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WO2019088095A1 - Analgésique et son utilisation - Google Patents

Analgésique et son utilisation Download PDF

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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
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receptor
adrenergic
astrocytes
analgesic
spinal
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Japanese (ja)
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誠 津田
雄太 高露
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Kyushu University NUC
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Kyushu University NUC
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    • 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.

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Abstract

La présente invention concerne : un analgésique qui contient, en tant que principe actif, une substance qui inhibe le signal provenant des récepteurs α1 adrénergiques des astrocytes ; et une méthode de criblage d'analgésiques caractérisée par le fait qu'elle sélectionne des substances qui inhibent un signal provenant des récepteurs α1 adrénergiques des astrocytes.
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