WO2019088095A1 - Analgesic and use thereof - Google Patents

Analgesic and use thereof Download PDF

Info

Publication number
WO2019088095A1
WO2019088095A1 PCT/JP2018/040319 JP2018040319W WO2019088095A1 WO 2019088095 A1 WO2019088095 A1 WO 2019088095A1 JP 2018040319 W JP2018040319 W JP 2018040319W WO 2019088095 A1 WO2019088095 A1 WO 2019088095A1
Authority
WO
WIPO (PCT)
Prior art keywords
receptor
adrenergic
astrocytes
analgesic
spinal
Prior art date
Application number
PCT/JP2018/040319
Other languages
French (fr)
Japanese (ja)
Inventor
誠 津田
雄太 高露
Original Assignee
国立大学法人九州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人九州大学 filed Critical 国立大学法人九州大学
Publication of WO2019088095A1 publication Critical patent/WO2019088095A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to analgesics and their use.
  • Priority is claimed on Japanese Patent Application No. 2017-213196, filed Nov. 2, 2017, and Japanese Patent Application No. 2018-18653, filed Feb. 5, 2018, on November 2, 2017. The contents of which are incorporated herein by reference.
  • Somatosensory information including noxious stimuli from peripheral tissues is input to the spinal dorsal horn via the primary afferent sensory nerves, appropriately processed by the neural circuit at the same site, and transmitted to the brain.
  • this transmission has been described using only nerve cells, but in recent years glial cells have been shown to greatly affect nerve activity and to be involved in modulation of sensory communication (Non-patent Document 1).
  • astrocytes which are the most numerous among glial cells, express neurotransmitter receptors and are spatially in contact with synapses, and thus involvement in synaptic activity is assumed (non-patent literature) 2).
  • the role of astrocytes in the spinal cord in sensory signal transduction is still unknown.
  • Adrenergic alpha receptors include alpha 1 receptors and alpha 2 receptors. Among these, there are three subtypes of adrenaline ⁇ 1 receptors, ⁇ 1A, ⁇ 1B and ⁇ 1D receptors, and it is known that they are involved in vasoconstriction, pupil dilation, pistatic hair, prostate contraction and the like. However, the role of adrenergic alpha 1 receptors in astrocytes is unknown.
  • the present invention aims to provide a new analgesic that targets astrocytes in the spinal cord.
  • the present invention includes the following aspects.
  • An analgesic containing, as an active ingredient, a substance that inhibits a signal from astrocyte adrenergic ⁇ 1 receptor.
  • the analgesic according to [1] wherein the substance that inhibits the signal from the adrenergic ⁇ 1 receptor is an adrenergic ⁇ 1 receptor antagonist or an adrenergic ⁇ 1 receptor expression inhibitor.
  • a screening method for an analgesic agent which comprises selecting a substance that inhibits a signal from adrenergic ⁇ 1 receptor of astrocytes.
  • FIG. 7 shows astrocytes expressing Hes5 in spinal dorsal horn in Experimental Example 1.
  • Experimental Examples 2 and 3 it is the figure which showed the activity of the astrocyte of the spinal dorsal horn by capsaicin, and the effect of a noradrenaline neuroselective toxin and an adrenergic alpha 1A receptor antagonist.
  • the left figure shows that hM3Dq receptor is expressed in Hes5-positive astrocytes in the spinal dorsal horn surface layer in Experimental Example 4.
  • the right figure is a figure which showed the behavioral analysis of the mouse
  • FIG. 10 is a diagram showing mild mechanical stimulation hyperalgesia when an adrenaline ⁇ 1 receptor agonist was administered intrathecally in Experimental Example 6;
  • FIG. 17 shows the effect on phenylephrine-induced mild mechanical stimulation hyperalgesia when administration of an adrenergic ⁇ 1A receptor antagonist and suppression of adrenergic ⁇ 1A receptor expression in Experimental Examples 7 and 8.
  • Experimental Example 9 it is a figure showing involvement of descending noradrenaline nerve in capsaicin-induced mild mechanical stimulation hyperalgesia.
  • Experimental example 10 it is the figure which showed the effect of adrenergic-alpha 1A receptor expression suppression of the spinal dorsal horn astrocyte on the capsaicin-induced mild mechanical stimulation hyperalgesia.
  • analgesic in one embodiment, the present invention provides an analgesic containing, as an active ingredient, a substance that inhibits a signal from astrocyte ⁇ 1 receptor of astrocytes.
  • Pain is transmitted to the spinal cord when nerves in the skin are excited when pain stimulation is applied to the skin which is a peripheral tissue.
  • the information transmitted to the spinal cord is transmitted to the brain and only when it feels pain.
  • noradrenaline nerves extend downward from the brain to the spinal dorsal horn, and upon stimulation of this descending noradrenaline nerve by pain stimulation, noradrenaline is released from the nerve terminal.
  • Astrocytes which are non-neuronal cells, exist at the junction of nerves and nerves, and are involved in signal transmission between nerves.
  • the present inventors have found that among the dorsal horn of the spinal cord, there are an astrocyte subpopulation localized in the surface layer and an astrocytic subpopulation localized in the deep region of the spinal dorsal horn.
  • noradrenaline released from nerve terminals by excitation of descending noradrenaline was bound to astrocyte adrenergic ⁇ 1 receptors present on the surface layer of spinal dorsal horn, signals from adrenergic ⁇ 1 receptors were input from peripheral tissues It was found to enhance pain stimulation.
  • a substance that inhibits the signal from astrocyte adrenergic ⁇ 1 receptor can be used as an analgesic because it can suppress pain stimulation input from peripheral tissues.
  • the analgesic can be used for the treatment, alleviation and / or prevention of pain.
  • the substance that inhibits the signal from the adrenergic ⁇ 1 receptor is not particularly limited as long as it has an activity to inhibit the signal from the adrenergic ⁇ 1 receptor, for example, adrenergic ⁇ 1 receptor Examples include body antagonists, adrenaline ⁇ 1 receptor expression inhibitors and the like.
  • the adrenergic alpha 1 receptor antagonist is not particularly limited as long as it is a substance having an ability to act on adrenergic alpha 1 receptor and inhibit a signal from the adrenaline alpha 1 receptor, and it is called an adrenaline alpha 1 receptor blocker There is also.
  • the adrenergic ⁇ 1 receptor antagonist examples include silodosin, tamsulosin, naphthopidil, terazosin, urapidil, prazosin, alfuzosin, doxazosin, bunazosin and the like.
  • the substance that inhibits the signal from the adrenergic ⁇ 1 receptor may be a substance that inhibits the activity of the adrenergic ⁇ 1 receptor.
  • the substance that suppresses the activity of the adrenaline ⁇ 1 receptor examples include a specific binding substance to the adrenaline ⁇ 1 receptor, and the like.
  • specific binding substances include antibodies, antibody fragments, aptamers, low molecular weight compounds and the like.
  • the adrenaline ⁇ 1 receptor expression inhibitor is not particularly limited as long as it is a substance capable of suppressing the expression of adrenaline ⁇ 1 receptor, and, for example, sh (short hairpin) RNA, siRNA, miRNA which suppresses the expression of adrenaline ⁇ 1 receptor And ribozymes, antisense nucleic acids, low molecular weight compounds and the like.
  • shRNA short hairpin
  • siRNA siRNA
  • miRNA miRNA which suppresses the expression of adrenaline ⁇ 1 receptor And ribozymes, antisense nucleic acids, low molecular weight compounds and the like.
  • shRNA that suppresses the expression of adrenergic ⁇ 1 receptor include, for example, an shRNA that uses a DNA containing the base sequence represented by SEQ ID NO: 1 as a target sequence.
  • the astrocyte adrenergic ⁇ 1 receptor may be any of ⁇ 1A, ⁇ 1B and ⁇ 1D. In one embodiment, the adrenergic ⁇ 1 receptor is an ⁇ 1A receptor.
  • the astrocytes may be astrocytes present in the spinal cord.
  • the astrocytes are astrocytes present in the surface layer of the spinal dorsal horn. Examples of astrocytes present in the surface layer of the dorsal horn of the spinal cord include astrocytes expressing the transcription factor Hes5.
  • the analgesic according to the present embodiment is not particularly limited as long as it has an effect of suppressing pain, and even if the analgesic has an effect of suppressing transient pain, chronic pain treatment such as neuropathic pain It may be an analgesic that has an effect of suppressing persistent pain, such as an agent.
  • the analgesic of the present embodiment may contain a pharmaceutically acceptable carrier in addition to the substance that inhibits the signal from astrocyte ⁇ 1 receptor of astrocytes.
  • the analgesic according to this embodiment is orally administered, for example, in the form of tablets, capsules, elixirs, microcapsules or the like, or parenterally in the form of injections, suppositories, external preparations for skin, etc. be able to. More specifically, examples of the skin external preparation include dosage forms such as ointments and patches.
  • the analgesic agent of the present embodiment is preferably administered in the form of central transfer.
  • a method of administration in the form of central transfer for example, a method of designing an active ingredient, a substance that inhibits a signal from adrenergic ⁇ 1 receptor of astrocyte, by design so as to cross the blood-brain barrier or And the method etc. of administering the analgesic of this embodiment in the spinal cord.
  • those generally used for the preparation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injection such as water, ethanol and glycerin;
  • the pressure-sensitive adhesive include rubber-based pressure-sensitive adhesives and silicone-based pressure-sensitive adhesives.
  • the analgesic of the present embodiment may contain an additive.
  • additives such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and maltitol; flavoring agents such as peppermint and red mono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid Salts, buffers such as sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.
  • the analgesic according to the present embodiment is required for generally accepted pharmaceutical practice, using an appropriate combination of the above-mentioned substance that inhibits the astrocyte signal from the adrenergic ⁇ 1 receptor, a pharmaceutically acceptable carrier and an additive as described above. It can be formulated by blending in unit dose form.
  • the dose of the analgesic according to the present embodiment varies depending on the condition, body weight, age, sex, etc. of the subject, and can not be determined indiscriminately.
  • the substance that inhibits the signal from is preferably administered, for example, 0.1 to 100 mg / kg body weight, such as 1 to 50 mg / kg body weight, such as 1 to 20 mg / kg body weight, such as 1 to 10 mg / kg body weight, per dosage unit form. Just do it.
  • the dosage unit form is, for example, 0.01 to 50 mg, for example 0.1 to 30 mg, for example 0.1 to 20 mg, for example, 0.2 to 50 mg. 1 to 10 mg of the active ingredient may be administered.
  • the daily dose of the analgesic according to the present embodiment varies depending on the condition, body weight, age, sex, etc. of the subject and can not be generally determined, but for example, 0.1 to 10 mg / kg per day for adults
  • the active ingredient of body weight may be administered once a day or divided into 2 to 4 times.
  • the analgesic of this embodiment has an analgesic action using the evaluation system of the well-known analgesic action.
  • it can investigate by the following evaluation system using a capsaicin model animal.
  • evaluation system using a capsaicin model animal.
  • the test drug is administered to 8 to 12-week-old C57BL / 6J mice (manufactured by CLEA Japan, Inc.), 10 ⁇ L of a 0.16 ⁇ g / ⁇ L capsaicin solution is then administered to the hind footpads of the mice, and after administration, the administration side of the mice
  • a sensory test filament (0.02 to 2 g of von Frey Filament, manufactured by North Coast Medical) is pressed to the sole of the hind leg, and the up-down method [J. Evaluate with filaments of 5 to 9 different forces according to Neurosci Methods, 1994, 53 (1): 55-63].
  • the hindpaw withdrawal behavior of the animal in response to the light mechanical stimulation is observed over time, and the 50% withdrawal threshold is calculated
  • the present invention provides a method of screening for analgesics, which comprises selecting a substance that inhibits the signal from astrocyte adrenergic ⁇ 1 receptor.
  • the substance that inhibits the signal from the adrenergic ⁇ 1 receptor is not particularly limited as long as it has an activity to inhibit the signal from the adrenergic ⁇ 1 receptor, for example, an adrenergic ⁇ 1 receptor antagonist, adrenergic receptor expression An inhibitor etc. are mentioned.
  • the adrenergic ⁇ 1 receptor antagonist is not particularly limited as long as it has an activity capable of inhibiting a signal from the adrenergic ⁇ 1 receptor by acting on the adrenergic ⁇ 1 receptor, and is referred to as an adrenergic ⁇ 1 receptor blocker There is also.
  • the adrenaline ⁇ 1 receptor expression inhibitor is not particularly limited as long as it is a substance capable of suppressing the expression of adrenaline ⁇ 1 receptor, and examples thereof include sh (short hairpin) RNA that suppresses the expression of adrenaline ⁇ 1 receptor and the like .
  • the astrocyte adrenergic ⁇ 1 receptor may be any of ⁇ 1A, ⁇ 1B and ⁇ 1D. In one embodiment, the adrenergic ⁇ 1 receptor is an ⁇ 1A receptor.
  • the astrocytes may be astrocytes present in the spinal cord.
  • the astrocytes are astrocytes present in the surface layer of the spinal dorsal horn. Examples of astrocytes present in the surface layer of the dorsal horn of the spinal cord include astrocytes expressing the transcription factor Hes5.
  • the analgesic according to the present embodiment is not particularly limited as long as it has an effect of suppressing pain, and even if the analgesic has an effect of suppressing transient pain, chronic pain treatment such as neuropathic pain It may be an analgesic that has an effect of suppressing persistent pain, such as an agent.
  • the screening method of the present embodiment is not particularly limited as long as it is a method capable of selecting a substance that inhibits the signal from adrenergic ⁇ 1 receptor of astrocytes, for example, to cells expressing adrenergic ⁇ 1 receptors of astrocytes
  • a method for selecting a test substance that inhibits a signal from the adrenergic ⁇ 1 receptor, which is generated when a test substance is added and an agonist of the adrenergic ⁇ 1 receptor is added, to a cell expressing the adrenergic ⁇ 1 receptor of astrocytes In the presence of a test substance, a selective ligand labeled with a radioactive substance or the like is bound, and a method of selecting a test substance that inhibits the binding between the ligand and the receptor can be mentioned.
  • a method using a method of DREADD Designer Receptors Exclusively Activated by Designer Drug
  • DREADD Designer Receptors Exclusively Activated by Designer Drug
  • CNO clozapine-N-oxide
  • pain-like behavior (allodinia) to mild mechanical stimulation induced by activation of spinal dorsal horn astrocytes is suppressed when the test substance is administered in advance.
  • Analgesia by evaluating the substance and selecting a substance that suppresses the pain-like behavior It can be screened.
  • the substance that inhibits the signal from the adrenergic ⁇ 1 receptor of astrocytes obtained by the screening method of the present embodiment can be used as an analgesic because it can suppress pain stimulation inputted from peripheral tissues.
  • the analgesic can be used for the treatment, alleviation and / or prevention of pain.
  • the present invention provides a method for treating, alleviating and / or preventing pain, comprising the step of administering to a subject an effective amount of a substance that inhibits a signal from astrocyte ⁇ 1 receptor of astrocytes.
  • the pain is not particularly limited, and may be transient pain or persistent pain such as chronic pain such as neuropathic pain.
  • the substance that inhibits the signal from astrocyte adrenergic ⁇ 1 receptor the above-mentioned substances can be mentioned.
  • a process of administering to a subject a process of administering in the form transferred to the center is preferable.
  • the step of administering in a form that moves to the center includes, for example, a step of designing and administering an active ingredient, a substance that inhibits a signal from adrenergic ⁇ 1 receptor of astrocyte, so as to cross the blood-brain barrier or And the step of administering to the spinal cord a substance that inhibits the signal from the astrocyte adrenergic ⁇ 1 receptor.
  • the present invention provides a substance that inhibits the signal from astrocyte adrenergic alpha 1 receptor for use in the treatment, alleviation and / or prevention of pain.
  • the pain is not particularly limited, and may be transient pain or persistent pain such as chronic pain such as neuropathic pain.
  • the substance that inhibits the signal from astrocyte adrenergic ⁇ 1 receptor the above-mentioned substances can be mentioned.
  • the present invention provides the use of a substance that inhibits a signal from astrocyte adrenergic ⁇ 1 receptor for producing an analgesic.
  • the analgesic is not particularly limited as long as it has an effect of suppressing pain, and even an analgesic having an effect of suppressing transient pain, chronic pain such as neuropathic pain It may be an analgesic that has an effect of suppressing persistent pain, such as a therapeutic agent.
  • the substance that inhibits the signal from astrocyte adrenergic ⁇ 1 receptor the above-mentioned substances can be mentioned.
  • the analgesic can be used for the treatment, alleviation and / or prevention of pain.
  • the mouse was anesthetized by intraperitoneal administration of pentobarbital sodium preparation (Somnopentyl (R) injection, manufactured by Kyoritsu Pharmaceutical Co., Ltd.), and after confirming disappearance of the tortuous reflex, the abdomen was immediately followed.
  • the heart was dissociated by perfusion with 20 mL of phosphate buffer (PBS).
  • PBS phosphate buffer
  • the whole body tissues were fixed by perfusion with 50 mL of ice-cold 4% paraformaldehyde (PFA) solution, and then the animals were ice-cold for about 1 hour.
  • the spinal cord was removed and the fourth lumbar spinal cord was isolated under a stereomicroscope.
  • the isolated spinal cords were subjected to immersion fixation in a 4% PFA solution until 4 to 5 hours after standing in ice. Thereafter, the spinal cord was transferred into cold 30% sucrose solution and shaken at 4 ° C. for 24 hours.
  • the embedded spinal cord stored at -80.degree. C. was allowed to stand for about 60 minutes in a cryostat (manufactured by Leica) at -20.degree. Then, after making a 30 ⁇ m-thick section using a cryostat, it was transferred into PBS and O.S. C. T. The compound was dissolved. The resulting spinal cord sections were washed in 0.3% Triton X-100 / PBS (PBST) with shaking. After washing, it was blocked for 2 hours at room temperature in 3% normal donkey serum / PBST.
  • PBST Triton X-100 / PBS
  • a primary antibody reaction was carried out by diluting a polyclonal goat-anti Sox9 antibody (diluted 1: 1000, manufactured by R & D) in 3% normal donkey serum / PBST and reacting at 4 ° C. for 48 hours. After completion of the reaction, wash with PBST solution, dilute donkey anti-goat IgG (H + L) Alexa Fluor 488 antibody (1000-fold dilution, Invitrogen) in 3% normal donkey serum / PBST, and react for 3 hours in the dark at room temperature To perform a secondary antibody reaction. After completion of the reaction, it was washed with PBST solution and PBS solution in the dark.
  • the spinal cord section was attached to a slide glass (manufactured by Matsunami Glass Industry Co., Ltd.) coated with aminopropyltriethoxysilane (APS), excess PBS solution around the section was removed, and dried.
  • the anti-fading agent VectaShield manufactured by Vector Labolatories
  • a confocal laser microscope (LSM700, manufactured by Zeiss) was used for observation.
  • FIG. 1 As shown in the left panel of FIG. 1, tdTomato positive cells were expressed in the surface layer of the dorsal horn of the spinal cord. Also, as shown in the right figure in FIG. 1, the astrocyte marker Sox9 was widely expressed in the spinal dorsal horn. From this, it was confirmed that the astrocytes are widely present in the spinal dorsal horn, but the Hes5-positive astrocytes are present in the surface layer of the spinal dorsal horn.
  • GCaMP6m The sequence of GCaMP6m (manufactured by addgene, # 40754) was subcloned into pENTR plasmid (manufactured by Themofisher). Next, this GCaMP6m cassette was inserted into an AAV shuttle vector (pZac2.1, obtained from Vector core of the University of Pennsylvania) loaded with gfaABC 1 D promoter (manufactured by addgene, # 19974).
  • AAV shuttle vector pZac2.1, obtained from Vector core of the University of Pennsylvania
  • Virus vector production using the above plasmid was performed according to a standard method (Sci Rep., 2015 Sep 21; 5: 14306).
  • the above plasmid, Rep / Cap plasmid (pAAV 2/9) and helper plasmid (pAd Delta F 6) (Rep / Cap and pAd Delta F 6 obtained from Vector core of the University of Pennsylvania) are transfected into Human embryonic kidney 293 (HEK 293) cells And purified by cesium chloride density gradient centrifugation (Gene Ther., 2010 Apr; 17 (4): 503-10).
  • the virus vector was dialyzed against PBS solution containing 0.001% (v / v) Pluronic-F68 using Amicon Ultra 100K filter unit (manufactured by Millipore). Titration of the viral vector was performed using Pico Green fluorometric reagent (manufactured by Molecular Probe) and stored at -80 ° C until use.
  • Physiological saline or DSP-4 [N- (2-chloroethyl) -N-ethyl-2 which is a noradrenaline neuroselective toxin is intraperitoneally injected into C57BL / 6J mice (manufactured by CLEA Japan, Inc.) of 8 to 12 weeks of age. -Bromobenzylamine] (manufactured by Sigma) was administered at 50 mg / kg.
  • the mixture is anesthetized by subcutaneous administration of 0.005 mL / g of a mixture of ketamine (100 mg / kg; KETALAL (R) for intramuscular injection) and xylazine (10 mg / kg; SERACTAL (R)).
  • a mixture of ketamine 100 mg / kg; KETALAL (R) for intramuscular injection
  • xylazine 10 mg / kg; SERACTAL (R)
  • Xylocaine registered trademark
  • the muscle tissue 1 mm lateral to the rostral tip of the first lumbar spine of the first lumbar vertebra was incised with a diameter of about 2 mm to expose the membrane between the thirteenth thoracic spinal cord and the first lumbar spinal vertebra bone.
  • Adeno-associated virus vector AAV2 / 9-gfaABC 1 D-GCaMP6m-WPRE to express the fluorescent protein GCaMP6m was microinjected.
  • Anesthetic treatment for animals is a mixed solution of carprofen (Limadile (registered trademark) injection, 5 mg / kg as carprofen), dexamethasone (Dexat (registered trademark) injection, 0.2 mg / kg as dexamethasone) for analgesia and inflammation prevention.
  • carprofen Limadile (registered trademark) injection, 5 mg / kg as carprofen
  • dexamethasone (Dexat (registered trademark) injection, 0.2 mg / kg as dexamethasone) for analgesia and inflammation prevention.
  • anesthesia was performed by subcutaneously administering a mixture of ketamine (100 mg / kg, KETALAL (registered trademark) for intramuscular injection) and xylazine (10 mg / kg, SERACTAL (registered trademark)) at 0.005 mL / g.
  • ketamine 100 mg / kg, KETALAL (registered trademark) for intramuscular injection
  • xylazine 10 mg / kg, SERACTAL (registered trademark)
  • Xylocaine registered trademark
  • Application of an animal eye ointment chloramphenicol 2 mg / 0.1 g, Mycochlorin (registered trademark) was performed to prevent dryness of the eye during observation window preparation and imaging.
  • the skin was incised about 3 cm along the median centering on the thoracic spinal curve.
  • the connective tissue was dissected to expose the dorso-lumbar dorsal aspect from the thirteenth thoracic spinal cord.
  • the muscles were stripped as much as possible without cutting.
  • the exposed vertebral spine was fixed using a dedicated fixture and a laminectomy was performed.
  • a dedicated stainless steel instrument was fixed to cover the exposed spinal cord.
  • the gap between the tissue and the instrument was filled with a non-cytotoxic, clear silicon elastomer and the observation window was covered with a coverslip.
  • the skin other than the part where the observation instrument was installed was sewn together.
  • Peak value of fluorescence signal ⁇ F / F 100 * (Ft ⁇ F 0 ) / F 0 t: fluorescent signal observation time at which peaked Ft: Peak of fluorescence signal F 0: Mean value of the fluorescence signal in the no-treatment of 50 seconds from the observation start
  • hM3Dq addgene, # 45547
  • pENTR plasmid Themofisher
  • a vector pAAV-EF1 ⁇ -FLEX in which the CA promoter was replaced with EF1 ⁇ was also prepared using pAAV-CA-FLEX (manufactured by addgene, # 38042), and the hM3Dq cassette was inserted into pAAV-EF1 ⁇ -FLEX.
  • Virus vector production using the above plasmid was performed according to a standard method (Sci Rep., 2015 Sep 21; 5: 14306). Transfect the above plasmid and Rep / Cap plasmid (pAAV2 / 5) and helper plasmid (pAd DeltaF6) (Rep / Cap and pAd DeltaF6 from Vector core of the University of Pennsylvania) into Human embryonic kidney 293 (HEK 293) cells , And purified by cesium chloride density gradient centrifugation (Gene Ther., 2010 Apr; 17 (4): 503-10).
  • the obtained adeno-associated virus vector AAV2 / 5-FLEX-EF1 ⁇ -hM3Dq-WPRE was treated with Amicon Ultra 100K filter unit (Millipore) in PBS solution containing 0.001% (v / v) Pluronic-F68 I dialyzed. Titration of the viral vector was performed using Pico Green fluorometric reagent (manufactured by Molecular Probe) and stored at -80 ° C until use.
  • Anesthesia was performed using a mixed solution of ketamine and xylazine subcutaneously administered at 0.005 mL / g using 8 to 12 week old Hes 5 Cre ERT 2 mice (Nat Commun., 2012 Feb 14; 3: 670). After confirming the effect of anesthesia, the back hair was shaved and a local anesthetic (Xylocaine (registered trademark) 2%) was applied. The skin was incised about 3 cm along the median centering on the thoracic spinal curve.
  • the muscle tissue 1 mm lateral to the rostral tip of the 13th and 1st lumbar vertebrae of the vertebra is incised with a diameter of about 2 mm, and the 12th and 13th spinal cords, the 13th and 25th spinal cords The lumbar and the membrane between the first lumbar and the second lumbar spinal bone were exposed.
  • 0.5 to 0.6 ⁇ L each was prepared at a concentration of 1 ⁇ 10 12 GC / mL at a total of three locations.
  • the Adeno-associated virus vector AAV2 / 5-FLEX-EF1 ⁇ -hM3Dq-WPRE which expresses the hM3Dq receptor specifically in Hes5 positive astrocytes
  • the skin was stitched with 5-0 silk thread.
  • the suture was applied with a local anesthetic (Xylocaine (registered trademark) 2%) to reduce animal pain.
  • diluted iodine tincture and gentacin (registered trademark) ointment 0.1%) were applied to prevent infection.
  • Cre recombinase activity is obtained by intraperitoneally administering 2 mg / 0.1 mL of tamoxifen (manufactured by Sigma) solution (prepared at 20 mg / mL using corn oil as solvent) to mice once a day for 10 days. And hM3Dq receptors were specifically expressed in Hes5 positive astrocytes.
  • the hM3Dq gene is placed retrogradely in the adeno-associated virus vector AAV2 / 5-FLEX-EF1 ⁇ -hM3Dq-WPRE, when tamoxifen is administered, Cre recombinase activity is induced in Hes5-positive astrocytes, A portion of the hM3Dq sequence in the viral vector is inverted and the hM3Dq receptor is expressed, but in the Hes5-negative astrocytes, the hM3Dq receptor is not expressed because the portion of the hM3Dq sequence in the viral vector is not inverted.
  • hM3Dq receptors were expressed on Hes5-positive astrocytes in the dorsal horn surface layer.
  • clozapine-N-oxide (Clozapine-N-oxide (CNO; Enzo Life Science), which is a selective stimulator of hM3Dq receptor, is expressed in a mouse in which hM3Dq receptor is specifically expressed in Hes5 positive astrocytes.
  • CNO Cerclozapine-N-oxide
  • 10 mg / kg of a solution containing the compound of the formula I) was intraperitoneally administered, and a perceptual test filament (von Frey Filament 0.02 to 2 g; manufactured by North Coast Medical) was pushed to the plantar region of the hind limbs of the animal, According to Neurosci Methods, 1994, 53 (1): 55-63], the filament was evaluated with 5 to 9 different force filaments.
  • mice for mild mechanical stimulation during Hes 5 negative spinal astrocyte stimulation an adeno-associated virus vector AAV2 / 5-FLEX-gfaABC 1 D-hM3Dq-WPRE that specifically expresses hM3Dq receptor in Hes5 negative astrocytes was constructed as follows.
  • the FLEX-hM3Dq in which the hM3Dq gene is arranged in anterograde manner is used as the pENTR plasmid (made by Themofisher) It was subcloned. This cassette was inserted into pZac2.1 loaded with the gfaABC 1 D promoter.
  • Virus vector production using the above plasmid was performed according to a standard method (Sci Rep., 2015 Sep 21; 5: 14306).
  • the above plasmid, Rep / Cap plasmid (pAAV2 / 5) and helper plasmid (pAd DeltaF6) (Rep / Cap and pAd DeltaF6 are obtained from Vector core of the University of Pennsylvania) are transfected into Human embryonic kidney 293 (HEK 293) cells. And purified by cesium chloride density gradient centrifugation (Gene Ther., 2010 Apr; 17 (4): 503-10).
  • the obtained adeno-associated virus vector AAV2 / 5-FLEX-gfaABC 1 D-hM3Dq-WPRE was PBS containing 0.001% (v / v) Pluronic-F68 using Amicon Ultra 100K filter unit (Millipore). The solution was dialyzed. Titration of the viral vector was performed using Pico Green fluorometric reagent (manufactured by Molecular Probe) and stored at -80 ° C until use.
  • Anesthesia was performed using a mixed solution of ketamine and xylazine subcutaneously administered at 0.005 mL / g using 8- to 12-week-old Hes5-CreERT2 mice (Nat Commun., 2012 Feb 14; 3: 670). After confirming the effect of anesthesia, the back hair was shaved and a local anesthetic (Xylocaine (registered trademark) 2%) was applied. The skin was incised about 3 cm along the median centering on the thoracic spinal curve.
  • the muscle tissue 1 mm lateral to the rostral tip of the 13th and 1st lumbar vertebrae of the vertebra is incised with a diameter of about 2 mm, and the 12th and 13th spinal cords, the 13th and 25th spinal cords The lumbar and the membrane between the first lumbar and the second lumbar spinal bone were exposed.
  • 0.5 to 0.6 ⁇ L each was prepared at a concentration of 1 ⁇ 10 12 GC / mL at a total of three locations.
  • Hes5-negative astrocytes were microinjected with the adeno-associated virus vector AAV2 / 5-FLEX-gfaABC 1 D-hM3Dq-WPRE, which expresses hM3Dq receptors specifically. After treatment, the skin was stitched with 5-0 silk thread. The suture was applied with a local anesthetic (Xylocaine (registered trademark) 2%) to reduce animal pain. At the same time, diluted iodine tincture and gentacin (registered trademark) ointment (0.1%) were applied to prevent infection.
  • Xylocaine registered trademark
  • diluted iodine tincture and gentacin (registered trademark) ointment 0.1%) were applied to prevent infection.
  • HM3Dq receptor was specifically expressed in Hes5 negative astrocytes. Since the adeno-associated virus vector AAV2 / 5-FLEX-gfaABC 1 D-hM3Dq arranges the hM3Dq gene anteroposteriorly, when corn oil is administered, the Astro does not depend on the presence or absence of Cre recombinase activity. HM3Dq receptor is expressed at the site.
  • hM3Dq receptors were expressed in astrocytes in the dorsal horn in the dorsal horn in the corn oil-administered group, but hM3Dq reception was only in Hes5-negative astrocytes present in the dorsal horn in the spinal cord in the tamoxifen-administered group. The body expressed.
  • Clozapine-N-oxide [Clozapine-N-oxide (CNO; Enzo Life Science)], which is a selective stimulator of hM3Dq receptor, is included in mice in which Hes5 negative astrocytes specifically express hM3Dq receptor.
  • a sensory test filament von Frey Filament 0.02 to 2 g; manufactured by North Coast Medical
  • the filament was evaluated with 5 to 9 different force filaments.
  • the hindlimb withdrawal behavior of the animals to the mild mechanical stimulation was observed over time at 0, 30, 60, 90, 120, 180 minutes after administration, and 50% withdrawal threshold (Paw withdrawal threshold) was calculated.
  • Example 6 Mild mechanical stimulation hyperalgesia by intrathecal administration of adrenergic ⁇ 1 receptor agonist
  • phenylephrine (R)-(-)-phenylephrine hydrochloride is obtained using a Hamilton microsyringe with a 30 G needle attached to a C57BL / 6J mouse (manufactured by CLEA Japan, Inc.) of 8 to 12 weeks of age. Wako Pure Chemical Industries, Ltd.] was administered 15 pmol / 5 ⁇ L saline and 50 pmol / 5 ⁇ L saline into the spinal subarachnoid space.
  • a filament for perceptual test (von Frey Filament 0.02 to 2 g; manufactured by North Coast Medical) is pushed onto the plantar region of the hindlimb of the animal, and the up-down method [J. According to Neurosci Methods, 1994, 53 (1): 55-63], the filament was evaluated with 5 to 9 different force filaments.
  • the hindpaw withdrawal behavior of the animals to the mild mechanical stimulation was observed at 0 and 30 minutes after administration, and 50% withdrawal threshold was calculated.
  • the results are shown in FIG.
  • FIG. 5 when the adrenaline ⁇ 1 receptor agonist phenylephrine was administered into the spinal subarachnoid space of wild-type mice, a dose-dependent decrease in hindlimb withdrawal threshold was observed. Thereby, it was confirmed that activation of the adrenergic ⁇ 1 receptor in the spinal cord induces pain-like behavior (allodinia) to mild mechanical stimulation.
  • Example 8 (Involvement of the adrenergic ⁇ 1A receptor on phenylephrine-induced mild mechanical hyperalgesia 2)
  • the adrenergic ⁇ 1A receptor selective knockdown vector AAV-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE and the control vector AAV-FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE are prepared as follows: Made.
  • the sequences of AcGFP and mir30-shRNA were subcloned into pENTR plasmid (manufactured by Themofisher).
  • pENTR-AcGFP-mir30 was replaced with a sequence consisting of the nucleotide sequence shown in SEQ ID NO: 1.
  • the obtained AcGFP-mir30-shRNA cassette was inserted into pAAV-CA-FLEX to obtain pAAV-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE.
  • a portion of the target sequence of pENTR-AcGFP-mir30 is replaced with a sequence consisting of the nucleotide sequence shown in SEQ ID NO: 2, and the obtained AcGFP-mir30-shRNA cassette is inserted into pAAV-CA-FLEX, pAAV- FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE was obtained.
  • Virus vector production using the above plasmid was performed according to a standard method (Sci Rep., 2015 Sep 21; 5: 14306).
  • human embryonic kidney 293 (HEK 293) cells the pAAV-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE or pAAV-FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE and Rep / Cap obtained above It was prepared by transfecting plasmid (pAAV2 / 5) and helper plasmid (pAd Delta F6) (Rep / Cap and pAd Delta F6 were obtained from Vector core of the University of Pennsylvania), and cesium chloride density gradient centrifugation (Gene Ther.
  • AAV2 / 5-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE and control vector AAV2 / 5-FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE are Amicon Ultra 100K filter unit (manufactured by Millipore) ) was dialyzed against a PBS solution containing 0.001% (v / v) Pluronic-F68. Titration of the viral vector was performed using Pico Green fluorometric reagent (manufactured by Molecular Probe) and stored at -80 ° C until use.
  • Anesthesia was performed using a mixed solution of ketamine and xylazine subcutaneously administered at 0.005 mL / g using 8- to 12-week-old Hes5-CreERT2 mice (Nat Commun., 2012 Feb 14; 3: 670). After confirming the effect of anesthesia, the back hair was shaved and a local anesthetic (Xylocaine (registered trademark) 2%) was applied. The skin was incised about 3 cm along the median centering on the thoracic spinal curve.
  • a muscle tissue of 1 mm lateral to the rostral tip of the first lumbar spine of the first lumbar spinal cord was incised with a diameter of about 2 mm to expose the membrane between the thirteenth thoracic spinal cord and the first lumbar spinal disc bone. It is represented by the above-mentioned SEQ ID NO: 1 at two sites of 0.5 to 0.6 ⁇ L each at a virus concentration of 1 ⁇ 10 12 GC / mL at both ends between each left vertebra via a glass capillary (manufactured by Eppendorf).
  • ⁇ 1A receptor selective knockdown vector containing the nucleotide sequence AAV2 / 5-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE is microinjected to knockdown the adrenergic ⁇ 1A receptor of Hes5-positive astrocytes in mice was produced.
  • a vector AAV2 / 5-FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE containing a base sequence represented by SEQ ID NO: 2 was microinjected to prepare a control mouse. After treatment, the skin was stitched with 5-0 silk thread.
  • the suture was applied with a local anesthetic (Xylocaine (registered trademark) 2%) to reduce animal pain.
  • a local anesthetic Xylocaine (registered trademark) 2%) to reduce animal pain.
  • diluted iodine tincture and gentacin (registered trademark) ointment 0.1%) were applied to prevent infection.
  • Example 9 Involvement of descending noradrenaline in capsaicin-induced mild mechanical hyperalgesia Physiological saline or DSP-4 [N- (2-chloroethyl) -N-ethyl-2-bromobenzylamine] (manufactured by Sigma) into the abdominal cavity of 8 to 12 week-old C57BL / 6J mice (manufactured by CLEA Japan, Inc.) was administered at 50 mg / kg. Three days after the administration, 0.16 ⁇ g / ⁇ L of capsaicin solution was administered to 10 ⁇ L of the sole, and then 50% escape threshold was calculated in the same manner as in Experimental Example 4. The results are shown in FIG.
  • noradrenaline nerve was destroyed by administering DSP-4, which is a noradrenaline nerve selective toxin, and the decrease in capsaicin-induced hindpaw withdrawal threshold was suppressed. This revealed that noradrenaline is involved in pain-like behavior (allodinia) to mild mechanical stimulation.
  • an analgesic agent containing, as an active ingredient, a substance that inhibits a signal from astrocyte adrenergic ⁇ 1 receptor, and to select a substance that inhibits a signal from astrocyte adrenergic ⁇ 1 receptor. It is possible to provide a screening method for analgesics, which is characterized.

Landscapes

  • Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pain & Pain Management (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Epidemiology (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention provides: an analgesic that contains, as an active ingredient, a substance that inhibits a signal from an adrenaline α1 receptor of astrocyte; and an analgesic screening method characterized by selecting a substance that inhibits a signal from the adrenaline α1 receptor of astrocyte.

Description

鎮痛剤及びその使用Analgesics and their use
　本発明は、鎮痛剤及びその使用に関する。
　本願は、２０１７年１１月２日に、日本に出願された特願２０１７－２１３１９６号、及び、２０１８年２月５日に、日本に出願された特願２０１８－１８６５３号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to analgesics and their use.
Priority is claimed on Japanese Patent Application No. 2017-213196, filed Nov. 2, 2017, and Japanese Patent Application No. 2018-18653, filed Feb. 5, 2018, on November 2, 2017. The contents of which are incorporated herein by reference.
　末梢組織からの侵害刺激を含む体性感覚情報は、一次求心性感覚神経を介して脊髄後角へと入力し、同部位の神経回路で適切に情報処理され脳へ伝達される。従来、この伝達は神経細胞のみで説明されてきたが、近年、グリア細胞が神経活動に大きく影響し、感覚情報伝達の変調に関与することが示されてきた（非特許文献１）。一方、グリア細胞の中で数的に最も多いアストロサイトは神経伝達物質受容体を発現し、空間的にはシナプスと接触していることからシナプス活動への関与が想定されている（非特許文献２）。しかしながら、感覚情報伝達における脊髄でのアストロサイトの役割は未だ分かっていない。 Somatosensory information including noxious stimuli from peripheral tissues is input to the spinal dorsal horn via the primary afferent sensory nerves, appropriately processed by the neural circuit at the same site, and transmitted to the brain. Conventionally, this transmission has been described using only nerve cells, but in recent years glial cells have been shown to greatly affect nerve activity and to be involved in modulation of sensory communication (Non-patent Document 1). On the other hand, astrocytes, which are the most numerous among glial cells, express neurotransmitter receptors and are spatially in contact with synapses, and thus involvement in synaptic activity is assumed (non-patent literature) 2). However, the role of astrocytes in the spinal cord in sensory signal transduction is still unknown.
　アドレナリンα受容体には、α１受容体とα２受容体がある。このうち、アドレナリンα１受容体にはα１Ａ、α１Ｂ及びα１Ｄ受容体の３つのサブタイプがあり、血管収縮、瞳孔散大、立毛、前立腺収縮等に関与していることが知られている。しかしながら、アストロサイトにおけるアドレナリンα１受容体の役割については知られていない。 Adrenergic alpha receptors include alpha 1 receptors and alpha 2 receptors. Among these, there are three subtypes of adrenaline α1 receptors, α1A, α1B and α1D receptors, and it is known that they are involved in vasoconstriction, pupil dilation, pistatic hair, prostate contraction and the like. However, the role of adrenergic alpha 1 receptors in astrocytes is unknown.
　このような背景のもと、新たなタイプの鎮痛剤の確立が求められている。本発明は、脊髄のアストロサイトを標的とした新たな鎮痛剤を提供することを目的とする。 Under such background, establishment of a new type of analgesic is required. The present invention aims to provide a new analgesic that targets astrocytes in the spinal cord.
　本発明は以下の態様を含む。
［１］アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を有効成分として含有する鎮痛剤。
［２］前記アドレナリンα１受容体からのシグナルを阻害する物質が、アドレナリンα１受容体拮抗剤又はアドレナリンα１受容体発現抑制剤である［１］に記載の鎮痛剤。
［３］前記アストロサイトが脊髄後角の表層に存在するアストロサイトである、［１］又は［２］に記載の鎮痛剤。
［４］前記脊髄後角の表層に存在するアストロサイトが、Ｈｅｓ５を発現しているアストロサイトである、［３］に記載の鎮痛剤。
［５］前記アドレナリンα１受容体が、アドレナリンα１Ａ受容体である、［１］～［４］のいずれか一項に記載の鎮痛剤。
［６］前記鎮痛剤が、慢性疼痛治療剤である、［１］～［５］のいずれか一項に記載の鎮痛剤。
［７］アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を選択することを特徴とする、鎮痛剤のスクリーニング方法。
［８］前記アドレナリンα１受容体からのシグナルを阻害する物質が、アドレナリンα１受容体拮抗剤又はアドレナリンα１受容体発現抑制剤である［７］に記載の方法。
［９］前記アストロサイトが脊髄後角の表層に存在するアストロサイトである、［７］又は［８］に記載の方法。
［１０］前記脊髄後角の表層に存在するアストロサイトが、Ｈｅｓ５を発現しているアストロサイトである、［９］に記載の方法。
［１１］前記アドレナリンα１受容体が、アドレナリンα１Ａ受容体である、［７］～［１０］のいずれか一項に記載の方法。
［１２］前記鎮痛剤が、慢性疼痛治療剤である、［７］～［１１］のいずれか一項に記載の方法。 The present invention includes the following aspects.
[1] An analgesic containing, as an active ingredient, a substance that inhibits a signal from astrocyte adrenergic α1 receptor.
[2] The analgesic according to [1], wherein the substance that inhibits the signal from the adrenergic α1 receptor is an adrenergic α1 receptor antagonist or an adrenergic α1 receptor expression inhibitor.
[3] The analgesic according to [1] or [2], wherein the astrocytes are astrocytes present in the surface layer of the spinal dorsal horn.
[4] The analgesic according to [3], wherein the astrocytes present in the surface layer of the spinal dorsal horn are astrocytes expressing Hes5.
[5] The analgesic according to any one of [1] to [4], wherein the adrenergic α1 receptor is an adrenergic α1A receptor.
[6] The analgesic according to any one of [1] to [5], wherein the analgesic is a chronic pain therapeutic agent.
[7] A screening method for an analgesic agent, which comprises selecting a substance that inhibits a signal from adrenergic α1 receptor of astrocytes.
[8] The method according to [7], wherein the substance that inhibits the signal from the adrenergic α1 receptor is an adrenergic α1 receptor antagonist or an adrenergic α1 receptor expression inhibitor.
[9] The method according to [7] or [8], wherein the astrocytes are astrocytes present in the surface layer of the spinal dorsal horn.
[10] The method according to [9], wherein the astrocytes present in the surface layer of the spinal dorsal horn are astrocytes expressing Hes5.
[11] The method according to any one of [7] to [10], wherein the adrenergic α1 receptor is an adrenergic α1A receptor.
[12] The method according to any one of [7] to [11], wherein the analgesic is a chronic pain therapeutic agent.
　本発明によれば、アストロサイトのアドレナリンα１受容体を標的とした新たな鎮痛剤を提供することができる。 According to the present invention, it is possible to provide a new analgesic that targets the astrocyte adrenergic α1 receptor.
実験例１において、脊髄後角において、Ｈｅｓ５を発現しているアストロサイトを示した図である。FIG. 7 shows astrocytes expressing Hes5 in spinal dorsal horn in Experimental Example 1. 実験例２及び３において、カプサイシンによる脊髄後角のアストロサイトの活動とノルアドレナリン神経選択的毒素およびアドレナリンα１Ａ受容体拮抗剤の効果を示した図である。In Experimental Examples 2 and 3, it is the figure which showed the activity of the astrocyte of the spinal dorsal horn by capsaicin, and the effect of a noradrenaline neuroselective toxin and an adrenergic alpha 1A receptor antagonist. 左図は、実験例４において、ｈＭ３Ｄｑ受容体が脊髄後角表層のＨｅｓ５陽性アストロサイトに発現していることを示した図である。右図は、実験例４において、Ｈｅｓ５陽性脊髄アストロサイト刺激時の軽度機械刺激に対するマウスの行動解析を示した図である。The left figure shows that hM3Dq receptor is expressed in Hes5-positive astrocytes in the spinal dorsal horn surface layer in Experimental Example 4. The right figure is a figure which showed the behavioral analysis of the mouse | mouth with respect to the light mechanical stimulation at the time of Hes 5 positive spinal astrocyte stimulation in Experimental example 4. FIG. 左図は、実験例５における、コーン油投与群及びタモキシフェン投与群のｈＭ３Ｄｑ受容体の発現を示した図である。右図は、実験例５において、Ｈｅｓ５陰性脊髄アストロサイト刺激時の軽度機械刺激に対するマウスの行動解析を示した図である。The left figure shows the expression of hM3Dq receptor in the corn oil administration group and the tamoxifen administration group in Experimental Example 5. The right figure is a figure which showed the behavioral analysis of the mouse | mouth with respect to the light mechanical stimulation at the time of Hes 5 negative spinal astrocyte stimulation in example 5 of an experiment. 実験例６において、アドレナリンα１受容体アゴニストを脊髄くも膜下腔内投与した時の軽度機械刺激痛覚過敏を示した図である。FIG. 10 is a diagram showing mild mechanical stimulation hyperalgesia when an adrenaline α1 receptor agonist was administered intrathecally in Experimental Example 6; 実験例７及び８において、アドレナリンα１Ａ受容体拮抗剤を投与した時、及びアドレナリンα１Ａ受容体発現を抑制した時のフェニレフリン誘発の軽度機械刺激痛覚過敏に対する効果を示した図である。FIG. 17 shows the effect on phenylephrine-induced mild mechanical stimulation hyperalgesia when administration of an adrenergic α1A receptor antagonist and suppression of adrenergic α1A receptor expression in Experimental Examples 7 and 8. 実験例９において、カプサイシン誘発の軽度機械刺激痛覚過敏に対する下行性ノルアドレナリン神経の関与を示した図である。In Experimental Example 9, it is a figure showing involvement of descending noradrenaline nerve in capsaicin-induced mild mechanical stimulation hyperalgesia. 実験例１０において、カプサイシン誘発の軽度機械刺激痛覚過敏に対する脊髄後角アストロサイトのアドレナリンα１Ａ受容体発現抑制の効果を示した図である。In Experimental example 10, it is the figure which showed the effect of adrenergic-alpha 1A receptor expression suppression of the spinal dorsal horn astrocyte on the capsaicin-induced mild mechanical stimulation hyperalgesia.
［鎮痛剤］
　１実施形態において、本発明は、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を有効成分として含有する鎮痛剤を提供する。 Analgesic
In one embodiment, the present invention provides an analgesic containing, as an active ingredient, a substance that inhibits a signal from astrocyte α1 receptor of astrocytes.
　痛みは末梢組織である皮膚等に痛み刺激が加わると皮膚の神経が興奮し、脊髄に伝わる。脊髄に伝えられた情報は、脳に伝わって、初めて痛みを感じる。この痛みを感じるルートの他に、脳からは脊髄後角へ下行性にノルアドレナリン神経が伸びており、痛み刺激により、この下行性ノルアドレナリン神経が興奮すると、神経末端からノルアドレナリンが放出される。 Pain is transmitted to the spinal cord when nerves in the skin are excited when pain stimulation is applied to the skin which is a peripheral tissue. The information transmitted to the spinal cord is transmitted to the brain and only when it feels pain. In addition to this painful route, noradrenaline nerves extend downward from the brain to the spinal dorsal horn, and upon stimulation of this descending noradrenaline nerve by pain stimulation, noradrenaline is released from the nerve terminal.
　非神経細胞であるアストロサイトは神経と神経のつなぎ目に存在し、神経と神経の間の情報伝達に関与する。本発明者らは、脊髄後角の中でも表層に局在するアストロサイト亜集団と脊髄後角深層部に局在するアストロサイト亜集団があることを見出した。さらに、下行性ノルアドレナリン神経の興奮によって神経末端から放出されたノルアドレナリンが、脊髄後角の表層に存在するアストロサイトのアドレナリンα１受容体に結合すると、アドレナリンα１受容体からのシグナルが末梢組織から入力した痛み刺激を増強させることを見出した。 Astrocytes, which are non-neuronal cells, exist at the junction of nerves and nerves, and are involved in signal transmission between nerves. The present inventors have found that among the dorsal horn of the spinal cord, there are an astrocyte subpopulation localized in the surface layer and an astrocytic subpopulation localized in the deep region of the spinal dorsal horn. Furthermore, when noradrenaline released from nerve terminals by excitation of descending noradrenaline was bound to astrocyte adrenergic α1 receptors present on the surface layer of spinal dorsal horn, signals from adrenergic α1 receptors were input from peripheral tissues It was found to enhance pain stimulation.
　従って、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質は、末梢組織から入力した痛み刺激を抑制することができるため、鎮痛剤として用いることができる。当該鎮痛剤は、痛みの治療、緩和及び／又は予防のために用いることができる。 Therefore, a substance that inhibits the signal from astrocyte adrenergic α1 receptor can be used as an analgesic because it can suppress pain stimulation input from peripheral tissues. The analgesic can be used for the treatment, alleviation and / or prevention of pain.
　本実施形態の鎮痛剤において、アドレナリンα１受容体からのシグナルを阻害する物質は、アドレナリンα１受容体からのシグナルを阻害する活性を有していれば、特に制限はないが、例えば、アドレナリンα１受容体拮抗剤、アドレナリンα１受容体発現抑制剤等が挙げられる。アドレナリンα１受容体拮抗剤としては、アドレナリンα１受容体に作用して、アドレナリンα１受容体からのシグナルを阻害できる能力を有する物質であれば、特に制限はなく、アドレナリンα１受容体遮断剤と呼ばれることもある。アドレナリンα１受容体拮抗剤の具体例としては、シロドシン、タムスロシン、ナフトピジル、テラゾシン、ウラピジル、プラゾシン、アルフゾシン、ドキサゾシン、ブナゾシン等が挙げられる。他に、アドレナリンα１受容体からのシグナルを阻害する物質は、アドレナリンα1受容体の活性を抑制する物質であってもよい。アドレナリンα1受容体の活性を抑制する物質としては、例えば、アドレナリンα1受容体に対する特異的結合物質等が挙げられる。ここで、特異的結合物質としては、抗体、抗体断片、アプタマー、低分子化合物等が挙げられる。 In the analgesic of the present embodiment, the substance that inhibits the signal from the adrenergic α1 receptor is not particularly limited as long as it has an activity to inhibit the signal from the adrenergic α1 receptor, for example, adrenergic α1 receptor Examples include body antagonists, adrenaline α1 receptor expression inhibitors and the like. The adrenergic alpha 1 receptor antagonist is not particularly limited as long as it is a substance having an ability to act on adrenergic alpha 1 receptor and inhibit a signal from the adrenaline alpha 1 receptor, and it is called an adrenaline alpha 1 receptor blocker There is also. Specific examples of the adrenergic α1 receptor antagonist include silodosin, tamsulosin, naphthopidil, terazosin, urapidil, prazosin, alfuzosin, doxazosin, bunazosin and the like. Alternatively, the substance that inhibits the signal from the adrenergic α1 receptor may be a substance that inhibits the activity of the adrenergic α1 receptor. Examples of the substance that suppresses the activity of the adrenaline α1 receptor include a specific binding substance to the adrenaline α1 receptor, and the like. Here, specific binding substances include antibodies, antibody fragments, aptamers, low molecular weight compounds and the like.
　アドレナリンα１受容体発現抑制剤としては、アドレナリンα１受容体の発現を抑制できる物質であれば、特に制限はなく、例えば、アドレナリンα１受容体の発現を抑制するｓｈ（ｓｈｏｒｔ　ｈａｉｒｐｉｎ）ＲＮＡ、ｓｉＲＮＡ、ｍｉＲＮＡ、リボザイム、アンチセンス核酸、低分子化合物等が挙げられる。アドレナリンα１受容体の発現を抑制するｓｈＲＮＡとしては、例えば、配列番号１で表される塩基配列を含むＤＮＡをターゲット配列とするｓｈＲＮＡ等が挙げられる。 The adrenaline α1 receptor expression inhibitor is not particularly limited as long as it is a substance capable of suppressing the expression of adrenaline α1 receptor, and, for example, sh (short hairpin) RNA, siRNA, miRNA which suppresses the expression of adrenaline α1 receptor And ribozymes, antisense nucleic acids, low molecular weight compounds and the like. Examples of the shRNA that suppresses the expression of adrenergic α1 receptor include, for example, an shRNA that uses a DNA containing the base sequence represented by SEQ ID NO: 1 as a target sequence.
　前記アストロサイトのアドレナリンα１受容体は、α１Ａ、α１Ｂ及びα１Ｄの何れでもよい。ある実施形態では、アドレナリンα１受容体は、α１Ａ受容体である。 The astrocyte adrenergic α1 receptor may be any of α1A, α1B and α1D. In one embodiment, the adrenergic α1 receptor is an α1A receptor.
　本実施形態において、アストロサイトは、脊髄に存在するアストロサイトであってもよい。ある実施形態では、アストロサイトは、脊髄後角の表層に存在するアストロサイトである。脊髄後角の表層に存在するアストロサイトとしては、転写因子であるＨｅｓ５を発現しているアストロサイト等が挙げられる。 In the present embodiment, the astrocytes may be astrocytes present in the spinal cord. In one embodiment, the astrocytes are astrocytes present in the surface layer of the spinal dorsal horn. Examples of astrocytes present in the surface layer of the dorsal horn of the spinal cord include astrocytes expressing the transcription factor Hes5.
　本実施形態の鎮痛剤としては、痛みを抑制する効果があれば、特に制限はなく、一過性の痛みを抑制する効果を有する鎮痛剤であっても、神経障害性疼痛等の慢性疼痛治療剤のような、持続的な痛みに対して抑制する効果を有する鎮痛剤であってもよい。 The analgesic according to the present embodiment is not particularly limited as long as it has an effect of suppressing pain, and even if the analgesic has an effect of suppressing transient pain, chronic pain treatment such as neuropathic pain It may be an analgesic that has an effect of suppressing persistent pain, such as an agent.
　本実施形態の鎮痛剤は、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質に加えて、薬学的に許容される担体を含有してもよい。 The analgesic of the present embodiment may contain a pharmaceutically acceptable carrier in addition to the substance that inhibits the signal from astrocyte α1 receptor of astrocytes.
　本実施形態の鎮痛剤は、例えば、錠剤、カプセル剤、エリキシル剤、マイクロカプセル剤等の形態で経口的に、あるいは、注射剤、坐剤、皮膚外用剤等の形態で非経口的に投与することができる。皮膚外用剤としては、より具体的には、軟膏剤、貼付剤等の剤型が挙げられる。 The analgesic according to this embodiment is orally administered, for example, in the form of tablets, capsules, elixirs, microcapsules or the like, or parenterally in the form of injections, suppositories, external preparations for skin, etc. be able to. More specifically, examples of the skin external preparation include dosage forms such as ointments and patches.
　本実施形態の鎮痛剤は、中枢に移行する形態で投与することが好ましい。中枢に移行する形態で投与する方法としては、例えば、有効成分である、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を、血液脳関門を通過するようにデザインして投与する方法や、本実施形態の鎮痛剤を脊髄内に投与する方法等が挙げられる。 The analgesic agent of the present embodiment is preferably administered in the form of central transfer. As a method of administration in the form of central transfer, for example, a method of designing an active ingredient, a substance that inhibits a signal from adrenergic α1 receptor of astrocyte, by design so as to cross the blood-brain barrier or And the method etc. of administering the analgesic of this embodiment in the spinal cord.
　薬学的に許容される担体としては、通常医薬組成物の製剤に用いられるものを特に制限なく用いることができる。より具体的には、例えば、ゼラチン、コーンスターチ、トラガントガム、アラビアゴム等の結合剤；デンプン、結晶性セルロース等の賦形剤；アルギン酸等の膨化剤；水、エタノール、グリセリン等の注射剤用溶剤；ゴム系粘着剤、シリコーン系粘着剤等の粘着剤等が挙げられる。 As the pharmaceutically acceptable carrier, those generally used for the preparation of pharmaceutical compositions can be used without particular limitation. More specifically, for example, binders such as gelatin, corn starch, tragacanth gum and gum arabic; excipients such as starch and crystalline cellulose; swelling agents such as alginic acid; solvents for injection such as water, ethanol and glycerin; Examples of the pressure-sensitive adhesive include rubber-based pressure-sensitive adhesives and silicone-based pressure-sensitive adhesives.
　本実施形態の鎮痛剤は添加剤を含んでいてもよい。添加剤としては、ステアリン酸カルシウム、ステアリン酸マグネシウム等の潤滑剤；ショ糖、乳糖、サッカリン、マルチトール等の甘味剤；ペパーミント、アカモノ油等の香味剤；ベンジルアルコール、フェノール等の安定剤；リン酸塩、酢酸ナトリウム等の緩衝剤；安息香酸ベンジル、ベンジルアルコール等の溶解補助剤；酸化防止剤；防腐剤等が挙げられる。 The analgesic of the present embodiment may contain an additive. As additives, lubricants such as calcium stearate and magnesium stearate; sweeteners such as sucrose, lactose, saccharin and maltitol; flavoring agents such as peppermint and red mono oil; stabilizers such as benzyl alcohol and phenol; phosphoric acid Salts, buffers such as sodium acetate; solubilizers such as benzyl benzoate and benzyl alcohol; antioxidants; preservatives and the like.
　本実施形態の鎮痛剤は、上述したアストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質、薬学的に許容される担体及び添加剤を適宜組み合わせて、一般に認められた製薬実施に要求される単位用量形態で混和することによって製剤化することができる。 The analgesic according to the present embodiment is required for generally accepted pharmaceutical practice, using an appropriate combination of the above-mentioned substance that inhibits the astrocyte signal from the adrenergic α1 receptor, a pharmaceutically acceptable carrier and an additive as described above. It can be formulated by blending in unit dose form.
　本実施形態の鎮痛剤の投与量は、対象の症状、体重、年齢、性別等によって異なり、一概には決定できないが、経口投与の場合には、有効成分である、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を、投与単位形態あたり、例えば０．１～１００ｍｇ／ｋｇ体重、例えば１～５０ｍｇ／ｋｇ体重、例えば、１～２０ｍｇ／ｋｇ体重、例えば１～１０ｍｇ／ｋｇ体重投与すればよい。また、非経口的に投与する場合には、例えば、注射剤の場合には、投与単位形態あたり、例えば０．０１～５０ｍｇ、例えば０．１～３０ｍｇ、例えば０．１～２０ｍｇ、例えば０．１～１０ｍｇの有効成分を投与すればよい。 The dose of the analgesic according to the present embodiment varies depending on the condition, body weight, age, sex, etc. of the subject, and can not be determined indiscriminately. The substance that inhibits the signal from is preferably administered, for example, 0.1 to 100 mg / kg body weight, such as 1 to 50 mg / kg body weight, such as 1 to 20 mg / kg body weight, such as 1 to 10 mg / kg body weight, per dosage unit form. Just do it. In the case of parenteral administration, for example, in the case of injections, the dosage unit form is, for example, 0.01 to 50 mg, for example 0.1 to 30 mg, for example 0.1 to 20 mg, for example, 0.2 to 50 mg. 1 to 10 mg of the active ingredient may be administered.
　また、本実施形態の鎮痛剤の１日あたりの投与量は、対象の症状、体重、年齢、性別等によって異なり、一概には決定できないが、例えば、成人１日あたり０．１～１０ｍｇ／ｋｇ体重の有効成分を１日１回又は２～４回程度に分けて投与すればよい。 The daily dose of the analgesic according to the present embodiment varies depending on the condition, body weight, age, sex, etc. of the subject and can not be generally determined, but for example, 0.1 to 10 mg / kg per day for adults The active ingredient of body weight may be administered once a day or divided into 2 to 4 times.
　本実施形態の鎮痛剤が、鎮痛作用を有することは、公知の鎮痛作用の評価系を用いて評価することができる。例えば、下記のカプサイシンモデル動物を用いた評価系により調べることができる。
［鎮痛作用の評価］
　８～１２週齢のＣ５７ＢＬ／６Ｊマウス（日本クレア社製）に被験薬を投与した後、０．１６μｇ／μＬのカプサイシン溶液１０μＬをマウスの後肢足裏に投与し、投与後、マウスの投与側後肢足底部に知覚試験用フィラメント(ｖｏｎ　Ｆｒｅｙ　Ｆｉｌａｍｅｎｔ　０．０２～２ｇ、Ｎｏｒｔｈ　Ｃｏａｓｔ　Ｍｅｄｉｃａｌ社製)を押しあて、アップダウン法［Ｊ．Ｎｅｕｒｏｓｃｉ　Ｍｅｔｈｏｄｓ，１９９４，５３（１）：５５－６３］に従って５回から９回の異なった力のフィラメントで評価する。その軽度機械刺激に対する動物の後肢逃避行動を経時的に観察し、下記式により５０％逃避閾値（Ｐａｗ　ｗｉｔｈｄｒａｗａｌ　ｔｈｒｅｓｈｏｌｄ）を算出する。 It can be evaluated that the analgesic of this embodiment has an analgesic action using the evaluation system of the well-known analgesic action. For example, it can investigate by the following evaluation system using a capsaicin model animal.
[Evaluation of analgesic activity]
The test drug is administered to 8 to 12-week-old C57BL / 6J mice (manufactured by CLEA Japan, Inc.), 10 μL of a 0.16 μg / μL capsaicin solution is then administered to the hind footpads of the mice, and after administration, the administration side of the mice A sensory test filament (0.02 to 2 g of von Frey Filament, manufactured by North Coast Medical) is pressed to the sole of the hind leg, and the up-down method [J. Evaluate with filaments of 5 to 9 different forces according to Neurosci Methods, 1994, 53 (1): 55-63]. The hindpaw withdrawal behavior of the animal in response to the light mechanical stimulation is observed over time, and the 50% withdrawal threshold is calculated by the following equation.
５０％逃避閾値（ｇ）＝１０（ｘｆ＋（ｋ×０．２２４））／１００００
ｘｆ：最後に用いたフィラメントの値
ｋ：逃避行動あり或いは逃避行動なしのパターンにより決定される値 50% escape threshold (g) = 10 (xf + (k × 0.224)) / 10000
xf: Value of last used filament k: Value determined by the pattern with or without escape behavior
［鎮痛薬のスクリーニング方法］
　１実施形態において、本発明は、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を選択することを特徴とする、鎮痛剤のスクリーニング方法を提供する。
　アドレナリンα１受容体からのシグナルを阻害する物質は、アドレナリンα１受容体からのシグナルを阻害する活性を有していれば、特に制限はないが、例えば、アドレナリンα１受容体拮抗剤、アドレナリン受容体発現抑制剤等が挙げられる。アドレナリンα１受容体拮抗剤としては、アドレナリンα１受容体に作用して、アドレナリンα１受容体からのシグナルを阻害できる活性を有していれば、特に制限はなく、アドレナリンα１受容体遮断剤と呼ばれることもある。アドレナリンα１受容体発現抑制剤としては、アドレナリンα１受容体の発現を抑制できる物質であれば、特に制限はなく、例えば、アドレナリンα１受容体の発現を抑制するｓｈ（ｓｈｏｒｔ　ｈａｉｒｐｉｎ）ＲＮＡ等が挙げられる。前記アストロサイトのアドレナリンα１受容体は、α１Ａ、α１Ｂ及びα１Ｄの何れでもよい。ある実施形態では、アドレナリンα１受容体は、α１Ａ受容体である。 [Method of screening for analgesics]
In one embodiment, the present invention provides a method of screening for analgesics, which comprises selecting a substance that inhibits the signal from astrocyte adrenergic α1 receptor.
The substance that inhibits the signal from the adrenergic α1 receptor is not particularly limited as long as it has an activity to inhibit the signal from the adrenergic α1 receptor, for example, an adrenergic α1 receptor antagonist, adrenergic receptor expression An inhibitor etc. are mentioned. The adrenergic α1 receptor antagonist is not particularly limited as long as it has an activity capable of inhibiting a signal from the adrenergic α1 receptor by acting on the adrenergic α1 receptor, and is referred to as an adrenergic α1 receptor blocker There is also. The adrenaline α1 receptor expression inhibitor is not particularly limited as long as it is a substance capable of suppressing the expression of adrenaline α1 receptor, and examples thereof include sh (short hairpin) RNA that suppresses the expression of adrenaline α1 receptor and the like . The astrocyte adrenergic α1 receptor may be any of α1A, α1B and α1D. In one embodiment, the adrenergic α1 receptor is an α1A receptor.
　本実施形態において、アストロサイトは、脊髄に存在するアストロサイトであってもよい。ある実施形態では、アストロサイトは、脊髄後角の表層に存在するアストロサイトである。脊髄後角の表層に存在するアストロサイトとしては、転写因子であるＨｅｓ５を発現しているアストロサイト等が挙げられる。 In the present embodiment, the astrocytes may be astrocytes present in the spinal cord. In one embodiment, the astrocytes are astrocytes present in the surface layer of the spinal dorsal horn. Examples of astrocytes present in the surface layer of the dorsal horn of the spinal cord include astrocytes expressing the transcription factor Hes5.
　本実施形態の鎮痛剤としては、痛みを抑制する効果があれば、特に制限はなく、一過性の痛みを抑制する効果を有する鎮痛剤であっても、神経障害性疼痛等の慢性疼痛治療剤のような、持続的な痛みに対して抑制する効果を有する鎮痛剤であってもよい。 The analgesic according to the present embodiment is not particularly limited as long as it has an effect of suppressing pain, and even if the analgesic has an effect of suppressing transient pain, chronic pain treatment such as neuropathic pain It may be an analgesic that has an effect of suppressing persistent pain, such as an agent.
　本実施形態のスクリーニング方法としては、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を選択できる方法であれば、特に制限はなく、例えば、アストロサイトのアドレナリンα１受容体を発現する細胞に、被験物質を添加し、アドレナリンα１受容体のアゴニストを添加したときに生じる、該アドレナリンα１受容体からのシグナルを阻害する被験物質を選択する方法、アストロサイトのアドレナリンα１受容体を発現する細胞に、被験物質存在下に、放射性物質等により標識した選択的なリガンドを結合させ、当該リガンドと前記受容体との結合を阻害する被験物質を選択する方法等が挙げられる。 The screening method of the present embodiment is not particularly limited as long as it is a method capable of selecting a substance that inhibits the signal from adrenergic α1 receptor of astrocytes, for example, to cells expressing adrenergic α1 receptors of astrocytes A method for selecting a test substance that inhibits a signal from the adrenergic α1 receptor, which is generated when a test substance is added and an agonist of the adrenergic α1 receptor is added, to a cell expressing the adrenergic α1 receptor of astrocytes In the presence of a test substance, a selective ligand labeled with a radioactive substance or the like is bound, and a method of selecting a test substance that inhibits the binding between the ligand and the receptor can be mentioned.
　また、本実施形態のスクリーニング方法としては、ＤＲＥＡＤＤ（Ｄｅｓｉｇｎｅｒ　Ｒｅｃｅｐｔｏｒｓ　Ｅｘｃｌｕｓｉｖｅｌｙ　Ａｃｔｉｖａｔｅｄ　ｂｙ　Ｄｅｓｉｇｎｅｒ　Ｄｒｕｇ）法を用いた方法も例示される。例えば、内因性アセチルコリンに不応答である、変異型ヒトムスカリンＭ３受容体ｈＭ３Ｄｑを組み込んだアデノ随伴ウイルス（ＡＡＶ）を、アストロサイトでＣｒｅが発現しているＧｆａｐ－Ｃｒｅマウス、Ｈｅｓ５－ＣｒｅＥＲＴ２マウス等の脊髄後角へ微量注入し、脊髄後角アストロサイト特異的にｈＭ３Ｄｑ受容体を発現させ、ｈＭ３Ｄｑ受容体を該受容体のアゴニストであるクロザピン－Ｎ－オキサイド（ｃｌｏｚａｐｉｎｅ－Ｎ－ｏｘｉｄｅ；ＣＮＯ）で活性化し、感覚刺激による行動の変化を測定し、脊髄後角アストロサイトの活性化により誘発される、軽度機械刺激に対する疼痛様行動（アロディニア）が、被験物質を予め投与した時に抑制されるか否かを評価し、該疼痛様行動を抑制する物質を選択することにより、鎮痛剤をスクリーニングすることができる。 In addition, as a screening method of the present embodiment, a method using a method of DREADD (Designer Receptors Exclusively Activated by Designer Drug) is also exemplified. For example, an adeno-associated virus (AAV) incorporating a mutant human muscarinic M3 receptor hM3Dq that is unresponsive to endogenous acetylcholine, such as Gfap-Cre mice and Hes5-CreERT2 mice expressing Cre at astrocytes It is microinjected into the dorsal horn of the spinal cord, and the hM3Dq receptor is expressed in a manner specific to the spinal dorsal horn astrocytes, and the hM3Dq receptor is activated with clozapine-N-oxide (CNO), an agonist of the receptor. Whether or not pain-like behavior (allodinia) to mild mechanical stimulation induced by activation of spinal dorsal horn astrocytes is suppressed when the test substance is administered in advance. Analgesia by evaluating the substance and selecting a substance that suppresses the pain-like behavior It can be screened.
　本実施形態のスクリーニング方法により得られるアストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質は、末梢組織から入力した痛み刺激を抑制することができるため、鎮痛剤として用いることができる。当該鎮痛剤は、痛みの治療、緩和及び／又は予防のために用いることができる。 The substance that inhibits the signal from the adrenergic α1 receptor of astrocytes obtained by the screening method of the present embodiment can be used as an analgesic because it can suppress pain stimulation inputted from peripheral tissues. The analgesic can be used for the treatment, alleviation and / or prevention of pain.
　１実施形態において、本発明は、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質の有効量を、対象に投与する工程を備える、痛みの治療、緩和及び／又は予防方法を提供する。本実施形態において、痛みとしては、特に制限はなく、一過性の痛みであっても、神経障害性疼痛等の慢性疼痛のような、持続的な痛みであってもよい。また、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質としては、上述した物質が挙げられる。
　対象に投与する工程としては、中枢に移行する形態で投与する工程が好ましい。中枢に移行する形態で投与する工程としては、例えば、有効成分である、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を、血液脳関門を通過するようにデザインして投与する工程や、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を脊髄内に投与する工程等が挙げられる。 In one embodiment, the present invention provides a method for treating, alleviating and / or preventing pain, comprising the step of administering to a subject an effective amount of a substance that inhibits a signal from astrocyte α1 receptor of astrocytes. In the present embodiment, the pain is not particularly limited, and may be transient pain or persistent pain such as chronic pain such as neuropathic pain. Further, as the substance that inhibits the signal from astrocyte adrenergic α1 receptor, the above-mentioned substances can be mentioned.
As a process of administering to a subject, a process of administering in the form transferred to the center is preferable. The step of administering in a form that moves to the center includes, for example, a step of designing and administering an active ingredient, a substance that inhibits a signal from adrenergic α1 receptor of astrocyte, so as to cross the blood-brain barrier or And the step of administering to the spinal cord a substance that inhibits the signal from the astrocyte adrenergic α1 receptor.
　１実施形態において、本発明は、痛みの治療、緩和及び／又は予防における使用ための、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を提供する。本実施形態において、痛みとしては、特に制限はなく、一過性の痛みであっても、神経障害性疼痛等の慢性疼痛のような、持続的な痛みであってもよい。また、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質としては、上述した物質が挙げられる。 In one embodiment, the present invention provides a substance that inhibits the signal from astrocyte adrenergic alpha 1 receptor for use in the treatment, alleviation and / or prevention of pain. In the present embodiment, the pain is not particularly limited, and may be transient pain or persistent pain such as chronic pain such as neuropathic pain. Further, as the substance that inhibits the signal from astrocyte adrenergic α1 receptor, the above-mentioned substances can be mentioned.
　１実施形態において、本発明は、鎮痛剤を製造するための、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質の使用を提供する。本実施形態において、鎮痛剤としては、痛みを抑制する効果があれば、特に制限はなく、一過性の痛みを抑制する効果を有する鎮痛剤であっても、神経障害性疼痛等の慢性疼痛治療剤のような、持続的な痛みに対して抑制する効果を有する鎮痛剤であってもよい。また、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質としては、上述した物質が挙げられる。当該鎮痛剤は、痛みの治療、緩和及び／又は予防のために用いることができる。 In one embodiment, the present invention provides the use of a substance that inhibits a signal from astrocyte adrenergic α1 receptor for producing an analgesic. In the present embodiment, the analgesic is not particularly limited as long as it has an effect of suppressing pain, and even an analgesic having an effect of suppressing transient pain, chronic pain such as neuropathic pain It may be an analgesic that has an effect of suppressing persistent pain, such as a therapeutic agent. Further, as the substance that inhibits the signal from astrocyte adrenergic α1 receptor, the above-mentioned substances can be mentioned. The analgesic can be used for the treatment, alleviation and / or prevention of pain.
　次に実験例を示して本発明を更に詳細に説明するが、本発明は以下の実験例に限定されるものではない。 The present invention will next be described in more detail by way of experimental examples, but the present invention is not limited to the following experimental examples.
［実験例１］
（脊髄後角の表層に存在するアストロサイト集団の同定）
　Ｈｅｓ５－ＣｒｅＥＲＴ２マウス（Ｎａｔ　Ｃｏｍｍｕｎ．，２０１２　Ｆｅｂ　１４；３：６７０）及びＲｏｓａ－ｔｄＴｏｍａｔｏマウス（Ｎａｔ　Ｎｅｕｒｏｓｃｉ．，２０１０　Ｊａｎ；１３（１）１３３－４０）を掛け合わせた８～１２週齢のＨｅｓ－５ＣｒｅＥＲＴ２／Ｒｏｓａ－ｔｄＴｏｍａｔｏマウスに、コーン油を使用し２０ｍｇ／ｍＬに調製したタモキシフェン（シグマ社製）溶液を２ｍｇ／０．１ｍＬを１日１回１０日間、マウスへ腹腔内投与することによりＣｒｅリコンビナーゼ活性を誘導した。 [Experimental Example 1]
(Identification of astrocyte population in the surface layer of spinal dorsal horn)
8-12 week-old Hes- combined with Hes5-CreERT2 mouse (Nat Commun., 2012 Feb 14; 3: 670) and Rosa-tdTomato mouse (Nat Neurosci., 2010 Jan; 13 (1) 133-40) Cre recombinase by intraperitoneal administration of 2 mg / 0.1 mL of tamoxifen (manufactured by Sigma) solution adjusted to 20 mg / mL using corn oil to mice once daily for 10 days to 5 CreERT2 / Rosa-tdTomato mice The activity was induced.
　タモキシフェンを投与終了時から１週間後に、ペントバルビタールナトリウム製剤（ソムノペンチル（登録商標）注射液、共立製薬社製）の腹腔内投与でマウスを麻酔し、翻尾反射の消失を確認した後、直ちに腹部を切開し、心臓よりリン酸緩衝液（ＰＢＳ）２０ｍＬを灌流して脱血した。続いて、氷冷した４％パラホルムアルデヒド（ｐａｒａｆｏｒｍａｌｄｅｈｙｄｅ；ＰＦＡ）溶液５０ｍＬを灌流することにより全身組織を固定後、動物を約１時間氷冷した。続いて、脊髄を摘出し、実体顕微鏡下で第４腰髄を単離した。単離した脊髄は氷中静置後４～５時間経過するまで４％ＰＦＡ溶液中にて浸漬固定を行った。その後、冷３０％ショ糖溶液中に脊髄を移し、４℃で２４時間振盪した。脊髄をＯ．Ｃ．Ｔ．Ｃｏｍｐｏｕｎｄ（サクラファインテックジャパン社製）に包埋し、ドライアイス上で急速凍結させ、使用するまで－８０℃で保存した。 One week after the end of administration of tamoxifen, the mouse was anesthetized by intraperitoneal administration of pentobarbital sodium preparation (Somnopentyl (R) injection, manufactured by Kyoritsu Pharmaceutical Co., Ltd.), and after confirming disappearance of the tortuous reflex, the abdomen was immediately followed. The heart was dissociated by perfusion with 20 mL of phosphate buffer (PBS). Subsequently, the whole body tissues were fixed by perfusion with 50 mL of ice-cold 4% paraformaldehyde (PFA) solution, and then the animals were ice-cold for about 1 hour. Subsequently, the spinal cord was removed and the fourth lumbar spinal cord was isolated under a stereomicroscope. The isolated spinal cords were subjected to immersion fixation in a 4% PFA solution until 4 to 5 hours after standing in ice. Thereafter, the spinal cord was transferred into cold 30% sucrose solution and shaken at 4 ° C. for 24 hours. O. spinal cord C. T. It was embedded in Compound (Sakura Finetech Japan Co., Ltd.), snap frozen on dry ice, and stored at -80 ° C. until use.
　上記－８０℃で保存していた包埋脊髄を、クライオスタット（Ｌｅｉｃａ社製）内で－２０℃の条件下、約６０分間静置した。その後、クライオスタットを用いて３０μｍ厚の切片を作製した後、ＰＢＳ中に移し、組織周辺のＯ．Ｃ．Ｔ．Ｃｏｍｐｏｕｎｄを溶解させた。得られた脊髄切片を０．３％ＴｒｉｔｏｎＸ－１００／ＰＢＳ（ＰＢＳＴ）中で振盪しながら洗浄した。洗浄後、３％ｎｏｒｍａｌ　ｄｏｎｋｅｙ　ｓｅｒｕｍ／ＰＢＳＴ中、室温で２時間ブロッキングした。次に、３％ｎｏｒｍａｌ　ｄｏｎｋｅｙ　ｓｅｒｕｍ／ＰＢＳＴにポリクローナルｇｏａｔ－ａｎｔｉ　Ｓｏｘ９抗体（１０００倍希釈、Ｒ＆Ｄ社製）を希釈し、４℃で４８時間反応させることにより、一次抗体反応を行った。反応終了後、ＰＢＳＴ溶液で洗浄し、３％ｎｏｒｍａｌ　ｄｏｎｋｅｙ　ｓｅｒｕｍ／ＰＢＳＴにｄｏｎｋｅｙ　ａｎｔｉ－ｇｏａｔ　ＩｇＧ（Ｈ＋Ｌ）Ａｌｅｘａ　Ｆｌｕｏｒ４８８抗体（１０００倍希釈、インビトロジェン社製）を希釈し、室温暗所で３時間反応させることにより、二次抗体反応を行った。反応終了後、暗所において、ＰＢＳＴ溶液およびＰＢＳ溶液で洗浄した。その後、脊髄切片をａｍｉｎｏｐｒｏｐｙｌｔｒｉｅｔｈｏｘｙｓｉｌａｎｅ（ＡＰＳ）でコートしたスライドグラス（松浪硝子工業社製）に貼り付け、切片周囲の余分なＰＢＳ溶液を除去した後、乾燥させた。続いて、退色防止剤ＶｅｃｔａＳｈｉｅｌｄ（Ｖｅｃｔｏｒ　Ｌａｂｏｌａｔｏｒｉｅｓ社製）をスライドグラスに滴下し、カバーガラスをかけ、ＶｅｃｔａＳｈｉｅｌｄが乾燥するまで暗所に静置し、その後４℃で保存した。観察には共焦点レーザー顕微鏡（ＬＳＭ７００、Ｚｅｉｓｓ社製）を使用した。 The embedded spinal cord stored at -80.degree. C. was allowed to stand for about 60 minutes in a cryostat (manufactured by Leica) at -20.degree. Then, after making a 30 μm-thick section using a cryostat, it was transferred into PBS and O.S. C. T. The compound was dissolved. The resulting spinal cord sections were washed in 0.3% Triton X-100 / PBS (PBST) with shaking. After washing, it was blocked for 2 hours at room temperature in 3% normal donkey serum / PBST. Next, a primary antibody reaction was carried out by diluting a polyclonal goat-anti Sox9 antibody (diluted 1: 1000, manufactured by R & D) in 3% normal donkey serum / PBST and reacting at 4 ° C. for 48 hours. After completion of the reaction, wash with PBST solution, dilute donkey anti-goat IgG (H + L) Alexa Fluor 488 antibody (1000-fold dilution, Invitrogen) in 3% normal donkey serum / PBST, and react for 3 hours in the dark at room temperature To perform a secondary antibody reaction. After completion of the reaction, it was washed with PBST solution and PBS solution in the dark. Thereafter, the spinal cord section was attached to a slide glass (manufactured by Matsunami Glass Industry Co., Ltd.) coated with aminopropyltriethoxysilane (APS), excess PBS solution around the section was removed, and dried. Subsequently, the anti-fading agent VectaShield (manufactured by Vector Labolatories) was dropped on a slide glass, a cover glass was put on it, and it was allowed to stand in the dark until the VectaShield dried, and then stored at 4 ° C. A confocal laser microscope (LSM700, manufactured by Zeiss) was used for observation.
　その結果を図１に示す。図１の左図に示したように、ｔｄＴｏｍａｔｏ陽性細胞は脊髄後角の表層に発現していた。また、図１に右図に示したようにアストロサイトマーカーであるＳｏｘ９は、脊髄後角広範に発現していた。このことから、アストロサイトは脊髄後角広範に存在するが、Ｈｅｓ５陽性のアストロサイトは、脊髄後角の表層に存在することが確認された。 The results are shown in FIG. As shown in the left panel of FIG. 1, tdTomato positive cells were expressed in the surface layer of the dorsal horn of the spinal cord. Also, as shown in the right figure in FIG. 1, the astrocyte marker Sox9 was widely expressed in the spinal dorsal horn. From this, it was confirmed that the astrocytes are widely present in the spinal dorsal horn, but the Hes5-positive astrocytes are present in the surface layer of the spinal dorsal horn.
［実験例２］
（カプサイシン誘発の脊髄アストロサイトのＣａ２＋応答と下行性ノルアドレナリン神経の関与）
　最初に、脊髄アストロサイト特異的にカルシウム感受性蛍光タンパク質ＧＣａＭＰ６ｍを発現させるアデノ随伴ウイルスベクターＡＡＶ２／９－ｇｆａＡＢＣ１Ｄ－ＧＣａＭＰ６ｍ－ＷＰＲＥを以下のようにして作製した。 [Experimental Example 2]
(Capsaicin-induced Ca 2+ response of spinal astrocytes and involvement of descending noradrenaline)
First, an adeno-associated virus vector AAV2 / 9-gfaABC 1 D-GCaMP6m-WPRE that specifically expresses the calcium-sensitive fluorescent protein GCaMP6m in the spinal cord astrocytes was constructed as follows.
　ＧＣａＭＰ６ｍ（ａｄｄｇｅｎｅ社製、＃４０７５４）の配列をｐＥＮＴＲプラスミド（Ｔｈｅｍｏｆｉｓｈｅｒ社製）にサブクローニングした。次に、このＧＣａＭＰ６ｍカセットをｇｆａＡＢＣ１Ｄプロモーター（ａｄｄｇｅｎｅ社製、＃１９９７４）を搭載したＡＡＶシャトルベクター（ｐＺａｃ２．１、Ｖｅｃｔｏｒ　ｃｏｒｅ　ｏｆ　ｔｈｅ　Ｕｎｉｖｅｒｓｉｔｙ　ｏｆ　Ｐｅｎｎｓｙｌｖａｎｉａより入手）に挿入した。 The sequence of GCaMP6m (manufactured by addgene, # 40754) was subcloned into pENTR plasmid (manufactured by Themofisher). Next, this GCaMP6m cassette was inserted into an AAV shuttle vector (pZac2.1, obtained from Vector core of the University of Pennsylvania) loaded with gfaABC 1 D promoter (manufactured by addgene, # 19974).
　上記プラスミドを用いたウイルスベクター作製は定法（Ｓｃｉ　Ｒｅｐ．，２０１５　Ｓｅｐ　２１；５：１４３０６）に従い実施した。Ｈｕｍａｎ　ｅｍｂｒｙｏｎｉｃ　ｋｉｄｎｅｙ　２９３（ＨＥＫ２９３）細胞に上記プラスミド、Ｒｅｐ／Ｃａｐプラスミド（ｐＡＡＶ２／９）及びヘルパープラスミド（ｐＡｄ　ＤｅｌｔａＦ６）（Ｒｅｐ／Ｃａｐ及びｐＡｄ　ＤｅｌｔａＦ６はＶｅｃｔｏｒ　ｃｏｒｅ　ｏｆ　ｔｈｅ　Ｕｎｉｖｅｒｓｉｔｙ　ｏｆ　Ｐｅｎｎｓｙｌｖａｎｉａより入手）をトランスフェクションすることにより作製し、塩化セシウム密度勾配遠心法（Ｇｅｎｅ　Ｔｈｅｒ．，２０１０　Ａｐｒ；１７（４）：５０３－１０）により精製した。ウイルスベクターはＡｍｉｃｏｎ　Ｕｌｔｒａ　１００Ｋ　ｆｉｌｔｅｒ　ｕｎｉｔ（Ｍｉｌｌｉｐｏｒｅ社製）を用いて０．００１％（ｖ／ｖ）Ｐｌｕｒｏｎｉｃ－Ｆ６８を含有したＰＢＳ溶液で透析した。ウイルスベクターの力価測定はＰｉｃｏ　Ｇｒｅｅｎ　ｆｌｕｏｒｏｍｅｔｒｉｃ　ｒｅａｇｅｎｔ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅ社製）を用いて行い、使用時まで－８０℃で保存した。 Virus vector production using the above plasmid was performed according to a standard method (Sci Rep., 2015 Sep 21; 5: 14306). The above plasmid, Rep / Cap plasmid (pAAV 2/9) and helper plasmid (pAd Delta F 6) (Rep / Cap and pAd Delta F 6 obtained from Vector core of the University of Pennsylvania) are transfected into Human embryonic kidney 293 (HEK 293) cells And purified by cesium chloride density gradient centrifugation (Gene Ther., 2010 Apr; 17 (4): 503-10). The virus vector was dialyzed against PBS solution containing 0.001% (v / v) Pluronic-F68 using Amicon Ultra 100K filter unit (manufactured by Millipore). Titration of the viral vector was performed using Pico Green fluorometric reagent (manufactured by Molecular Probe) and stored at -80 ° C until use.
　８～１２週齢のＣ５７ＢＬ／６Ｊマウス（日本クレア社製）の腹腔内に、生理食塩水、又はノルアドレナリン神経選択的毒素であるＤＳＰ－４［Ｎ－（２－ｃｈｌｏｒｏｅｔｈｙｌ）－Ｎ－ｅｔｈｙｌ－２－ｂｒｏｍｏｂｅｎｚｙｌａｍｉｎｅ］（シグマ社製）を５０ｍｇ／ｋｇで投与した。投与３～５日後に、ケタミン（１００ｍｇ／ｋｇ；ケタラール（登録商標）筋注用）とキシラジン（１０ｍｇ／ｋｇ；セラクタール(登録商標)）の混合液を０．００５ｍＬ／ｇ皮下投与することで麻酔を行った。麻酔の効果を確認後、背部の毛を剃り、局所麻酔薬(キシロカイン（登録商標）ゼリー ２％)を塗布した。胸髄湾曲部を中心に正中に沿って３ｃｍ程皮膚を切開した。椎骨の第１腰髄の棘突起吻側先端から１ｍｍ側方の筋組織を直径２ｍｍ程度で切開し第１３胸髄と第１腰髄椎骨間の膜を露出した。ガラスキャピラリー（エッペンドルフ社製）を介して左側各椎骨間に、ウイルス濃度８．５×１０１２ ＧＣ／ｍＬで、０．７μＬを１か所、上記で作製した、脊髄アストロサイト特異的にカルシウム感受性蛍光タンパク質のＧＣａＭＰ６ｍを発現させるアデノ随伴ウイルスベクターＡＡＶ２／９－ｇｆａＡＢＣ１Ｄ－ＧＣａＭＰ６ｍ－ＷＰＲＥを微量注入した。処置後は５－０絹糸を用いて皮膚を縫い合わせた。縫合部には局所麻酔薬（キシロカイン（登録商標）ゼリー ２％）を塗布し動物の苦痛の軽減を図った。同時に希ヨードチンキとゲンタシン（登録商標）軟膏（０．１％）の塗布も行い感染の防止を図った。さらに術後翌日および２日後に、カルプロフェン（リマダイル（登録商標）注射液、カルプロフェンとして５ｍｇ／ｋｇ）を腹腔投与することで術後の苦痛軽減を図った。 Physiological saline or DSP-4 [N- (2-chloroethyl) -N-ethyl-2 which is a noradrenaline neuroselective toxin is intraperitoneally injected into C57BL / 6J mice (manufactured by CLEA Japan, Inc.) of 8 to 12 weeks of age. -Bromobenzylamine] (manufactured by Sigma) was administered at 50 mg / kg. Three to five days after administration, the mixture is anesthetized by subcutaneous administration of 0.005 mL / g of a mixture of ketamine (100 mg / kg; KETALAL (R) for intramuscular injection) and xylazine (10 mg / kg; SERACTAL (R)). Did. After confirming the effect of anesthesia, the back hair was shaved and a local anesthetic (Xylocaine (registered trademark) 2%) was applied. The skin was incised about 3 cm along the median centering on the thoracic spinal curve. The muscle tissue 1 mm lateral to the rostral tip of the first lumbar spine of the first lumbar vertebra was incised with a diameter of about 2 mm to expose the membrane between the thirteenth thoracic spinal cord and the first lumbar spinal vertebra bone. Calcium sensitization specifically to spinal cord astrocytes, prepared above with 0.7 μL at one site, with a virus concentration of 8.5 × 10 12 GC / mL, between each vertebra on the left side via a glass capillary (manufactured by Eppendorf) Adeno-associated virus vector AAV2 / 9-gfaABC 1 D-GCaMP6m-WPRE to express the fluorescent protein GCaMP6m was microinjected. After treatment, the skin was stitched with 5-0 silk thread. The suture was applied with a local anesthetic (Xylocaine (registered trademark) 2%) to reduce animal pain. At the same time, diluted iodine tincture and gentacin (registered trademark) ointment (0.1%) were applied to prevent infection. Furthermore, postoperative pain relief was achieved by intraperitoneally administering carprofen (Limadile (registered trademark) injection solution, 5 mg / kg as carprofen) one day and two days after the operation.
　ウイルス投与から３～４週間後に２光子励起顕微鏡を用いて、生きたままの動物で脊髄腰髄部における脊髄アストロサイトＣａ２＋イメージングを行うための観察用窓作製手術を行った。動物への麻酔処置は、鎮痛・炎症予防のためカルプロフェン（リマダイル（登録商標）注射液、カルプロフェンとして５ｍｇ／ｋｇ）、デキサメタゾン（デキサート（登録商標）注射液、デキサメタゾンとして０．２ｍｇ／ｋｇ）混合溶液を０．０１ｍＬ／ｇ腹腔投与した。
　次に、ケタミン（１００ｍｇ／ｋｇ、ケタラール（登録商標）筋注用）とキシラジン（１０ｍｇ／ｋｇ、セラクタール（登録商標））の混合液を０．００５ｍＬ／ｇ皮下投与することで麻酔を行った。麻酔の効果を確認後、背部の毛を剃り、局所麻酔薬（キシロカイン（登録商標）ゼリー２％）を塗布した。観察窓作製中及びイメージング中の眼の乾燥予防のため動物用眼軟膏（クロラムフェニコール２ｍｇ／０．１ｇ、マイコクロリン（登録商標））の塗布を行った。胸髄湾曲部を中心に正中に沿って３ｃｍ程皮膚を切開した。椎骨の第１３胸髄から第１／２腰髄背側を露出するために結合組織を切り離した。筋肉はできる限り切断をさけて剥離した。露出させた椎骨背部を専用の固定具を用いて固定し、椎弓切除を行った。露出した脊髄を覆うように専用のステンレス製器具を固定した。組織と器具の間隙に細胞毒性のない透明なシリコンエストラマーを充填し観察用窓をカバーガラスで覆った。観察器具を設置した部分以外の皮膚を縫い合わせた。 Three to four weeks after virus administration, a two-photon excitation microscope was used to perform an observation window preparation procedure to perform spinal astrocytic Ca 2+ imaging in the lumbar spinal cord with live animals. Anesthetic treatment for animals is a mixed solution of carprofen (Limadile (registered trademark) injection, 5 mg / kg as carprofen), dexamethasone (Dexat (registered trademark) injection, 0.2 mg / kg as dexamethasone) for analgesia and inflammation prevention. Was intraperitoneally administered at 0.01 mL / g.
Next, anesthesia was performed by subcutaneously administering a mixture of ketamine (100 mg / kg, KETALAL (registered trademark) for intramuscular injection) and xylazine (10 mg / kg, SERACTAL (registered trademark)) at 0.005 mL / g. After confirming the effect of anesthesia, the back hair was shaved and a local anesthetic (Xylocaine (registered trademark) 2%) was applied. Application of an animal eye ointment (chloramphenicol 2 mg / 0.1 g, Mycochlorin (registered trademark)) was performed to prevent dryness of the eye during observation window preparation and imaging. The skin was incised about 3 cm along the median centering on the thoracic spinal curve. The connective tissue was dissected to expose the dorso-lumbar dorsal aspect from the thirteenth thoracic spinal cord. The muscles were stripped as much as possible without cutting. The exposed vertebral spine was fixed using a dedicated fixture and a laminectomy was performed. A dedicated stainless steel instrument was fixed to cover the exposed spinal cord. The gap between the tissue and the instrument was filled with a non-cytotoxic, clear silicon elastomer and the observation window was covered with a coverslip. The skin other than the part where the observation instrument was installed was sewn together.
　観察は観察用窓作製時に投与したケタミン／キシラジン混合溶液の麻酔から覚醒の後、イソフルラン吸入麻酔下（エスカイン（登録商標）；導入時５．０％、維持には１．２～１．５％混合空気）において２光子励起顕微鏡（Ｏｌｙｍｐｕｓ　ＦＶ１０００、オリンパス社製）を使用し、対物レンズはＯｌｙｍｐｕｓ　ＸＬＰｌａｎＮ（水浸２５倍、開口数１．０５）、ＧＣａＭＰ６ｍの励起には９００ｎｍの波長のレーザー（～２０ｍＷ）を用いた。レーザー照射に伴う光毒性を避けるために０．３Ｈｚの頻度で画像を取得した。３７℃に設定したｓｍａｌｌ　ｈｅａｔ　ｃｏｎｔｒｏｌｌｅｒ（ユニークメディカル社製）を用いてマウス直腸温が３４℃以上になるように体温を維持した。 Observation was awake from anesthesia of ketamine / xylazine mixed solution administered at the time of preparation of the observation window, then under isoflurane inhalation anesthesia (Escaine®; 5.0% at the time of introduction, 1.2 to 1.5% for maintenance) Two-photon excitation microscope (Olympus FV1000, manufactured by Olympus Corporation) is used in mixed air, and the objective lens is a laser with a wavelength of 900 nm (excitation of Olympus XLPlanN (25 × water immersion, 1.05 numerical aperture), GCaMP 6m) 20 mW was used. Images were acquired at a frequency of 0.3 Hz to avoid phototoxicity associated with laser irradiation. The body temperature was maintained so that the mouse rectal temperature was 34 ° C. or higher using a small heat controller (manufactured by Unique Medical) set at 37 ° C.
　次に、マウス足裏に０．１μｇ／μＬカプサイシン溶液１０μＬを投与し、投与後３０分後まで観察を行った。イメージング画像はＩｍａｇｅ　Ｊを用いて解析し、拍動に伴った観察画像のずれ補正はＴｕｒｂｏＲｅｇを用いて行った（ＩＥＥＥ　Ｔｒａｎｓ　Ｉｍａｇｅ　Ｐｒｏｃｅｓｓ．，１９９８；７（１）：２７－４１）。細胞の形態および下記式で表される蛍光シグナルのピーク値（Ｐｅａｋ　ａｍｐｌｉｔｕｄｅ）ΔＦ／Ｆの値が３０％以上を解析対象とした。解析対象領域は直径１０μｍの範囲で選択し、Ｉｍａｇｅ　Ｊのｍｕｌｔｉ　ｍｅａｓｕｒｅ　ｐｌｕｇｉｎを用いて解析を行った。 Next, 10 μL of a 0.1 μg / μL capsaicin solution was administered to the soles of mice, and observation was performed until 30 minutes after administration. The imaging image was analyzed using Image J, and the displacement correction of the observation image accompanying the pulsation was performed using TurboReg (IEEE Trans Image Process., 1998; 7 (1): 27-41). The analysis target was 30% or more of the cell morphology and the peak value (Peak amplitude) ΔF / F of the fluorescence signal represented by the following formula. The analysis area was selected in the range of 10 μm in diameter, and analysis was performed using Image J's multi measure plugin.
蛍光シグナルのピーク値ΔＦ／Ｆ＝１００＊（Ｆｔ－Ｆ０）／Ｆ０
ｔ：蛍光シグナルがピークに達した時の観察時間
Ｆｔ：ピーク時の蛍光シグナル
Ｆ０：観察開始時から５０秒の無処置時における蛍光シグナルの平均値 Peak value of fluorescence signal ΔF / F = 100 * (Ft−F 0 ) / F 0
t: fluorescent signal observation time at which peaked Ft: Peak of fluorescence signal F 0: Mean value of the fluorescence signal in the no-treatment of 50 seconds from the observation start
　その結果を、図２の左図に示す。図２の左図に示すように、マウス足裏へのカプサイシン投与時における脊髄アストロサイトのカルシウム応答が、ノルアドレナリン神経選択的毒素であるＤＳＰ－４により抑制された。このことから、カプサイシン誘発の痛み刺激により、脊髄アストロサイトのカルシウム応答が下行性アドレナリン神経を介して誘発されることが確認された。 The results are shown in the left view of FIG. As shown in the left panel of FIG. 2, the calcium response of spinal astrocytes upon administration of capsaicin to the mouse sole was suppressed by DSP-4, a noradrenaline neuroselective toxin. This confirms that capsaicin-induced pain stimulation elicits a calcium response of spinal astrocytes via the descending adrenergic nerve.
［実験例３］
（カプサイシン誘発の脊髄アストロサイトのＣａ２＋応答におけるアストロサイトのアドレナリンα１Ａ受容体拮抗剤の効果）
　実験例２において、ＤＳＰ－４を投与せず、又、カプサイシン投与前に、軽度のイソフルラン麻酔下において、ＰＢＳ溶液又はアドレナリンα１Ａ受容体拮抗剤であるシロドシン溶液（３ｎｍｏｌ／５μＬ、和光純薬社製）を３０Ｇの注射針を付けたＨａｍｉｌｔｏｎマイクロシリンジを用いて脊髄くも膜下腔内へ定法［Ｅｕｒ　Ｊ　Ｐｈａｒｍａｃｏｌ．，１９８０　Ｏｃｔ　１７；６７（２－３）：３１３－６］に従い投与する以外は、実験例２と同様に行った。カプサイシンは、ＰＢＳ溶液又はシロドシン溶液投与３０～４０分後に投与した。 [Experimental Example 3]
(Effect of astrocyte adrenergic α1A receptor antagonist on capsaicin-induced spinal cord astrocyte Ca 2+ response)
In Experimental Example 2, PBS-4 or Sirodosin solution (3 nmol / 5 μL, Wako Pure Chemical Industries, Ltd., an adrenergic α1A receptor antagonist) was administered under mild isoflurane anesthesia without administering DSP-4 and before capsaicin administration. ) Into the spinal subarachnoid space using a Hamilton microsyringe equipped with a 30 G injection needle [Eur J Pharmacol. , 1980 Oct 17; 67 (2-3): 313-6] except that administration was performed in the same manner as in Experimental Example 2. Capsaicin was administered 30 to 40 minutes after PBS solution or silodosin solution administration.
　その結果を図２右図に示す。図２の右図に示すように、マウス足裏へのカプサイシン投与時における脊髄アストロサイトのカルシウム応答が、アドレナリンα１Ａ受容体拮抗剤であるシロドシンにより抑制された。このことから、カプサイシン誘発の痛み刺激による下行性ノルアドレナリン神経の活性化は、アドレナリンα１Ａ受容体の活性化を阻害する物質により抑制されることが確認された。 The results are shown in the right of FIG. As shown in the right figure of FIG. 2, the calcium response of the spinal cord astrocytes upon administration of capsaicin to the mouse sole was suppressed by the adrenergic α1A receptor antagonist silodosin. From this, it was confirmed that the activation of the descending noradrenaline nerve by capsaicin-induced pain stimulation is suppressed by the substance that inhibits the activation of the adrenergic α1A receptor.
［実験例４］
（Ｈｅｓ５陽性脊髄アストロサイト刺激時の軽度機械刺激に対するマウスの行動解析）
　最初に、Ｈｅｓ５陽性アストロサイト特異的にｈＭ３Ｄｑ受容体を発現させるアデノ随伴ウイルスベクターＡＡＶ２／５－ＦＬＥＸ－ＥＦ１α-ｈＭ３Ｄｑ－ＷＰＲＥを以下のようにして作製した。 [Experimental Example 4]
Behavioral analysis of mice to mild mechanical stimulation during Hes5-positive spinal astrocyte stimulation
First, an adeno-associated virus vector AAV2 / 5-FLEX-EF1α-hM3Dq-WPRE that specifically expresses the hM3Dq receptor in Hes5 positive astrocytes was generated as follows.
　ｈＭ３Ｄｑ（ａｄｄｇｅｎｅ社製、＃４５５４７）の配列をｐＥＮＴＲプラスミド（Ｔｈｅｍｏｆｉｓｈｅｒ社製）にサブクローニングした。また、ｐＡＡＶ－ＣＡ－ＦＬＥＸ（ａｄｄｇｅｎｅ社製、＃３８０４２）を利用し、ＣＡプロモーターをＥＦ１αに置換したベクターｐＡＡＶ－ＥＦ１α－ＦＬＥＸも作製し、上記ｈＭ３ＤｑカセットをｐＡＡＶ－ＥＦ１α－ＦＬＥＸに挿入した。 The sequence of hM3Dq (addgene, # 45547) was subcloned into pENTR plasmid (Themofisher). In addition, a vector pAAV-EF1α-FLEX in which the CA promoter was replaced with EF1α was also prepared using pAAV-CA-FLEX (manufactured by addgene, # 38042), and the hM3Dq cassette was inserted into pAAV-EF1α-FLEX.
　上記プラスミドを用いたウイルスベクター作製は定法（Ｓｃｉ　Ｒｅｐ．，２０１５　Ｓｅｐ　２１；５：１４３０６）に従い実施した。Ｈｕｍａｎ　ｅｍｂｒｙｏｎｉｃ　ｋｉｄｎｅｙ　２９３（ＨＥＫ２９３）細胞に上記プラスミド、及びＲｅｐ／Ｃａｐプラスミド（ｐＡＡＶ２／５）及びヘルパープラスミド（ｐＡｄ　ＤｅｌｔａＦ６）（Ｒｅｐ／Ｃａｐ及びｐＡｄ　ＤｅｌｔａＦ６はＶｅｃｔｏｒ　ｃｏｒｅ　ｏｆ　ｔｈｅ　Ｕｎｉｖｅｒｓｉｔｙ　ｏｆ　Ｐｅｎｎｓｙｌｖａｎｉａより入手）をトランスフェクションすることにより作製し、塩化セシウム密度勾配遠心法（Ｇｅｎｅ　Ｔｈｅｒ．，２０１０　Ａｐｒ；１７（４）：５０３－１０）により精製した。得られたアデノ随伴ウイルスベクターＡＡＶ２／５－ＦＬＥＸ－ＥＦ１α-ｈＭ３Ｄｑ－ＷＰＲＥはＡｍｉｃｏｎ　Ｕｌｔｒａ　１００Ｋ　ｆｉｌｔｅｒ　ｕｎｉｔ（Ｍｉｌｌｉｐｏｒｅ社製）を用いて０．００１％（ｖ／ｖ）Ｐｌｕｒｏｎｉｃ－Ｆ６８を含有したＰＢＳ溶液で透析した。ウイルスベクターの力価測定はＰｉｃｏ　Ｇｒｅｅｎ　ｆｌｕｏｒｏｍｅｔｒｉｃ　ｒｅａｇｅｎｔ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅ社製）を用いて行い、使用時まで－８０℃で保存した。 Virus vector production using the above plasmid was performed according to a standard method (Sci Rep., 2015 Sep 21; 5: 14306). Transfect the above plasmid and Rep / Cap plasmid (pAAV2 / 5) and helper plasmid (pAd DeltaF6) (Rep / Cap and pAd DeltaF6 from Vector core of the University of Pennsylvania) into Human embryonic kidney 293 (HEK 293) cells , And purified by cesium chloride density gradient centrifugation (Gene Ther., 2010 Apr; 17 (4): 503-10). The obtained adeno-associated virus vector AAV2 / 5-FLEX-EF1α-hM3Dq-WPRE was treated with Amicon Ultra 100K filter unit (Millipore) in PBS solution containing 0.001% (v / v) Pluronic-F68 I dialyzed. Titration of the viral vector was performed using Pico Green fluorometric reagent (manufactured by Molecular Probe) and stored at -80 ° C until use.
　８～１２週齢のＨｅｓ５ＣｒｅＥＲＴ２マウス (Ｎａｔ　Ｃｏｍｍｕｎ．，　２０１２　Ｆｅｂ　１４；３：６７０)を用い、ケタミンとキシラジンの混合液を０．００５ｍＬ／ｇ皮下投与することで麻酔を行った。麻酔の効果を確認後、背部の毛を剃り、局所麻酔薬(キシロカイン（登録商標）ゼリー ２％)を塗布した。胸髄湾曲部を中心に正中に沿って３ｃｍ程皮膚を切開した。椎骨の第１３胸髄および第１／２腰髄の棘突起吻側先端から１ｍｍ側方の筋組織を直径２ｍｍ程度で切開し第１２胸髄と第１３胸髄、第１３胸髄と第１腰髄および第１腰髄と第２腰髄椎骨間の膜を露出した。ガラスキャピラリー（エッペンドルフ社製）を介して左側各椎骨間に、ウイルス濃度１×１０１２ ＧＣ／ｍＬで、０．５～０．６μＬをそれぞれ１か所、合計３か所に、上記で作製した、Ｈｅｓ５陽性アストロサイト特異的にｈＭ３Ｄｑ受容体を発現させるアデノ随伴ウイルスベクターＡＡＶ２／５－ＦＬＥＸ－ＥＦ１α-ｈＭ３Ｄｑ－ＷＰＲＥを微量注入した。処置後は５－０絹糸を用いて皮膚を縫い合わせた。縫合部には局所麻酔薬（キシロカイン（登録商標）ゼリー ２％）を塗布し動物の苦痛の軽減を図った。同時に希ヨードチンキとゲンタシン（登録商標）軟膏（０．１％）の塗布も行い感染の防止を図った。 Anesthesia was performed using a mixed solution of ketamine and xylazine subcutaneously administered at 0.005 mL / g using 8 to 12 week old Hes 5 Cre ERT 2 mice (Nat Commun., 2012 Feb 14; 3: 670). After confirming the effect of anesthesia, the back hair was shaved and a local anesthetic (Xylocaine (registered trademark) 2%) was applied. The skin was incised about 3 cm along the median centering on the thoracic spinal curve. The muscle tissue 1 mm lateral to the rostral tip of the 13th and 1st lumbar vertebrae of the vertebra is incised with a diameter of about 2 mm, and the 12th and 13th spinal cords, the 13th and 25th spinal cords The lumbar and the membrane between the first lumbar and the second lumbar spinal bone were exposed. Between each vertebra on the left side through a glass capillary (manufactured by Eppendorf), 0.5 to 0.6 μL each was prepared at a concentration of 1 × 10 12 GC / mL at a total of three locations. Then, the Adeno-associated virus vector AAV2 / 5-FLEX-EF1α-hM3Dq-WPRE, which expresses the hM3Dq receptor specifically in Hes5 positive astrocytes, was microinjected. After treatment, the skin was stitched with 5-0 silk thread. The suture was applied with a local anesthetic (Xylocaine (registered trademark) 2%) to reduce animal pain. At the same time, diluted iodine tincture and gentacin (registered trademark) ointment (0.1%) were applied to prevent infection.
　次に、タモキシフェン（シグマ社製）溶液（溶媒にコーン油を使用し２０ｍｇ／ｍＬで調製したもの）２ｍｇ／０．１ｍＬを１日１回１０日間、マウスへ腹腔内投与することによりＣｒｅリコンビナーゼ活性を誘導し、ｈＭ３Ｄｑ受容体をＨｅｓ５陽性アストロサイト特異的に発現させた。なお、アデノ随伴ウイルスベクターＡＡＶ２／５－ＦＬＥＸ－ＥＦ１α-ｈＭ３Ｄｑ－ＷＰＲＥは、ｈＭ３Ｄｑ遺伝子を逆行性に配置しているため、タモキシフェンを投与した場合は、Ｈｅｓ５陽性アストロサイトでＣｒｅリコンビナーゼ活性が誘導され、ウイルスベクター内のｈＭ３Ｄｑ配列の部分が反転し、ｈＭ３Ｄｑ受容体が発現するが、Ｈｅｓ５陰性アストロサイトでは、ウイルスベクター内のｈＭ３Ｄｑ配列の部分が反転しないためｈＭ３Ｄｑ受容体が発現しない。 Next, Cre recombinase activity is obtained by intraperitoneally administering 2 mg / 0.1 mL of tamoxifen (manufactured by Sigma) solution (prepared at 20 mg / mL using corn oil as solvent) to mice once a day for 10 days. And hM3Dq receptors were specifically expressed in Hes5 positive astrocytes. In addition, since the hM3Dq gene is placed retrogradely in the adeno-associated virus vector AAV2 / 5-FLEX-EF1α-hM3Dq-WPRE, when tamoxifen is administered, Cre recombinase activity is induced in Hes5-positive astrocytes, A portion of the hM3Dq sequence in the viral vector is inverted and the hM3Dq receptor is expressed, but in the Hes5-negative astrocytes, the hM3Dq receptor is not expressed because the portion of the hM3Dq sequence in the viral vector is not inverted.
　その結果を図３の左図に示す。図３左図に示すように、ｈＭ３Ｄｑ受容体は、脊髄後角表層のＨｅｓ５陽性アストロサイトに発現した。 The results are shown in the left view of FIG. As shown in the left panel of FIG. 3, hM3Dq receptors were expressed on Hes5-positive astrocytes in the dorsal horn surface layer.
　次に、ｈＭ３Ｄｑ受容体をＨｅｓ５陽性アストロサイト特異的に発現させたマウスに、ｈＭ３Ｄｑ受容体の選択的刺激薬であるクロザピン－Ｎ－オキサイド［Ｃｌｏｚａｐｉｎｅ－Ｎ－ｏｘｉｄｅ（ＣＮＯ；Ｅｎｚｏ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅ社製）］を含む溶液１０ｍｇ／ｋｇを腹腔内投与し、動物の後肢足底部に知覚試験用フィラメント（ｖｏｎ　Ｆｒｅｙ　Ｆｉｌａｍｅｎｔ　０．０２～２ｇ；Ｎｏｒｔｈ　Ｃｏａｓｔ　Ｍｅｄｉｃａｌ社製）を押しあて、アップダウン法［Ｊ．Ｎｅｕｒｏｓｃｉ　Ｍｅｔｈｏｄｓ，１９９４，５３（１）：５５－６３］に従って５回から９回の異なった力のフィラメントで評価した。その軽度機械刺激に対する動物の後肢逃避行動を投与０分後、３０分後、６０分後、９０分後、１２０分後、１８０分後に経時的に観察し、５０％逃避閾値（Ｐａｗ　ｗｉｔｈｄｒａｗａｌ　ｔｈｒｅｓｈｏｌｄ）を算出した。コントロールとして、それぞれのマウスにＣＮＯの代わりに、生理食塩水を投与し、上記と同様に５０％逃避閾値を算出した。 Next, clozapine-N-oxide [Clozapine-N-oxide (CNO; Enzo Life Science), which is a selective stimulator of hM3Dq receptor, is expressed in a mouse in which hM3Dq receptor is specifically expressed in Hes5 positive astrocytes. 10 mg / kg of a solution containing the compound of the formula I) was intraperitoneally administered, and a perceptual test filament (von Frey Filament 0.02 to 2 g; manufactured by North Coast Medical) was pushed to the plantar region of the hind limbs of the animal, According to Neurosci Methods, 1994, 53 (1): 55-63], the filament was evaluated with 5 to 9 different force filaments. The hindlimb withdrawal behavior of the animals to the mild mechanical stimulation was observed over time at 0, 30, 60, 90, 120, 180 minutes after administration, and 50% withdrawal threshold (Paw withdrawal threshold) Was calculated. As a control, physiological saline was administered to each mouse instead of CNO, and 50% escape threshold was calculated as described above.
　その結果を図３右図に示す。図３右図に示すように、脊髄後角表層のＨｅｓ５陽性アストロサイトに発現したｈＭ３Ｄｑ受容体をＣＮＯで刺激することにより、マウス左後肢に逃避閾値の低下が観察された。なお、ウイルスベクターを投与していない右側脊髄に投射している右後肢においては逃避閾値の低下は観察されなかった。図３の結果から、脊髄後角表層に存在するＨｅｓ５陽性アストロサイトを刺激することにより、軽度機械刺激に対する疼痛様行動（アロディニア）が誘発されることが明らかとなった。 The results are shown in the right of FIG. As shown in the right panel of FIG. 3, a decrease in escape threshold was observed in the left hind leg of the mouse by stimulating the hM3Dq receptor expressed in Hes5-positive astrocytes in the dorsal horn of the spinal cord with CNO. In the right hind limb projecting to the right spinal cord not receiving the viral vector, no decrease in escape threshold was observed. From the results shown in FIG. 3, it became clear that stimulation of Hes5 positive astrocytes present in the surface layer of the dorsal horn of the spinal cord induces pain-like behavior (allodynia) to mild mechanical stimulation.
［実験例５］
（Ｈｅｓ５陰性脊髄アストロサイト刺激時の軽度機械刺激に対するマウスの行動解析）
　最初に、Ｈｅｓ５陰性アストロサイト特異的にｈＭ３Ｄｑ受容体を発現させるアデノ随伴ウイルスベクターＡＡＶ２／５－ＦＬＥＸ－ｇｆａＡＢＣ１Ｄ－ｈＭ３Ｄｑ－ＷＰＲＥを以下のようにして作製した。 [Experimental Example 5]
Behavioral analysis of mice for mild mechanical stimulation during Hes 5 negative spinal astrocyte stimulation
First, an adeno-associated virus vector AAV2 / 5-FLEX-gfaABC 1 D-hM3Dq-WPRE that specifically expresses hM3Dq receptor in Hes5 negative astrocytes was constructed as follows.
　ｈＭ３Ｄｑ（ａｄｄｇｅｎｅ社製、＃４５５４７）の配列およびｐＡＡＶ－ＣＡ－ＦＬＥＸ（ａｄｄｇｅｎｅ社製、＃３８０４２）を利用し、ｈＭ３Ｄｑ遺伝子を順行性に配置したＦＬＥＸ－ｈＭ３ＤｑをｐＥＮＴＲプラスミド（Ｔｈｅｍｏｆｉｓｈｅｒ社製）にサブクローニングした。このカセットを、ｇｆａＡＢＣ１Ｄプロモーターを搭載したｐＺａｃ２．１へ挿入した。 Using the sequence of hM3Dq (made by addgene, # 45547) and pAAV-CA-FLEX (made by addgene, # 38042), the FLEX-hM3Dq in which the hM3Dq gene is arranged in anterograde manner is used as the pENTR plasmid (made by Themofisher) It was subcloned. This cassette was inserted into pZac2.1 loaded with the gfaABC 1 D promoter.
　上記プラスミドを用いたウイルスベクター作製は定法（Ｓｃｉ　Ｒｅｐ．，２０１５　Ｓｅｐ　２１；５：１４３０６）に従い実施した。Ｈｕｍａｎ　ｅｍｂｒｙｏｎｉｃ　ｋｉｄｎｅｙ　２９３（ＨＥＫ２９３）細胞に上記プラスミド、Ｒｅｐ／Ｃａｐプラスミド（ｐＡＡＶ２／５）及びヘルパープラスミド（ｐＡｄ　ＤｅｌｔａＦ６）（Ｒｅｐ／Ｃａｐ及びｐＡｄ　ＤｅｌｔａＦ６はＶｅｃｔｏｒ　ｃｏｒｅ　ｏｆ　ｔｈｅ　Ｕｎｉｖｅｒｓｉｔｙ　ｏｆ　Ｐｅｎｎｓｙｌｖａｎｉａより入手）をトランスフェクションすることにより作製し、塩化セシウム密度勾配遠心法（Ｇｅｎｅ　Ｔｈｅｒ．，２０１０　Ａｐｒ；１７（４）：５０３－１０）により精製した。得られたアデノ随伴ウイルスベクターＡＡＶ２／５－ＦＬＥＸ－ｇｆａＡＢＣ１Ｄ－ｈＭ３Ｄｑ－ＷＰＲＥはＡｍｉｃｏｎ　Ｕｌｔｒａ　１００Ｋ　ｆｉｌｔｅｒ　ｕｎｉｔ（Ｍｉｌｌｉｐｏｒｅ社製）を用いて０．００１％（ｖ／ｖ）Ｐｌｕｒｏｎｉｃ－Ｆ６８を含有したＰＢＳ溶液で透析した。ウイルスベクターの力価測定はＰｉｃｏ　Ｇｒｅｅｎ　ｆｌｕｏｒｏｍｅｔｒｉｃ　ｒｅａｇｅｎｔ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅ社製）を用いて行い、使用時まで－８０℃で保存した。 Virus vector production using the above plasmid was performed according to a standard method (Sci Rep., 2015 Sep 21; 5: 14306). The above plasmid, Rep / Cap plasmid (pAAV2 / 5) and helper plasmid (pAd DeltaF6) (Rep / Cap and pAd DeltaF6 are obtained from Vector core of the University of Pennsylvania) are transfected into Human embryonic kidney 293 (HEK 293) cells. And purified by cesium chloride density gradient centrifugation (Gene Ther., 2010 Apr; 17 (4): 503-10). The obtained adeno-associated virus vector AAV2 / 5-FLEX-gfaABC 1 D-hM3Dq-WPRE was PBS containing 0.001% (v / v) Pluronic-F68 using Amicon Ultra 100K filter unit (Millipore). The solution was dialyzed. Titration of the viral vector was performed using Pico Green fluorometric reagent (manufactured by Molecular Probe) and stored at -80 ° C until use.
　８～１２週齢のＨｅｓ５－ＣｒｅＥＲＴ２マウス (Ｎａｔ　Ｃｏｍｍｕｎ．，　２０１２　Ｆｅｂ　１４；３：６７０)を用い、ケタミンとキシラジンの混合液を０．００５ｍＬ／ｇ皮下投与することで麻酔を行った。麻酔の効果を確認後、背部の毛を剃り、局所麻酔薬(キシロカイン（登録商標）ゼリー ２％)を塗布した。胸髄湾曲部を中心に正中に沿って３ｃｍ程皮膚を切開した。椎骨の第１３胸髄および第１／２腰髄の棘突起吻側先端から１ｍｍ側方の筋組織を直径２ｍｍ程度で切開し第１２胸髄と第１３胸髄、第１３胸髄と第１腰髄および第１腰髄と第２腰髄椎骨間の膜を露出した。ガラスキャピラリー（エッペンドルフ社製）を介して左側各椎骨間に、ウイルス濃度１×１０１２ ＧＣ／ｍＬで、０．５～０．６μＬをそれぞれ１か所、合計３か所に、上記で作製した、Ｈｅｓ５陰性アストロサイト特異的にｈＭ３Ｄｑ受容体を発現させるアデノ随伴ウイルスベクターＡＡＶ２／５－ＦＬＥＸ－ｇｆａＡＢＣ１Ｄ－ｈＭ３Ｄｑ－ＷＰＲＥを微量注入した。処置後は５－０絹糸を用いて皮膚を縫い合わせた。縫合部には局所麻酔薬（キシロカイン（登録商標）ゼリー ２％）を塗布し動物の苦痛の軽減を図った。同時に希ヨードチンキとゲンタシン（登録商標）軟膏（０．１％）の塗布も行い感染の防止を図った。 Anesthesia was performed using a mixed solution of ketamine and xylazine subcutaneously administered at 0.005 mL / g using 8- to 12-week-old Hes5-CreERT2 mice (Nat Commun., 2012 Feb 14; 3: 670). After confirming the effect of anesthesia, the back hair was shaved and a local anesthetic (Xylocaine (registered trademark) 2%) was applied. The skin was incised about 3 cm along the median centering on the thoracic spinal curve. The muscle tissue 1 mm lateral to the rostral tip of the 13th and 1st lumbar vertebrae of the vertebra is incised with a diameter of about 2 mm, and the 12th and 13th spinal cords, the 13th and 25th spinal cords The lumbar and the membrane between the first lumbar and the second lumbar spinal bone were exposed. Between each vertebra on the left side through a glass capillary (manufactured by Eppendorf), 0.5 to 0.6 μL each was prepared at a concentration of 1 × 10 12 GC / mL at a total of three locations. , Hes5-negative astrocytes were microinjected with the adeno-associated virus vector AAV2 / 5-FLEX-gfaABC 1 D-hM3Dq-WPRE, which expresses hM3Dq receptors specifically. After treatment, the skin was stitched with 5-0 silk thread. The suture was applied with a local anesthetic (Xylocaine (registered trademark) 2%) to reduce animal pain. At the same time, diluted iodine tincture and gentacin (registered trademark) ointment (0.1%) were applied to prevent infection.
　次に、コーン油又はタモキシフェン（シグマ社製）溶液（溶媒にコーン油を使用し２０ｍｇ／ｍｌで調製したもの）２ｍｇ／０．１ｍＬを１日１回１０日間、マウスへ腹腔内投与することによりＣｒｅリコンビナーゼ活性を誘導し、ｈＭ３Ｄｑ受容体をＨｅｓ５陰性アストロサイト特異的に発現させた。なお、アデノ随伴ウイルスベクターＡＡＶ２／５－ＦＬＥＸ－ｇｆａＡＢＣ１Ｄ－ｈＭ３Ｄｑは、ｈＭ３Ｄｑ遺伝子を順行性に配置しているため、コーン油を投与した場合は、Ｃｒｅリコンビナーゼ活性の有無に関わらず、アストロサイトでｈＭ３Ｄｑ受容体が発現する。一方、タモキシフェンを投与した場合は、Ｈｅｓ５陽性アストロサイトでＣｒｅリコンビナーゼ活性が誘導されるため、ウイルスベクター内のｈＭ３Ｄｑ配列の部分が反転し、ｈＭ３Ｄｑ受容体が発現しなくなるが、Ｈｅｓ５陰性アストロサイトではＣｒｅリコンビナーゼが誘導されないため、ウイルスベクター内のｈＭ３Ｄｑ配列の部分が反転せず、ｈＭ３Ｄｑ受容体が発現する。 Next, by intraperitoneal administration of 2 mg / 0.1 mL of corn oil or tamoxifen (manufactured by Sigma) solution (prepared at 20 mg / ml using corn oil as solvent) to mice once a day for 10 days Cre recombinase activity was induced, and hM3Dq receptor was specifically expressed in Hes5 negative astrocytes. Since the adeno-associated virus vector AAV2 / 5-FLEX-gfaABC 1 D-hM3Dq arranges the hM3Dq gene anteroposteriorly, when corn oil is administered, the Astro does not depend on the presence or absence of Cre recombinase activity. HM3Dq receptor is expressed at the site. On the other hand, when tamoxifen is administered, since Cre recombinase activity is induced in Hes5 positive astrocytes, the hM3Dq sequence part in the viral vector is reversed and hM3Dq receptor is not expressed, but in Hes5 negative astrocytes, Cre Since recombinase is not induced, the hM3Dq receptor in the viral vector is not inverted and the hM3Dq receptor is expressed.
　その結果を、図４の左図に示す。図４左図に示すように、コーン油投与群において脊髄後角広範のアストロサイトにｈＭ３Ｄｑ受容体が発現したが、タモキシフェン投与群では脊髄後角深層部に存在するＨｅｓ５陰性アストロサイトにのみｈＭ３Ｄｑ受容体が発現した。 The results are shown in the left view of FIG. As shown in FIG. 4 left, hM3Dq receptors were expressed in astrocytes in the dorsal horn in the dorsal horn in the corn oil-administered group, but hM3Dq reception was only in Hes5-negative astrocytes present in the dorsal horn in the spinal cord in the tamoxifen-administered group. The body expressed.
　ｈＭ３Ｄｑ受容体をＨｅｓ５陰性アストロサイト特異的に発現させたマウスに、ｈＭ３Ｄｑ受容体の選択的刺激薬であるクロザピン－Ｎ－オキサイド［Ｃｌｏｚａｐｉｎｅ－Ｎ－ｏｘｉｄｅ（ＣＮＯ；Ｅｎｚｏ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅ社製）］を含む溶液１０ｍｇ／ｋｇを腹腔内投与し、動物の後肢足底部に知覚試験用フィラメント（ｖｏｎ　Ｆｒｅｙ　Ｆｉｌａｍｅｎｔ　０．０２～２ｇ；Ｎｏｒｔｈ　Ｃｏａｓｔ　Ｍｅｄｉｃａｌ社製）を押しあて、アップダウン法［Ｊ．Ｎｅｕｒｏｓｃｉ　Ｍｅｔｈｏｄｓ，１９９４，５３（１）：５５－６３］に従って５回から９回の異なった力のフィラメントで評価した。その軽度機械刺激に対する動物の後肢逃避行動を投与０分後、３０分後、６０分後、９０分後、１２０分後、１８０分後に経時的に観察し、５０％逃避閾値（Ｐａｗ　ｗｉｔｈｄｒａｗａｌ　ｔｈｒｅｓｈｏｌｄ）を算出した。 Clozapine-N-oxide [Clozapine-N-oxide (CNO; Enzo Life Science)], which is a selective stimulator of hM3Dq receptor, is included in mice in which Hes5 negative astrocytes specifically express hM3Dq receptor A solution of 10 mg / kg was intraperitoneally administered, and a sensory test filament (von Frey Filament 0.02 to 2 g; manufactured by North Coast Medical) was pushed to the plantar region of the hindlimb of the animal, and the up-down method [J. According to Neurosci Methods, 1994, 53 (1): 55-63], the filament was evaluated with 5 to 9 different force filaments. The hindlimb withdrawal behavior of the animals to the mild mechanical stimulation was observed over time at 0, 30, 60, 90, 120, 180 minutes after administration, and 50% withdrawal threshold (Paw withdrawal threshold) Was calculated.
　その結果を図４右図に示す。図４右図に示すように、脊髄後角広範に存在するアストロサイトに発現しているｈＭ３Ｄｑ受容体をＣＮＯで刺激すると、マウスの後肢逃避閾値が低下したが、Ｈｅｓ５陰性アストロサイトに発現しているｈＭ３Ｄｑ受容体をＣＮＯで刺激しても、マウスの後肢逃避閾値に大きな変化はなかった。なお、ウイルスベクターを投与していない右側脊髄に投射する右後肢においては逃避閾値の低下は観察されなかった。図４の結果から、脊髄後角深層部に存在するＨｅｓ５陰性アストロサイトを刺激しても、軽度機械刺激に対する疼痛様行動（アロディニア）は誘発されないことが明らかとなった。 The results are shown in the right of FIG. As shown in the right panel of Figure 4, stimulation of hNO3Dq receptor expressed in astrocytes in the spinal dorsal horn widely with CNO reduced hindlimb withdrawal threshold in mice but expressed in Hes5 negative astrocytes Stimulation of the hM3Dq receptor with CNO did not significantly alter the hindpaw withdrawal threshold in mice. In the right hind limb projecting to the right spinal cord not receiving the viral vector, no decrease in escape threshold was observed. From the results in FIG. 4, it is clear that stimulation of Hes5 negative astrocytes present in the deep area of the dorsal horn does not induce pain-like behavior (allodynia) to mild mechanical stimulation.
［実験例６］
（アドレナリンα１受容体アゴニストの脊髄くも膜下腔内投与による軽度機械刺激痛覚過敏）
　軽度のイソフルラン麻酔下において、８～１２週齢のＣ５７ＢＬ／６Ｊマウス（日本クレア社製）に３０Ｇの注射針を付けたハミルトンマイクロシリンジを用いて、フェニレフリン［（Ｒ）－（－）－ｐｈｅｎｙｌｅｐｈｒｉｎｅ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ；和光純薬社製］を１５ｐｍｏｌ／５μＬ生理食塩水、及び５０ｐｍｏｌ／５μＬ生理食塩水を脊髄くも膜下腔内に投与した。 [Experimental Example 6]
(Mild mechanical stimulation hyperalgesia by intrathecal administration of adrenergic α1 receptor agonist)
Under mild isoflurane anesthesia, phenylephrine [(R)-(-)-phenylephrine hydrochloride is obtained using a Hamilton microsyringe with a 30 G needle attached to a C57BL / 6J mouse (manufactured by CLEA Japan, Inc.) of 8 to 12 weeks of age. Wako Pure Chemical Industries, Ltd.] was administered 15 pmol / 5 μL saline and 50 pmol / 5 μL saline into the spinal subarachnoid space.
　次に、動物の後肢足底部に知覚試験用フィラメント（ｖｏｎ　Ｆｒｅｙ　Ｆｉｌａｍｅｎｔ　０．０２～２ｇ；Ｎｏｒｔｈ　Ｃｏａｓｔ　Ｍｅｄｉｃａｌ社製）を押しあて、アップダウン法［Ｊ．Ｎｅｕｒｏｓｃｉ　Ｍｅｔｈｏｄｓ，１９９４，５３（１）：５５－６３］に従って５回から９回の異なった力のフィラメントで評価した。その軽度機械刺激に対する動物の後肢逃避行動を投与０分後、及び３０分後に観察し、５０％逃避閾値（Ｐａｗ　ｗｉｔｈｄｒａｗａｌ　ｔｈｒｅｓｈｏｌｄ）を算出した。
　その結果を図５に示す。図５に示したように、野生型マウスの脊髄くも膜下腔内へアドレナリンα１受容体アゴニストであるフェニレフリンを投与すると、用量依存的な後肢逃避閾値の低下が観察された。これにより、脊髄のアドレナリンα１受容体の活性化により軽度機械刺激に対する疼痛様行動（アロディニア）が誘発されることが確認された。 Next, a filament for perceptual test (von Frey Filament 0.02 to 2 g; manufactured by North Coast Medical) is pushed onto the plantar region of the hindlimb of the animal, and the up-down method [J. According to Neurosci Methods, 1994, 53 (1): 55-63], the filament was evaluated with 5 to 9 different force filaments. The hindpaw withdrawal behavior of the animals to the mild mechanical stimulation was observed at 0 and 30 minutes after administration, and 50% withdrawal threshold was calculated.
The results are shown in FIG. As shown in FIG. 5, when the adrenaline α1 receptor agonist phenylephrine was administered into the spinal subarachnoid space of wild-type mice, a dose-dependent decrease in hindlimb withdrawal threshold was observed. Thereby, it was confirmed that activation of the adrenergic α1 receptor in the spinal cord induces pain-like behavior (allodinia) to mild mechanical stimulation.
［実験例７］
（フェニレフリン誘発の軽度機械刺激痛覚過敏に対するアドレナリンα１Ａ受容体の関与１）
　８～１２週齢のＣ５７ＢＬ／６Ｊマウス（日本クレア社製）を、軽度のイソフルラン麻酔下において、リン酸緩衝液（ＰＢＳ）又は３ｎｍｏｌ／５μＬシロドシン溶液（和光純薬社製）を３０Ｇの注射針を付けたハミルトンマイクロシリンジを用いて、定法［Ｅｕｒ　Ｊ　Ｐｈａｒｍａｃｏｌ．，１９８０　Ｏｃｔ　１７；６７（２－３）：３１３－６］に従い、脊髄くも膜下腔内へ投与した。ＰＢＳ又はシロドシン投与３０分後に、フェニレフリン［（Ｒ）－（－）－ｐｈｅｎｙｌｅｐｈｒｉｎｅ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ；和光純薬社製］を５０ｐｍｏｌ／５μＬ生理食塩水を脊髄くも膜下腔内に投与した。 [Experimental Example 7]
(Involvement of the adrenergic α1A receptor in phenylephrine-induced mild mechanical hyperalgesia 1)
A 30 G injection needle of phosphate buffer (PBS) or 3 nmol / 5 μL silodosin solution (Wako Pure Chemical Industries, Ltd.) under mild isoflurane anesthesia with a C57BL / 6J mouse (manufactured by CLEA Japan) for 8-12 weeks old [Eur J Pharmacol. , 1980 Oct 17; 67 (2-3): 313-6], and were administered into the spinal subarachnoid space. Thirty minutes after PBS or silodosin administration, 50 pmol / 5 μL saline solution of phenylephrine [(R)-(-)-phenylephrine hydrochloride; manufactured by Wako Pure Chemical Industries, Ltd.] was administered into the spinal cord intrathecal space.
　次に、実験例５と同様にして、マウスの行動解析を行い、５０％逃避閾値を算出した。その結果を図６左図に示す。図６左図に示したように、アドレナリンα１Ａ受容体選択的拮抗剤であるシロドシンを、脊髄くも膜下腔内に前投与すると、フェニレフリンによる脊髄くも膜下内腔投与で誘発される後肢逃避閾値の低下が抑制された。このことから、アドレナリンα１Ａ受容体拮抗剤は、フェニレフリンによる軽度機械刺激に対する疼痛様行動（アロディニア）を抑制することが確認された。 Next, behavior analysis of the mouse was performed in the same manner as in Experimental Example 5, and a 50% escape threshold was calculated. The result is shown in the left figure of FIG. As shown in the left panel of FIG. 6, pre-administration of adrenergic alpha 1A receptor selective antagonist silodosin into spinal cord intrathecal space reduces the hindlimb withdrawal threshold induced by spinal cord intrathecal administration with phenylephrine Was suppressed. From this, it was confirmed that an adrenergic α1A receptor antagonist suppresses pain-like behavior (allodynia) to mild mechanical stimulation by phenylephrine.
［実験例８］
（フェニレフリン誘発の軽度機械刺激痛覚過敏に対するアドレナリンα１Ａ受容体の関与２）
　最初に、アドレナリンα１Ａ受容体選択的ノックダウンベクターＡＡＶ－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ＡＤＲＡ１Ａ－ｓｈＲＮＡ－ＷＰＲＥ及びコントロールベクターＡＡＶ－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ｓｃｒａｍｂｌｅ－ｓｈＲＮＡ－ＷＰＲＥを以下のようにして作製した。 [Experimental Example 8]
(Involvement of the adrenergic α1A receptor on phenylephrine-induced mild mechanical hyperalgesia 2)
First, the adrenergic α1A receptor selective knockdown vector AAV-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE and the control vector AAV-FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE are prepared as follows: Made.
　ＡｃＧＦＰおよびｍｉｒ３０－ｓｈＲＮＡ（ｐＧＩＰＺ－ｍｉｒ３０－ｓｈＧｒａｐｄｈ、Ｔｈｅｍｏｆｉｓｈｅｒ社製）の配列をｐＥＮＴＲプラスミド（Ｔｈｅｍｏｆｉｓｈｅｒ社製）にサブクローニングした。次に、ｐＥＮＴＲ－ＡｃＧＦＰ－ｍｉｒ３０のターゲット配列の部分を配列番号１で表される塩基配列からなる配列に置換した。得られたＡｃＧＦＰ－ｍｉｒ３０－ｓｈＲＮＡカセットはｐＡＡＶ－ＣＡ－ＦＬＥＸに挿入し、ｐＡＡＶ－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ＡＤＲＡ１Ａ－ｓｈＲＮＡ－ＷＰＲＥを得た。 The sequences of AcGFP and mir30-shRNA (pGIPZ-mir30-shGrapdh, manufactured by Themofisher) were subcloned into pENTR plasmid (manufactured by Themofisher). Next, a part of the target sequence of pENTR-AcGFP-mir30 was replaced with a sequence consisting of the nucleotide sequence shown in SEQ ID NO: 1. The obtained AcGFP-mir30-shRNA cassette was inserted into pAAV-CA-FLEX to obtain pAAV-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE.
　また、ｐＥＮＴＲ－ＡｃＧＦＰ－ｍｉｒ３０のターゲット配列の部分を配列番号２で表される塩基配列からなる配列に置換し、得られたＡｃＧＦＰ－ｍｉｒ３０－ｓｈＲＮＡカセットはｐＡＡＶ－ＣＡ－ＦＬＥＸに挿入し、ｐＡＡＶ－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ｓｃｒａｍｂｌｅ－ｓｈＲＮＡ－ＷＰＲＥを得た。 In addition, a portion of the target sequence of pENTR-AcGFP-mir30 is replaced with a sequence consisting of the nucleotide sequence shown in SEQ ID NO: 2, and the obtained AcGFP-mir30-shRNA cassette is inserted into pAAV-CA-FLEX, pAAV- FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE was obtained.
　上記プラスミドを用いたウイルスベクター作製は定法（Ｓｃｉ　Ｒｅｐ．，２０１５　Ｓｅｐ　２１；５：１４３０６）に従い実施した。Ｈｕｍａｎ　ｅｍｂｒｙｏｎｉｃ　ｋｉｄｎｅｙ　２９３（ＨＥＫ２９３）細胞に、上記で得られたｐＡＡＶ－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ＡＤＲＡ１Ａ－ｓｈＲＮＡ－ＷＰＲＥ又はｐＡＡＶ－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ｓｃｒａｍｂｌｅ－ｓｈＲＮＡ－ＷＰＲＥ及びＲｅｐ／Ｃａｐプラスミド（ｐＡＡＶ２／５）及びヘルパープラスミド（ｐＡｄ　ＤｅｌｔａＦ６）（Ｒｅｐ／Ｃａｐ及びｐＡｄ　ＤｅｌｔａＦ６はＶｅｃｔｏｒ　ｃｏｒｅ　ｏｆ　ｔｈｅ　Ｕｎｉｖｅｒｓｉｔｙ　ｏｆ　Ｐｅｎｎｓｙｌｖａｎｉａより入手）をトランスフェクションすることにより作製し、塩化セシウム密度勾配遠心法（Ｇｅｎｅ　Ｔｈｅｒ．，２０１０　Ａｐｒ；１７（４）：５０３－１０）により精製した。得られたＡＡＶ２／５－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ＡＤＲＡ１Ａ－ｓｈＲＮＡ－ＷＰＲＥ及びコントロールベクターＡＡＶ２／５－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ｓｃｒａｍｂｌｅ－ｓｈＲＮＡ－ＷＰＲＥはＡｍｉｃｏｎ　Ｕｌｔｒａ　１００Ｋ　ｆｉｌｔｅｒ　ｕｎｉｔ（Ｍｉｌｌｉｐｏｒｅ社製）を用いて０．００１％（ｖ／ｖ）Ｐｌｕｒｏｎｉｃ－Ｆ６８を含有したＰＢＳ溶液で透析した。ウイルスベクターの力価測定はＰｉｃｏ　Ｇｒｅｅｎ　ｆｌｕｏｒｏｍｅｔｒｉｃ　ｒｅａｇｅｎｔ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅ社製）を用いて行い、使用時まで－８０℃で保存した。 Virus vector production using the above plasmid was performed according to a standard method (Sci Rep., 2015 Sep 21; 5: 14306). In human embryonic kidney 293 (HEK 293) cells, the pAAV-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE or pAAV-FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE and Rep / Cap obtained above It was prepared by transfecting plasmid (pAAV2 / 5) and helper plasmid (pAd Delta F6) (Rep / Cap and pAd Delta F6 were obtained from Vector core of the University of Pennsylvania), and cesium chloride density gradient centrifugation (Gene Ther. , 2010 Apr; 17 (4): 503-10). The obtained AAV2 / 5-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE and control vector AAV2 / 5-FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE are Amicon Ultra 100K filter unit (manufactured by Millipore) ) Was dialyzed against a PBS solution containing 0.001% (v / v) Pluronic-F68. Titration of the viral vector was performed using Pico Green fluorometric reagent (manufactured by Molecular Probe) and stored at -80 ° C until use.
　８～１２週齢のＨｅｓ５－ＣｒｅＥＲＴ２マウス (Ｎａｔ　Ｃｏｍｍｕｎ．，　２０１２　Ｆｅｂ　１４；３：６７０)を用い、ケタミンとキシラジンの混合液を０．００５ｍＬ／ｇ皮下投与することで麻酔を行った。麻酔の効果を確認後、背部の毛を剃り、局所麻酔薬(キシロカイン（登録商標）ゼリー ２％)を塗布した。胸髄湾曲部を中心に正中に沿って３ｃｍ程皮膚を切開した。椎骨の第１腰髄の棘突起吻側先端から１ｍｍ側方の筋組織を直径２ｍｍ程度で切開し、第１３胸髄と第１腰髄椎骨間の膜を露出した。ガラスキャピラリー（エッペンドルフ社製）を介して左側各椎骨間の両端に、ウイルス濃度１×１０１２ ＧＣ／ｍＬで、０．５～０．６μＬをそれぞれ２か所、上記の配列番号１で表される塩基配列を含むアドレナリンα１Ａ受容体選択的ノックダウンベクターＡＡＶ２／５－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ＡＤＲＡ１Ａ－ｓｈＲＮＡ－ＷＰＲＥを微量注入し、Ｈｅｓ５陽性アストロサイトのアドレナリンα１Ａ受容体をノックダウンしたマウスを作製した。また、コントロールベクターとして、上記の配列番号２で表される塩基配列を含むベクターＡＡＶ２／５－ＦＬＥＸ－ＣＡ－ＡｃＧＦＰ－ｍｉｒ３０－ｓｃｒａｍｂｌｅ－ｓｈＲＮＡ－ＷＰＲＥを微量注入し、コントロールマウスを作製した。処置後は５－０絹糸を用いて皮膚を縫い合わせた。縫合部には局所麻酔薬（キシロカイン（登録商標）ゼリー ２％）を塗布し動物の苦痛の軽減を図った。同時に希ヨードチンキとゲンタシン（登録商標）軟膏（０．１％）の塗布も行い感染の防止を図った。 Anesthesia was performed using a mixed solution of ketamine and xylazine subcutaneously administered at 0.005 mL / g using 8- to 12-week-old Hes5-CreERT2 mice (Nat Commun., 2012 Feb 14; 3: 670). After confirming the effect of anesthesia, the back hair was shaved and a local anesthetic (Xylocaine (registered trademark) 2%) was applied. The skin was incised about 3 cm along the median centering on the thoracic spinal curve. A muscle tissue of 1 mm lateral to the rostral tip of the first lumbar spine of the first lumbar spinal cord was incised with a diameter of about 2 mm to expose the membrane between the thirteenth thoracic spinal cord and the first lumbar spinal disc bone. It is represented by the above-mentioned SEQ ID NO: 1 at two sites of 0.5 to 0.6 μL each at a virus concentration of 1 × 10 12 GC / mL at both ends between each left vertebra via a glass capillary (manufactured by Eppendorf). Α1A receptor selective knockdown vector containing the nucleotide sequence AAV2 / 5-FLEX-CA-AcGFP-mir30-ADRA1A-shRNA-WPRE is microinjected to knockdown the adrenergic α1A receptor of Hes5-positive astrocytes in mice Was produced. Also, as a control vector, a vector AAV2 / 5-FLEX-CA-AcGFP-mir30-scramble-shRNA-WPRE containing a base sequence represented by SEQ ID NO: 2 was microinjected to prepare a control mouse. After treatment, the skin was stitched with 5-0 silk thread. The suture was applied with a local anesthetic (Xylocaine (registered trademark) 2%) to reduce animal pain. At the same time, diluted iodine tincture and gentacin (registered trademark) ointment (0.1%) were applied to prevent infection.
　次に、タモキシフェン（シグマ社製）溶液（溶媒にコーン油を使用し２０ｍｇ／ｍＬで調製したもの）２ｍｇ／０．１ｍＬを１日１回１０日間、上記で作製したマウスへ腹腔内投与することによりＣｒｅリコンビナーゼ活性を誘導した。アデノ随伴ウイルスベクター投与２８日後に、フェニレフリン［（Ｒ）－（－）－ｐｈｅｎｙｌｅｐｈｒｉｎｅ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ；和光純薬社製］を５０ｐｍｏｌ／５μＬ生理食塩水を脊髄くも膜下腔内に投与し、実験例６と同様にして、マウスの行動解析を行い、５０％逃避閾値を算出した。その結果を図６右図に示す。 Next, intraperitoneally administer tamoxifen (manufactured by Sigma) solution (prepared at 20 mg / mL using corn oil as solvent) 2 mg / 0.1 mL once a day for 10 days to the mouse prepared above Induced Cre Recombinase activity. 28 days after administration of the adeno-associated virus vector, 50 pmol / 5 μL saline solution of phenylephrine [(R)-(-)-phenylephrine hydrochloride; manufactured by Wako Pure Chemical Industries, Ltd.] is administered into the spinal cord intrathecally, as in Example 6. Then, mouse behavior analysis was performed and 50% escape threshold was calculated. The results are shown in the right of FIG.
　図６右図に示したように、Ｈｅｓ５陽性アストロサイト特異的アドレナリンα１Ａ受容体をノックダウンすることにより、フェニレフリンによる脊髄くも膜下腔内投与で誘発される後肢逃避閾値の低下が抑制された。このことから、アストロサイトのアドレナリンα１Ａ受容体を発現抑制することにより、フェニレフリンによる軽度機械刺激に対する疼痛様行動（アロディニア）が抑制されることが確認された。 As shown in the right panel of FIG. 6, knockdown of the Hes5-positive astrocyte-specific adrenergic α1A receptor suppressed the decrease in hindlimb withdrawal threshold induced by intrathecal administration of phenylephrine by spinal cord. From this, it was confirmed that the pain-like behavior (allodynia) to mild mechanical stimulation by phenylephrine is suppressed by suppressing the expression of astrocyte adrenergic α1A receptor.
［実験例９］
（カプサイシン誘発の軽度機械刺激痛覚過敏に対する下行性ノルアドレナリン神経の関与）
　８～１２週齢のＣ５７ＢＬ／６Ｊマウス（日本クレア社製）の腹腔内に生理食塩水、又はＤＳＰ－４［Ｎ－（２－ｃｈｌｏｒｏｅｔｈｙｌ）－Ｎ－ｅｔｈｙｌ－２－ｂｒｏｍｏｂｅｎｚｙｌａｍｉｎｅ］（シグマ社製）を５０ｍｇ／ｋｇで投与した。投与３日後に、カプサイシン溶液０．１６μｇ／μＬを１０μＬ足裏に投与し、その後、実験例４と同様にして、５０％逃避閾値を算出した。その結果を図７に示す。 [Experimental Example 9]
Involvement of descending noradrenaline in capsaicin-induced mild mechanical hyperalgesia
Physiological saline or DSP-4 [N- (2-chloroethyl) -N-ethyl-2-bromobenzylamine] (manufactured by Sigma) into the abdominal cavity of 8 to 12 week-old C57BL / 6J mice (manufactured by CLEA Japan, Inc.) Was administered at 50 mg / kg. Three days after the administration, 0.16 μg / μL of capsaicin solution was administered to 10 μL of the sole, and then 50% escape threshold was calculated in the same manner as in Experimental Example 4. The results are shown in FIG.
　図７に示したように、ノルアドレナリン神経選択的毒素であるＤＳＰ－４を投与することにより、ノルアドレナリン神経を破壊すると、カプサイシン誘発の後肢逃避閾値の低下が抑制された。このことにより、ノルアドレナリン神経が、軽度機械刺激に対する疼痛様行動（アロディニア）に関与することが明らかとなった。 As shown in FIG. 7, the noradrenaline nerve was destroyed by administering DSP-4, which is a noradrenaline nerve selective toxin, and the decrease in capsaicin-induced hindpaw withdrawal threshold was suppressed. This revealed that noradrenaline is involved in pain-like behavior (allodinia) to mild mechanical stimulation.
［実験例１０］
（カプサイシン誘発の軽度機械刺激痛覚過敏に対するＨｅｓ５陽性アストロサイトのアドレナリンα１Ａ受容体の関与）
　実験例８で作製したＨｅｓ５陽性アストロサイト特異的アドレナリンα１Ａ受容体ノックダウンマウスに、カプサイシン溶液０．１６μｇ／μＬを１０μＬ足裏に投与し、実験例４と同様にして、５０％逃避閾値を算出した。その結果を図８に示す。 [Experimental Example 10]
(Involvement of adrenergic α1A receptor of Hes5 positive astrocytes on capsaicin-induced mild mechanical hyperalgesia)
0.16 μg / μL of capsaicin solution is administered to 10 μL of the footpad to Hes5-positive astrocyte-specific adrenergic α1A receptor knockdown mice prepared in Experimental Example 8 and 50% escape threshold is calculated in the same manner as Experimental Example 4. did. The results are shown in FIG.
　図８に示したように、脊髄後角のＨｅｓ５陽性アストロサイト選択的にアドレナリンα１Ａ受容体をノックダウンすることにより、カプサイシン誘発の後肢逃避閾値が顕著に抑制された。このことにより、Ｈｅｓ５陽性アストロサイトのアドレナリンα１Ａ受容体の発現を抑制することにより、軽度機械刺激に対する疼痛様行動（アロディニア）が抑制されることが明らかとなった。 As shown in FIG. 8, by selectively knocking down the adrenergical α1A receptor Hes5-positive astrocytes in the dorsal horn of the spinal cord, the capsaicin-induced hindpaw withdrawal threshold was significantly suppressed. This revealed that suppressing the expression of the adrenergic α1A receptor of Hes5 positive astrocytes suppresses pain-like behavior (allodinia) to mild mechanical stimulation.
　本発明によれば、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を有効成分として含有する鎮痛剤、及び、アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を選択することを特徴とする、鎮痛剤のスクリーニング方法を提供することができる。 According to the present invention, it is possible to select an analgesic agent containing, as an active ingredient, a substance that inhibits a signal from astrocyte adrenergic α1 receptor, and to select a substance that inhibits a signal from astrocyte adrenergic α1 receptor. It is possible to provide a screening method for analgesics, which is characterized.

Claims (12)

  1. 　アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を有効成分として含有する鎮痛剤。 Analgesic agent containing as active ingredient a substance that inhibits the signal from astrocyte adrenergic α1 receptor.
  2. 　前記アドレナリンα１受容体からのシグナルを阻害する物質が、アドレナリンα１受容体拮抗剤又はアドレナリンα１受容体発現抑制剤である請求項１に記載の鎮痛剤。 The analgesic according to claim 1, wherein the substance that inhibits the signal from the adrenergic α1 receptor is an adrenergic α1 receptor antagonist or an adrenergic α1 receptor expression inhibitor.
  3. 　前記アストロサイトが脊髄後角の表層に存在するアストロサイトである、請求項１又は２に記載の鎮痛剤。 The analgesic according to claim 1 or 2, wherein the astrocytes are astrocytes present in the surface layer of the spinal dorsal horn.
  4. 　前記脊髄後角の表層に存在するアストロサイトが、Ｈｅｓ５を発現しているアストロサイトである、請求項３に記載の鎮痛剤。 The analgesic according to claim 3, wherein the astrocytes present in the surface layer of the spinal dorsal horn are astrocytes expressing Hes5.
  5. 　前記アドレナリンα１受容体が、アドレナリンα１Ａ受容体である、請求項１～４のいずれか一項に記載の鎮痛剤。 The analgesic according to any one of claims 1 to 4, wherein the adrenergic α1 receptor is an adrenergic α1A receptor.
  6. 　前記鎮痛剤が、慢性疼痛治療剤である、請求項１～５のいずれか一項に記載の鎮痛剤。 The analgesic according to any one of claims 1 to 5, wherein the analgesic is a chronic pain therapeutic agent.
  7. 　アストロサイトのアドレナリンα１受容体からのシグナルを阻害する物質を選択することを特徴とする、鎮痛剤のスクリーニング方法。 A screening method for an analgesic agent, comprising selecting a substance that inhibits a signal from astrocyte adrenergic α1 receptor.
  8. 　前記アドレナリンα１受容体からのシグナルを阻害する物質が、アドレナリンα１受容体拮抗剤又はアドレナリンα１受容体発現抑制剤である請求項７に記載の方法。 The method according to claim 7, wherein the substance that inhibits the signal from the adrenergic α1 receptor is an adrenergic α1 receptor antagonist or an adrenergic α1 receptor expression inhibitor.
  9. 　前記アストロサイトが脊髄後角の表層に存在するアストロサイトである、請求項７又は８に記載の方法。 The method according to claim 7 or 8, wherein the astrocytes are astrocytes present in the surface layer of the spinal dorsal horn.
  10. 　前記脊髄後角の表層に存在するアストロサイトが、Ｈｅｓ５を発現しているアストロサイトである、請求項９に記載の方法。 The method according to claim 9, wherein the astrocytes present in the surface layer of the spinal dorsal horn are astrocytes expressing Hes5.
  11. 　前記アドレナリンα１受容体が、アドレナリンα１Ａ受容体である、請求項７～１０のいずれか一項に記載の方法。 The method according to any one of claims 7 to 10, wherein the adrenalin α1 receptor is an adrenalin α1A receptor.
  12. 　前記鎮痛剤が、慢性疼痛治療剤である、請求項７～１１のいずれか一項に記載の方法。 The method according to any one of claims 7 to 11, wherein the analgesic is a chronic pain therapeutic agent.
PCT/JP2018/040319 2017-11-02 2018-10-30 Analgesic and use thereof WO2019088095A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2017-213196 2017-11-02
JP2017213196 2017-11-02
JP2018018653 2018-02-05
JP2018-018653 2018-02-05

Publications (1)

Publication Number Publication Date
WO2019088095A1 true WO2019088095A1 (en) 2019-05-09

Family

ID=66333094

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/040319 WO2019088095A1 (en) 2017-11-02 2018-10-30 Analgesic and use thereof

Country Status (1)

Country Link
WO (1) WO2019088095A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000078716A1 (en) * 1999-06-24 2000-12-28 Toray Industries, Inc. α1B-ADRENERGIC RECEPTOR ANTAGONISTS
JP2002504523A (en) * 1998-02-27 2002-02-12 シンクロニュ−ロン，エルエルシ− Methods for treating painful conditions in the anus and compositions therefor
JP2004507527A (en) * 2000-08-31 2004-03-11 エフ．ホフマン−ラ　ロシュ　アーゲー Quinazoline derivatives as alpha-1 adrenergic antagonists
WO2004022045A1 (en) * 2002-09-03 2004-03-18 Yamanouchi Pharmaceutical Co., Ltd. Hypogastric and/or perineal pain-relieving agent
JP2008505173A (en) * 2004-06-30 2008-02-21 アラーガン、インコーポレイテッド Methods and compositions for treating pain and other α2 adrenergic mediated conditions

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002504523A (en) * 1998-02-27 2002-02-12 シンクロニュ−ロン，エルエルシ− Methods for treating painful conditions in the anus and compositions therefor
WO2000078716A1 (en) * 1999-06-24 2000-12-28 Toray Industries, Inc. α1B-ADRENERGIC RECEPTOR ANTAGONISTS
JP2004507527A (en) * 2000-08-31 2004-03-11 エフ．ホフマン−ラ　ロシュ　アーゲー Quinazoline derivatives as alpha-1 adrenergic antagonists
WO2004022045A1 (en) * 2002-09-03 2004-03-18 Yamanouchi Pharmaceutical Co., Ltd. Hypogastric and/or perineal pain-relieving agent
JP2008505173A (en) * 2004-06-30 2008-02-21 アラーガン、インコーポレイテッド Methods and compositions for treating pain and other α2 adrenergic mediated conditions

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CAMPOS, M. ET AL.: "Effecct of cyproterone acetate on alphal-adrenoceptor subtypes in rat vas deferens", BRAZILIAN JOURNAL OF MEDICAL AND BIOLOGICAL RESEARCH, vol. 36, 2003, pages 1571 - 1581, XP055612226 *
GANGKAK, G. ET AL.: "A single oral dose of Silodosin and Diclofenac sodium is effective in reducing pain after ureteric stent removal: a prospective, randomized, double blind placebo-controlled study", SPRINGERPLUS, vol. 5, no. 23, 2016, pages 1 - 6, XP021232357 *
HOLDEN, J.E. ET AL.: "Microinjection of morphine in the A7 catecholamine cell group produces opposing effects on nociception that are mediated by al-and a2-adrenoceptors", NEUROSCIENCE, vol. 91, no. 3, 1999, pages 979 - 990, XP055612227 *
KAUSHAL, R. ET AL.: "GABA-A receptor activity in the noradrenergic locus coeruleus drives trigeminal neuropathic pain in the rat; contribution of NAOE1 receptors in the medial prefrontal cortex", NEUROSCIENCE, vol. 334, 2016, pages 148 - 159, XP029786458, DOI: doi:10.1016/j.neuroscience.2016.08.005 *
MATOS, R. ET AL.: "Bladder pain induced by prolonged peripheral alpha 1A adrenoceptor stimulation involves the enhancement of transient receptor potential vanilloid 1 activity and an increase of urothelial adenosine triphosphate release", ACTA PHYSIOLOGICA, vol. 218, no. 4, 2016, pages 265 - 275, XP055612224 *
MATOS, R. ET AL.: "The water avoidance stress induces bladder pain due to a prolonged alphalA adrenoceptor stimulation", NAUNYN-SCHMIEDEBERG'S ARCHIVES OF PHARMACOLOGY, vol. 390, no. 8, 31 May 2017 (2017-05-31), pages 839 - 844, XP036275918, DOI: doi:10.1007/s00210-017-1384-1 *
MEISNER, J. G. ET AL.: "alpha1-adrenergic receptors augment P2X3 receptor-mediated nociceptive responses in the uninjured state", THE JOURNAL OF PAIN, vol. 8, no. 7, 2007, pages 556 - 562, XP022129263, DOI: doi:10.1016/j.jpain.2007.02.434 *
ZHANG, Y. ET AL.: "Different roles of alpha1- adrenoceptor subtypes in mediating cardiomyocyte protein synthesis in neonatal rats", CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, vol. 31, 2004, pages 626 - 633, XP055612229 *

Similar Documents

Publication Publication Date Title
Rodrigues et al. The sympathetic nervous system regulates skeletal muscle motor innervation and acetylcholine receptor stability
US11529359B2 (en) Use of GABAA receptor modulators for treatment of itch
JP6590828B2 (en) Combination of modified receptor vector and its exogenous agonist in the treatment of seizures
US20210254101A1 (en) Methods for treating spinal cord injury
Ralvenius et al. Itch suppression in mice and dogs by modulation of spinal α2 and α3GABAA receptors
Feng et al. The entorhinal cortex modulates trace fear memory formation and neuroplasticity in the mouse lateral amygdala via cholecystokinin
Xiang et al. Primary sensory neuron-specific interference of TRPV1 signaling by adeno-associated virus-encoded TRPV1 peptide aptamer attenuates neuropathic pain
Zhang et al. Plic-1, a new target in repressing epileptic seizure by regulation of GABAAR function in patients and a rat model of epilepsy
JP2005536438A (en) Treatment of pain by targeting hyperpolarization-activated cyclic nucleotide-gated channels
Yu et al. Endophilin A1 mediates seizure activity via regulation of AMPARs in a PTZ-kindled epileptic mouse model
Ferrari et al. Rabphilin-3A as a novel target to reverse α-synuclein-induced synaptic loss in Parkinson’s disease
Sun et al. Protein tyrosine phosphatase receptor type D regulates neuropathic pain after nerve injury via the STING-IFN-I pathway
Zhou et al. Dezocine attenuates the remifentanil-induced postoperative hyperalgesia by inhibition of phosphorylation of CaMKⅡα
US10047377B2 (en) Methods for modulating KLHL1 levels, methods for modulating current activity in T-type calcium channels, molecules therefor, and methods for identifying molecules therefor
Liang et al. TBI-induced nociceptive sensitization is regulated by histone acetylation
WO2019088095A1 (en) Analgesic and use thereof
Ferreira et al. Evidence of a functional α7-neuronal nicotinic receptor subtype located on motoneurons of the dorsal motor nucleus of the vagus
US20240366736A1 (en) Method for managing pain
Steyaert et al. Qualitative analysis of substance P, NK1-receptor and nerve ingrowth in substance P-treated ruptured rat Achilles tendon.
López-Canul et al. Selective Enhancement of REM Sleep in Male Rats through Activation of MT1 Receptors Located in the Locus Coeruleus Norepinephrine neurons
ES2869887T3 (en) Blt2 agonists for pain management
RU2781636C2 (en) Use of gabaa-receptor modulators for treatment of itch
US10597655B2 (en) Methods for modulating KLHL1 levels, methods for modulating current activity in T-type calcium channels, molecules therefor, and methods for identifying molecules therefor
Salvatierra The role of Nav1. 9 and Nav1. 1 in somatosensory perception
JP2009232705A (en) Method for discriminating glioma origin and glioma therapeutic agent

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18873346

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18873346

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP