的消耗量(对水瓶称重)，共记录48小时。
慢性束缚应激(chronic restraint stress，CRS)小鼠抑郁模型构建
动物随机分成两组，一组每天11:00到14:00间放入50ml离心管束缚 2h，连续束缚14天，为了利于动物呼吸，所用50ml的离心管上钻有2mm直径的数个小孔；另一组对照动物则不接受束缚应激。每天的束缚结束后，动物放回自己的笼子并与对照组动物饲养在同一饲养室。在第15天进行强迫游泳和糖水偏好测试去评估动物的抑郁表型。
自由活动清醒小鼠光遗传行为测试
该部分所有动物行为学检测都是动物处于生物节律的黑暗时期，且在病毒表达至少三周后进行。植入的光纤通过陶瓷套管连接到插线电缆(陶瓷套管购自于中国杭州NEWDOON公司)，插线电缆接通过一个FC/PC适配器连接到到一个转动接头(购自于加拿大Doric,Québec)上，使得动物可以无限制地自由运动。另一根插线电缆通过FC/PC适配器连接到电脑和473nm的DPSS激光器(Aurora-220-473)或者589nm DPSS激光器(Aurora-220-589)，激光器均购自于中国杭州NEWDOON公司。
实时位置回避(Real-time place aversion，RTPA)测试
基于前人的实验方法(Matthews et al.,2016；Zhu et al.,2016)，一个中间连通的52x26x23厘米敞口箱子，分左右两个相同大小的箱子(26x26x23厘米)用于行为学测试。小鼠被放置到箱内自由活动20分钟，用于评估小鼠对两侧箱子的基础偏好水平。接下来的20分钟测试阶段，将小鼠平均分配到左右侧箱子，并以这一侧作为刺激箱，另一侧则为每只小鼠对应的非刺激箱。将小鼠放到非刺激箱开始实验。小鼠一旦进入刺激箱便能激活黄光刺激(eNpHR3.0:589nm,1Hz,16mW,100ms间隔)，直到小鼠返回非刺激侧结束。回避箱子的正上方安装有摄像头，记录每次实验动物的活动情况。并用Any-maze软件(美国Stoelting公司)分析动物的相关行为指标。回避分数＝后20min刺激侧与非刺激侧停留时间差值-前20min刺激侧与非刺激侧停留时间差值。
旷场测试：基于前人研究的实验方法(Matthews et al.,2016)，所用旷场测试箱大小为：45x45x45厘米，四个壁和底部均为白色树脂材料。动物先在旷场内一共自由探索9分钟，中间3分钟给予激光刺激(eNpHR3.0:589nm,1Hz,16mW,100ms间隔)。旷场正上方摄像头记录每次实验动物的运动情况，用Any-maze软件分析动物的相关行为指标。
强迫游泳测试：基于已有的强迫游泳测试方法(Li et al.,2013)。实验在 正常光照(约100lux)下进行。小鼠强迫游泳圆柱形透明容器的直径为12cm，高25cm。测试水深为14cm，水温22.5-23℃。当小鼠放入水中后，激光刺激即开始，持续6分钟(eNpHR3.0:589nm,1Hz,16mW,100ms间隔)。摄像头从侧边记录小鼠在6min内的游泳情况。实验结束后，采用双盲方式统计视频中小鼠游泳6min内后4min的不动时间(动物的漂浮姿势或者四肢完全没有活动的时间)。
统计分析
所有的数据都以平均值±SEM。对于所有的行为数据，采用two-tailed Student's t-tests。
实施例2 在大鼠的外侧缰核局部给药NMDA受体抑制剂可产生快速抗抑郁效果
通过对cLH抑郁症大鼠在外侧缰核双侧套管植入后给予氯胺酮来观察抑郁表型的变化。图1的A是cLH大鼠外侧缰核双侧套管植入示意图，白色虚线指示缰核位置。图1的B-G给出实验的结果：局部双侧施加不同的NMDAR抑制剂氯胺酮(25μg每侧，图1的B-D)和AP5(40nmol每侧，图1的E-G)到LHb，在短时间内(0.5或1小时)能有效逆转cLH大鼠的抑郁表型，包括显著降低强迫游泳中的不动时间(图1的C和F)，显著增高抑郁动物对糖水的偏好性(图1的D和G)。
在实验中还观察到，LHb双侧施加NMDAR抑制剂氯胺酮的抗抑郁效果可以持续到给药后第14天(如图1的H-I所示)。
以上实验证明，在大鼠的外侧缰核局部对NMDA受体进行抑制处理，可产生快速和持久的抗抑郁效果。
这是在本领域第一次发现在脑部的局部组织中给予NMDA受体抑制剂，例如氯胺酮，能够产生快速和持久的抗抑郁效果。
实施例3 外侧缰核中三种神经元自发放电模式(不放电，单个放电和簇状放电)在抑郁动物中的发放特性
运用全细胞膜片钳技术在离体脑切片观察抑郁症动物外侧缰核神经元的放电模式。图2的A显示全细胞膜片钳记录的记录位点，记录位点分布 于外侧缰核的不同亚区。发现外侧缰核中神经元存在三种自发放模式典型图，分别是不放电(silent)(图2的B所示)，单个放电(tonic)(图2的C所示)和簇状放电(burst)(图2的D所示)。
图2的E(散点图)和F(累积曲线)显示静息膜电位(RMPs)的均值和分布。结果显示，相较于不发放的细胞，单个放电细胞静息膜电位表现出去极化而簇状放电细胞表现为超级化。
另外，实验发现，在大鼠和小鼠抑郁动物模型中，外侧缰核自发簇状放电的神经元比例显著高于对照正常动物，且NMDAR抑制剂氯胺酮可显著降低抑郁动物自发簇状放电的神经元比例。提示抑郁症中外侧缰核神经元簇状放电增强。如图2的I和L(饼状统计图)显示，大鼠和小鼠抑郁模型中，簇状放电神经元数目增加。图2的J和M(柱状统计图)显示所有发放细胞中单个放电和簇状放电的细胞比例。图2的K和N(柱状统计图)显示不动抑郁动物缰核中神经元峰电位间隔的分布。
实施例4 体内实验观察抑郁症动物外侧缰核神经元的放电模式
为了进一步确证外侧缰核神经元簇状放电对抑郁症的作用，运用在体多通道电生理记录方法，一种更好模拟动物生理状态的记录方法，在清醒的小鼠外侧缰核置入多通道电生理记录电极，记录外侧缰核神经元的发放，包括小鼠簇状放电活性和θ波段的同步化活动等。结果如图3所示。
图3的A显示在体记录电极在对照和CRS抑郁症小鼠(CRS，chronic restrain stress)的LHb中的记录位点。图3的B为在体记录对照，CRS和CRS+氯胺酮的小鼠LHb神经元放电的代表性示例(左)和平均发放波形(右)，通过分析峰电位间隔(ISI)来分离簇状放电。图3的C-D显示，CRS小鼠LHb神经元簇状放电比例和每分钟簇状放电的个数都显著高于对照小鼠，且可以被氯胺酮所反转。图3的E显示，对照小鼠和CRS小鼠在氯胺酮注射前后峰电位间隔的累积分布曲线(对照组：143ms，CRS组：33ms，CRS+氯胺酮组：121ms)。虚线指示峰电位50％变化的点。
已知簇状放电可以增强神经元网络的同步化发放。我们通过计算神经元发放相关场电位(spike-triggered averages，STAs)，去检测发放和场电位间振荡的同步化效应。图3的F显示对照小鼠和CRS小鼠在氯胺酮注射前后 神经元发放相关场电位，对照小鼠发放相关场电位分布呈较平缓的趋势，提示没有神经元同步化效应。CRS组则出现7Hz的发放相关场电位分布，提示CRS抑郁小鼠外侧缰核神经元网络放电呈现出θ波段(4-10Hz)节律，且这种同步化效应可被氯胺酮所阻断。
图3的G通过分析每个放电单位的神经元放电与场电位的相关性(SFC，左)()，平均的SFC(中)和θ波段(4-10Hz)中SFC百分比(右)，进一步证实了CRS抑郁小鼠外侧缰核神经元网络放电呈现出可被氯胺酮所阻断的θ波段(4-10Hz)节律。
以上结果显示，慢性束缚应激诱导的抑郁小鼠模型中，外侧缰核神经元簇状放电频率和簇内发放个数都显著高于对照正常小鼠，这种增高可被NMDAR抑制剂氯胺酮所反转。
实施例5 离体实验证明，NMDA受体的激活是LHb神经元产生簇状放电的充分必要条件
已有在其他脑区的研究结果显示，簇状放电的产生需要有NMDA受体介导的钙离子内流。本发明进一步研究为了阐明外侧缰核中NMDA受体在簇状放电产生过程中的作用，首先确证了LHb有NMDAR的表达：膜片钳记录到LHb脑片中NMDA电流。图4的A是将神经元钳制在-80mV时产生的兴奋性突触后电流典型图。通过在无Mg 2+的人工脑脊液(ACSF)中加入GABA受体抑制剂(picrotoxin)和AMPA受体阻断剂(NBQX)来分离NMDA受体介导的兴奋性突触后电流(NMDAR-EPSCS)，并用NMDA受体阻断剂AP5来确认电流。
图4的B显示LHb神经元钳制在不同电压下记录到的NMDAR-EPSCs，该电流可被NMDAR抑制剂AP5完全阻断。这确证了LHb有NMDAR的表达。
图4的C-H显示外侧缰核中氯胺酮(C-D)，AP5(E-F)和NBQX(G-H)对自发簇状放电的影响。左侧为典型图，右侧为统计图。以图4的C-D为例，可以看到氯胺酮不影响神经元的静息膜电位(resting membrane potentials，RMPs)，但是几乎完全阻断了自发簇状放电。如4的C显示，在以氯胺酮处理后10秒，LHb的簇状放电转化为了单个放电。结果显示，NMDA受体抑 制剂氯胺酮和AP5均有效降低自发簇状放电的频率，而阻断另一种谷氨酸受体AMPA，对自发簇状放电的影响远比NMDA受体抑制剂的为弱。
进一步的实验在脑片中直接灌流NMDA，发现可使LHb中不发放的细胞产生簇状放电，而且这种簇状放电能被氯胺酮所阻断。如图4的I和J所示，NMDA灌流可使不发放的细胞产生簇状放电，这种诱发的簇状放电可被氯胺酮所抑制。NMDA可诱发大的兴奋性突触后电位和簇状放电。
结果证明，LHb中NMDA受体的激活是LHb神经元产生簇状放电的充分必要条件。
实施例6 LHb簇状放电需要神经元膜超级化和T型电压敏感型干通道的参与
实施例3中所发现的现象，即LHb中不同发放模式的神经元有不同的静息膜电位(RMP)，自发簇状放电的神经元表现出超级化的静息膜电位。
发明人进一步确认静息膜电位与神经元发放模式间的关系。如图5所示，首先，给记录的神经元注入一个递增的斜坡电流，使细胞的静息膜电位从-80到-40mV间变化。超级化电流注入，使细胞产生簇状放电。簇状放电的簇内个数的峰值在-56～-60mV之间，与自发簇状放电细胞的静息膜电位相近。同时对自放放电的细胞给予超级化或去极化的电流注入也可以使细胞在单个发放和簇状发放之间相互转化。
图5中的A是斜坡电流注射诱导LHb神经元从簇状放电向单个放电转化的典型图。如图所示，神经元在相对超级化的状态下容易产生簇状放电，而在相对去极化的状态下产生单个放电。图5中的B为统计图，显示大小鼠中LHb神经元注入超级化电流后能诱导产生簇状放电的神经元比例。图5中的C-E为：电流钳加记录到的簇状放电频率(C)，簇状放电持续时间(D)和簇内放电个数(E)与神经元静息膜电位的相关性。
由于NMDA受体是一个去极化条件下才激活的通道，而产生簇状放电的神经元静息膜电位是超级化的，本申请发明人那么进一步研究超级化条件下NMDA受体是怎么被激活而参与到簇状放电中。
本申请的发明人发现了在神经元超级化时被激活并能使神经元去极化的离子通道：T型电压敏感的钙离子通道。T型电压敏感的钙离子通道是一类 超级化下激活的钙通道，通道激活后使钙离子内流而导致神经元去极化。该通道有三个亚型，Cav3.1，Cav3.2和Cav3.3，三个亚型在LHb中均有表达。
发明人通过实验证明了在LHb中T型电压敏感的钙离子通道的激活能引发簇状放电。图5中的F是超级化下自发单个放电神经元向簇状放电转化的典型示例图。图5中的G是去极化下自发簇状放电向单个放电转化的典型示例图。
发明人测试了T-VSCC对LHb自发或诱发的簇状放电的影响。另外，测试另一个离子通道-HCN通道(hyperpolarization-activated cyclic nucleotide–gated channel)对LHb自发或诱发的簇状放电的影响。实验通过测试T-VSCC阻断剂和HCN的阻断剂对LHb自发或诱发的簇状放电的影响来进行。图5中的H，I显示，T-VSCC阻断剂Mibefradil(H)和HCN通道阻断剂ZD7288(I)对LHb神经元自发簇状放电的影响。左侧为典型图，右侧为统计图。结果证明，T-VSCC阻断剂能显著抑制LHb自发或诱发的簇状放电频率。而HCN通道阻断剂对LHb自发或诱发的簇状放电的影响远小于T-VSCC阻断剂。
由此，发明人首次发现了NMDA受体和T-VSCCs协同引起外侧缰核中的自发簇状放电。该簇状放电生理过程如图5中的J所示：激活的T-VSCC使得阻断NMDA受体的镁离子被移开，T-VSCC和NMDA受体通道的打开驱动神经元膜电位超簇状放电阈值方向变化。当快速失活T-VSCC和NMDA受体通道时，神经元静息膜电位恢复到-55mV一下，起始另一个簇状放电周期。
实验的电生理记录和模型的数据都证明了T-VSCC和NMDA受体协同介导LHb神经元的簇状放电。
实施例7 在动物模型的外侧缰核局部给药，阻断外侧缰核的T-VSCC，消除抑郁症症状
发明人在先天抑郁大鼠外侧缰核双侧置入套管，给予T-VSCC的阻断剂Mibefradil(10nmol/ul/侧)，如图6所示。图6的A为LHb注射CTB确定套管的注射位点图。观察到Mibefradil作用1h即可有快速的抗抑郁效果：在FST(图6的B)和SPT(图6的C)行为中均表现出快速的抗抑 郁效果。证明了局部阻断LHb T-VSCC可快速抗抑郁。
实施例8簇状放电而不是整个发放频率的增加贡献于抑郁症的产生
发明人首次发现，簇状放电模式而不是整个发放频率的增加贡献于抑郁症的产生。
发明人通过激活抑制性的光敏感通道eNpHR3.0，在离体脑片上能检测到回弹的簇状放电。同时，发明人通过体内实验，在动物中光激活外侧缰核表达的eNpHR3.0光敏感通道，发现也可快速介导厌恶情绪和抑郁表型的产生。
图7的A给出了eNpHR病毒表达载体构建示意图(上图)，光电及记录示意图(下图)。
图7的B，C是在AAV2/9-eNpHR病毒表达的小鼠LHb中，黄光激活的脑片神经元(B)和在体记录到的神经元(C)反弹簇状放电的典型图。成功诱导出簇状放电的细胞百分比显示于图C的右侧统计图。(D)点阵图和刺激后时间柱图显示在体光电极记录中一个代表性的LHb神经元对100ms黄光刺激的反应。黄光结束后神经元有一个反弹式的放电频率增高。1Hz黄光照射eNpHR3.0所产生的簇内动作电位的发放频率与CRS抑郁动物LHb中记录到的频率相当，提示1Hz黄光照射eNpHR3.0可模拟抑郁状态下的簇状放电水平。同时行为学研究结果也发现，eNpHR光激活诱导的反弹式簇状放电使动物表现出厌恶和抑郁表型。而图7的F，G，H表明，eNpHR光激活引起的反弹式簇状放电可诱发实时位置厌恶(RTPA)和抑郁表型。以上结果说明，增加LHb簇状放电足以产生抑郁样表型。
与此相对，运用5Hz的光激活oChIEF光通道产生5Hz的单个放电(图8A)，且放电中频率与1Hz的光激活eNpHR3.0激活产生的总放电频率(每秒产生一个簇状放电，每簇内有5个发放，总放电频率为5Hz)相当(图7B)，但不能诱导出抑郁表型(图8C)。与表达对照非光通道的eGFP小鼠相比，光激活并不改变动物的运动能力(图8B)。
以上结果证明，簇状放电模式而不是整个发放频率的增加贡献于抑郁症的产生。
实施例9体外实验，证明部分阻断诱发簇状放电浓度下的NMDA受体抑制剂与T-VSCC受体抑制剂联合给药，能完全阻断诱发簇状放电的产生。
为了验证NMDAR抑制剂与T-VSCC抑制剂联合给药对诱发簇状放电的影响，分别测试NMDAR抑制剂与T-VSCC抑制剂单独及联合用药对诱发簇状放电的影响。
给予外侧缰核神经元注射-100pA的超级化电流100ms，使神经元处于超极化状态，超级化状态诱发外侧缰核神经元簇状放电。外侧缰核脑片单独灌流氯胺酮(100μM，图10A)，AP5(100μM，图10B)或Mibefradil(10μM,图10C)时，神经元超级化诱发簇状放电的概率被部分降低为0.19，0.12和0.05。
当氯胺酮(100μM)与Mibefradil(10μM)联合给药(图10D)或AP5(100μM)与Mibefradil(10μM)联合给药(图10E)，都能使超级化诱发的簇状放电的概率进一步降到0，完全阻断超级化诱发的簇状放电。
实施例10体内实验，证明低于单独给药起效剂量的NMDA受体抑制剂与低于单独给药起效剂量的T-VSCC受体抑制剂联合给药，能够有效产生抗抑郁效果，并且降低两种药物的副作用
1.测试NMDA受体抑制剂氯胺酮产生抗抑郁效果的最低生效浓度。
对3mg/kg氯胺酮在C57BL/6小鼠上注射1hr后产生抗抑郁效果的实验进行验证。实验结果显示，强迫游泳测试中，注射3mg/kg氯胺酮(1hr)，C57BL/6小鼠与注射安慰剂生理盐水(1hr)的C57BL/6小鼠相比，放弃挣扎的时间有降低趋势(P＝0.097，n＝8each group)。相反，2.5mg/kg ketamine注射1hr后，在强迫游泳行为范式中，不能够降低小鼠放弃挣扎的时间。
2.测试T-VSCC受体抑制剂ethosuximide产生抗抑郁效果的最低生效浓度。
对200mg/kg ethosuximide在C57BL/6小鼠上注射1hr后产生抗抑郁效果的实验进行测试。实验结果显示，强迫游泳测试中，注射200mg/kg ethosuximide(1hr)C57BL/6小鼠与注射安慰剂生理盐水(1hr)C57BL/6小鼠相比，放弃挣扎的时间显著降低(P＝0.04，n＝8each group)。糖水偏好测试中，注射200mg/kg ethosuximide(1hr)C57BL/6小鼠与注射安慰剂生理盐水(1hr)C57BL/6小鼠相比，对于糖水的偏好显著升高(P＝0.01，n＝8each group)。
相反，100mg/kg ethosuximide(ETH)注射1hr后，在强迫游泳行为范式 中，不能够降低小鼠放弃挣扎的时间。
3.联合使用低于单独给药起效剂量的低剂量NMDA受体抑制剂与低剂量T-VSCC受体抑制剂
如图9所示，通过对C57BL/6小鼠进行2.5mg/kg ketamine+100mg/kg ethosuximide注射1hr后，在强迫游泳行为范式中，能够降低小鼠放弃挣扎的时间。
以上实验证明，低于单独给药起效剂量的低剂量NMDA受体抑制剂与低剂量T-VSCC受体抑制剂联合的方式，能够产生快速抗抑郁效果，并且降低两种药物的副作用。
结论
本发明首次和意外发现外侧缰核的神经元的簇状放电在抑郁症的产生中具有重要作用，并发现了影响外侧缰核的簇状放电的关键因素，包括NMDA受体的激活是LHb神经元产生簇状放电的充分必要条件，以及LHb簇状放电需要神经元膜超级化和T型电压敏感型干通道的参与。特别是意外发现了是簇状放电而不是整个发放频率的增加贡献于抑郁症的产生。由此发明人提供了通过抑制外侧缰核的簇状放电来治疗(抑制)抑郁症的方法和药物，特别是快速治疗(抑制)抑郁症的方法和药物。
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。
如无特别表示，本文中出现的温度的单位“度”是指摄氏度，即℃。

Claims (20)

  1. 治疗对象中的抑郁症的方法，其中包括向所述对象施用抑制外侧缰核中簇状放电的试剂。
  2. 权利要求1的方法，其中所述抑制外侧缰核中簇状放电的试剂为N-甲基-D-天冬氨酸受体抑制剂，优选的，所述N-甲基-D-天冬氨酸受体抑制剂为：
    1)NMDA受体的竞争性抑制剂，例如AP5，AP7，CPPene，塞福太(Selfotel)；
    2)NMDA受体的非竞争性抑制剂，例如阿替加奈(Aptiganel)，氯胺酮，美金刚(memantine)、Huperzine A，伊博格碱(Ibogaine)，HU-211，加巴喷丁(Gabapentin)，PD-137889；
    3)NMDA受体反竞争性的通道阻断剂，例如金刚胺(Amantadine)，阿托西汀(Atomoxetine)，AZD6765，右美沙芬(Dextromethorphan)，盐酸镁金刚胺，MK801(Dizocilpine)；或
    4)甘氨酸结合位点抑制剂，例如TK-40，犬尿酸(Kynurenic acid)。
  3. 权利要求1的方法，其中所述抑制外侧缰核中簇状放电的试剂为T型钙离子通道抑制剂，优选的，所述T型钙离子通道抑制剂为：
    琥珀酰亚胺类(Succinimides)，例如乙琥胺(ethosuximide)、甲琥胺(methsuximide)；乙内酰脲类(hydantoins)；唑尼沙胺(zonisamide)；丙戊酸钠(valproate sodium)；phenytoin；Mibefradil；苯妥英(Phenytoin)；sipatrigine；哌嗪类似物如Flunarizine、Z941；哌啶类似物如Z944和Fluoropiperidine；TTA-P1；TTA-P2；喹唑啉酮(quinazolinone)；匹莫齐特(Pimozide)；三甲双酮(Trimethadione)和二甲双酮；TTA-Q4；ML218等。
  4. 权利要求1的方法，其中包括向所述对象施用组分(a)N-甲基-D-天冬氨酸受体(NMDAR)抑制剂和组分(b)T型钙离子通道(T-VSCC) 抑制剂的组合。
  5. 如权利要求4所述的方法，其中施用于所述对象的组分(a)和组分(b)的其中至少一种的治疗有效量低于不存在另一组分时施用的所述组分的治疗有效量；优选的，所述对象的组分(a)和组分(b)的治疗有效量都低于不存在另一组分时施用的所述组分的治疗有效量。
  6. 如权利要求4所述的方法，其中施用于所述对象的组分(a)和组分(b)的其中至少一种，特别是两种的治疗有效量比不存在另一组分时施用的所述组分的治疗有效量低至少10％，优选为低至少25％，更优选为低至少50％，例如，其中施用于所述对象的组分(a)和组分(b)的其中至少一种，优选两种的治疗有效量比不存在另一组分时施用的所述组分的治疗有效量低10％至90％，优选为低25％至90％，更优选为低50％至90％。
  7. 权利要求1的方法，其中所述抑制簇状放电的试剂不抑制单个放电(tonic pulse)。
  8. 权利要求1的方法，其中在外侧缰核局部给药。
  9. 权利要求1的方法，其中所述药物是快速起效治疗抑郁症的药物，优选的，所述药物是中效和长效的治疗抑郁症的药物。
  10. 权利要求1所述的方法，其中所述抑郁症是外侧缰核簇状放电介导的抑郁症。
  11. 治疗抑郁症的药物组合物，其中包含抑制外侧缰核中簇状放电的试剂。
  12. 权利要求11的药物组合物，其中所述抑制外侧缰核中簇状放电的试剂为N-甲基-D-天冬氨酸受体抑制剂，优选的，所述N-甲基-D-天冬氨酸受体抑制剂为：
    1)NMDA受体的竞争性抑制剂，例如AP5，AP7，CPPene，塞福太 (Selfotel)；
    2)NMDA受体的非竞争性抑制剂，例如阿替加奈(Aptiganel)，氯胺酮，美金刚(memantine)、Huperzine A，伊博格碱(Ibogaine)，HU-211，加巴喷丁(Gabapentin)，PD-137889；
    3)NMDA受体反竞争性的通道阻断剂，例如金刚胺(Amantadine)，阿托西汀(Atomoxetine)，AZD6765，右美沙芬(Dextromethorphan)，盐酸镁金刚胺，MK801(Dizocilpine)；或
    4)甘氨酸结合位点抑制剂，例如TK-40，犬尿酸(Kynurenic acid)。
  13. 权利要求11的药物组合物，其中所述抑制外侧缰核中簇状放电的试剂为T型钙离子通道抑制剂，优选的，所述T型钙离子通道抑制剂为：
    琥珀酰亚胺类(Succinimides)，例如乙琥胺(ethosuximide)、甲琥胺(methsuximide)；乙内酰脲类(hydantoins)；唑尼沙胺(zonisamide)；丙戊酸钠(valproate sodium)；phenytoin；Mibefradil；苯妥英(Phenytoin)；sipatrigine；哌嗪类似物如Flunarizine、Z941；哌啶类似物如Z944和Fluoropiperidine；TTA-P1；TTA-P2；喹唑啉酮(quinazolinone)；匹莫齐特(Pimozide)；三甲双酮(Trimethadione)和二甲双酮；TTA-Q4；ML218等。
  14. 权利要求11的药物组合物，其包括组分(a)N-甲基-D-天冬氨酸受体(NMDAR)抑制剂和组分(b)T型钙离子通道(T-VSCC)抑制剂的组合。
  15. 权利要求11的药物组合物，其为在外侧缰核局部给药的剂型。
  16. 权利要求11所述的药物组合物，其中所述抑郁症是外侧缰核簇状放电介导的抑郁症。
  17. 抑制外侧缰核中簇状放电的试剂在制备治疗抑郁症的药物中的用途，优选的，所述治疗抑郁症的药物为在外侧缰核局部给药的剂型。
  18. 权利要求17的用途，其中所述抑制外侧缰核中簇状放电的试剂 为N-甲基-D-天冬氨酸受体抑制剂，优选的，所述N-甲基-D-天冬氨酸受体抑制剂为：
    1)NMDA受体的竞争性抑制剂，例如AP5，AP7，CPPene，塞福太(Selfotel)；
    2)NMDA受体的非竞争性抑制剂，例如阿替加奈(Aptiganel)，氯胺酮，美金刚(memantine)、Huperzine A，伊博格碱(Ibogaine)，HU-211，加巴喷丁(Gabapentin)，PD-137889；
    3)NMDA受体反竞争性的通道阻断剂，例如金刚胺(Amantadine)，阿托西汀(Atomoxetine)，AZD6765，右美沙芬(Dextromethorphan)，盐酸镁金刚胺，MK801(Dizocilpine)；或
    4)甘氨酸结合位点抑制剂，例如TK-40，犬尿酸(Kynurenic acid)。
  19. 权利要求17的用途，其中所述抑制外侧缰核中簇状放电的试剂为T型钙离子通道抑制剂，优选的，所述T型钙离子通道抑制剂为：
    琥珀酰亚胺类(Succinimides)，例如乙琥胺(ethosuximide)、甲琥胺(methsuximide)；乙内酰脲类(hydantoins)；唑尼沙胺(zonisamide)；丙戊酸钠(valproate sodium)；phenytoin；Mibefradil；苯妥英(Phenytoin)；sipatrigine；哌嗪类似物如Flunarizine、Z941；哌啶类似物如Z944和Fluoropiperidine；TTA-P1；TTA-P2；喹唑啉酮(quinazolinone)；匹莫齐特(Pimozide)；三甲双酮(Trimethadione)和二甲双酮；TTA-Q4；ML218等。
  20. 权利要求19的用途，其中所述抑制外侧缰核中簇状放电的试剂为组分(a)N-甲基-D-天冬氨酸受体抑制剂和组分(b)T型钙离子通道抑制剂的组合。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020088270A1 (zh) * 2018-10-29 2020-05-07 中国药科大学 犬尿喹啉酸或其衍生物在制备改善慢性精神应激相关病理损伤药物中的应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115006534B (zh) * 2017-05-09 2023-11-21 浙江大学 钾离子通道Kir4.1抑制剂治疗抑郁症的用途和药物组合物
JP2021050161A (ja) 2019-09-25 2021-04-01 武田薬品工業株式会社 複素環化合物及びその用途
JP2021080177A (ja) 2019-11-14 2021-05-27 武田薬品工業株式会社 複素環化合物及びその用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4694010A (en) * 1985-08-16 1987-09-15 New York University Anticonvulsant compositions and method
WO2007137247A2 (en) 2006-05-22 2007-11-29 Vanda Pharmaceuticals, Inc. Treatment for depressive disorders
CN102512682A (zh) * 2012-01-12 2012-06-27 王玉丰 治疗噪音性听力损伤的药物组合物及其应用
US20160375000A1 (en) * 2015-06-26 2016-12-29 Korea Advanced Institute Of Science And Technology Pharmaceutical composition for prevention and treatment of mental disease with enhanced nmdar function
CN106562952A (zh) * 2015-10-09 2017-04-19 北京安珀科技有限责任公司 氯胺酮在治疗重性抑郁障碍中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090029690A (ko) * 2006-03-22 2009-03-23 마운트 시나이 스쿨 오브 메디신 오브 뉴욕 유니버시티 우울증을 치료하기 위한 케타민의 투여 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4694010A (en) * 1985-08-16 1987-09-15 New York University Anticonvulsant compositions and method
WO2007137247A2 (en) 2006-05-22 2007-11-29 Vanda Pharmaceuticals, Inc. Treatment for depressive disorders
CN102512682A (zh) * 2012-01-12 2012-06-27 王玉丰 治疗噪音性听力损伤的药物组合物及其应用
US20160375000A1 (en) * 2015-06-26 2016-12-29 Korea Advanced Institute Of Science And Technology Pharmaceutical composition for prevention and treatment of mental disease with enhanced nmdar function
CN106562952A (zh) * 2015-10-09 2017-04-19 北京安珀科技有限责任公司 氯胺酮在治疗重性抑郁障碍中的应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CAO, QIUYUN: "The Clinical Features and Treatment of Late-Onset Depression", CHINESE JOURNAL OF BRAIN DISEASES AND REHABILITATION, vol. 5, no. 3, 30 June 2015 (2015-06-30), pages 141 - 144, XP009517789, ISSN: 2095-123X *
D. SCHULZM. M. MIRRIONEF. A. HENN, NEUROBIOL LEARN MEM, vol. 93, February 2010 (2010-02-01), pages 291
LI ET AL., SCIENCE, vol. 341, 2013, pages 1016 - 1020
LI, B. ET AL., NATURE, vol. 470, 2011, pages 535 - 539
YANG HONGBO; ZHANG JICHUAN: "Research Progress on Effect of Lateral Habenular Nucleus in Depression", CHINESE JOURNAL OF GERONTOLOGY, vol. 36, no. 5, 31 March 2016 (2016-03-31), pages 1246 - 1248, XP009517781, ISSN: 1005-9202 *
ZEYNEP ATES-ALAGOZ ET AL.: "NMDA Receptor Antagonists for Treatment of Depression", PHARMACEUTICALS, vol. 6, no. 4, 3 April 2013 (2013-04-03), pages 480 - 499, XP055479845, ISSN: 1424-8247 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020088270A1 (zh) * 2018-10-29 2020-05-07 中国药科大学 犬尿喹啉酸或其衍生物在制备改善慢性精神应激相关病理损伤药物中的应用

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