JP2002540063A - Binding partner for 5-HT5 receptor for migraine treatment - Google Patents
Binding partner for 5-HT5 receptor for migraine treatmentInfo
- Publication number
- JP2002540063A JP2002540063A JP2000593096A JP2000593096A JP2002540063A JP 2002540063 A JP2002540063 A JP 2002540063A JP 2000593096 A JP2000593096 A JP 2000593096A JP 2000593096 A JP2000593096 A JP 2000593096A JP 2002540063 A JP2002540063 A JP 2002540063A
- Authority
- JP
- Japan
- Prior art keywords
- binding
- receptor
- binding partner
- affinity
- activity
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 230000027455 binding Effects 0.000 title claims abstract description 195
- 108010006590 serotonin 5 receptor Proteins 0.000 title claims abstract description 74
- 238000011282 treatment Methods 0.000 title claims description 29
- 208000019695 Migraine disease Diseases 0.000 title claims description 27
- 206010027599 migraine Diseases 0.000 title claims description 25
- 238000000034 method Methods 0.000 claims abstract description 44
- 238000012216 screening Methods 0.000 claims abstract description 17
- 208000026106 cerebrovascular disease Diseases 0.000 claims abstract description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 5
- 150000001875 compounds Chemical class 0.000 claims description 45
- 210000004027 cell Anatomy 0.000 claims description 42
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 claims description 42
- 102000005962 receptors Human genes 0.000 claims description 41
- 108020003175 receptors Proteins 0.000 claims description 41
- 230000000694 effects Effects 0.000 claims description 32
- 239000011575 calcium Substances 0.000 claims description 20
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 18
- 230000003834 intracellular effect Effects 0.000 claims description 18
- 229910052791 calcium Inorganic materials 0.000 claims description 17
- 108091032151 5-hydroxytryptamine receptor family Proteins 0.000 claims description 16
- 102000040125 5-hydroxytryptamine receptor family Human genes 0.000 claims description 12
- WKZLNEWVIAGNAW-UHFFFAOYSA-N 5-Carboxyamidotryptamine Chemical compound C1=C(C(N)=O)C=C2C(CCN)=CNC2=C1 WKZLNEWVIAGNAW-UHFFFAOYSA-N 0.000 claims description 10
- 108091006027 G proteins Proteins 0.000 claims description 10
- 102000030782 GTP binding Human genes 0.000 claims description 10
- 108091000058 GTP-Binding Proteins 0.000 claims description 10
- 102000014384 Type C Phospholipases Human genes 0.000 claims description 7
- 108010079194 Type C Phospholipases Proteins 0.000 claims description 7
- 230000003491 cAMP production Effects 0.000 claims description 7
- 102100027499 5-hydroxytryptamine receptor 1B Human genes 0.000 claims description 6
- 230000001154 acute effect Effects 0.000 claims description 6
- 230000001270 agonistic effect Effects 0.000 claims description 6
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 5
- 238000000338 in vitro Methods 0.000 claims description 5
- 230000006698 induction Effects 0.000 claims description 5
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 5
- 102100027493 5-hydroxytryptamine receptor 1D Human genes 0.000 claims description 4
- 208000032612 Glial tumor Diseases 0.000 claims description 4
- 206010018338 Glioma Diseases 0.000 claims description 4
- 101150050738 HTR1B gene Proteins 0.000 claims description 2
- 101100321769 Takifugu rubripes htr1d gene Proteins 0.000 claims description 2
- 210000003292 kidney cell Anatomy 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims 1
- 241000894007 species Species 0.000 claims 1
- 206010019233 Headaches Diseases 0.000 abstract description 6
- 231100000869 headache Toxicity 0.000 abstract description 6
- 238000012512 characterization method Methods 0.000 abstract description 3
- 239000000556 agonist Substances 0.000 description 15
- 235000001465 calcium Nutrition 0.000 description 15
- XOFLBQFBSOEHOG-UUOKFMHZSA-N γS-GTP Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=S)[C@@H](O)[C@H]1O XOFLBQFBSOEHOG-UUOKFMHZSA-N 0.000 description 13
- 239000000872 buffer Substances 0.000 description 12
- 210000004379 membrane Anatomy 0.000 description 12
- 239000012528 membrane Substances 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 210000004556 brain Anatomy 0.000 description 10
- 230000005764 inhibitory process Effects 0.000 description 10
- 230000006870 function Effects 0.000 description 9
- 239000003446 ligand Substances 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 208000024891 symptom Diseases 0.000 description 8
- 238000003556 assay Methods 0.000 description 7
- 210000001130 astrocyte Anatomy 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 208000035475 disorder Diseases 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 230000000638 stimulation Effects 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 101710138068 5-hydroxytryptamine receptor 1D Proteins 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 210000000170 cell membrane Anatomy 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 5
- 229960000367 inositol Drugs 0.000 description 5
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- 102000035038 5-HT1 receptors Human genes 0.000 description 4
- 108091005478 5-HT1 receptors Proteins 0.000 description 4
- 101710138639 5-hydroxytryptamine receptor 1B Proteins 0.000 description 4
- 102000014630 G protein-coupled serotonin receptor activity proteins Human genes 0.000 description 4
- RLJFTICUTYVZDG-UHFFFAOYSA-N Methiothepine Chemical compound C12=CC(SC)=CC=C2SC2=CC=CC=C2CC1N1CCN(C)CC1 RLJFTICUTYVZDG-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000000654 additive Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000008878 coupling Effects 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000005859 coupling reaction Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000002287 radioligand Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000002821 scintillation proximity assay Methods 0.000 description 4
- 230000007480 spreading Effects 0.000 description 4
- 238000003892 spreading Methods 0.000 description 4
- KQKPFRSPSRPDEB-UHFFFAOYSA-N sumatriptan Chemical compound CNS(=O)(=O)CC1=CC=C2NC=C(CCN(C)C)C2=C1 KQKPFRSPSRPDEB-UHFFFAOYSA-N 0.000 description 4
- ZISSAWUMDACLOM-UHFFFAOYSA-N triptane Chemical compound CC(C)C(C)(C)C ZISSAWUMDACLOM-UHFFFAOYSA-N 0.000 description 4
- -1 5-HT5 Proteins 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 3
- 230000005856 abnormality Effects 0.000 description 3
- 230000008499 blood brain barrier function Effects 0.000 description 3
- 210000001218 blood-brain barrier Anatomy 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000006957 competitive inhibition Effects 0.000 description 3
- 238000012937 correction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000004064 dysfunction Effects 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011321 prophylaxis Methods 0.000 description 3
- 229960003708 sumatriptan Drugs 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 210000005166 vasculature Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 101710138638 5-hydroxytryptamine receptor 1A Proteins 0.000 description 2
- 102100022738 5-hydroxytryptamine receptor 1A Human genes 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 206010010904 Convulsion Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 206010015866 Extravasation Diseases 0.000 description 2
- OZLGRUXZXMRXGP-UHFFFAOYSA-N Fluo-3 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C2=C3C=C(Cl)C(=O)C=C3OC3=CC(O)=C(Cl)C=C32)N(CC(O)=O)CC(O)=O)=C1 OZLGRUXZXMRXGP-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 229930193140 Neomycin Natural products 0.000 description 2
- SNIOPGDIGTZGOP-UHFFFAOYSA-N Nitroglycerin Chemical compound [O-][N+](=O)OCC(O[N+]([O-])=O)CO[N+]([O-])=O SNIOPGDIGTZGOP-UHFFFAOYSA-N 0.000 description 2
- 239000000006 Nitroglycerin Substances 0.000 description 2
- 208000002193 Pain Diseases 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- BKRGVLQUQGGVSM-KBXCAEBGSA-N Revanil Chemical compound C1=CC(C=2[C@H](N(C)C[C@H](C=2)NC(=O)N(CC)CC)C2)=C3C2=CNC3=C1 BKRGVLQUQGGVSM-KBXCAEBGSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229930002945 all-trans-retinaldehyde Natural products 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000000730 antalgic agent Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 102000036109 cAMP binding proteins Human genes 0.000 description 2
- 108091010966 cAMP binding proteins Proteins 0.000 description 2
- 229910002091 carbon monoxide Inorganic materials 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000012612 commercial material Substances 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 229960003133 ergot alkaloid Drugs 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 230000036251 extravasation Effects 0.000 description 2
- 108700025906 fos Genes Proteins 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 229940014259 gelatin Drugs 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 229960003711 glyceryl trinitrate Drugs 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 150000003949 imides Chemical class 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical group [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 210000004498 neuroglial cell Anatomy 0.000 description 2
- 230000009251 neurologic dysfunction Effects 0.000 description 2
- 208000015015 neurological dysfunction Diseases 0.000 description 2
- 239000002858 neurotransmitter agent Substances 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000611 regression analysis Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 235000020945 retinal Nutrition 0.000 description 2
- 239000011604 retinal Substances 0.000 description 2
- 230000002207 retinal effect Effects 0.000 description 2
- NCYCYZXNIZJOKI-OVSJKPMPSA-N retinal group Chemical group C\C(=C/C=O)\C=C\C=C(\C=C\C1=C(CCCC1(C)C)C)/C NCYCYZXNIZJOKI-OVSJKPMPSA-N 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 229940076279 serotonin Drugs 0.000 description 2
- 239000000952 serotonin receptor agonist Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 210000000427 trigeminal ganglion Anatomy 0.000 description 2
- 230000024883 vasodilation Effects 0.000 description 2
- NCYCYZXNIZJOKI-UHFFFAOYSA-N vitamin A aldehyde Natural products O=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-UHFFFAOYSA-N 0.000 description 2
- QDZOEBFLNHCSSF-PFFBOGFISA-N (2S)-2-[[(2R)-2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-1-[(2R)-2-amino-5-carbamimidamidopentanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-N-[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2S)-1-amino-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]pentanediamide Chemical compound C([C@@H](C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CCCNC(N)=N)C1=CC=CC=C1 QDZOEBFLNHCSSF-PFFBOGFISA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 101710138085 5-hydroxytryptamine receptor 1E Proteins 0.000 description 1
- 102100036312 5-hydroxytryptamine receptor 1E Human genes 0.000 description 1
- 102100024959 5-hydroxytryptamine receptor 2C Human genes 0.000 description 1
- 101710138093 5-hydroxytryptamine receptor 2C Proteins 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 108091023037 Aptamer Proteins 0.000 description 1
- 206010003225 Arteriospasm coronary Diseases 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 208000006561 Cluster Headache Diseases 0.000 description 1
- 208000003890 Coronary Vasospasm Diseases 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 241001269524 Dura Species 0.000 description 1
- 206010013886 Dysaesthesia Diseases 0.000 description 1
- 208000012661 Dyskinesia Diseases 0.000 description 1
- 241000156978 Erebia Species 0.000 description 1
- 206010017711 Gangrene Diseases 0.000 description 1
- 206010018341 Gliosis Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101150087426 Gnal gene Proteins 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000879758 Homo sapiens Sjoegren syndrome nuclear autoantigen 1 Proteins 0.000 description 1
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000012839 Krebs-Henseleit buffer Substances 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- VAYOSLLFUXYJDT-RDTXWAMCSA-N Lysergic acid diethylamide Chemical compound C1=CC(C=2[C@H](N(C)C[C@@H](C=2)C(=O)N(CC)CC)C2)=C3C2=CNC3=C1 VAYOSLLFUXYJDT-RDTXWAMCSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- AWEZYKMQFAUBTD-UHFFFAOYSA-N Naratriptan hydrochloride Chemical compound [H+].[Cl-].C12=CC(CCS(=O)(=O)NC)=CC=C2NC=C1C1CCN(C)CC1 AWEZYKMQFAUBTD-UHFFFAOYSA-N 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 108090000189 Neuropeptides Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 208000005056 Ophthalmoplegic Migraine Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102100024304 Protachykinin-1 Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 1
- 102000007568 Proto-Oncogene Proteins c-fos Human genes 0.000 description 1
- 241000144290 Sigmodon hispidus Species 0.000 description 1
- 102100037330 Sjoegren syndrome nuclear autoantigen 1 Human genes 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 101800003906 Substance P Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- NIJJYAXOARWZEE-UHFFFAOYSA-N Valproic acid Chemical compound CCCC(C(O)=O)CCC NIJJYAXOARWZEE-UHFFFAOYSA-N 0.000 description 1
- 206010057469 Vascular stenosis Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- KRMDCWKBEZIMAB-UHFFFAOYSA-N amitriptyline Chemical group C1CC2=CC=CC=C2C(=CCCN(C)C)C2=CC=CC=C21 KRMDCWKBEZIMAB-UHFFFAOYSA-N 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 230000002460 anti-migrenic effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229940005513 antidepressants Drugs 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical compound NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 description 1
- 239000002876 beta blocker Substances 0.000 description 1
- 229940097320 beta blocking agent Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004781 brain capillary Anatomy 0.000 description 1
- 230000006931 brain damage Effects 0.000 description 1
- 231100000874 brain damage Toxicity 0.000 description 1
- 208000029028 brain injury Diseases 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 229960001714 calcium phosphate Drugs 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 229960003340 calcium silicate Drugs 0.000 description 1
- 235000012241 calcium silicate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 208000015114 central nervous system disease Diseases 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000004035 construction material Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 150000004696 coordination complex Chemical class 0.000 description 1
- 201000011634 coronary artery vasospasm Diseases 0.000 description 1
- 210000000877 corpus callosum Anatomy 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- KPHWPUGNDIVLNH-UHFFFAOYSA-M diclofenac sodium Chemical compound [Na+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KPHWPUGNDIVLNH-UHFFFAOYSA-M 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000001079 digestive effect Effects 0.000 description 1
- 229960004704 dihydroergotamine Drugs 0.000 description 1
- HESHRHUZIWVEAJ-JGRZULCMSA-N dihydroergotamine Chemical compound C([C@H]1C(=O)N2CCC[C@H]2[C@]2(O)O[C@@](C(N21)=O)(C)NC(=O)[C@H]1CN([C@H]2[C@@H](C3=CC=CC4=NC=C([C]34)C2)C1)C)C1=CC=CC=C1 HESHRHUZIWVEAJ-JGRZULCMSA-N 0.000 description 1
- 230000010339 dilation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003221 ear drop Substances 0.000 description 1
- 229940047652 ear drops Drugs 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 229960004943 ergotamine Drugs 0.000 description 1
- OFKDAAIKGIBASY-VFGNJEKYSA-N ergotamine Chemical compound C([C@H]1C(=O)N2CCC[C@H]2[C@]2(O)O[C@@](C(N21)=O)(C)NC(=O)[C@H]1CN([C@H]2C(C3=CC=CC4=NC=C([C]34)C2)=C1)C)C1=CC=CC=C1 OFKDAAIKGIBASY-VFGNJEKYSA-N 0.000 description 1
- XCGSFFUVFURLIX-UHFFFAOYSA-N ergotaminine Natural products C1=C(C=2C=CC=C3NC=C(C=23)C2)C2N(C)CC1C(=O)NC(C(N12)=O)(C)OC1(O)C1CCCN1C(=O)C2CC1=CC=CC=C1 XCGSFFUVFURLIX-UHFFFAOYSA-N 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003889 eye drop Substances 0.000 description 1
- 229940012356 eye drops Drugs 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- SMANXXCATUTDDT-QPJJXVBHSA-N flunarizine Chemical compound C1=CC(F)=CC=C1C(C=1C=CC(F)=CC=1)N1CCN(C\C=C\C=2C=CC=CC=2)CC1 SMANXXCATUTDDT-QPJJXVBHSA-N 0.000 description 1
- 229960000326 flunarizine Drugs 0.000 description 1
- SYTBZMRGLBWNTM-UHFFFAOYSA-N flurbiprofen Chemical compound FC1=CC(C(C(O)=O)C)=CC=C1C1=CC=CC=C1 SYTBZMRGLBWNTM-UHFFFAOYSA-N 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- YFHXZQPUBCBNIP-UHFFFAOYSA-N fura-2 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=3OC(=CC=3C=2)C=2OC(=CN=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 YFHXZQPUBCBNIP-UHFFFAOYSA-N 0.000 description 1
- 210000000609 ganglia Anatomy 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 239000003349 gelling agent Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000007387 gliosis Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 239000000416 hydrocolloid Substances 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- AFJSFHAKSSWOKG-UHFFFAOYSA-N hydron;n-[1-[2-(1h-indol-3-yl)ethyl]piperidin-4-yl]benzamide;chloride Chemical compound Cl.C1CN(CCC=2C3=CC=CC=C3NC=2)CCC1NC(=O)C1=CC=CC=C1 AFJSFHAKSSWOKG-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000007914 intraventricular administration Methods 0.000 description 1
- 238000001616 ion spectroscopy Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- DKYWVDODHFEZIM-UHFFFAOYSA-N ketoprofen Chemical compound OC(=O)C(C)C1=CC=CC(C(=O)C=2C=CC=CC=2)=C1 DKYWVDODHFEZIM-UHFFFAOYSA-N 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000011344 liquid material Substances 0.000 description 1
- 238000005567 liquid scintillation counting Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- HYYBABOKPJLUIN-UHFFFAOYSA-N mefenamic acid Chemical group CC1=CC=CC(NC=2C(=CC=CC=2)C(O)=O)=C1C HYYBABOKPJLUIN-UHFFFAOYSA-N 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 206010052787 migraine without aura Diseases 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 230000004766 neurogenesis Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000006959 non-competitive inhibition Effects 0.000 description 1
- 229940121367 non-opioid analgesics Drugs 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 210000000956 olfactory bulb Anatomy 0.000 description 1
- 239000003605 opacifier Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229960005489 paracetamol Drugs 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 208000035824 paresthesia Diseases 0.000 description 1
- 230000001314 paroxysmal effect Effects 0.000 description 1
- 239000004031 partial agonist Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- FIADGNVRKBPQEU-UHFFFAOYSA-N pizotifen Chemical compound C1CN(C)CCC1=C1C2=CC=CC=C2CCC2=C1C=CS2 FIADGNVRKBPQEU-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000013492 plasmid preparation Methods 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- DBABZHXKTCFAPX-UHFFFAOYSA-N probenecid Chemical compound CCCN(CCC)S(=O)(=O)C1=CC=C(C(O)=O)C=C1 DBABZHXKTCFAPX-UHFFFAOYSA-N 0.000 description 1
- 229960003081 probenecid Drugs 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000007398 protein translocation Effects 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000002331 radioactive microsphere Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000012056 semi-solid material Substances 0.000 description 1
- 230000000862 serotonergic effect Effects 0.000 description 1
- 108010053303 serotonin 1F receptor Proteins 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 208000027765 speech disease Diseases 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000002511 suppository base Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000002636 symptomatic treatment Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- HHLJUSLZGFYWKW-UHFFFAOYSA-N triethanolamine hydrochloride Chemical compound Cl.OCCN(CCO)CCO HHLJUSLZGFYWKW-UHFFFAOYSA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000002227 vasoactive effect Effects 0.000 description 1
- 239000005526 vasoconstrictor agent Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 229940063674 voltaren Drugs 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- ULSDMUVEXKOYBU-ZDUSSCGKSA-N zolmitriptan Chemical compound C1=C2C(CCN(C)C)=CNC2=CC=C1C[C@H]1COC(=O)N1 ULSDMUVEXKOYBU-ZDUSSCGKSA-N 0.000 description 1
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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
- A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/48—Ergoline derivatives, e.g. lysergic acid, ergotamine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
-
- 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
- A61P25/06—Antimigraine agents
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Neurology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Neurosurgery (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Pain & Pain Management (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
(57)【要約】 本発明は、5−HT5受容体に対する選択的結合パートナー、かかる結合パートナーの同定及び特徴付けのための方法、特にスクリーニング法、並びに前記の結合パートナーを含有する医薬品組成物及び脳血管障害、例えば偏頭痛の治療におけるその使用に関する。 (57) [Summary] The present invention relates to selective binding partners for the 5-HT5 receptor, methods for the identification and characterization of such binding partners, in particular screening methods, and pharmaceutical compositions and cerebrovascular disorders containing said binding partners, for example, It relates to its use in treating headache.
Description
【0001】 本発明は5−HT5受容体に対する結合パートナー、このタイプの結合パート
ナーの同定及び特徴付けのための方法、並びにまたこれらを含有する医薬品組成
物及び脳血管障害、例えば偏頭痛の治療のためのその使用に関する。The present invention relates to a binding partner for the 5-HT5 receptor, a method for the identification and characterization of this type of binding partner, and also pharmaceutical compositions containing them and for the treatment of cerebrovascular disorders such as migraine. Regarding its use for.
【0002】 少なくとも7種の異なる受容体クラスが神経伝達物質のセロトニン(5−ヒド
ロキシトリプタミン、略して5−HT)の関連に起因する多様な生理学的活性を
媒介する。国際的に認められた分類によれば、これらは5−HT1、5−HT2
、5−HT3、5−HT4、5−HT5、5−HT6及び5−HT7で表される
。更にこれらのクラスの殆どは更に分類できる受容体型を含む。このように5−
HT1クラスは少なくとも5種のサブクラスに分けられる受容体を含み、これら
は5−HT1A、5−HT1B、5−HT1C、5−HT1D及び5−HT1E
で表される(Boess F. G. and Martin I. L., Neuropharmacology 33:275-317 1
994)。[0002] At least seven different receptor classes mediate a variety of physiological activities due to the involvement of the neurotransmitter serotonin (5-hydroxytryptamine, abbreviated 5-HT). According to internationally accepted classifications, these are 5-HT1, 5-HT2
, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7. Furthermore, most of these classes contain receptor types that can be further classified. Thus, 5-
The HT1 class contains receptors divided into at least five subclasses, which are 5-HT1A, 5-HT1B, 5-HT1C, 5-HT1D and 5-HT1E.
(Boess FG and Martin IL, Neuropharmacology 33: 275-317 1
994).
【0003】 5−HT5クラスはプラサット他(Plassat et al)によって初めて記載され
た(The EMBO Journal Vol. 11 No. 13, pp. 4779-4786 (1992))。5−HT5
A受容体及び5−HT5B受容体が区別されている(Erlander et al., Proc. N
atl. Acad. Sci. USA 90:3452-3456 (1993))。5−HT5受容体と他の5−H
T受容体との間には僅かな配列ホモロジーしか存在しない。これらの受容体の薬
理学的プロフィールは著しく異なる。分子生物学的な技術を使用して、5−HT
5受容体の所在を嗅球、海馬、コルテックス、脳室、脳梁及び小脳に突き止める
ことができた。免疫組織学的手法によって、5−HT5受容体は原則的に星状細
胞で発現されることを示すことができた(Carson et al., GLIA 17:317-326 (19
96))。星状細胞は、血液脳関門の脳毛細血管の基底膜に直接隣接している。異
常な星状細胞の内皮構造は血液脳関門の損失を伴う。星状細胞の厳密な意味は明
らかでない。これらは輸送作業及び結合機能を行うと考えられている。反応性の
星状細胞はかなりの病理学的な脳の変化及び神経精神障害において反応性グリオ
ーシスに関連して観察された。脳の損傷の結果として、これらはその形態を変化
させる。タンパク質発現パターンが変化し、かつ成長因子が産生される。培養し
た星状細胞におけるインビトロの調査において、5−HT5受容体に媒介される
応答を検出することができた。こうして一方ではこれらが障害後の脳の回復プロ
セスに関連することを推測すべきであるが、他方ではこれらは損傷を引き起こし
、損傷を増やす一因となることも無視すべきでない。The 5-HT5 class was first described by Prasat et al. (The EMBO Journal Vol. 11 No. 13, pp. 4779-4786 (1992)). 5-HT5
A receptor and 5-HT5B receptor have been distinguished (Erlander et al., Proc. N
atl. Acad. Sci. USA 90: 3452-3456 (1993)). 5-HT5 receptor and other 5-H
There is little sequence homology with the T receptor. The pharmacological profiles of these receptors differ markedly. Using molecular biology techniques, 5-HT
The location of the 5 receptors could be located in the olfactory bulb, hippocampus, cortex, ventricle, corpus callosum and cerebellum. Immunohistological techniques could show that the 5-HT5 receptor was expressed in principle in astrocytes (Carson et al., GLIA 17: 317-326 (19
96)). Astrocytes are directly adjacent to the basement membrane of the brain capillaries of the blood-brain barrier. Abnormal astrocyte endothelial structure is associated with loss of the blood-brain barrier. The exact meaning of astrocytes is not clear. They are believed to perform transport operations and coupling functions. Reactive astrocytes have been observed in association with reactive gliosis in considerable pathological brain changes and neuropsychiatric disorders. As a result of brain damage, they change their morphology. The protein expression pattern changes and growth factors are produced. In vitro studies on cultured astrocytes were able to detect 5-HT5 receptor mediated responses. Thus, on the one hand, it should be speculated that they are involved in the brain recovery process after the injury, but on the other hand, it should not be ignored that they cause damage and contribute to increasing damage.
【0004】 偏頭痛は大部分の人口が患うCNS障害である。殆どの場合、何度も繰り返さ
れる頭痛が現れ、それに大雑把に見積もっても8百万人、すなわち全子供の3〜
5%、全男性の7%及び全女性の14%が患っている。遺伝的素質も伝わるが、
原因は複雑であると考えられている(Diener H.C. et al., Arzneimitteltherap
ie 15:387-394 (1997))。[0004] Migraine is a CNS disorder that affects most populations. In most cases, headaches are repeated over and over, and a rough estimate is 8 million, or 3 to 3 of all children.
It affects 5%, 7% of all men and 14% of all women. Genetic disposition is transmitted,
The cause is thought to be complex (Diener HC et al., Arzneimitteltherap
ie 15: 387-394 (1997)).
【0005】 2つの仮説が有力である。既に長く知られた血管理論(vascular theory)は
原因として内脳血管系及び外脳血管系の拡張プロセスを提案している。神経原説
は脳組織を刺激し、炎症反応に導き、こうして痛みを引き起こすであろう一定の
神経節の刺激の結果として血管作動性の神経伝達物質、原則的に神経ペプチド、
例えばサブスタンスP及びニューロキニンの脈管構造の軸索からの分泌に基づく
。[0005] Two hypotheses are influential. The vascular theory, which has been known for a long time, has proposed a dilation process of the inner cerebral vasculature and the outer cerebral vasculature as a cause. Neurogenesis evokes vasoactive neurotransmitters, essentially neuropeptides, as a result of the stimulation of certain ganglia that stimulate brain tissue and lead to an inflammatory response, thus causing pain.
For example, it is based on the secretion of substance P and neurokinin from the axons of the vasculature.
【0006】 現在、依然として偏頭痛の治療のための原因治療は存在しない。現在、2種の
治療法として第一に偏頭痛発作の再発防止のための予防的治療、及び第二に発作
の間の急性症状の抑制のための対症治療が存在する。偏頭痛特異的な有効化合物
、例えばSanmigran(R)、Nocerton(R)、Deserni
l(R)及びVidora(R)、また他の指標のために通常使用される有効化
合物、例えばβ−遮断薬、制吐性の有効化合物、例えばSibelium(R) 、抗鬱薬、例えばLaroxyl(R)、又は抗てんかん性有効化合物、例えば
Depakin(R)を予防的に投与する。急性治療の過程においては、鎮痛薬
、例えばアスピリン、パラセタモール又はOptalidon(R)、非ステロ
イド性抗炎症薬、例えばCebutid(R)、Voltaren(R)、Br
ufen(R)、Ponstyl(R)、Profenid(R)、Apran
x(R)及びNaprosin(R)が痛み及び炎症に対して投与され、かつ麦
角アルカロイド、例えば血管狭窄を誘導できるエルゴタミン、ジヒドロエルゴタ
ミン、又は5−HT1D受容体に高い親和性を有するトリプタンファミリーの物
質、例えばスマトリプタン、Naramig(R)及びAscoTop(R)が
投与される。後者の物質はアゴニストとして作用し、血管拡張をブロックする。[0006] Currently, there is still no causal treatment for the treatment of migraine. Currently, there are two types of treatment, firstly prophylactic treatment to prevent recurrence of migraine attacks and secondly symptomatic treatment to control acute symptoms during attacks. Migraine-specific active compounds, for example Sanmigran (R), Nocerton (R ), Deserni
l (R) and Vidora (R), also active compounds normally used for other indicators, for example, β- blockers, active compounds of the braking吐性, e.g. Sibelium (R), antidepressants, for example Laroxyl (R ), or anti-epileptic active compound administered, for example Depakin the (R) prophylactically. In the course of acute treatment, analgesics, such as aspirin, paracetamol or Optalidon (R), non-steroidal anti-inflammatory drugs, for example Cebutid (R), Voltaren (R ), Br
ufen (R) , Ponstyl (R) , Profenid (R) , Apran
x (R) and Naprosin (R) are administered for pain and inflammation, and substances of the ergot alkaloids, such as ergotamine, dihydroergotamine, which are capable of inducing vascular stenosis, or a triptan family having a high affinity for the 5-HT1D receptor, For example sumatriptan, Naramig (R) and AscoTop (R) is administered. The latter act as agonists and block vasodilation.
【0007】 しかしながら挙げられる有効化合物は偏頭痛の治療のために最適ではない。非
オピオイド鎮痛薬はしばしば副作用を有する。強力な末梢血管拡張の結果として
、麦角アルカロイドの作用の複雑なメカニズムが副作用、例えば高血圧及び壊疽
を引き起こす。トリプタンファミリーに属する化合物は完全に満足のいくような
ものではない(Pfaffenrath V., Muench. med. Wschr. 625-626 (1998))。However, the active compounds mentioned are not optimal for the treatment of migraine. Non-opioid analgesics often have side effects. As a result of strong peripheral vasodilation, a complex mechanism of action of ergot alkaloids causes side effects such as hypertension and gangrene. Compounds belonging to the triptan family are not entirely satisfactory (Pfaffenrath V., Muench. Med. Wschr. 625-626 (1998)).
【0008】 スマトリプタン(Imigran(R))、急性偏頭痛発作に対する最も効果
的かつ頻繁に使用される有効化合物は顕著な親水性のため血管脳関門を通過しな
い。患者の28%においては、この有効化合物は効果的でなく、かつ50〜10
0mgの経口用量は非常に多い。知られている禁忌は冠状血管痙攣、高血圧、腎
疾患及び肝疾患である。[0008] Sumatriptan (Imigran (R)), the most effective and active compound which is frequently used for acute migraine attacks does not pass through the blood-brain barrier for the outstanding hydrophilicity. In 28% of patients, the active compound is not effective and 50 to 10
The 0 mg oral dose is very high. Known contraindications are coronary vasospasm, hypertension, kidney disease and liver disease.
【0009】 今に至るまで、5−HT1受容体に対する選択的な親和性を有する化合物は偏
頭痛の治療のために考慮されてきた(Goadsby P.J., CNS Drugs 10:271-286 (19
98); Saxena P. R., Exp. Opin. Invest. Drugs 581-593 (1996))。例えばスマ
トリプタンは5−HT1D受容体に対する非常に選択的なリガンドとして数えら
れ、そのため、業界ではこれらの結合特性及びそれによって媒介される血管収縮
活性を最適化させることに時間が費やされている。その幾つかは5−HT1B受
容体リガンド及び5−HT1F受容体リガンドに当てはまる。こうして一連の有
効化合物が開発されたが、概説した問題を改善することは不可能であった。To date, compounds with a selective affinity for the 5-HT1 receptor have been considered for the treatment of migraine (Goadsby PJ, CNS Drugs 10: 271-286 (19
98); Saxena PR, Exp. Opin. Invest. Drugs 581-593 (1996)). For example, sumatriptan is counted as a very selective ligand for the 5-HT1D receptor, so the industry spends time optimizing these binding properties and the vasoconstrictor activity mediated thereby. . Some of them apply to 5-HT1B receptor ligands and 5-HT1F receptor ligands. A series of active compounds has thus been developed, but it has not been possible to ameliorate the problems outlined.
【0010】 従って、本発明の課題は、適当な効率をもって、かつ副作用を殆ど有さずに偏
頭痛様の脳血管障害を急性治療(acute treatment)することを可能にすること
である。[0010] It is therefore an object of the present invention to make it possible to acutely treat migraine-like cerebrovascular disorders with adequate efficiency and with few side effects.
【0011】 意想外にも、5−HT5受容体に対して比較的高い結合親和性を有する物質が
所望の効果を提供できることが判明した。It has surprisingly been found that substances having a relatively high binding affinity for the 5-HT5 receptor can provide the desired effect.
【0012】 従って、本発明は5−HT5受容体に対する選択的結合パートナーに関し、そ
の5−HT5受容体に対する結合親和性は5−HT5以外の1種以上の5−HT
受容体に対するものよりも大きい。[0012] Accordingly, the present invention relates to a selective binding partner for the 5-HT5 receptor, wherein the binding affinity for the 5-HT5 receptor is one or more of 5-HT5 other than 5-HT5.
Larger than for the receptor.
【0013】 用語“5−HT5受容体に対する結合パートナー”は5−HT5受容体に結合
する物質を記載しており、従って5−HT5受容体リガンドとも呼ぶことができ
る。The term “binding partner for the 5-HT5 receptor” describes a substance that binds to the 5-HT5 receptor, and thus can also be called a 5-HT5 receptor ligand.
【0014】 または金属錯体のような配位結合を有する。前記の可逆的な分子相互作用の他
に、結合パートナーと受容体間の不可逆的な相互作用、例えば共有結合も可能で
ある。Alternatively, it has a coordination bond such as a metal complex. In addition to the reversible molecular interactions described above, irreversible interactions between the binding partner and the receptor, such as covalent bonds, are also possible.
【0015】 選択性は、結合パートナーの、有利には5−HT5受容体に結合する特性と解
されるべきである。[0015] Selectivity is to be understood as the property of a binding partner, advantageously binding to the 5-HT5 receptor.
【0016】 このように、結合パートナーは5−HT5受容体に対して結合親和性を有し、
これらは5−HT5以外の1種以上の5−HT受容体、すなわち前記の5−HT
受容体クラス、5−HT1、5−HT2、5−HT3、5−HT4、5−HT6
及び5−HT7に割り当てられるべき受容体に対するよりも大きい。5−HT5
受容体に対する結合パートナーの結合親和性が5−HT5以外の5−HT受容体
のそれよりも高い場合、5−HT5以外の5−HT受容体に対して、これらの結
合パートナーが5−HT5受容体により選択的に結合するといえる。特定の結合
パートナーは、5−HT5受容体に対する結合親和性が少なくとも1種及び、特
に全ての5−HT1受容体、特に5−HT1D受容体および/または5−HT1
B受容体に対するより大きいものである。5−HT5受容体に対する結合親和性
が5−HT5以外の全ての5HT受容体に対するよりも大きい結合パートナーは
本発明による他の特定の結合パートナーのクラスである。Thus, the binding partner has a binding affinity for the 5-HT5 receptor,
These are one or more 5-HT receptors other than 5-HT5, ie, the aforementioned 5-HT receptors.
Receptor class, 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT6
And 5-HT7 for the receptor to be assigned. 5-HT5
If the binding affinity of the binding partners for the receptor is higher than that of the 5-HT receptors other than 5-HT5, then for those 5-HT receptors other than 5-HT5, these binding partners will It can be said that it is selectively bound by the body. Particular binding partners have at least one binding affinity for the 5-HT5 receptor and in particular all 5-HT1 receptors, in particular the 5-HT1D receptor and / or 5-HT1
Greater for the B receptor. Binding partners whose binding affinity for the 5-HT5 receptor is greater than for all 5HT receptors other than 5-HT5 are another particular class of binding partners according to the invention.
【0017】 前記に概説した選択性に関しては、一方では5−HT5受容体に対する及び他
方では5−HT5以外の1種以上の5−HT受容体に対する結合親和性が十分に
異なることが重要である。親和性の差異は、少なくとも2、より有利には少なく
とも5,特に有利には少なくとも10、有利には少なくとも20、特に有利には
少なくとも50及び特に少なくとも100の結合親和性比が存在すると有利であ
る。With regard to the selectivities outlined above, it is important that the binding affinities on the one hand to the 5-HT5 receptor and on the other hand to one or more 5-HT receptors other than 5-HT5 are sufficiently different. . The affinity difference is advantageously such that a binding affinity ratio of at least 2, more advantageously at least 5, particularly advantageously at least 10, advantageously at least 20, particularly advantageously at least 50 and especially at least 100 is present. .
【0018】 1つの態様によれば、本発明による結合パートナーは比較結合パートナー、例
えば5−HT(5−ヒドロキシトリプタミン)又は5−CT(5−カルボキシア
ミドトリプタミン)が5−HT5受容体に結合するのを競合的に阻害する。According to one embodiment, the binding partner according to the invention is a comparative binding partner, for example 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine) binds to the 5-HT5 receptor. Competitively.
【0019】 競合阻害とは本発明による結合パートナーが比較結合パートナー、有利には5
−HT又は5−CTと、受容体への結合に関して競合する、すなわち1つが結合
するとその他の結合が妨害されると解されるべきである。Competition inhibition means that the binding partner according to the invention is a comparative binding partner, preferably 5
It should be understood that -HT or 5-CT compete for binding to the receptor, i.e., binding of one interferes with the other.
【0020】 他の態様によれば、本発明による結合パートナーは比較結合パートナー、例え
ば5−HT(5−ヒドロキシトリプタミン)又は5−CT(5−カルボキシアミ
ドトリプタミン)が5−HT5受容体に結合するのを非競合的に阻害する。According to another aspect, the binding partner according to the invention is a comparative binding partner, for example 5-HT (5-hydroxytryptamine) or 5-CT (5-carboxamidotryptamine) binds to the 5-HT5 receptor. Non-competitively.
【0021】 非競合阻害は、本発明による結合パートナーが比較結合パートナー、有利には
例えば5−HTまたは5−CTの結合を変え、特にその結合親和性をその受容体
への結合を介して低減させることを意味すると解されるべきである。Non-competitive inhibition means that the binding partner according to the invention alters the binding of a comparative binding partner, advantageously for example 5-HT or 5-CT, and in particular reduces its binding affinity via its binding to its receptor Should be understood to mean
【0022】 少なくとも競合阻害、すなわち可逆的結合の場合には、1つの結合パートナー
の他のものへの置き換えが受容体に関して、一方の結合親和性が低減するか、ま
たは他方の結合親和性が増加するのに伴い増大するという原理を利用している。
。更に適当には、従って本発明により使用できる結合パートナーは5−HT5受
容体に対して高い結合親和性を有する。このタイプの結合親和性は一方では5−
HT5受容体に対する天然産生結合パートナー、例えばセロトニン(5−ヒドロ
キシトリプタミン、5−HT)それ自体の効果的な置き換えを可能にし、その際
、本発明により使用できる結合パートナーの、この結合パートナーの一定の量が
5−HT5受容体に結合するために必要な濃度は結合親和性の増加とともに減少
する。医療での使用に関しては、結合パートナーは従って有利であり、その結合
親和性はこれらを有効化合物として効果的な医学的治療の過程で調節可能な量で
投与できることが優れている。本発明による結合パートナーは従って有利には約
0.01〜100mg/体重のkg及び、特に非経口投与の場合は0.1〜15
mg/体重のkg及び経口投与の場合には1〜30mg/体重のkgの日用量で
投与される。[0022] At least in the case of competitive inhibition, ie reversible binding, replacement of one binding partner by another reduces the binding affinity of one for the receptor or increases the binding affinity of the other It utilizes the principle that it increases as you do.
. More suitably, therefore, the binding partners which can be used according to the invention have a high binding affinity for the 5-HT5 receptor. This type of binding affinity on the other hand is 5-
It allows the efficient replacement of naturally occurring binding partners for the HT5 receptor, such as serotonin (5-hydroxytryptamine, 5-HT) itself, whereby certain of the binding partners which can be used according to the invention The concentration required for the amount to bind to the 5-HT5 receptor decreases with increasing binding affinity. For use in medicine, the binding partners are therefore advantageous, and their binding affinity is excellent in that they can be administered as active compounds in an amount which can be adjusted in the course of an effective medical treatment. The binding partner according to the invention is therefore advantageously of about 0.01 to 100 mg / kg of body weight and in particular for parenteral administration 0.1 to 15
The daily dose of mg / kg of body weight and 1 to 30 mg / kg of body weight in case of oral administration.
【0023】 50%の別の比較結合パートナーを受容体結合部位から追い出す本発明による
結合パートナーの濃度(IC50値)をインビトロで測定する前記に参照した競
合試験は結合親和性を表現する1つの可能性を提供する。こうして5−CTの5
−HT5受容体への結合の競合阻害を、有利には本発明により使用できる結合パ
ートナーが10- 7M未満、有利には10- 8M未満、特に10- 9M未満の半最
大阻害定数(half-maximal inhibition)IC50を有するという趣旨で評価す
ることができる。The competition test referred to above, which measures in vitro the concentration (IC 50 value) of a binding partner according to the invention, which drives off 50% of another comparative binding partner from the receptor binding site, is one of the ones that express binding affinity. Offer the possibility. Thus, 5-CT5
-HT5 binding of competitive inhibition to the receptor, preferably binding partner 10 which can be used according to the invention - less than 7 M, preferably 10 - 8 M less, particularly 10 - 9 M less than half maximal inhibition constant ( can be evaluated the effect that with a half-maximal inhibition) IC 50.
【0024】 本発明による結合パートナーの結合親和性を阻害定数Kiによって表現でき、
これは一般に競合試験を使用して同様に測定される。5−HT5受容体の結合の
ために、本発明による結合パートナーは、有利には10- 8M未満、有利には1
0- 9M未満及び、特に10- 10M未満のKi値を有する。本発明による化合物
のKi値は、例えば1・10- 7M〜7・10- 7Mの範囲であるか、又は1・1
0- 8M〜1・10- 7Mの範囲内である。The binding affinity of the binding partner according to the invention can be expressed by an inhibition constant K i ,
This is generally measured similarly using competition tests. For 5-HT5 receptor binding, binding partners according to the invention is advantageously 10 - less than 8 M, preferably 1
0 - less than 9 M and, in particular, 10 - with a 10 K i value of less than M. K i values for the compounds according to the invention, for example, 1 · 10 - 7 M~7 · 10 - or in the range of 7 M, or 1-1
0 - 8 M~1 · 10 - 7 are within the scope of the M.
【0025】 更なる態様によれば、本発明による結合パートナーは、5−HT5以外の1種
以上の5−HT受容体に対して、前記の有利な結合親和性を有する5−HT5受
容体により選択的に結合する。According to a further aspect, the binding partner according to the invention is provided by a 5-HT5 receptor having an advantageous binding affinity as described above for one or more 5-HT receptors other than 5-HT5. Selectively combine.
【0026】 更なる態様によれば、本発明による結合パートナーは5−HT5以外の全ての
5−HT受容体に対して、前記の有利な結合親和性を有する5−HT5受容体に
より選択的に結合する。According to a further aspect, the binding partner according to the invention is selective for all 5-HT receptors other than 5-HT5 by means of a 5-HT5 receptor having said favorable binding affinity. Join.
【0027】 前記の親和性及び選択性を伴ってグリア細胞及び特に星状細胞で発現される5
−HT5受容体に結合する結合パートナーが特に有利である。5 expressed in glial cells and especially astrocytes with the affinity and selectivity described above
Particularly preferred are binding partners that bind to the -HT5 receptor.
【0028】 本発明よれば、ヒトの受容体の変異体は、本発明により使用される結合パート
ナーのための有利なターゲットである。According to the invention, variants of the human receptor are advantageous targets for the binding partners used according to the invention.
【0029】 本発明による結合パートナーの5−HT5受容体への結合はエフェクター機能
に結びつくものである。結合パートナーはアゴニスト的又はアンタゴニスト的に
、かつ部分的にアゴニスト的に又は部分的にアンタゴニスト的に作用する。The binding of the binding partner according to the invention to the 5-HT5 receptor is linked to effector functions. The binding partner acts agonistically or antagonistically and partially agonistically or partially antagonistically.
【0030】 アゴニストは、完全に又は部分的に5−HT5受容体での5−HT活性に似て
いる本発明による化合物とする。Agonists are compounds according to the invention that mimic wholly or partly 5-HT activity at the 5-HT5 receptor.
【0031】 アンタゴニストは5−HT5受容体での5−HTのアゴニスト活性をブロック
することができる本発明による化合物とする。An antagonist is a compound according to the invention capable of blocking the agonist activity of 5-HT at the 5-HT5 receptor.
【0032】 本発明の特定の態様によれば、5−HT5アゴニストは結合パートナーとして
提供される。表現“5−HT5アゴニスト”は部分的ないし完全にアゴニスト作
用を誘導する結合パートナーを示す。5−HT5受容体で部分的にアゴニスト作
用を誘導する結合パートナーは本発明による十分なアゴニスト活性を有するので
、効果的な医学的治療の過程において調節可能な量で投与するのを可能にする。
この態様に関連する有利な結合パートナーは少なくとも50%のアゴニスト作用
を有するものである。特に有利な結合パートナーは少なくとも80%のアゴニス
ト作用を有するものであり、かつ特に実質的に完全なアゴニスト作用を有する(
Emax)ものである。According to a particular aspect of the present invention, a 5-HT5 agonist is provided as a binding partner. The expression "5-HT5 agonist" denotes a binding partner that induces a partial or complete agonist effect. A binding partner that induces partial agonist action at the 5-HT5 receptor has sufficient agonist activity according to the present invention, allowing it to be administered in an adjustable amount in the course of an effective medical treatment.
Advantageous binding partners associated with this embodiment are those that have at least 50% agonistic effect. Particularly advantageous binding partners are those that have at least 80% of agonistic activity, and in particular have substantially complete agonistic activity (
E max ).
【0033】 本発明の特定の態様によれば、少なくともh5−HT5をトランスフェクショ
ンさせたCHO細胞の5−HT5受容体への結合が膜結合Gタンパク質に結合す
るGTPの刺激、細胞内カルシウム濃度のアゴニストに誘導される変化、ホスホ
リパーゼC活性の誘導および/またはcAMP産生のアゴニストに誘導される変
化をもたらす結合パートナーを得ることを可能にする。細胞内のカルシウム濃度
の変化が考慮される限り、細胞内カルシウム濃度の増大をもたらす結合パートナ
ーの使用は本発明の特定の態様である。According to a particular aspect of the present invention, the binding of at least h5-HT5 transfected CHO cells to the 5-HT5 receptor stimulates GTP binding to membrane-bound G protein, reducing intracellular calcium concentration. It makes it possible to obtain a binding partner which results in an agonist-induced change, an induction of phospholipase C activity and / or an agonist-induced change in cAMP production. The use of a binding partner that results in an increase in intracellular calcium concentration is a particular aspect of the invention, as long as changes in intracellular calcium concentration are considered.
【0034】 また本態様は脳血管障害の公知の動物モデル、特に偏頭痛のための動物モデル
において活性であり、かつ/又は脳領域で一定のインビボ作用、特に脳内のゲノ
ム応答、例えば転写因子、例えばc−fos、c−jun、zif269又はH
omer遺伝子のイソ型(Brakeman P.R. et al., Nature 386:284-288 (1997)
)の発現を誘導する結合パートナーを含む。The present embodiments are also active in known animal models of cerebrovascular disorders, especially for migraine, and / or have certain in vivo effects in brain regions, especially genomic responses in the brain, such as transcription factors. E.g., c-fos, c-jun, zif269 or H
isoform of the omer gene (Brakeman PR et al., Nature 386: 284-288 (1997)
)).
【0035】 有利な結合パートナーは、そのエフェクター機能に関しても前記の認識で5−
HT5受容体に選択的なものである。[0035] Advantageous binding partners are also known from the above recognition as to their effector functions.
It is selective for the HT5 receptor.
【0036】 1つの態様によれば、5−HT5結合パートナーは低分子量であり、通常は合
成化合物である。According to one embodiment, the 5-HT5 binding partner is of low molecular weight, usually a synthetic compound.
【0037】 更なる態様によれば、本発明による5−HT5結合パートナーは5−HT5特
異抗体である。これらはポリクローナル抗血清、モノクローナル抗体、抗体断片
、例えばF(ab)、Fcなど、キメラ抗体及び組み換え抗体である。かかる抗
体は自体公知の方法で製造できる。使用される免疫原は5−HT5受容体自体又
は抗原断片、一般に慣用の担体タンパク質にカップリングされた断片であってよ
い。According to a further aspect, the 5-HT5 binding partner according to the invention is a 5-HT5 specific antibody. These are polyclonal antisera, monoclonal antibodies, antibody fragments, such as F (ab), Fc, etc., chimeric antibodies and recombinant antibodies. Such an antibody can be produced by a method known per se. The immunogen used may be the 5-HT5 receptor itself or an antigenic fragment, generally a fragment coupled to a conventional carrier protein.
【0038】 更なる態様によれば、本発明による5−HT5結合パートナーはアプタマー、
すなわち5−HT5受容体に対して十分な親和性を有する核酸、一般にはオリゴ
ヌクレオチドである。According to a further aspect, the 5-HT5 binding partner according to the invention is an aptamer,
That is, it is a nucleic acid having a sufficient affinity for the 5-HT5 receptor, generally an oligonucleotide.
【0039】 本発明による結合パートナーの5−HT5受容体への結合親和性の測定のため
のアッセイは原則的に公知である。これは、例えば比較結合パートナーの5−H
T5受容体への結合の、調査されるべき物質による競合阻害を評価することによ
って実施できる。適当な結合パートナーは5−HT受容体に対する公知のリガン
ド、例えば5−HT又は5−CT又はLSDである。これらは適宜に、これらの
5−HT受容体への結合を標準的な方法を使用して分析的にモニターできるよう
に標識する。5−HT5受容体での結合研究の場合には、5−CT又はLSD、
特に[3H]−LSDを本発明によれば使用する。結合親和性は半最大阻害定数
IC50又は阻害定数Kiとして表現できる。この方法は、有利には一次スクリ
ーニングのために使用される。SPA又はフラッシュプレート技術(FlashPlate
technology)が有利には使用される。Assays for measuring the binding affinity of a binding partner according to the invention to the 5-HT5 receptor are known in principle. This is for example the comparative binding partner 5-H
This can be done by assessing the competitive inhibition of binding to the T5 receptor by the substance to be investigated. Suitable binding partners are known ligands for the 5-HT receptor, for example 5-HT or 5-CT or LSD. They are suitably labeled so that their binding to the 5-HT receptor can be monitored analytically using standard methods. For binding studies at the 5-HT5 receptor, 5-CT or LSD,
In particular, [ 3 H] -LSD is used according to the invention. Binding affinity can be expressed as a half-maximal inhibition constant IC 50 or inhibition constant K i. This method is advantageously used for primary screening. SPA or FlashPlate technology
technology) is advantageously used.
【0040】 調査されるべき結合パートナーへの結合を5−HT受容体上で直接測定するこ
ともできる。結合親和性を表現する阻害定数Kiは、例えば測熱的に、すなわち
放出される結合エネルギーの測定によって決定できる。The binding to the binding partner to be investigated can also be measured directly on the 5-HT receptor. The inhibition constant K i describing the binding affinity can be determined, for example, thermometrically, ie by measuring the released binding energy.
【0041】 選択性の測定のために、他の5−HT受容体に関して調査されるべき結合パー
トナーの結合親和性を同様に(所望であればそれぞれの受容体に特異的なリガン
ドを使用して)測定し、かつ得られた値を比較する。For the determination of selectivity, the binding affinities of the binding partners to be investigated for the other 5-HT receptors can likewise be determined (if desired using ligands specific for the respective receptor). ) Measure and compare the values obtained.
【0042】 またエフェクター機能を、公知の機能的アッセイを使用してインビトロ及びイ
ンビボの両者で定性的又は定量的に評価することもできる。[0042] Effector function can also be assessed qualitatively or quantitatively, both in vitro and in vivo, using known functional assays.
【0043】 アゴニスト活性の評価は、5−HTの5−HT5受容体への結合によって惹起
される全ての前記の効果に基づいてよい。本発明によれば細胞内カルシウム濃度
、ホスホリパーゼC活性および/またはcAMP産生におけるGTPのGタンパ
ク質への結合の作用を評価することが有利である。これらの方法は、有利には二
次スクリーニングのために使用される。また本願でもSPA又はフラッシュ技術
が有利に使用される。Evaluation of agonist activity may be based on any of the aforementioned effects elicited by the binding of 5-HT to the 5-HT5 receptor. According to the present invention, it is advantageous to assess the effect of GTP binding to G protein on intracellular calcium concentration, phospholipase C activity and / or cAMP production. These methods are advantageously used for secondary screening. Also in this application, SPA or flash technology is advantageously used.
【0044】 Gタンパク質に結合するGTPはGTPの加水分解不可能な類似体、例えば[ 35 S]GTPγSを使用することによって調査でき、その結合は放射線学的に
調査できる。この調査は有利には5−HT5受容体を有する膜上で実施される。GTP binding to G proteins is a non-hydrolysable analog of GTP, such as [ 35 [S] GTPγS and its binding can be determined radiologically.
Can investigate. The investigation is advantageously performed on a membrane having the 5-HT5 receptor.
【0045】 細胞内カルシウム濃度の測定のために、適当なカルシウムプローブ、一般にカ
ルシウムキレート、例えば蛍光性化合物、例えばFura 2−アセチルメチル
エステル又はFluo−3−AMを使用できる、この調査は有利には5−HT5
受容体を有する細胞培養中、特に個々の細胞中で実施できる。For the measurement of intracellular calcium concentration, a suitable calcium probe, generally a calcium chelate, such as a fluorescent compound, such as Fura 2-acetylmethyl ester or Fluo-3-AM, can be used. 5-HT5
It can be carried out in cell culture with the receptor, in particular in individual cells.
【0046】 ホスホリパーゼC活性はその触媒反応、例えば検出目的のために有利には[3 H]−ミオイノシトールとして放射線標識されたミオイノシトールの混入又はP
PIP2のIP3への反応によって測定でき、その際、有利にはPPIP2は[ 32 P]PIP2として放射線標識されている。これらの調査は、有利には5−
HT5受容体を有する個々の細胞で実施する。The phospholipase C activity is preferably determined for its catalytic reaction, eg for detection purposes [3 H] -contamination of myo-inositol radiolabeled as myo-inositol or P
PIP2IP3To PPIP, preferably with PPIP2Is [ 32 P] PIP2As a radiolabel. These studies are advantageously
Performed on individual cells with the HT5 receptor.
【0047】 cAMP産生はcAMP結合タンパク質を使用して測定できる。この調査は有
利には5−HT5受容体を有する個々の細胞で実施される。[0047] cAMP production can be measured using a cAMP binding protein. This investigation is advantageously performed on individual cells having the 5-HT5 receptor.
【0048】 適当であれば、エフェクター機能、すなわち本発明による結合パートナーの、
他の5−HT受容体に対する活性を測定する。これは適宜に5−HT5と他の5
−HT受容体に関して測定される結合親和性を考慮して、すなわち選択性を考慮
して実施される。If appropriate, the effector function, ie of the binding partner according to the invention,
Activity on other 5-HT receptors is measured. This can be done with 5-HT5 and other 5
-Is performed taking into account the binding affinity measured for the HT receptor, ie taking into account the selectivity.
【0049】 従って本発明は本発明による結合パートナーの同定及び特徴付けのための方法
に関する。前記の、かつ他の同様に適当な方法はインビトロスクリーニング法の
ための基礎を形成してよく、該方法によって多くの種々の化合物から、将来の使
用に関して最も有望だと思われるものを選択することができる。例えば組合せ化
学によって無数の潜在的な有効化合物からなる大規模な物質バンクを作成するこ
とができる。所望の活性を有する物質に関する組合せ物質ライブラリーの構築は
自動化できる。スクリーニングロボットを個々のアッセイの効率的な分析のため
に使用でき、これらは有利にはマイクロタイタープレート上で編成される。こう
して本発明はスクリーニング法、すなわち一次スクリーニング法及び二次スクリ
ーニング法の両者に関し、該方法においては、有利には前記の方法の少なくとも
1つが使用される。幾つかの方法を使用する場合、異なる時間で、又は1つの同
じ試料において同時にか、又は調査されるべき物質の異なる試料で実施できる。The invention therefore relates to a method for the identification and characterization of a binding partner according to the invention. These and other equally suitable methods may form the basis for in vitro screening methods by which to select from a number of different compounds that are likely to be most promising for future use. Can be. For example, combinatorial chemistry can create large substance banks of countless potential active compounds. Construction of a combinatorial substance library for substances having the desired activity can be automated. Screening robots can be used for efficient analysis of individual assays, which are advantageously organized on microtiter plates. The invention thus relates to screening methods, both primary and secondary screening methods, wherein at least one of the above-mentioned methods is advantageously used. If several methods are used, they can be performed at different times or simultaneously in one and the same sample or with different samples of the substance to be investigated.
【0050】 この種の方法を実施するために特に有用な技術は、シンチレーション近接アッ
セイ(sintillation Proximity Assay)、略してSPAであり、これは有効化合
物スクリーニングの分野では公知である。このアッセイを実施するためのキット
及び構成材料は、例えばアマシャム ファルマシア バイオテク(Amersham Pharm
acia Biotech)から市販されている。原則的に可溶化または膜結合された受容体
はシンチレーターを有する小さな微小球上に固定される。例えばラジオリガンド
が、固定化された受容体に結合する場合、シンチレーターが励起されて発光し、
シンチレーターとラジオリガンド間の空間的近接(spatial proximity)が与え
られる。A particularly useful technique for performing such methods is the Scintillation Proximity Assay, or SPA for short, which is known in the field of active compound screening. Kits and components for performing this assay are available, for example, from Amersham Pharmacia Biotech.
acia Biotech). In principle, the solubilized or membrane-bound receptor is immobilized on small microspheres with scintillators. For example, when a radioligand binds to an immobilized receptor, the scintillator is excited to emit light,
Spatial proximity between the scintillator and the radioligand is provided.
【0051】 前記のタイプの方法を実施するための他の特に効果的な技術は有効化合物スク
リーニングの分野で公知のFlashPlate(R)技術である。このアッセ
イを実施するためのキット及び構成材料は、例えばNEN(R)ライフサイエン
スプロダクツ(Life Science Products)から市販されている。この原理は同様
にシンチレーターで被覆されたマイクロタイタープレート(96ウェル又は38
4ウェル)をベースとしている。[0051] Another particularly effective technique for implementing the types of methods are known in FlashPlate (R) technology in the field of active compound screening. Kits and construction materials for carrying out this assay is commercially available for example from NEN (R) Life Science Products (Life Science Products). This principle also applies to microtiter plates (96 wells or 38 wells) coated with scintillators.
4 wells).
【0052】 前記のアッセイは原則的に当業者に公知である。The above assays are known in principle to the person skilled in the art.
【0053】 本発明による第一の方法は、5−HT5受容体に対する結合パートナーの親和
性および/または選択性の測定のために使用される。この目的のために結合パー
トナーを5−HT5受容体に接触させ、結合親和性を測定する。The first method according to the invention is used for measuring the affinity and / or selectivity of a binding partner for the 5-HT5 receptor. For this purpose, the binding partner is contacted with the 5-HT5 receptor and the binding affinity is determined.
【0054】 本発明による他の方法は5−HT5受容体に対する結合パートナーの活性、す
なわちアゴニスト作用、部分的なアゴニスト作用、アンタゴニスト作用および/
または部分的なアンタゴニスト作用の測定に関する。この目的のために結合パー
トナーを5−HT5受容体に接触させ、結合によってもたらされる効果を評価す
る。Another method according to the present invention relates to the activity of a binding partner for the 5-HT5 receptor, ie agonistic, partial agonistic, antagonistic and / or
Or for the measurement of partial antagonism. For this purpose, the binding partner is contacted with the 5-HT5 receptor and the effect produced by the binding is evaluated.
【0055】 有利な態様によれば、結合パートナーに、前記の[3H]−5−CT又は[3 H]−LSD競合試験を使用してそれらの5−HT5受容体への結合親和性を測
定することによって一次スクリーニングを実施する。10- 6M未満の範囲内の
阻害定数IC50を有する結合パートナーに、次いで前記のようにそのエフェク
ター機能を評価することによって、特にGTP結合および/または細胞内カルシ
ウム濃度に関して二次スクリーニングを実施する。最後に、前記のようにして選
択された結合パートナーに、実質的に前記のように(場合によりそれぞれの受容
体に特異的なリガンドを使用して)他の5−HT受容体へのそれらの結合親和性
を測定することによって選択性の測定のために対向スクリーニング(counter-sc
reening)を実施する。例えば[3H]−8−ヒドロキシジプロピルアミノテト
ラリン([3H]−8−DPAT)を5−HT1A受容体への結合研究のために
使用でき、一方5−HT1B及び5−HT1D受容体は[3H]−5−CTを使
用して調査できる。According to an advantageous embodiment, the binding partners have their binding affinity to the 5-HT5 receptor using the [ 3 H] -5-CT or [ 3 H] -LSD competition test described above. Primary screening is performed by measuring. 10 - 6 binding partner having an inhibition constant IC 50 of the range of less than M, by evaluating its effector function as described above is then carried out secondary screening for particular GTP binding and / or intracellular calcium concentration . Finally, the binding partners selected in the manner described above are provided with their binding to other 5-HT receptors substantially as described above (optionally using ligands specific for the respective receptor). Counter-screen (counter-sc) for determination of selectivity by measuring binding affinity
reening). For example, [ 3 H] -8-hydroxydipropylaminotetralin ([ 3 H] -8-DPAT) can be used for binding studies to the 5-HT1A receptor, while the 5-HT1B and 5-HT1D receptors are [ 3 H] -5-CT can be used to investigate.
【0056】 5−HT5受容体は、有利には細胞系の形において、すなわち5−HT5受容
体を有する膜、細胞、細胞コロニー、組織又は器官の形で得ることを可能にする
。この種の細胞系は元々5−HT5受容体を発現できるが、これらは適当な遺伝
子操作、例えばトランスフェクションによって5−HT5を発現するように誘導
することもできる。h5−HT5に関する本発明の有利な態様に関しては、Re
es S.et al,FEBS Letters 335:242-246(19
94)に記載されるコーディング配列を特にこのために使用できる(アクセッシ
ョン番号X81411)。ヒトのグリオーマ細胞系統は5−HT5受容体を有す
る天然細胞系として有利である。h5−HT5をトランスフェクションさせた異
種細胞系統のうち、h5−HT5遺伝子を発現するものが有利である。例えばh
5−HT5をトランスフェクションさせたCHO細胞、h5−HT5をトランス
フェクションされたヒトの腎臓細胞、特にh5−HT5をトランスフェクション
されたHEK293細胞又はh5−HT5をトランスフェクションされたC−6
グリオーマ細胞が挙げられる。The 5-HT5 receptor advantageously makes it possible to obtain it in the form of a cell line, ie in the form of a membrane, a cell, a cell colony, a tissue or an organ having the 5-HT5 receptor. Although such cell lines are naturally capable of expressing the 5-HT5 receptor, they can also be induced to express 5-HT5 by appropriate genetic manipulation, such as transfection. For an advantageous embodiment of the invention relating to h5-HT5, see Re.
es S.S. et al, FEBS Letters 335: 242-246 (19
94) can be used especially for this (accession number X81411). The human glioma cell line is advantageous as a natural cell line with the 5-HT5 receptor. Of the heterologous cell lines transfected with h5-HT5, those expressing the h5-HT5 gene are advantageous. For example, h
5-HT5 transfected CHO cells, h5-HT5 transfected human kidney cells, especially h5-HT5 transfected HEK293 cells or h5-HT5 transfected C-6
Glioma cells.
【0057】 本発明による結合パートナーの選択性、親和性及び活性を測定するために、ま
た脳組織切片及び脳の一部からの自生の膜を使用してもよい。ラジオラベルを使
用する場合、組織切片の評価は有利にはオートラジオグラフィーによって行う。To determine the selectivity, affinity and activity of a binding partner according to the invention, brain tissue sections and native membranes from parts of the brain may also be used. If radiolabels are used, the evaluation of the tissue sections is advantageously performed by autoradiography.
【0058】 本発明による結合パートナーの偏頭痛の治療に関する有効性は有利には、偏頭
痛の形成の根底であり得るメカニズムをベースとする動物モデルで測定される。The efficacy of a binding partner according to the invention for treating migraine is advantageously measured in animal models based on mechanisms that may underlie the formation of migraine.
【0059】 例えば本発明による結合パートナーによって誘導されるタンパク質溢出を測定
することができる。このアッセイは自体公知のように、例えば蛍光染色(Johnso
n K. W. and Phebus L.A. (1998) J. Neurosci. Methods 81: 19-24)又はアイ
ソトープ標識したアルブミン(Petty M. A. et al., (1997) Eur. J. Pharmacol
. 336: 127-136)を使用することによって実施できる。このために、前記の動物
を連続的に試験化合物及び[125I]−アルブミン又は蛍光色素で処理する。
次いで三叉神経節を電気的に刺激する。脳硬膜中に蓄積した放射活性又は蛍光を
測定する。本発明による結合パートナーの抗偏頭痛活性を三叉神経節の刺激によ
って誘導されるタンパク質溢出で判断する。For example, protein extravasation induced by a binding partner according to the invention can be measured. This assay is known per se, for example by fluorescent staining (Johnso
n KW and Phebus LA (1998) J. Neurosci. Methods 81: 19-24) or isotope-labeled albumin (Petty MA et al., (1997) Eur. J. Pharmacol
336: 127-136). For this, the animals are treated successively with a test compound and [ 125 I] -albumin or a fluorescent dye.
The trigeminal ganglia are then electrically stimulated. The radioactivity or fluorescence accumulated in the dura is measured. The antimigraine activity of the binding partner according to the invention is determined by protein extravasation induced by stimulation of the trigeminal ganglion.
【0060】 他のモデルは、頸動脈血流の分布に基づく(Saxena P. R. et al., (1998) Eu
r. J. Pharmacol. 351: 329-339)。このために、放射活性の微小球を頸動脈中
に注入する。安定期(stabilization phase)の後に、動物を試験化合物で処理
し、かつ脳内に蓄積される放射活性をマイクロ−β−カウンター(micro-β-cou
nter)中で測定する。Another model is based on the distribution of carotid blood flow (Saxena PR et al., (1998) Eu
r. J. Pharmacol. 351: 329-339). For this, radioactive microspheres are injected into the carotid artery. After the stabilization phase, the animals are treated with the test compound and the radioactivity accumulated in the brain is measured using a micro-β-couper.
nter).
【0061】 ニトログリセリンで誘導されるc−fos遺伝子発現及びトランスロケーショ
ンの測定も適当である。このために、動物をニトログリセリン及び試験されるべ
き化合物で処理する。c−fos遺伝子発現及び核内でのタンパク質トランスロ
ケーションはインサイチュー・ハイブリダイゼーションを使用するか、又は免疫
組織化学的に測定する(Garcia-Ladona F. J. et al., (1994) Mol. Brain Res.
21: 75-84; Garcia-Ladona F. J. et al (1997) J. Neurosci. Res 50: 50-61
)。Measurement of nitroglycerin-induced c-fos gene expression and translocation is also appropriate. For this, the animals are treated with nitroglycerin and the compound to be tested. c-fos gene expression and protein translocation in the nucleus are measured using in situ hybridization or immunohistochemically (Garcia-Ladona FJ et al., (1994) Mol. Brain Res.
21: 75-84; Garcia-Ladona FJ et al (1997) J. Neurosci. Res 50: 50-61
).
【0062】 他のモデルは、Fernandes de Lima V.M. et al.(
1993) Brain Res.614:445−51(網膜拡延性抑圧(Reti
nal Spreading depression))及びKawahara N.et al.(199
9) Exp.Neurol.108:27−36(皮質拡延性抑圧(Cortical
Spreading Depression))から公知である。Other models are described in Fernandes de Lima V. M. et al. (
1993) Brain Res. 614: 445-51 (Retinal Spreading Depression (Reti
nal Spreading depression)) and Kawahara N. et al. et al. (199
9) Exp. Neurol. 108: 27-36 (Cortical spreading depression)
Spreading Depression)).
【0063】 5−HT5結合パートナーの本発明による使用は、治療に関して1つのプロセ
スを有する。このプロセスにおいて、概して製薬学的かつ獣医学的な実施によっ
て配合される1種以上の5−HT5結合パートナーの有効量を治療されるべき個
体、有利にはホニュウ類、特にヒト、有用動物又はペットに投与する。かかる治
療を指示し、その形で実施するかどうかは、個々のケースに依存し、かつ医学的
評価(診断)、徴候、症状および/または機能不全の提示、一定の徴候、症状お
よび/または機能不全の発生の危険に依存し、かつ更に他の要素を含む。The use of a 5-HT5 binding partner according to the invention has one process for treatment. In this process, an individual to be treated, preferably a frog, especially a human, useful animal or pet, is to be treated with an effective amount of one or more 5-HT5 binding partners, generally formulated by pharmaceutical and veterinary practice. To be administered. Whether such treatment is indicated and performed in that manner depends on the individual case and on the medical evaluation (diagnosis), presentation of signs, symptoms and / or dysfunction, certain signs, symptoms and / or functions. Depends on the risk of failure to occur and also includes other factors.
【0064】 一般に、治療は1回の投与又は繰り返しの毎日の投与によって、適当であれば
一緒に又は他の有効化合物又は有効化合物含有調剤と交互に、約0.001g〜
10g、有利には約0.001g〜約1gの日用量が治療されるべき個体に投与
されるように実施する。In general, treatment will be from about 0.001 g to a single dose or repeated daily doses, if appropriate together or alternating with other active compounds or active compound-containing preparations.
The practice is such that a daily dose of 10 g, advantageously from about 0.001 g to about 1 g, is administered to the individual to be treated.
【0065】 また本発明は個体、有利にはホニュウ類、特にヒト、有用動物又はペットの治
療のための薬剤の製造に関する。The present invention also relates to the manufacture of a medicament for the treatment of an individual, advantageously a flies, especially humans, useful animals or pets.
【0066】 本発明による結合パートナーは通常、製薬学的に認容性の添加剤を少なくとも
1種の本発明による結合パートナー及び、適当であれば他の有効化合物と一緒に
含有する医薬品組成物の形で投与する。これらの組成物は、例えば経口、直腸内
、経皮的、皮下的、静脈内、筋内又は鼻腔内の経路で適用できる。The binding partners according to the invention are usually in the form of pharmaceutical compositions containing a pharmaceutically acceptable excipient together with at least one binding partner according to the invention and, if appropriate, other active compounds. To be administered. These compositions can be applied, for example, by the oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, or intranasal routes.
【0067】 適当な医薬品製剤の例は固体の医薬品形、例えば粉剤、顆粒剤、錠剤、香錠、
サッシェ、カシェ剤、コーティング剤、カプセル剤、例えば硬質ゼラチンカプセ
ル剤及び軟質ゼラチンカプセル剤、坐剤又は経膣製剤形、半固体の医薬品形、例
えば軟膏剤、クリーム剤、ヒドロゲル剤、ペースト剤又は貼付剤、並びに液体医
薬品形、例えば液剤、エマルジョン、特に水中油エマルジョン、懸濁液、例えば
ローション剤、注入剤及び輸注調剤、点眼剤及び点耳剤である。埋植された送出
装置を本発明による結合パートナーの投与のために使用してもよい。更にリポソ
ーム、微小球またはポリマーマトリクスを使用してもよい。Examples of suitable pharmaceutical formulations are solid pharmaceutical forms such as powders, granules, tablets, lozenges,
Sachets, cachets, coatings, capsules such as hard and soft gelatin capsules, suppositories or vaginal preparations, semisolid pharmaceutical forms such as ointments, creams, hydrogels, pastes or patches And liquid pharmaceutical forms, such as solutions, emulsions, especially oil-in-water emulsions, suspensions, such as lotions, injection and infusion preparations, eye drops and ear drops. An implanted delivery device may be used for administration of a binding partner according to the invention. In addition, liposomes, microspheres or polymer matrices may be used.
【0068】 組成物の製造において、本発明による結合パートナーを通常、添加剤と混合す
るか、又は添加剤で希釈する。添加剤は、固体、半固体又は液体の材料であって
よく、これらは有効化合物のための賦形剤又は媒体として使用される。In the preparation of the compositions, the binding partners according to the invention are usually mixed with or diluted with additives. Additives may be solid, semi-solid or liquid materials, which are used as excipients or vehicles for the active compound.
【0069】 適当な添加剤は、例えばラクトース、デキストロース、スクロース、ソルビト
ール、マンニトール、デンプン、アカシアゴム、リン酸カルシウム、アルギネー
ト、トラガカント、ゼラチン、ケイ酸カルシウム、微結晶セルロース、ポリビニ
ルピロリドン、セルロース、水、糖蜜及びメチルセルロースである。更に、製剤
は製薬学的に認容性の賦形剤又は慣用の添加剤、例えば滑沢剤、例えば獣脂、ス
テアリン酸マグネシウム及び鉱油;湿潤剤;乳化剤及び懸濁剤;保存剤、例えば
ヒドロキシ安息香酸メチル及びプロピル;酸化防止剤;抗刺激薬(antistimulan
t);キレート剤;被覆助剤;エマルジョン安定剤;皮膜形成剤;ゲル化剤;フ
レーバーマスキング剤(flavor-masking agent);フレーバー改善剤(flavor c
orrigent);樹脂;ヒドロコロイド;溶剤;可溶化剤;中和剤;浸透促進剤(pe
rmeation accelerator);顔料;第4級アンモニウム化合物;加脂剤(refattin
g agent)及び過脂剤(superfatting agent);軟膏;クリーム又はオイルベー
ス;シリコーン誘導体:拡散助剤;安定化剤;滅菌剤;坐剤基剤;錠剤添加剤、
例えば結合剤、増量剤、滑沢剤、崩壊剤又はコーティング;噴射剤;乾燥剤;乳
白剤;増粘剤;ワックス;可塑剤;ホワイトオイルを含む。関連の態様はFiedle
r, H.P., Lexikon der Hilfsstoffe fuer Pharmazie, Kosmetik und angrenzend
e gebiete[Encyclopedia of excipients for pharmacy, cosmetics and related
areas], 4th Edition, Aulendorf:ECV-Editio-Kantor-Verlag, 1996に表されて
いるような専門知識に基づくものである。Suitable additives include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, molasses and Methyl cellulose. In addition, the formulation may be a pharmaceutically acceptable excipient or a conventional additive such as a lubricant such as tallow, magnesium stearate and mineral oil; a wetting agent; an emulsifying and suspending agent; a preservative such as hydroxybenzoic acid. Methyl and propyl; antioxidants; antistimulans
chelating agents; coating aids; emulsion stabilizers; film-forming agents; gelling agents; flavor-masking agents;
orrigent); resin; hydrocolloid; solvent; solubilizer; neutralizing agent;
rmeation accelerator); pigment; quaternary ammonium compound;
ointments; cream or oil bases; silicone derivatives: diffusion aids; stabilizers; sterilants; suppository bases;
For example, binders, fillers, lubricants, disintegrants or coatings; propellants; desiccants; opacifiers; thickeners; waxes; Related aspects are Fiedle
r, HP, Lexikon der Hilfsstoffe fuer Pharmazie, Kosmetik und angrenzend
e gebiete [Encyclopedia of excipients for pharmacy, cosmetics and related
areas], 4th Edition, Aulendorf: Based on expertise as expressed in ECV-Editio-Kantor-Verlag, 1996.
【0070】 また本発明は5−HT5受容体のための結合パートナーの、かつ特に前記の特
定かつ有利な本発明による結合パートナーの態様の偏頭痛性脳血管障害の治療、
特に偏頭痛及び他の血管に関連する頭痛、特に偏頭痛型の発作性の頭痛のための
使用に関する。これらは神経学的な機能障害を併発するか、又は併発しない単純
な偏頭痛及び典型的な偏頭痛、真性偏頭痛及び普通型偏頭痛、及びこの種のより
特異的な障害、例えば関連偏頭痛(associated migraine)、いわゆる偏頭痛代
理症、消化性偏頭痛(digestive migraine)、眼性偏頭痛、眼筋麻痺性偏頭痛、
赤色偏頭痛(migraine rouge)として記載される障害、群発頭痛(ホートン症候
群)及び頸性偏頭痛として記載される疾患を含む。治療とは、予防的治療、特に
発作の再発防止、例えば区間治療(interval treatment)及び急性症状、特に頭
痛相の間の治療の両者を意味すると解されるべきである。優れた治療は症状、特
に頭痛、神経学的機能障害、例えば視力障害、知覚障害、運動不全及び言語障害
、悪心及びむかつきおよび/または発作の頻度の低減をもたらす。偏頭痛の、結
合パートナーによる急性治療が有利である。特に本発明は前記の結合パートナー
を、その形成および/または経路において5−HT5受容体が関連する前記の障
害の形、すなわち5−HT5受容体活性によって調節される障害の治療のための
使用に関する。The present invention also relates to the treatment of migraine cerebrovascular disorders of the binding partner for the 5-HT5 receptor, and in particular of the specific and advantageous embodiments of the binding partner according to the invention as defined above.
It particularly relates to the use for migraine and other blood vessel related headaches, especially migraine-type paroxysmal headaches. These are simple and typical migraines with or without neurological dysfunction, true and common migraines, and more specific disorders of this type, such as associated migraines (Associated migraine), so-called migraine surrogate, digestive migraine, ocular migraine, ophthalmoplegic migraine,
Includes disorders described as migraine rouge, cluster headaches (Houghton's syndrome), and diseases described as cervical migraines. Treatment is to be taken to mean both prophylactic treatment, in particular prevention of seizure recurrence, for example interval treatment and treatment during acute symptoms, especially during the headache phase. Good treatment results in a reduction in the frequency of symptoms, in particular headaches, neurological dysfunctions, such as impaired vision, dysesthesia, dyskinesia and speech disorders, nausea and upset and / or seizures. Acute treatment of migraine with a binding partner is advantageous. In particular, the present invention relates to the use of said binding partners for the treatment of the above-mentioned forms of disorders in which 5-HT5 receptors are involved in their formation and / or pathway, ie disorders which are modulated by 5-HT5 receptor activity. .
【0071】 用語“障害”は本発明による意味においては一般に病理学的状態とされる異常
を表しており、かつ一定の徴候、症状および/または機能不全の形で明らかにす
ることができる。本発明による治療は個々の障害、すなわち異常又は病理学的状
態に向けられるが、原因的に互いに関連する幾つかの異常をまとめて、本発明に
より治療できるパターン、すなわち症候群を提供しうる。The term “disorder”, in the sense of the present invention, refers to an abnormality generally referred to as a pathological condition and can be manifested in the form of certain signs, symptoms and / or dysfunctions. Although treatment according to the present invention is directed to individual disorders, ie abnormalities or pathological conditions, several abnormalities that are causally related to one another may be combined to provide a pattern, ie, a syndrome, treatable by the present invention.
【0072】 本発明による意味の範囲内の治療とは急性または慢性の徴候、症状および/ま
たは機能不全の治療だけでなく、特に再発防止又は相防止(phase prophylaxis
)としての予防的治療(予防)を含む。治療は症候的に、例えば症状の抑制とし
て達成できる。これは短時間で実施でき、中期間で達成され、又は、例えば持続
的治療に関して長期治療であってよい。The treatment within the meaning according to the invention includes not only the treatment of acute or chronic signs, symptoms and / or dysfunctions but also, in particular, prevention of relapse or phase prophylaxis.
)). Treatment can be achieved symptomatically, for example, as suppression of symptoms. This can be performed in a short period of time, achieved in a medium period, or can be a long-term treatment, for example for a continuous treatment.
【0073】 偏頭痛の治療のために使用できる結合パートナーは、5−HT5とは異なる特
異的受容体に対するよりも低い親和性、実質的に同じ親和性又は高い親和性で5
−HTに結合できる。[0073] Binding partners that can be used for the treatment of migraine include those with lower, substantially the same, or higher affinity than 5-HT5 for specific receptors that are different.
-Can bind to HT.
【0074】 本発明による使用に関する5−HT5受容体のための結合パートナーはこのよ
うに、特に5−HT1受容体、特に5−HT1Bおよび/または5−HT1Dに
対する親和性に匹敵する5−HT5受容体に対する結合親和性が、これらが本発
明による使用のために有利には適当であるほど高い。このことは必要に応じて5
−HT5受容体のために選択的な結合パートナーが本発明の特定の態様であって
も5−HT5受容体への比較的より選択的な結合を前提としていない。The binding partner for the 5-HT5 receptor for use according to the invention is thus especially a 5-HT5 receptor which is comparable in affinity to the 5-HT1 receptor, in particular to 5-HT1B and / or 5-HT1D. The binding affinity for the body is so high that they are advantageously suitable for use according to the invention. This may be 5 if necessary.
A selective binding partner for the -HT5 receptor does not presuppose relatively more selective binding to the 5-HT5 receptor, even in certain embodiments of the invention.
【0075】 例えば特に5−HT1Bおよび/または5−HT1Dのために5−HT5受容
体及び5−HT1受容体の両者に対して高い親和性を有する結合パートナーを使
用してよい。本願での高い親和性は一般に1・10- 9M〜1・10- 6Mの範囲
内のKi値を意味する。特定の態様によれば、その高い親和性範囲で使用可能な
結合パートナーは、5−HT受容体に対して5−HT5への結合親和性によって
顕著な結合プロフィールを有し、これは、前記の範囲の他の結合親和性と比較し
て実質的に同じであるか、又はほんの僅かに低いだけである。10倍以下が有利
であってよい。For example, a binding partner with high affinity for both the 5-HT5 and 5-HT1 receptors may be used, especially for 5-HT1B and / or 5-HT1D. High affinity herein generally 1 - 10 - means K i values within the range of 6 M - 9 M~1 · 10. According to a particular aspect, the binding partners usable in its high affinity range have a pronounced binding profile due to the binding affinity to 5-HT5 for the 5-HT receptor, It is substantially the same or only slightly lower compared to the other binding affinities of the range. A factor of 10 or less may be advantageous.
【0076】 本発明の他の態様によれば、前記の選択的な5−HT5結合パートナーが使用
される。According to another aspect of the present invention, a selective 5-HT5 binding partner as described above is used.
【0077】 本発明の特定の態様によれば、h5−HT5をトランスフェクションされたC
HO細胞の少なくとも5−HT5受容体への結合は膜結合Gタンパク質に結合す
るGTP結合のアゴニストに誘導される刺激の変化、細胞内カルシウム濃度の変
化、ホスホリパーゼC活性のアゴニストに誘導される誘導の変化および/または
cAMP産生の変化をもたらす。細胞内カルシウム濃度を考慮する限り、細胞内
カルシウム濃度の増大をもたらす結合パートナーの使用は本発明の特定の態様で
ある。According to a particular aspect of the invention, h5-HT5 transfected C
The binding of HO cells to at least the 5-HT5 receptor is induced by an agonist-induced change in GTP binding to a membrane-bound G protein, a change in intracellular calcium concentration, or an agonist-induced induction of phospholipase C activity. Resulting in altered and / or altered cAMP production. As far as intracellular calcium concentration is considered, the use of a binding partner that results in an increase in intracellular calcium concentration is a particular aspect of the present invention.
【0078】 本発明を以下の実施例によってより詳細に説明するが、これは本発明を制限す
るものではない。The present invention is described in more detail by the following examples, which do not limit the invention.
【0079】 参照例1 h5−HT5受容体を発現するHEK293細胞及びCHO細胞 ヒトの5−HT5受容体をコードする遺伝子をヒトの組織から公知のように3
′−5′−RT−PCR(RACEシステム、ベーリンガー・マンハイム)によ
って単離した。次いでその遺伝子配列をネオマイシン耐性遺伝子を有するプラス
ミド(pcDNA3;インビトロジェン、ドイツ)中に挿入し、製造者の指示に
従ってE.coli中で増幅させた。得られたプラスミドの調製物をLipof
ectamin(R)(ギブコ ライフ−サイエンシズ、ドイツ)と混合し、H
EK293細胞をペトリ皿(2.5cm)中でトランスフェクション混合物を薄
層にしてインキュベートした。次いでトランスフェクション混合物をネオマイシ
ン含有培養培地に取り換えた。生存している細胞を更に10%のウシ胎仔血清、
2mMのグルタミン及び抗生物質(90mgのストレプトマイシン、90mgの
ペニシリン)を補ったDMEM−F12培地中で培養した。細胞を5%CO2、
95%の大気湿度及び37℃で集密状態まで増殖させた。Reference Example 1 HEK293 Cells and CHO Cells Expressing h5-HT5 Receptor The gene encoding the human 5-HT5 receptor was cloned from a human tissue as described in US Pat.
Isolated by '-5'-RT-PCR (RACE system, Boehringer Mannheim). The gene sequence was then inserted into a plasmid carrying the neomycin resistance gene (pcDNA3; Invitrogen, Germany) and E. coli was used according to the manufacturer's instructions. E. coli. The resulting plasmid preparation is transferred to Lipof
ectamin® ( Gibco Life Sciences, Germany)
EK293 cells were incubated in a thin layer of transfection mixture in Petri dishes (2.5 cm). The transfection mixture was then replaced with a culture medium containing neomycin. Surviving cells were further supplemented with 10% fetal bovine serum,
Cultures were performed in DMEM-F12 medium supplemented with 2 mM glutamine and antibiotics (90 mg streptomycin, 90 mg penicillin). Cells are 5% CO 2 ,
Grow to confluence at 95% atmospheric humidity and 37 ° C.
【0080】 h5−HT5受容体を発現するCHO細胞を同様に得る。[0080] CHO cells expressing the h5-HT5 receptor are similarly obtained.
【0081】 参照例2 細胞膜の調製 ここで使用される方法は、実質的に細胞から細胞膜を調製するための公知の方
法に従うものである(Findlay J.B.C. and Evans W.H., Biological Membranes,
Practical Approach (1987))。参照例1によって培養された細胞を培養容器の
表面から慎重に剥離し、180×gで10分間DMEM−F12培地中で遠心分
離した。得られる細胞ペレットを5mMのEDTA、5mMのEGTA、0.1
mMのPMSF及び3mMのベンズアミジンを含有する5mMのトリスHClバ
ッファー(pH:7.6;バッファーA)中に再懸濁し、4℃で15分間インキ
ュベートした。細胞懸濁液をUltraturrax(R)(15000rpm
)中で均質化(6×3秒)し、1000×g及び4℃で1分間遠心分離した。ペ
レットをバッファーA中に再懸濁し、かつ前記のように均質化及び遠心分離した
。両工程からの上清を回収し、40000×g及び4℃で20分間遠心分離した
。ペレットをバッファーA中に再懸濁し、均質化した(1×15秒)。膜懸濁液
を40000×g及び4℃で20分間遠心分離した。得られたペレットを10%
のグリセロール及び1%のウシ血清アルブミンを含有するバッファーA中に再懸
濁した。アリコートを凍結させ、−80℃で使用するまで貯蔵した。Reference Example 2 Preparation of Cell Membrane The method used here substantially follows the known method for preparing cell membranes from cells (Findlay JBC and Evans WH, Biological Membranes,
Practical Approach (1987)). The cells cultured according to Reference Example 1 were carefully detached from the surface of the culture vessel and centrifuged at 180 × g for 10 minutes in DMEM-F12 medium. The resulting cell pellet was treated with 5 mM EDTA, 5 mM EGTA, 0.1
Resuspended in 5 mM Tris HCl buffer (pH: 7.6; buffer A) containing mM PMSF and 3 mM benzamidine and incubated at 4 ° C. for 15 minutes. The cell suspension was treated with Ultraturrax® (15000 rpm ).
)), And centrifuged at 1000 × g and 4 ° C. for 1 minute. The pellet was resuspended in buffer A and homogenized and centrifuged as described above. Supernatants from both steps were collected and centrifuged at 40000 × g and 4 ° C. for 20 minutes. The pellet was resuspended in buffer A and homogenized (1 × 15 seconds). The membrane suspension was centrifuged at 40000 × g and 4 ° C. for 20 minutes. 10% of the obtained pellets
Glycerol and 1% bovine serum albumin. Aliquots were frozen and stored at -80 C until use.
【0082】 参照例3 [3H]−5−CTの飽和結合のキネティクス 方法論は実質的に公知である(Ress S. et al., FEBS Letter 335:242-246(19
94))。参照例2により得られる膜(200μl)を全容量600μlで1mM
のEDTAを含有する100mMのトリスHCl(pH:7.7;バッファーB
)中で[3H]−5−CT(96Ci/ミリモル)の濃度を増大させてインキュ
ベートし、その際、10μMのメチオテピンを特異的結合の測定のために添加し
、一方でメチオテピンを全結合の測定のために添加しなかった。混合物を30℃
で90分間インキュベートした。次いで試料を、Skatron(R)濾過シス
テム及び0.3%ポリエチレンイミド中に埋め込まれたGF/Bフィルタを使用
して濾過した。フィルターを4℃で9mlのバッファーBを使用して洗浄した。
フィルター中に残留する放射活性を液体シンチレーション計数によって測定した
。その際5mlのUltima−Gold(パッカード)を使用した。Reference Example 3 Kinetics of Saturated Binding of [ 3 H] -5-CT The methodology is substantially known (Ress S. et al., FEBS Letter 335: 242-246 (19
94)). The membrane (200 μl) obtained according to Reference Example 2 was diluted with 1 mM
100 mM Tris HCl (pH: 7.7; buffer B)
)) And incubated with increasing concentrations of [< 3 > H] -5-CT (96 Ci / mmol), with the addition of 10 [mu] M methiothepin for measuring specific binding, while adding methiothepin to total binding. Not added for measurement. 30 ° C the mixture
For 90 minutes. Then sample was filtered using a Skatron (R) GF / B filters embedded in the filter system and 0.3% polyethylene imide. The filters were washed at 4 ° C. using 9 ml of buffer B.
Radioactivity remaining in the filters was measured by liquid scintillation counting. At that time, 5 ml of Ultima-Gold (Packard) was used.
【0083】 参照例4 [3H]−5−CT結合の競合 結合の競合についての実験を実質的に公知の研究に従って実施した(Rees et
al., 1994)。例2によって得られる膜(200μl)を2nMの[3H]−5
−CTの存在下にバッファーB中の600μlの全容量で選択された化合物の濃
度を増大させてインキュベートした。30℃で75分間のインキュベート後に、
これらの試料を4℃でポリエチレンイミド中に埋め込まれたGF/Bフィルター
を通してバッファーBを使用して濾過した。フィルターを9mlのバッファーB
で洗浄した。フィルター上に残留する放射活性を参照例3でのように測定した。
全結合は、他の化合物を添加しないで観察されるラジオリガンドの結合として定
義した。この非特異的結合を、10μMのメチオテピンの存在下に観察される[ 3 H]−5−CTの結合として定義した。またマイクロタイタープレートの使用
の結果として高い試料処理量及び二次スクリーニングを可能にする類似のシステ
ムを使用してもよい。Reference Example 43H] -5-CT Binding Competition Experiments on binding competition were performed substantially according to known studies (Rees et al.
al., 1994). The membrane (200 μl) obtained according to Example 2 was added at 2 nM [3H] -5
-Concentration of selected compounds in a total volume of 600 μl in buffer B in the presence of CT
Incubate at increasing degrees. After incubation at 30 ° C. for 75 minutes,
GF / B filters embedded in polyethylene imide at 4 ° C.
And filtered using buffer B. Filter with 9 ml buffer B
And washed. The radioactivity remaining on the filter was measured as in Reference Example 3.
Total binding is defined as radioligand binding observed without the addition of other compounds.
Justified. This non-specific binding is observed in the presence of 10 μM methiothepin [ 3 H] -5-CT. Use of microtiter plates
A similar system that allows high sample throughput and secondary screening as a result of
May be used.
【0084】 [3H]−5−CT結合の飽和パラメーターを非線形回帰分析及びSigma
Plotソフトウェア(Jandel Scientific, Germany)を使用する線形プロット
の両方から測定した。放射活性結合を全結合のパーセンテージ割合として表現す
る競合曲線を作成した。半最大阻害定数IC50及びヒル係数(nH)を非線形
回帰分析によって測定した。The saturation parameters of [ 3 H] -5-CT binding were determined by nonlinear regression analysis and Sigma.
Measured from both linear plots using Plot software (Jandel Scientific, Germany). A competition curve expressing radioactive binding as a percentage of total binding was generated. The half-maximal inhibition constant IC 50 and Hill coefficient (n H ) were determined by non-linear regression analysis.
【0085】 b)FlashPlate技術を使用するHTSによるh5−HT5受容体リ
ガンドの同定 h5−HT5膜で被覆された96ウェルのフラッシュプレートをBio Si
gnal Inc(カナダ)から入手した。[3H]−LSDを10mMのMg
Cl2、0.5mMのEDTA及び0.5%のBSAを含有するトリスHClバ
ッファー中で適当な濃度に希釈した。ラジオリガンド溶液をウェル(25ml)
に添加した。これらはそれぞれ試験化合物を含有させるか、または試験化合物を
含有させていなかった。プレートを室温で180分間インキュベートし、放射活
性シグナルをマイクロ β−カウンター(Wallac)を使用して測定した。
非特異的結合をメチオテピンを使用して測定した。[3H]−LSDは12nM
の親和性を有していた。試験化合物の結合親和性が増大すると、[3H]−LS
Dの放射活性シグナルが減少した。B) Identification of h5-HT5 Receptor Ligands by HTS Using FlashPlate Technology A 96-well flash plate coated with an h5-HT5 membrane was placed on a Bio Si
gnal Inc (Canada). [ 3 H] -LSD was added to 10 mM Mg.
Cl 2, and diluted to an appropriate concentration in Tris HCl buffer containing 0.5mM EDTA and 0.5% BSA. Radioligand solution (25 ml)
Was added. These each contained the test compound or no test compound. The plates were incubated at room temperature for 180 minutes and the radioactivity signal was measured using a micro β-counter (Wallac).
Non-specific binding was measured using methiothepin. [ 3 H] -LSD is 12 nM
Had an affinity of As the binding affinity of the test compound increases, [ 3 H] -LS
The radioactivity signal of D decreased.
【0086】 参照例5 [35S]GTPγS結合のアゴニストに誘導される刺激の測定 [35S]GTPγS結合アッセイは公知である。本アッセイは従来記載され
ているヒルフ及びヤコブ(Hilf, G. and Jakobs, K. H.)の方法(Eur. J. Mol.
Pharmacol. 225:245-252(1992))に従って実施した。h5−HT5受容体遺伝
子を安定トランスフェクションさせたHEK293細胞の膜への[35S]GT
PγS結合における有効化合物に誘導される変化を測定した(参照例1及び参照
例2を参照のこと)。細胞膜(12μg)を6.75mMのMgCl2、150
mMのNaCl、1mMのDTT、1mMのEDTA、10μMのGDP及び[ 35 S]GTPγSを含有する50mMのトリエタノールアミンHClバッファ
ー(pH:7.5)とインキュベートした。試験されるべき有効化合物を添加す
るか、又は添加せずに30℃で60分間インキュベートした後に、試験混合物(
100μl)を迅速にGF−Bフィルターを通してSkatron(R)濾過装
置を使用して濾過した。フィルターを迅速に、100mMのNaCl及び5mM
のMgCl2を含有する50mMのトリスHClバッファー(9ml;pH:7
.5;4℃)で洗浄した。フィルター上に残留している放射活性を、Ultim
a Goldシンチレーション液を使用してシンチレーション分光法(sintillat
ion spectrometry)によって測定した。マイクロタイタープレートの使用の結果
として高い処理量及び二次スクリーニングを可能にする類似のシステムを同様に
使用してよい。Reference Example 5 [35S] Measurement of stimulation induced by agonists of GTPγS binding [35[S] GTPγS binding assays are known. This assay has been described previously.
Hilf, G. and Jakobs, K. H. (Eur. J. Mol.
Pharmacol. 225: 245-252 (1992)). h5-HT5 receptor inheritance
HEK293 cells stably transfected with35S] GT
Active compound-induced changes in PγS binding were measured (see Reference Example 1 and Reference Example 1).
See Example 2). Cell membrane (12 μg) was washed with 6.75 mM MgCl2, 150
mM NaCl, 1 mM DTT, 1 mM EDTA, 10 μM GDP and [ 35 S] 50 mM triethanolamine HCl buffer containing GTPγS
(PH: 7.5). Add active compound to be tested
After incubation at 30 ° C. for 60 minutes with or without addition, the test mixture (
100 μl) was quickly passed through a GF-B filter to Skatron.(R)Filtration equipment
And filtered. Filter quickly with 100 mM NaCl and 5 mM
MgCl250 mM Tris HCl buffer (9 ml; pH: 7
. 5; 4 ° C). The radioactivity remaining on the filter is determined by Ultim
a Scintillation spectroscopy using Gold scintillation fluid (sintillat
ion spectrometry). Results of using microtiter plates
As well as similar systems that allow high throughput and secondary screening
May be used.
【0087】 有効化合物活性を有効化合物の不在下に測定した基本結合のパーセンテージ割
合として表現した。曲線の整合を非線形回帰分析のためのソフトウェア(SigmaP
lot, Jandel Scientific, Germany)を使用して、一般式 E=(L×Emax)/(L+EC50) [式中、Eは効力、Lはリガンド濃度、Emaxは最大効力及びEC50は最大
効力の50%を誘導するその濃度である]に従って実施した。Active compound activity was expressed as a percentage of basal binding measured in the absence of active compound. Software for nonlinear regression analysis of curve matching (SigmaP
lot, Jandel Scientific, Germany) using the general formula E = (L × E max ) / (L + EC 50 ) where E is potency, L is ligand concentration, E max is max potency and EC 50 is max Its concentration that induces 50% of potency].
【0088】 参照例6 細胞内カルシウム濃度におけるアゴニストに誘導される変化の測定 該方法は公知である(Kao J. P. Y., Methods in Cell Biology 40:155-181(1
994))。参照例1に記載のように、h5−HT5受容体を発現するHEK293
細胞を培養容器中で増殖させた。これらの細胞を、これらが集密状態になる前に
慎重に剥離した。これらの細胞を、Fura 2−アセチルメチルエステル(シ
グマ)と室温においてインキュベートすることによってFura2で標識した。
これらの細胞を180×gで10分間遠心分離し、血清を含有しないDMEM−
F12培地中に再懸濁し、37℃、5%CO2及び95%の大気湿度において4
5分間インキュベートした。Reference Example 6 Measurement of Agonist-Induced Change in Intracellular Calcium Concentration The method is known (Kao JPY, Methods in Cell Biology 40: 155-181 (1
994)). HEK293 expressing h5-HT5 receptor as described in Reference Example 1
Cells were grown in culture vessels. The cells were carefully detached before they became confluent. These cells were labeled with Fura2 by incubating with Fura 2-acetylmethyl ester (Sigma) at room temperature.
These cells were centrifuged at 180 xg for 10 minutes, and serum-free DMEM-
Resuspend in F12 medium and add 4% at 37 ° C., 5% CO 2 and 95% atmospheric humidity.
Incubated for 5 minutes.
【0089】 細胞内カルシウム濃度を適当なフィルタ交換システム(オリンパス/ハママツ
)を備えている蛍光顕微鏡を使用して測定した。蛍光比(340nm/380n
m)をArgus(R)ソフトウェアを使用して測定した。細胞内カルシウム濃
度を個々の細胞中で有効化合物を添加せずに短時間観察し、次いで試験されるべ
き有効化合物の添加の30分後に観察した。マイクロタイタープレートの使用の
結果として高い処理量及び二次スクリーニングを可能にする類似のシステムを同
様に使用してよい。Intracellular calcium concentration was measured using a fluorescence microscope equipped with a suitable filter exchange system (Olympus / Hamamatsu). Fluorescence ratio (340 nm / 380n
m) was measured using the Argus (R) software. The intracellular calcium concentration was observed briefly in individual cells without addition of the active compound, and then 30 minutes after addition of the active compound to be tested. Similar systems that allow high throughput and secondary screening as a result of the use of microtiter plates may be used as well.
【0090】 同様に、細胞内Ca2+濃度の調節をHTSで評価した。このために、h5−
HT5受容体を発現するCHO細胞を96ウェルプレート中で一晩培養した(3
00000〜80000細胞/ウェル)。細胞を、1mMのFluo−3−AM
、10%のプルロニック酸(pluronic acid)及び2.5mMのプロベネシド(p
robencid)を含有するHEPESバッファーを使用して1時間標識した。試験化
合物をそれぞれのウェルに添加した。カルシウム濃度の測定のために、蛍光強度
を、蛍光測定作業をするプレートリーダー(Fluorometric Imaging Plate Reade
r;FLIPR)を使用して読みとった。Similarly, regulation of intracellular Ca 2+ concentration was assessed by HTS. For this, h5-
CHO cells expressing the HT5 receptor were cultured overnight in 96-well plates (3
0000-80000 cells / well). Cells were treated with 1 mM Fluo-3-AM.
10% pluronic acid and 2.5 mM probenecid (p
robencid) using HEPES buffer for 1 hour. Test compounds were added to each well. To measure the calcium concentration, the fluorescence intensity is measured using a plate reader (Fluorometric Imaging Plate Reade).
r; FLIPR).
【0091】 参照例7 アゴニストに誘導されるホスホリパーゼC活性の測定 該方法は実質的に公知である(Garcia-Ladona F. J. et al., Neuroreport 4:
691-694 (1993))。これらの細胞を0.125μMの[3H]ミオイノシトール
と24時間インキュベートした。導入されない[3H]ミオイノシトールを培地
から除去し、10mMのLiClを含有するクレブス−ヘンゼライトバッファー
に取り換えた。10分間のインキュベート後に、試験されるべき有効化合物を添
加した。45分後に反応を、刺激培地を蒸留水に取り換えることによって停止さ
せた。組織試料を使用する場合、同様の手順が使用される(Garcia-Ladona et a
l., 1993)。細胞を凍結させ、−80℃で貯蔵した。[3H]イノシトール一リ
ン酸の産生を公知のクロマトグラフィー法によって測定した。組織ミニプリズム
(tissue miniprism)に関しては同様の方法を使用してよい。同様に、ホスホリ
パーゼC刺激の測定は参照例2に記載のように膜フラクションを調製し、かつ[ 32 P]PIP2及び有効化合物とインキュベートすることによって同様に実施
した。この場合、IP3の産生が測定された。また公知の方法を最適化して、マ
イクロタイタープレートをベースとするシステムを使用してもよい。市販の材料
によって高い処理量及び二次スクリーニングの実施を伴う分析に拡張できる。Reference Example 7 Determination of Agonist-Induced Phospholipase C Activity The method is substantially known (Garcia-Ladona F.J. et al., Neuroreport 4:
691-694 (1993)). These cells were treated with 0.125 μM [3H] Myo-inositol
For 24 hours. Not introduced [3H] Myo-inositol as a medium
Krebs-Henseleit buffer containing 10 mM LiCl
Was replaced. After a 10-minute incubation, the active compound to be tested is added.
Added. After 45 minutes the reaction was stopped by replacing the stimulation medium with distilled water.
I let you. A similar procedure is used when using tissue samples (Garcia-Ladona et a
l., 1993). Cells were frozen and stored at -80C. [3H] Inositol
Acid production was measured by known chromatographic methods. Tissue mini prism
A similar method may be used for (tissue miniprism). Similarly, phosphorylation
Measurement of Pase C stimulation was performed by preparing a membrane fraction as described in Reference Example 2 and [ 32 P] PIP2And also by incubating with the active compound
did. In this case, IP3Production was measured. Also, by optimizing known methods,
A system based on the microtiter plate may be used. Commercial materials
This can be extended to analysis involving high throughput and performing secondary screening.
【0092】 参照例8 cAMP産生におけるアゴニストに誘導される変化の測定 使用される方法は実質的に公知である(Strada S. S. et al., Methods in Ne
urotransmission receptor analysis: 89-110(1990))。これらの細胞を血清及
び抗生物質を含有しない培地中で10分間インキュベートした。培地を95℃で
15分間加熱して、反応を停止させた。細胞試料を凍結させて、−80℃で貯蔵
した。cAMP濃度を、cAMP結合タンパク質を使用する市販のキットを使用
して測定した。公知の方法を最適化して、マイクロタイタープレートをベースと
するシステムを使用した。市販の材料によって、高い処理量及び二次スクリーニ
ングの実施を伴う分析に拡張できる。Reference Example 8 Determination of Agonist-Induced Changes in cAMP Production The methods used are substantially known (Strada SS et al., Methods in Neighborhood).
urotransmission receptor analysis: 89-110 (1990)). The cells were incubated for 10 minutes in medium without serum and antibiotics. The reaction was stopped by heating the medium at 95 ° C. for 15 minutes. Cell samples were frozen and stored at -80 <0> C. The cAMP concentration was measured using a commercially available kit using cAMP binding protein. The known method was optimized to use a microtiter plate based system. Commercial materials can be extended to analyzes involving high throughput and performing secondary screening.
【0093】 参照例9 組織調製 有効化合物の投与(経口、腹腔内、静脈内又は脳室内)の90分後に、試験動
物を断頭した。全脳を迅速に頭蓋から取り出し、ドライアイス上で凍結させ、−
80℃で貯蔵した。ラットの脳切片(15μm)をクリオスタット中で−20℃
で得て、ゼラチン被覆したスライドガラスに適用し、かつ−30℃で使用するま
で貯蔵した。Reference Example 9 Tissue Preparation The test animals were decapitated 90 minutes after administration of the active compound (oral, intraperitoneal, intravenous or intraventricular). The whole brain is quickly removed from the skull, frozen on dry ice,
Stored at 80 ° C. Rat brain sections (15 μm) were placed in a cryostat at −20 ° C.
And applied to gelatin-coated glass slides and stored at -30 ° C until use.
【0094】 例1 参照例3により、5−HT5受容体への[3H]−5−CTの結合親和性を測
定した。図1は結合した[3H]−5−CTのプロットを[3H]−5−CT濃
度の関数として示している。Kd=0.570の解離定数が測定された。クロー
ン細胞系統に依存して、受容体結合密度(B)は900〜28000fmol/
タンパク質のmgの範囲で変化した。Example 1 According to Reference Example 3, the binding affinity of [ 3 H] -5-CT to the 5-HT5 receptor was determined. FIG. 1 shows a plot of bound [ 3 H] -5-CT as a function of [ 3 H] -5-CT concentration. A dissociation constant of K d = 0.570 was measured. Depending on the clonal cell line, the receptor binding density (B) is 900-28000 fmol /
It varied in the mg protein range.
【0095】 例2 参照例4により、セロトニン作動性化合物の結合親和性を[3H]−5−CT
結合競合によって測定した。以下のIC50値が得られた:Example 2 According to Reference Example 4, the binding affinity of serotonergic compounds was determined by [ 3 H] -5-CT
It was determined by binding competition. The following IC 50 values were obtained:
【0096】[0096]
【表1】 [Table 1]
【0097】 更なる試験列において、以下の化合物の阻害定数Kiを測定した(Ki=IC 50 /(1+C/Kd)。その際、Cは[3H]−5−CTの濃度であり、Kd は例1により測定された):In a further test series, the inhibition constants K of the following compoundsiWas measured (Ki= IC 50 / (1 + C / Kd). At that time, C3H] -5-CT.d Was measured according to Example 1):
【0098】[0098]
【表2】 [Table 2]
【0099】 例3 参照例5により、Gタンパク質へのGTPの、有効化合物に誘導される結合を
調査した。HEK293細胞中でのGタンパク質への5−HT5受容体のカップ
リングは顕著であった。典型的なセロトニン作動性のアゴニスト5−HT及び5
−CTは、基本値の40%を上回る細胞膜への[35S]GTPγS結合の増大
を誘導した(図2参照)。5−HT5受容体はGDPをGタンパク質へのカップ
リングのために必要であり、これはアゴニストによって媒介される(図3A参照
)。5−HT効果は用量依存性であり(図4参照)、EC50は2.6μMであ
った。Example 3 Reference example 5 investigated the active compound-induced binding of GTP to G proteins. Coupling of the 5-HT5 receptor to the G protein in HEK293 cells was prominent. Typical serotonergic agonists 5-HT and 5
-CT induced an increase in [ 35 S] GTPγS binding to cell membranes above 40% of the basal value (see FIG. 2). The 5-HT5 receptor is required for coupling GDP to G proteins, which is mediated by agonists (see FIG. 3A). The 5-HT effect was dose-dependent (see FIG. 4), with an EC 50 of 2.6 μM.
【0100】 例4 細胞内カルシウム濃度についての有効化合物に誘導される効果を、参照例6に
従って調査した。HEK293細胞における5−HT5受容体のR(+)−リス
リド(1μM)での刺激は細胞内Ca2+に増大をもたらした(図5参照)。Example 4 The effect induced by active compounds on intracellular calcium concentration was investigated according to Reference Example 6. Stimulation of the 5-HT5 receptor with R (+)-lisuride (1 μM) in HEK293 cells resulted in an increase in intracellular Ca 2+ (see FIG. 5).
【図1】 図1は結合した[3H]−5−CTのプロットを[3H]−5−CT濃度の関
数として示している。FIG. 1 shows a plot of bound [ 3 H] -5-CT as a function of [ 3 H] -5-CT concentration.
【図2】 図2は典型的なセロトニン作動性のアゴニスト5−HT及び5−CTによる基
本値に対する細胞膜への[35S]GTPγS結合の誘導を示すグラフである。FIG. 2 is a graph showing the induction of [ 35 S] GTPγS binding to cell membranes against basal values by typical serotonergic agonists 5-HT and 5-CT.
【図3】 図3はアゴニストによって媒介されるGDPのGタンパク質へのカップリング
を示すグラフである。FIG. 3 is a graph showing agonist-mediated coupling of GDP to G protein.
【図4】 図4は用量に依存する5−HT効果を示すグラフである。FIG. 4 is a graph showing the dose-dependent 5-HT effect.
【図5】 HEK293細胞における5−HT5受容体のR(+)−リスリド(1μM)
での刺激による細胞内Ca2+の変化を表すグラフである。FIG. 5: R (+)-lisuride of 5-HT5 receptor in HEK293 cells (1 μM)
4 is a graph showing changes in intracellular Ca 2+ due to stimulation in Example 1.
【手続補正書】特許協力条約第34条補正の翻訳文提出書[Procedural Amendment] Submission of translation of Article 34 Amendment
【提出日】平成13年5月31日(2001.5.31)[Submission date] May 31, 2001 (2001.5.31)
【手続補正1】[Procedure amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】特許請求の範囲[Correction target item name] Claims
【補正方法】変更[Correction method] Change
【補正の内容】[Contents of correction]
【特許請求の範囲】[Claims]
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI テーマコート゛(参考) C12Q 1/02 C12Q 1/02 1/44 1/44 G01N 33/15 G01N 33/15 Z 33/50 33/50 Z (81)指定国 EP(AT,BE,CH,CY, DE,DK,ES,FI,FR,GB,GR,IE,I T,LU,MC,NL,PT,SE),OA(BF,BJ ,CF,CG,CI,CM,GA,GN,GW,ML, MR,NE,SN,TD,TG),AP(GH,GM,K E,LS,MW,SD,SL,SZ,TZ,UG,ZW ),EA(AM,AZ,BY,KG,KZ,MD,RU, TJ,TM),AE,AL,AM,AT,AU,AZ, BA,BB,BG,BR,BY,CA,CH,CN,C R,CU,CZ,DE,DK,DM,EE,ES,FI ,GB,GD,GE,GH,GM,HR,HU,ID, IL,IN,IS,JP,KE,KG,KP,KR,K Z,LC,LK,LR,LS,LT,LU,LV,MA ,MD,MG,MK,MN,MW,MX,NO,NZ, PL,PT,RO,RU,SD,SE,SG,SI,S K,SL,TJ,TM,TR,TT,TZ,UA,UG ,US,UZ,VN,YU,ZA,ZW Fターム(参考) 2G045 BB10 BB14 BB46 BB50 BB51 CB01 FB08 4B063 QA01 QA05 QA18 QQ08 QQ13 QQ20 QQ32 QQ63 QQ89 QR48 QR51 QR77 QR80 QS36 QX02 QX07 4C084 AA17 ZA08 ZA36 ZC41 4C086 AA01 AA02 BC13 CB22 MA02 MA05 NA14 ZA08 ZA36 ZC42──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification code FI Theme coat ゛ (Reference) C12Q 1/02 C12Q 1/02 1/44 1/44 G01N 33/15 G01N 33/15 Z 33/50 33 / 50 Z (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, H, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZWF terms (reference) 2G045 BB10 BB14 BB46 BB50 BB51 CB01 FB08 4B063 QA01 QA05 QA18 QQ08 QQ13 QQ20 QQ32 QQ63 QQ89 QR48 QR51 QR77 QR80 QS36 QX02 QX07 4C084 AA17 ZA08 ZA36 ZC41 4C086 AA01 AA02 BC13 CB22 MA02 MA05 NA14 ZA08 ZA36 ZC42
Claims (18)
種以上の5−HT受容体に対するよりも大きい、5−HT5受容体に対する選択
的結合パートナー。1. A binding affinity for a 5-HT5 receptor other than 5-HT5.
A selective binding partner for the 5-HT5 receptor that is greater than for more than one species of the 5-HT receptor.
および/または5−HT1B受容体に対するよりも大きい、請求項1記載の結合
パートナー。2. The binding partner according to claim 1, wherein the binding affinity for the 5-HT5 receptor is greater than for the 5-HT1D and / or 5-HT1B receptors.
請求項1又は2記載の結合パートナー。3. Competitively inhibiting the binding of 5-CT to the 5-HT5 receptor.
A binding partner according to claim 1.
未満、有利には10- 9M未満、特に10- 10M未満である、請求項1から3ま
でのいずれか1項記載の結合パートナー。4. A 5-HT5 K i value of 10 for that binding to the receptor - 8 M
Less, advantageously 10 - less than 9 M, in particular 10 - less than 10 M, binding partner of any one of claims 1 to 3.
結合を刺激する、請求項1から4までのいずれか1項記載の結合パートナー。5. The binding partner according to claim 1, wherein its binding to the 5-HT5 receptor stimulates the binding of GTP to G proteins.
大をもたらす、請求項1から5までのいずれか1項記載の結合パートナー。6. The binding partner according to claim 1, wherein its binding to the 5-HT5 receptor results in an increase in intracellular calcium concentration.
導をもたらす、請求項1から6までのいずれか1項記載の結合パートナー。7. The binding partner according to claim 1, wherein its binding to the 5-HT5 receptor results in the induction of phospholipase C activity.
らす、請求項1から7までのいずれか1項記載の結合パートナー。8. The binding partner according to any one of claims 1 to 7, wherein its binding to the 5-HT5 receptor results in the induction of cAMP production.
結合パートナー及び製薬学的に認容性の添加剤、及び場合により他の有効化合物
を含有する医薬品組成物。9. A pharmaceutical composition comprising at least one binding partner according to claim 1 and a pharmaceutically acceptable excipient, and optionally other active compounds.
5受容体に対する結合パートナーの使用。10. A 5-HT for producing a medicament for treating a cerebrovascular disorder.
Use of binding partners for 5 receptors.
10記載の使用。11. Use according to claim 10, for the treatment of migraine, especially for the acute treatment of migraine.
の請求項10又は11記載の使用。12. Use according to claim 10 or 11, of a binding partner according to any one of claims 1 to 8.
触させ、かつ結合親和性を測定して、5−HT5受容体に対する結合パートナー
の親和性を測定するための方法。13. A method for measuring the affinity of a binding partner for the 5-HT5 receptor by contacting the binding partner with a cell line having a 5-HT5 receptor and measuring the binding affinity.
触させ、少なくとも1つの結合パートナーに誘導されるアゴニスト作用を測定し
て、5−HT5受容体に対する結合パートナーの活性を測定するための方法。14. The activity of a binding partner at the 5-HT5 receptor is determined by contacting the binding partner with a cell line having a 5-HT5 receptor and measuring the agonistic activity induced by at least one binding partner. Way to do.
ホスホリパーゼC活性および/またはcAMP産生を測定する、請求項14記載
の方法。15. GTP binding to G protein, intracellular calcium concentration,
15. The method according to claim 14, wherein phospholipase C activity and / or cAMP production is measured.
ェクションさせた異種細胞系統を使用する、請求項14又は15記載の方法。16. The method according to claim 14, wherein a human glioma cell line or a heterologous cell line transfected with h5-HT5 is used.
h5−HT5をトランスフェクションさせたヒトの腎臓細胞又はh5−HT5を
トランスフェクションさせたC−6グリオーマ細胞を使用する、請求項16記載
の方法。17. CHO cells transfected with h5-HT5,
17. The method according to claim 16, wherein human kidney cells transfected with h5-HT5 or C-6 glioma cells transfected with h5-HT5 are used.
する、5−HT5受容体結合パートナーの同定のためのインビトロスクリーニン
グ法。18. An in vitro screening method for identifying a 5-HT5 receptor binding partner using the method of any one of claims 13 to 17.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19900674A DE19900674A1 (en) | 1999-01-11 | 1999-01-11 | Binding partner for 5-HT5 receptors for migraine treatment |
DE19900674.1 | 1999-01-11 | ||
PCT/EP2000/000142 WO2000041472A2 (en) | 1999-01-11 | 2000-01-11 | Binding partners for 5-ht5-receptors for the treatment of migraine |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2002540063A true JP2002540063A (en) | 2002-11-26 |
JP2002540063A5 JP2002540063A5 (en) | 2007-03-01 |
Family
ID=7893919
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2000593096A Pending JP2002540063A (en) | 1999-01-11 | 2000-01-11 | Binding partner for 5-HT5 receptor for migraine treatment |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP1144050A2 (en) |
JP (1) | JP2002540063A (en) |
CN (1) | CN1321635C (en) |
AU (1) | AU2289100A (en) |
CA (1) | CA2359360C (en) |
DE (1) | DE19900674A1 (en) |
MX (1) | MXPA01006986A (en) |
WO (1) | WO2000041472A2 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102004008141A1 (en) | 2004-02-19 | 2005-09-01 | Abbott Gmbh & Co. Kg | Guanidine compounds and their use as binding partners for 5-HT5 receptors |
WO2007022947A2 (en) | 2005-08-21 | 2007-03-01 | Abbott Gmbh & Co. Kg | 5-ring heteroaromatic compounds and their use as binding partners for 5-ht5 receptors |
US9296697B2 (en) | 2005-08-24 | 2016-03-29 | Abbott Laboratories | Hetaryl-substituted guanidine compounds and use thereof as binding partners for 5-HT5-receptors |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06505879A (en) * | 1992-01-08 | 1994-07-07 | シナプティック・ファーマシューティカル・コーポレーション | DNA encoding human 5-HT↓1↓f receptor and its use |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU8770282A (en) * | 1981-08-27 | 1983-03-03 | Shaun R. Coughlin | Composition for the treatment of atherosclerosis |
FR2693200B1 (en) * | 1992-07-01 | 1994-08-19 | Inst Nat Sante Rech Med | New polypeptides having serotonergic receptor activity, nucleic acids encoding these polypeptides and uses. |
FR2701265B1 (en) * | 1993-02-09 | 1995-04-07 | Inst Nat Sante Rech Med | New polypeptides having serotoninergic receptor activity, nucleic acids encoding these polypeptides and uses. |
-
1999
- 1999-01-11 DE DE19900674A patent/DE19900674A1/en not_active Withdrawn
-
2000
- 2000-01-11 AU AU22891/00A patent/AU2289100A/en not_active Abandoned
- 2000-01-11 CA CA2359360A patent/CA2359360C/en not_active Expired - Fee Related
- 2000-01-11 EP EP00901526A patent/EP1144050A2/en not_active Ceased
- 2000-01-11 JP JP2000593096A patent/JP2002540063A/en active Pending
- 2000-01-11 MX MXPA01006986A patent/MXPA01006986A/en not_active IP Right Cessation
- 2000-01-11 WO PCT/EP2000/000142 patent/WO2000041472A2/en active Application Filing
- 2000-01-11 CN CNB008046107A patent/CN1321635C/en not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06505879A (en) * | 1992-01-08 | 1994-07-07 | シナプティック・ファーマシューティカル・コーポレーション | DNA encoding human 5-HT↓1↓f receptor and its use |
Non-Patent Citations (7)
Title |
---|
JPN6010001088, Trends Pharmacol Sci, 1998, Vol.19, No.8, Page.311−316 * |
JPN6010001125, Clin Neurosci, 2003, Vol.21, No.6, Page.631−634 * |
JPN6010001127, Ann N Y Acad Sci, 1998, Vol.861, Page.85−90 * |
JPN6010001131, EMBO J, 1992, Vol.11, No.13, Page.4779−4786 * |
JPN6010001135, 基礎と臨床, 1993, Vol.27, No.9, Page.3593−3607 * |
JPN6010001137, J Med Chem, 1997, Vol.40, No.22, Page.3501−3503 * |
JPN6010001140, ペインクリニック, 1998, Vol.19, No.8, Page.1136−1143 * |
Also Published As
Publication number | Publication date |
---|---|
EP1144050A2 (en) | 2001-10-17 |
MXPA01006986A (en) | 2002-09-18 |
DE19900674A1 (en) | 2000-07-13 |
WO2000041472A3 (en) | 2001-10-11 |
CA2359360C (en) | 2012-11-27 |
WO2000041472A2 (en) | 2000-07-20 |
CN1321635C (en) | 2007-06-20 |
AU2289100A (en) | 2000-08-01 |
CA2359360A1 (en) | 2000-07-20 |
CN1342070A (en) | 2002-03-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103399144B (en) | Breast cancer treatment medicine is selected with antibody array | |
JP5085529B2 (en) | Biological markers predicting anticancer responses to epidermal growth factor receptor kinase inhibitors | |
JP5240739B2 (en) | Biological markers that predict anticancer responses to kinase inhibitors | |
Chen et al. | The 4.1 protein coracle mediates subunit-selective anchoring of Drosophila glutamate receptors to the postsynaptic actin cytoskeleton | |
JP2010527232A (en) | Biological markers predicting anticancer responses to epidermal growth factor receptor kinase inhibitors | |
US20080188461A1 (en) | Compositions and methods for detecting, preventing and treating seizures and seizure related disorders | |
JP2002504687A (en) | Cancer Treatment | |
CN109790582A (en) | Breast cancer treatment treatment based on C-MAF state | |
JP2006510008A (en) | Diagnostic, therapeutic and useful agents for conditions characterized by modulation of activin βC levels | |
US20030170747A1 (en) | Peripheral marker of blood brain barrier permeability | |
JP2020535125A (en) | TRPV2 antagonist | |
CN114828890A (en) | Treatment of diabetes with stem cell migration agent | |
JP2005538065A (en) | Immunoregulation of serotonin family receptors and blood-brain barrier and effects on cellular processes | |
US20080311578A1 (en) | System for screening eukaryotic membrane proteins | |
Aldrich et al. | Neurochemical studies of human narcolepsy: alpha-adrenergic receptor autoradiography of human narcoleptic brain and brainstem | |
US6750221B1 (en) | Use of 5-ht5-ligands in the treatment of neurodegenerative and neuropsychiatric disturbances | |
US6906179B2 (en) | Antibody to SNIP1 | |
JP2002540063A (en) | Binding partner for 5-HT5 receptor for migraine treatment | |
EP3244911B1 (en) | A method for determining a likely effect of a treatment to improve male infertility | |
JP4591599B2 (en) | Gastrointestinal motility improving screening method | |
WO2007123274A1 (en) | Novel marker for sensitivity against sulfonamide compound | |
JPH06500775A (en) | Regulation of malignant cell proliferation via a novel 5HT1a receptor | |
US20220273751A1 (en) | Gpcr heteromer inhibitors and uses thereof | |
JP2006510590A (en) | Use of histamine H4 receptor modulators for the treatment of allergies and asthma |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20070110 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20070110 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20100115 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20100415 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20100422 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20100517 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20100526 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20100615 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20100622 |
|
A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100715 |
|
A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20100818 |
|
RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20101228 |
|
RD13 | Notification of appointment of power of sub attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7433 Effective date: 20121120 |
|
A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20121120 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20130730 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20130802 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20130828 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20130903 |
|
A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20130927 |
|
A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20131002 |