WO2002057782A1 - Procede de criblage - Google Patents

Procede de criblage Download PDF

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Publication number
WO2002057782A1
WO2002057782A1 PCT/JP2002/000293 JP0200293W WO02057782A1 WO 2002057782 A1 WO2002057782 A1 WO 2002057782A1 JP 0200293 W JP0200293 W JP 0200293W WO 02057782 A1 WO02057782 A1 WO 02057782A1
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WO
WIPO (PCT)
Prior art keywords
cart
peptide
cart peptide
receptor
peptide receptor
Prior art date
Application number
PCT/JP2002/000293
Other languages
English (en)
Japanese (ja)
Inventor
Shigeyuki Chaki
Takeo Funakoshi
Naoya Kawashima
Yuichi Oshida
Yoshiko Suzuki
Tomokazu Ueki
Original Assignee
Taisho Pharmaceutical Co., Ltd.
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 Taisho Pharmaceutical Co., Ltd. filed Critical Taisho Pharmaceutical Co., Ltd.
Priority to JP2002558012A priority Critical patent/JPWO2002057782A1/ja
Publication of WO2002057782A1 publication Critical patent/WO2002057782A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor

Definitions

  • the present invention relates to a method for screening agonists and angiogonists of CART peptide receptor, a method for screening a therapeutic agent for anxiety and depression, and a novel therapeutic agent for anxiety and depression.
  • CRF corticotropin-releasing 'factor
  • HPA hypothalamus-pituitary-adrenal
  • Stress increases the expression of CRFmRNA in the paraventricular nucleus of the hypothalamus, which is thought to be the cause of activation of the HPA system.
  • the hypothalamic paraventricular nucleus has a projection system in the pituitary gland and plays an important role in stress-induced HPA activity. Stress changes the expression of various peptides in addition to CRF in the paraventricular nucleus of the hypothalamus.
  • CART cocaine- and amphetami ne- regulated transcript
  • CART is a gene that has been cloned by PCR differential display as a gene whose expression is induced in the nucleus accumbens upon administration of cocaine or amphetamine. It has been suggested that the active peptide in vivo encodes 128 amino acids and is a CART (55-102) consisting of 48 amino acids (FEBS Lett., 428, 263). , 1998). Recently, one of the physiological functions of the CART peptide has been elucidated. CART peptide caused appetite suppression by intracerebroventricular administration, whereas anti-CART peptide antibody enhanced appetite (Nature, 393, 72, 1998).
  • CART peptide has a strong anxiogenic effect in experimental animals.
  • IC 5 Is selected as a therapeutic agent for depression or anxiety neurosis.
  • a therapeutic agent for depression or anxiety neurosis which comprises the compound obtained by the screening of (10) or (11).
  • a therapeutic agent for depression or anxiety neurosis comprising an effective amount of a CAR T peptide receptor antagonist for improving a therapeutic agent for depression or anxiety neurosis;
  • a warm-blooded animal is a mouse (16) an anxiogenic model
  • FIG. 1 shows the amino acid sequence of the CART peptide.
  • FIG. 2 shows the effects of restraint stress on rat hypothalamic CART mRNA expression.
  • FIG. 3 shows the effect of CAR Ding peptide on locus coeruleus nerve activity.
  • FIG. 4 shows the relationship between the protein concentration of the membrane fraction and the specific binding amount of [ 125 I] CART peptide-CART peptide receptor.
  • Figure 5 shows the relationship between incubation time and specific binding of [ 125I ] CART peptide-CART peptide receptor.
  • Figure 6 shows the relationship between the [125 I] CART peptide concentration and [l25 I] specific binding of CART peptide one CART peptide receptors.
  • FIG. 1 shows the amino acid sequence of the CART peptide.
  • FIG. 2 shows the effects of restraint stress on rat hypothalamic CART mRNA expression.
  • FIG. 3 shows the effect of CAR Ding peptide on locus coerul
  • FIG. 7 shows the results of Scatchyard analysis of [ 125 I] CART peptide binding.
  • FIG. 8 shows the effect of PS AO 961 on [ 125 I] CART peptide binding to rat pituitary membrane.
  • FIG. 9 shows an anxiety-inducing effect of the CART peptide in a mouse elevated plus maze test and the effect of an anxiolytic drug PSA0961 on it.
  • the CART peptide is a peptide having the amino acid sequence represented by SEQ ID NO: 1, or one or several amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 1.
  • Preferred is a peptide comprising the amino acid sequence represented by SEQ ID NO: 2.
  • the peptide represented by SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 3 lacks one or several amino acids, It is preferable to use a substituted or modified amino acid sequence (FEBS Letter, 428, 263-268 (1998)).
  • the peptide represented by SEQ ID NO: 3 has a secondary structure as shown in FIG.
  • the labeled CART peptide includes, for example, those obtained by labeling CART peptide with fluorescein isotianate, biotin, an enzyme, or a radioisotope.
  • the enzyme include alkaline phosphatase and peroxidase.
  • Radioisotope Examples of the element include [ 3 H] and [ , 25 I]. Labeling of the CART peptide can be performed by a method known per se.
  • the CART peptide receptor can be prepared from animal fibers or cells. Preferably, it is a CART peptide receptor prepared from E ⁇ of a warm-blooded animal. For example, after homogenizing rat pituitary gland in a buffer such as Tris-HCl buffer, the precipitate obtained by centrifugation is suspended in Tris-HCl buffer, and ffij ⁇ g products containing CART peptide receptor are suspended in Tris-HCl buffer. Obtainable.
  • the screening method of the present invention is a screening method characterized by using a CART peptide receptor, and is preferably characterized in that a CART peptide and a test substance are used as a CART peptide receptor. More preferably, (a) mixing a labeled CART peptide and a CART peptide receptor and incubating at 0 to 42 ° C (b) binding to the CART peptide receptor (C) The step (c) of (a) and (b) is performed in the presence and absence of a test substance, and the two steps are compared.
  • the determination of the amount of labeled CART peptide can be measured by a method known per se. For example, those labeled with a radioisotope are radiated by a liquid scintillation counter or an aquarium. It can be calculated by measuring tongue properties, and the enzyme-labeled product can be calculated by reacting with a chromogenic substrate and then measuring by colorimetry.
  • CART peptide receptor antagonist means a compound having an action of antagonizing the binding of CARPT peptide to the receptor, and includes competitive antagonists and non-competitive drugs.
  • a concentration-dependent manner in receptor binding experiments using cells or brain tissue membrane [1 2 5 I] shows the CART peptide bond 3 ⁇ 4 ⁇ 3 ⁇ 4 action, physiological action of Ru caused by CART peptides (e.g. c- fos induction activity) Is a compound that inhibits IC 5 In even more preferably [1 2 5 I] CAR T peptide binding experiments. Is a compound of 10 M or less.
  • the remedy for anxiety and depression according to the present invention is usually formulated and used.
  • Formulations containing the CART peptide receptor antagonist as an active ingredient used in the present invention are mixed with carriers, excipients, and other additives that are pharmaceutical auxiliaries usually used in the formulation of pharmaceuticals. And prepared. ,
  • Administration may be any of the following: oral administration by! ⁇ , pill, capsule, mu powder, il, etc., or parenteral administration by injection such as intravenous injection or intramuscular injection, suppository, transdermal, etc. .
  • the effective amount of the drug for the treatment of depression or anxiety neurosis is determined according to the individual case, taking into account the symptoms, age, sex, etc. of the administration subject. In the range of 1 to 100 mg, preferably 5 to 20 mg per day per person, this may be administered once to several times a day or in the case of parenteral administration.
  • a smaller dose than the above dose range may be sufficient.
  • the anxiety-inducing model of the present invention is obtained by administering a CART peptide into a ventricle, and can be used for a test system for a drug for treating anxiety neuropathy and depression, for example, an elevated plus maze.
  • Industrial applicability The present invention provides a compound useful for suppressing appetite based on a novel mechanism of action, a method for screening a compound effective for treating anxiety and depression, and a model for triggering anxiety. It has become possible to provide new therapeutic agents.
  • the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
  • the animals were 9-week-old SD rats (Japanese charrriver) and subjected to 1 hour of restraint stress. After the stress, the rats were sacrificed by decapitation. After quickly removing the brain, the hypothalamus was separated and immediately frozen with liquid nitrogen. Total RA was prepared from the hypothalamus using RNeasy Mini Kit (QIAGEN).
  • Pre-hybridization buffer (6XSSC, 50% formamide, 0.5% sodium dodecyl sulfate (SDS), 5 XDenhardt solution)
  • Signal detection includes DIG wash and block buffer set, AP-labeled anti-digoxy Genin Fab fragment (both Roche Diagnostics) and CDP-siar (TR0PIX) were used.
  • the membrane was boiled in a 0.1% SDS solution for 10 minutes, and the CART probe was stripped.
  • the membrane was placed in prehybridization buffer and incubated at 42 ° C for 3 hours. Thereafter, the mixture was incubated overnight at 42 ° C. in a hybridization buffer containing a G 3 PDH probe prepared using a PCP DIG probe synthesis kit (Roche Diagnostics). Wash the membrane twice in 2XSS C buffer containing 0.1% SDS for 5 minutes at room temperature, then in 42, 0.1% SDS in 0.1 XSSC buffer for 15 minutes Was washed twice. Signal detection was performed as in CART.
  • the signal intensities of CART and G3PDH were measured by Lumi's Imager (Roche's Diagnostics), respectively, and the CART signal intensities were corrected by the internal standard G3PDH signal intensity.
  • the experiment was performed using Wistar male rats (Nippon Charlsliver) under anesthesia with urethane (1.5 g / kg, ip). Leaching of noradrenaline nerve activity in the locus coeruleus (LC) was performed as follows. In other words, after fixing the animal on the brain fixation device (Narimo), the scalp was incised and the skull was exposed, paying attention to bleeding. A 2mm square hole was drilled at a position (A:-3mm, L: 1.2mm, V: -7.5mm) from the lambda suture. From this position, a micro glass electrode for recording the action potential of a single nerve was inserted. The electrode had a resistance of 2-8 M ⁇ and was filled with 2M NaC1.
  • the glass electrode was inserted into the brain at an angle of 20 ° forward to avoid the sinus on the LC.
  • Identification of noradrenaline neuronal activity in the locus coeruleus is performed by retrograde fie by the action potential waveform and electrical stimulation of the dorsal noradrenergic bundle (A: +1.5 mm, L: 0.8 mm, V: ⁇ 5.7 mm from lambda suture).
  • Indicator of Id potential Went as.
  • CAR T peptide (3 g: SEQ ID NO: 3) was intracerebroventricularly administered. Changes in nerve activity were observed for about 10 to 15 minutes after administration.
  • a solvent administration group was provided as a control group. The effect of the CART peptide was indicated by an increase rate after administration with respect to the firing frequency of nerve activity before administration.
  • the symbol * in FIG. 3 indicates that there is a significant difference compared to the control group at p ⁇ 0.05 when a significant difference test was performed by the Dunnett test.
  • Rat pituitary gland is homogenized in 5 OmM Tris-HCl buffer (pH 7.4 or pH 9.0) containing 0.5 mM phenylmethylsulfonylfurylleolide, which is harmful to 2 OmlZg, and then 48, OOOXg for 20 min. And centrifuged at 4 ° C for minutes. The precipitate obtained by centrifugation was re-homogenized with the same buffer, and the homogenate was centrifuged at 48 ° C. for 20 minutes at 4 ° C. with OOXg. This operation was repeated twice.
  • the precipitate was suspended in 50 mM Tris-HCl buffer (pH 7.4 or 9.0) containing 0.5 mM phenylmethylsulfonyl fluoride and 0.1% serum albumin and bound as a crude advisory product. Used for experiments.
  • the ffiE ⁇ product (0.5 ml) was incubated with [ 125 I] CART peptide (Amersham: SEQ ID NO: 3) at 25 ° C. After the incubation was completed, the reaction solution was filtered through a GF / B glass fiber filter paper which had been immersed in a 0.3% polyethyleneimine solution for 2 hours in advance using a cell binding tester for receptor binding experiments. The filter paper was washed three times with 3 ml of 5 OmM Tris-HCl buffer (pH 7.4 or pH 9.0) containing 0.1% serum albumin and 50 mM sodium chloride. The radioactivity of the filter paper was measured with a counter. The amount of binding in the presence of 1 M CART peptide was defined as non-specific binding, and the value obtained by subtracting non-specific binding from the total binding in the absence of 1 M CART peptide was defined as specific binding.
  • the high-affinity specific binding obtained in this [ 125 I] CART peptide binding test was determined by CART. It revealed that the peptide receptor was labeled.
  • the [ 125 I] CART peptide concentration was set to 0. InM
  • the protein concentration was set to 50 Hg / m 1
  • the incubation time was set to 2 hours.
  • the test substance was dissolved in a 100% DMSO solution and added to the restaurant simultaneously with the [ 125 I] CART peptide. 1 0 IC 5 from inhibition curves in one 8 M to 10- 5 M concentration. Values were calculated.
  • PSAO961 the compound represented by (1) (hereinafter referred to as PSAO961)
  • the animals used were ICR male mice weighing 24-33 g.
  • the elevated cross maze used in the test consisted of an open path ( ⁇ 5 cm, length 30 cm) and a closed path (width 5 cm, length 30 cm), with an open path of 0.3 cm,
  • the closed road was covered with 20 cm high clear plexiglass.
  • the maze was fixed at 38.5 cm above the floor. Illuminance was 50-60 lux at the center of the device.
  • the mouse was placed with its head facing the open lane in the center of the cross maze, measurement was started, and the time spent in the open lane for 5 minutes was measured. The results are shown in FIG.
  • the CART peptide was dissolved in phosphate-buffered saline containing 0.02% KC1 and 0.1% serum albumin and administered intracerebroventricularly at a rate of 0.1 g / mouse 1 hour before the test.
  • the anxiolytic was administered intraperitoneally 30 minutes before the intracerebroventricular administration of the CART peptide.
  • the CART peptide receptor antagonist was administered intraventricularly at the same time as the CART peptide.
  • Fig. 9 indicates that when a significant difference test was performed by the Dunnet test, The symbol ## indicates that there is a significant difference compared with the group, and the symbol ## indicates that there is a significant difference compared with the CART peptide administration group at P ⁇ 0.01 when the significant difference test is performed by the Dunnett test.
  • the CART peptide-administered group markedly and significantly reduced the time spent on the open road.
  • this reduced open-stay time was significantly and dose-dependently antagonized by intraperitoneal administration of 0.3 mgZkg, lmg / kg or 3 mgZkg of diazebam or buspirone.
  • PS AO961 was significantly antagonized by the shortening of the residence time on open roads by administration of the CART peptide (Fig. 9 (c)).
  • the CART peptide causes anxiety-like symptoms and stress response, and a compound that acts as a CART peptide receptor antagonist has an action to suppress anxiety-like symptoms. Useful.

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Abstract

L'invention concerne un procédé de criblage d'un agoniste ou d'un antagoniste du récepteur peptidique CART, ce procédé étant caractérisé par l'utilisation du récepteur peptidique CART.
PCT/JP2002/000293 2001-01-18 2002-01-17 Procede de criblage WO2002057782A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2002558012A JPWO2002057782A1 (ja) 2001-01-18 2002-01-17 新規スクリーニング方法

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JP2001009904 2001-01-18
JP2001-9904 2001-01-18

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WO2002057782A1 true WO2002057782A1 (fr) 2002-07-25

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034619A1 (fr) * 1995-05-04 1996-11-07 Amgen Inc. Procedes de prevention de la degenerescence des neurones et de promotion de la regeneration de ceux-ci

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034619A1 (fr) * 1995-05-04 1996-11-07 Amgen Inc. Procedes de prevention de la degenerescence des neurones et de promotion de la regeneration de ceux-ci

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ASAKAWA ET AL., HORMONE AND METABOLIC RESEARCH, vol. 33, no. 9, 2001, pages 554 - 558, XP002950548 *
KASK ET AL., BRAIN RESEARCH, vol. 857, 2000, pages 283 - 285, XP002950547 *
KIMMEL ET AL., JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, vol. 294, no. 2, 2000, pages 784 - 792, XP002950546 *

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