JP2004534531A - Method - Google Patents
Method Download PDFInfo
- Publication number
- JP2004534531A JP2004534531A JP2002589805A JP2002589805A JP2004534531A JP 2004534531 A JP2004534531 A JP 2004534531A JP 2002589805 A JP2002589805 A JP 2002589805A JP 2002589805 A JP2002589805 A JP 2002589805A JP 2004534531 A JP2004534531 A JP 2004534531A
- Authority
- JP
- Japan
- Prior art keywords
- aqp8
- activity
- ibd
- measuring
- compound
- 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
- 238000000034 method Methods 0.000 title claims abstract description 43
- 102100029463 Aquaporin-8 Human genes 0.000 claims abstract description 85
- 101000771417 Homo sapiens Aquaporin-8 Proteins 0.000 claims abstract description 85
- 230000000694 effects Effects 0.000 claims abstract description 29
- 150000001875 compounds Chemical class 0.000 claims abstract description 25
- 238000003556 assay Methods 0.000 claims abstract description 17
- 238000013518 transcription Methods 0.000 claims abstract description 12
- 230000035897 transcription Effects 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- 238000002405 diagnostic procedure Methods 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 6
- 238000013519 translation Methods 0.000 claims abstract description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 108700028369 Alleles Proteins 0.000 claims description 12
- 102000004891 aquaporin 8 Human genes 0.000 claims description 9
- 239000003550 marker Substances 0.000 claims description 9
- 108700008625 Reporter Genes Proteins 0.000 claims description 8
- 108090001000 aquaporin 8 Proteins 0.000 claims description 8
- 238000005259 measurement Methods 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 6
- 230000001404 mediated effect Effects 0.000 claims description 6
- 108020004999 messenger RNA Proteins 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 230000002759 chromosomal effect Effects 0.000 claims 1
- 230000001105 regulatory effect Effects 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 102000010637 Aquaporins Human genes 0.000 description 45
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 40
- 210000004027 cell Anatomy 0.000 description 35
- 230000014509 gene expression Effects 0.000 description 32
- 208000002551 irritable bowel syndrome Diseases 0.000 description 31
- 108010063290 Aquaporins Proteins 0.000 description 27
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 22
- 102100034414 Aquaporin-2 Human genes 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 108010036221 Aquaporin 2 Proteins 0.000 description 16
- 241000282414 Homo sapiens Species 0.000 description 14
- 102000004888 Aquaporin 1 Human genes 0.000 description 13
- 208000011231 Crohn disease Diseases 0.000 description 13
- 241000700159 Rattus Species 0.000 description 13
- 210000000349 chromosome Anatomy 0.000 description 13
- 238000004458 analytical method Methods 0.000 description 12
- 230000035699 permeability Effects 0.000 description 12
- 108090001004 Aquaporin 1 Proteins 0.000 description 11
- 206010009900 Colitis ulcerative Diseases 0.000 description 11
- 201000006704 Ulcerative Colitis Diseases 0.000 description 11
- 230000032258 transport Effects 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 208000024191 minimally invasive lung adenocarcinoma Diseases 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 108091023040 Transcription factor Proteins 0.000 description 8
- 102000040945 Transcription factor Human genes 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 108010036280 Aquaporin 4 Proteins 0.000 description 7
- 108090000976 Aquaporin 5 Proteins 0.000 description 7
- 102100037276 Aquaporin-4 Human genes 0.000 description 7
- 102000004392 Aquaporin 5 Human genes 0.000 description 6
- 230000027455 binding Effects 0.000 description 6
- 210000000170 cell membrane Anatomy 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 238000003205 genotyping method Methods 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 238000011813 knockout mouse model Methods 0.000 description 6
- 210000004739 secretory vesicle Anatomy 0.000 description 6
- 101150062140 Aqp8 gene Proteins 0.000 description 5
- 101001125026 Homo sapiens Nucleotide-binding oligomerization domain-containing protein 2 Proteins 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 102100029441 Nucleotide-binding oligomerization domain-containing protein 2 Human genes 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 210000001072 colon Anatomy 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 201000008638 inflammatory bowel disease 1 Diseases 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 210000002700 urine Anatomy 0.000 description 5
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 4
- 102000009016 Cholera Toxin Human genes 0.000 description 4
- 108010049048 Cholera Toxin Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 230000004064 dysfunction Effects 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 230000003204 osmotic effect Effects 0.000 description 4
- 210000003079 salivary gland Anatomy 0.000 description 4
- 208000011317 telomere syndrome Diseases 0.000 description 4
- 238000011830 transgenic mouse model Methods 0.000 description 4
- 102000004363 Aquaporin 3 Human genes 0.000 description 3
- 108090000991 Aquaporin 3 Proteins 0.000 description 3
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000000636 Northern blotting Methods 0.000 description 3
- 208000021386 Sjogren Syndrome Diseases 0.000 description 3
- 102100026383 Vasopressin-neurophysin 2-copeptin Human genes 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010230 functional analysis Methods 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 238000012252 genetic analysis Methods 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 230000002485 urinary effect Effects 0.000 description 3
- WDGXQSVUSMTYNG-UHFFFAOYSA-N 2-Methyl-3-(2-methylphenyl)-3,4-dihydroquinazolin-4-one [2-(diphenylmethoxy)ethyl]dimethylamine hydrochloride Chemical compound Cl.C=1C=CC=CC=1C(OCCN(C)C)C1=CC=CC=C1.CC1=CC=CC=C1N1C(=O)C2=CC=CC=C2N=C1C WDGXQSVUSMTYNG-UHFFFAOYSA-N 0.000 description 2
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 description 2
- 102100029406 Aquaporin-7 Human genes 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 206010007559 Cardiac failure congestive Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 208000011514 Familial renal glucosuria Diseases 0.000 description 2
- 208000034826 Genetic Predisposition to Disease Diseases 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 206010019280 Heart failures Diseases 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000771402 Homo sapiens Aquaporin-7 Proteins 0.000 description 2
- 208000019025 Hypokalemia Diseases 0.000 description 2
- WHXSMMKQMYFTQS-UHFFFAOYSA-N Lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 108091092878 Microsatellite Proteins 0.000 description 2
- 201000005118 Nephrogenic diabetes insipidus Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 208000004608 Ureteral Obstruction Diseases 0.000 description 2
- 241000269370 Xenopus <genus> Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 238000000149 argon plasma sintering Methods 0.000 description 2
- 230000001363 autoimmune Effects 0.000 description 2
- 108010005774 beta-Galactosidase Proteins 0.000 description 2
- 230000002146 bilateral effect Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- -1 cationic lipid Chemical class 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000112 colonic effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 102000052557 human AQP1 Human genes 0.000 description 2
- 210000003917 human chromosome Anatomy 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000003246 kidney medulla Anatomy 0.000 description 2
- 229910052744 lithium Inorganic materials 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 210000000287 oocyte Anatomy 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 208000024896 potassium deficiency disease Diseases 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 208000007278 renal glycosuria Diseases 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 1
- 101150047512 AQP2 gene Proteins 0.000 description 1
- 108091093088 Amplicon Proteins 0.000 description 1
- 101150073415 Aqp4 gene Proteins 0.000 description 1
- 102000004391 Aquaporin 6 Human genes 0.000 description 1
- 108090000977 Aquaporin 6 Proteins 0.000 description 1
- 102100037332 Aquaporin-3 Human genes 0.000 description 1
- 108700003860 Bacterial Genes Proteins 0.000 description 1
- 108010023063 Bacto-peptone Proteins 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010007558 Cardiac failure chronic Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 108010062580 Concanavalin A Proteins 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
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 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 1
- 241000283086 Equidae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150107049 Exoc3 gene Proteins 0.000 description 1
- 102100030860 Exocyst complex component 3 Human genes 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 101000684063 Homo sapiens Aquaporin-1 Proteins 0.000 description 1
- 101000923631 Homo sapiens Aquaporin-2 Proteins 0.000 description 1
- 101000771413 Homo sapiens Aquaporin-9 Proteins 0.000 description 1
- 102100021711 Ileal sodium/bile acid cotransporter Human genes 0.000 description 1
- 206010053198 Inappropriate antidiuretic hormone secretion Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100023487 Lens fiber major intrinsic protein Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101100055974 Mus musculus Aqp8 gene Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 235000011464 Pachycereus pringlei Nutrition 0.000 description 1
- 240000006939 Pachycereus weberi Species 0.000 description 1
- 235000011466 Pachycereus weberi Nutrition 0.000 description 1
- 206010033645 Pancreatitis Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102000009572 RNA Polymerase II Human genes 0.000 description 1
- 108010009460 RNA Polymerase II Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 101150049811 SEC6 gene Proteins 0.000 description 1
- 108091006614 SLC10A2 Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 229930006000 Sucrose Natural products 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
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 1
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 1
- 102000002852 Vasopressins Human genes 0.000 description 1
- 108010004977 Vasopressins Proteins 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010018755 aquaporin 0 Proteins 0.000 description 1
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 1
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 1
- 238000012550 audit Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000009134 cell regulation Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 210000004922 colonic epithelial cell Anatomy 0.000 description 1
- 210000004953 colonic tissue Anatomy 0.000 description 1
- 210000002777 columnar cell Anatomy 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000001446 dark-field microscopy Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 201000010064 diabetes insipidus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 210000004921 distal colon Anatomy 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000001378 electrochemiluminescence detection Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000036397 gastrointestinal physiology Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002727 hyperosmolar Effects 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 201000008284 inappropriate ADH syndrome Diseases 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000003156 inflammatory bowel disease 7 Diseases 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000005305 interferometry Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000001630 jejunum Anatomy 0.000 description 1
- 210000000231 kidney cortex Anatomy 0.000 description 1
- 238000004556 laser interferometry Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000004923 pancreatic tissue Anatomy 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000002135 phase contrast microscopy Methods 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 238000009428 plumbing Methods 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 108010030416 proteoliposomes Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000003156 radioimmunoprecipitation Methods 0.000 description 1
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose 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[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000037425 regulation of transcription Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000002416 scanning tunnelling spectroscopy Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000000492 total internal reflection fluorescence microscopy Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect 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
- 229940035893 uracil Drugs 0.000 description 1
- 229960003726 vasopressin Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000001262 western blot Methods 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Abstract
本発明は、ＩＢＤの治療に有用な化合物を特定する方法を提供する。該方法は、ＡＱＰ８の活性または量を調節するその能力について該化合物をアッセイすることを含む。該アッセイは、ＡＱＰ８を発現する細胞株を用いるかまたは精製ＡＱＰ８タンパク質を用いるＡＱＰ８活性の測定、およびＡＱＰ８を発現する細胞株でのＡＱＰ８の転写または翻訳の測定から選択される。本発明はまた、医薬組成物の製造方法、ＩＢＤに対する感受性を測定するための診断方法、ＩＢＤまたはその素因の診断方法、およびＡＱＰ８の活性または量を調節することができる化合物のＩＢＤ治療用医薬の製造における使用を提供する。The present invention provides methods for identifying compounds that are useful for treating IBD. The method comprises assaying the compound for its ability to modulate the activity or amount of AQP8. The assay is selected from measuring AQP8 activity using a cell line expressing AQP8 or using purified AQP8 protein and measuring transcription or translation of AQP8 in a cell line expressing AQP8. The present invention also provides a method for producing a pharmaceutical composition, a diagnostic method for measuring sensitivity to IBD, a method for diagnosing IBD or a predisposition thereto, and a medicament for treating IBD with a compound capable of regulating the activity or amount of AQP8. Provide use in manufacturing.
Description
【技術分野】
【０００１】
本発明は、ＩＢＤの治療に有用な化合物を特定する方法、医薬組成物の製造方法、ＩＢＤに対する感受性を測定するための診断方法、ＩＢＤまたはその素因の診断方法、およびＡＱＰ８の活性または量を調節することができる化合物のＩＢＤ治療用医薬の製造における使用を提供する。
【背景技術】
【０００２】
ＩＢＤは、胃腸管の慢性再発性の腸炎症を特徴とする。ＩＢＤは欧米の国々で１０００人に約１人が罹患し、平均発症年齢は成人初期である。今日まで該疾患の病因は不明である。臨床的および病理組織学的特色に基づいて、ＩＢＤは以下の２つの主なサブタイプに分類される：クローン病（ＣＤ）（オンラインヒトメンデル遺伝（“On Line Mendelian Inheritance in Man"）OMIM 266600）および潰瘍性大腸炎（ＵＣ）（OMIM 191390）。ＩＢＤの原因は不明であるが、該疾患の家族内集団発生および一卵性双生児における一致の増加は強い遺伝的感受性を示している。兄弟内発症の危険率の推定値（λｓ）は１０〜５０の範囲であり、遺伝的因子がＩＢＤ素因に重要な役割を果たすことを示唆している。疫学的データによれば、ＩＢＤは、環境因子と同様に多くの感受性遺伝子によって惹起される多因子性異常であると考えられる。本発明ではＩＢＤという用語は、クローン病および潰瘍性大腸炎に加えてＩＢＤも含むことを意図している。
【０００３】
以前のゲノムワイドな連鎖解析によってＩＢＤに対する多数の感受性遺伝子座、例えばＩＢＤ１（OMIM 266600）（Hugot et al. 1996; Brant et al. 1998; Curran et al. 1998; Hampe et al. 1999a）、ＩＢＤ２（OMIM 601458）（Duerr et al. 1998; Parkes et al. 2000）、ＩＢＤ３（OMIM 604519）（Hampe et al. 1999b）、ＩＢＤ７（OMIM 605225）（Cho et al. 1998; Cho et al. 2000）が特定された。ＩＢＤ１遺伝子座は、ヒト染色体１６のペリセントロメア領域に位置し、１６ｐ１２−ｑ１３領域にマッピングされる。
【０００４】
ＡＱＰ８は水分チャネルタンパク質（アクアポリン）群に属する。アクアポリン（ＡＱＰ）は不可欠の膜タンパク質を構成する主要な固有スーパーファミリーに属するメンバーであり、動物、植物および細菌で細胞膜の水分通過を促進する特殊なチャネルとして機能する。これまでのところ０から９の番号を付された１０個のＡＱＰホモログが哺乳類でクローニングされた。これらは広範囲に分布し、２つ以上のＡＱＰが同じ細胞に存在することができる（Echevarria and Ilundain, 1998）。いくつかのヒトＡＱＰ遺伝子がクローニングされ、多くの障害がこれらのタンパク質の機能異常に関連していることが判明した（Echevarria and Ilundain, 1998）。
【０００５】
先ず初めにマウスのＡＱＰ８をコードするｃＤＮＡがMa et al.(1997a)によってクローニングされた。ノザンブロット分析は、多数の組織（胎盤、結腸、肝臓、心臓、膵臓、肺臓、腎臓、精巣、脾臓、胃および脳を含む）での発現を示した。
【０００６】
ヒトのＡＱＰ８をコードするｃＤＮＡは１９９８年にクローニングされ、細胞液輸送に重要であることが示された（Koyama et al. 1998）。該遺伝子の発現はノザンブロットによってヒトの膵臓および結腸の組織で示された（Koyama et al. 1998）。
【０００７】
Koyama et al.（1999）は、ラットではＡＱＰ８は空腸の吸収性柱状細胞、結腸の近位部および遠位部で発現されることをin situハイブリダイゼーションで示し、ＡＱＰ８は胃腸管で水分の取り込みおよび／または排出に役割を有すると提唱した。マウスでは、ＡＱＰ３、ＡＱＰ４およびＡＱＰ８が結腸上皮表面で発現され、液輸送におけるこれらアクアポリンの共同的役割が示唆された（Ma and Verkman 1999）。
【０００８】
ラット肝細胞におけるＡＱＰ８の発現、非細胞局在および調節がGarcia et al.（2001）によってさらに詳細に調べられた。最近になって、ヒト結腸におけるＡＱＰ８の発現がFischer et al.（2001）によって実証され、さらに正常な増殖中の結腸上皮細胞のマーカーであると提唱された。
【０００９】
アクアポリンは小型で膜に広がる非常に疎水性の固有の膜タンパク質（モノマーサイズは約３０ｋＤａ）であり、液輸送に必要な多くの細胞タイプの細胞質膜で発現される。アクアポリンの構造の基本的特色は、変異誘発、エピトープ付加、並びにスペクトロスコープおよび凍結割断電子顕微鏡法を用いて明らかにされた。アクアポリンは膜でホモテトラマーとして集合するように思われる。このホモテトラマーでは、各モノマー（アミノおよびカルボキシ末端が細胞質に向いている６つのメンブレンスパンニングα−らせんドメインから成る）は明瞭な水の孔を含む。
【００１０】
アクアポリンの構造および機能はVerkman and Mitra（2000）およびKing et al.（2000）が概論している。
稀なコルトン−ヌル（Colton-null）表現型を有するヒトはＡＱＰ１に欠損を有するが、明瞭な臨床的表現型を示さず（Preston et al1994）、尿濃縮能の低下のみが認められる（Mathai et al. 1996）。対照的に、ＡＱＰ１ノックアウトマウスでは顕著な尿濃縮機能不全が示された（Ma et al. 1998）。同様に、ＡＱＰ３ノックアウトマウスでは、重篤な尿濃縮機構の機能不全を示す腎性尿崩症を発症する（Ma et al. 2000）。
【００１１】
ＡＱＰ２の変異は、劇的な臨床的表現型である希な遺伝性腎性尿崩症を惹起する（ＮＤＩ）（Deen et al. 1994）。ＮＤＩは、バソプレッシンに対する腎の耐性が病因の疾患であり、その臨床的特徴は大量の希薄尿の排出である。これはまた、ある環境下でのアクアポリンの発現および／または機能状態がホルモンの影響下にあることを示している。ＡＱＰ２遺伝子に変異を有する常染色体の劣性ＮＤＩをもつ患者は、アクアポリンは水分輸送について律速因子であろうという最初の明瞭な例を提供する。後天的ＮＤＩは遺伝型よりもいっそう一般的であり、多様な原因を有する。リチウム（Marples et al. 1995a）、両側性尿管閉塞（Frφkiaer 1996）および慢性低カリウム血症（Marples et al. 1995b）（公知のＮＤＩの原因である）は全て、動物で顕著なＡＱＰ２発現の低下を生じ、同時に尿濃縮能力を低下させる。水分の不均衡スペクトルのまた逆の面では、ＡＱＰ２発現の増加が、うっ血性心不全（Nielsen et al. 1997; Xu et al. 1997）、肝硬変（Fujita et al. 1995）および妊娠（Ohara et al. 1998）を含む液体停留状態で認められた。
【００１２】
マウスではＡＱＰ４遺伝子を破壊することによって中等度の尿濃縮機能不全が生じたが（Ma et al. 1997b）、結腸の液排出または糞便の脱水に対してはほとんどまたは全く影響は認められなかった（Wang et al. 2000）。
【００１３】
ＡＱＰ１およびＡＱＰ４ノックアウトマウスにおける肺臓の液体輸送がBai et l.（1999）によって調べられた。野生型マウスと比較してＡＱＰ１ノックアウトマウスでは気腔−毛細管の水分透過性が１０倍低下したにもかかわらず、等浸透圧の肺胞液の吸収速度は顕著には相違しなかった。気道の水分チャネルＡＱＰ４の欠損の影響はほとんど存在しなかった。
【００１４】
Ma et al.（1999）はＡＱＰ５を欠くトランスジェニックマウスの唾液分泌が低下することを報告し、ＡＱＰ５の唾液腺機能における生理学的関連が示唆された。Steinfeld et al.（2001）は、ヒト唾液腺にＡＱＰ５が局在することを報告し、ショーグレン症候群患者のＡＱＰ５の唾液腺における異常分布を示し、この異常分布はショーグレン症候群患者の唾液分泌低下の原因であろう。
【００１５】
現在哺乳類アクアポリンの２つの機能群が認識されている（Agre et al. 1998）。第一群（ＡＱＰ０、ＡＱＰ１、ＡＱＰ２、ＡＱＰ４およびＡＱＰ５を含む）は、古典的に定義されたとおり水に対してのみ浸透性を有する。第二群（ＡＱＰ３、ＡＱＰ７およびＡＱＰ９を含む）は、水に対して強い浸透性を有するが、さらにグリセロールおよび他の小分子も浸透させる。これらの相違の構造的説明および生理学的相関性は不明である。ＡＱＰ６の配列は水選択性群と同様であるが、ゲートコントロール陰イオンコンダクタンス（gated anion conductance）の最近の驚くべき観察は、機能的相違は以前に考えられたものよりも複雑であることを示した（Yasui et al. 1999）。ＡＱＰ８の配列は水に選択性を有する群とグリセロール透過群との中間で、機能的な定義は保留されている（Koyama et al. 1997）。したがって、アクアポリン（ＡＱＰ８を含む）は、水およびおそらくは他の小分子の腸および他の組織の上皮（高い近傍細胞透過性を有すると考えられる）におけるゲートコントロールに重要な役割を果たすであろう。
【００１６】
本発明は、ＡＱＰ８遺伝子の位置にきわめて近傍の染色体１６上の領域に位置する遺伝的マーカーに関連するＩＢＤの遺伝的素因の発見に基づいている。ＡＱＰ８の活性または量を調節することができる化合物を使用することは、本発明によるＩＢＤ治療の新規な治療概念として確認された。ＡＱＰ８遺伝子の配列分析はＩＢＤに対する感受性の新規な診断方法を提供するであろう。
【発明の開示】
【００１７】
本発明の特徴の１つにしたがえば、ＩＢＤ治療のためにＡＱＰ８の活性または量を調節することができる化合物の使用が提供される。化合物によるＡＱＰ８の量の調節は、例えば遺伝子発現レベルまたはメッセージの安定性の変更によって達成することができる。化合物によるＡＱＰ８の活性の調節は、例えば化合物をＡＱＰ８タンパク質に結合させることによって達成できる。ある実施態様では、ＡＱＰ８の調節は、ＡＱＰ８の活性または量を減少させることができる化合物を含む。別の実施態様では、ＡＱＰ８の調節は、ＡＱＰ８の活性または量を増加させることができる化合物を含む。
【００１８】
ＡＱＰ８の活性を調節することができる化合物の例は抗体である。抗体は任意の適当な方法を用いて調製することができる。例えば、精製ポリペプチドを利用して特異的抗体を調製することができる。“抗体”という用語はポリクローナル抗体、モノクローナル抗体および種々のタイプの抗体構築物、例えばＦ(ａｂ')2、Ｆａｂおよび単鎖Ｆｖを含むことを意図する。抗体は、約１０7Ｍ-1より大きいかまたは等しいＫaでＳＬＣ１０Ａ２の対立遺伝子変異体と結合する場合は特異的に結合すると定義される。結合親和性は、通常の技術（例えば文献（Scatchard et al., Ann. N.Y. Acad. Sci., 51:660(1949)に記載された技術）を用いて決定することができる。
【００１９】
ポリクローナル抗体は、多様な供給源（例えばウマ、ウシ、ヤギ、ヒツジ、イヌ、ニワトリ、ウサギ、マウスまたはラット）から当分野で周知の方法を用いて容易に作製することができる。一般に、抗原は宿主動物に典型的には腹腔注射により投与される。抗原の免疫原性は、アジュバント（例えばフロイントの完全または不完全アジュバント）を使用することによって高めることができる。ブースター免疫の後で、少量の血清サンプルを採集し、抗原に対する反応性を検査する。このような測定に有用な種々のアッセイの例には、以下の文献に記載されたもの（Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988）の他に、例えば交差免疫電気泳動（ＣＩＥＰ）、放射性イムノアッセイ、放射性免疫沈澱、酵素結合免疫吸着アッセイ（ＥＬＩＳＡ）、ドットブロットアッセイおよびサンドイッチアッセイ（米国特許第4,376,110号および4,486,530号を参照されたい）のような方法が含まれる。
【００２０】
モノクローナル抗体は周知の方法を用いて容易に製造できる。例えば以下の文献に記載された方法を参照されたい：米国特許RE32,011号、4,902,614号、4,543,439号および4,411,993号；Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn and Bechtol (eds.), (1980)。
【００２１】
本発明のモノクローナル抗体はまた別の技術、例えばAlting-Mees et al.が記載した技術（“Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas", Strategies in Molecular Biology 3:1-9(1990)、この文献は参照により本明細書に含まれる）を用いて製造することができる。同様に、結合パートナーはリコンビナントＤＮＡ技術を用いて構築し、特異的な結合抗体をコードする遺伝子の可変領域を取り込むことができる。そのような技術はLarrick et al.が記載している（Biotechnology, 7:394(1989)）。
いったん単離され精製されたら、その抗体を用いて、確立されたアッセイプロトコルにより抗原の存在を検出することができる（例えば以下を参照されたい：“A Practical Guide to ELISA" , D.M. Kemeny, Pergamon Press, Oxford, England）。
【００２２】
本発明の別の特徴にしたがえば、ＡＱＰ８の活性または量を調節することができる化合物のＩＢＤ治療用医薬の製造における使用が提供される。
【００２３】
本発明のさらにまた別の特徴にしたがえば、ＩＢＤの治療に潜在的に有用な化合物を特定する方法が提供され、該方法は、ＡＱＰ８の活性または量を調節する化合物の能力についてその化合物をアッセイすることを含む。好ましくは該アッセイは以下から選択される：
ｉ）ＡＱＰ８を発現する細胞株を用いるか、または精製ＡＱＰ８タンパク質を用いるＡＱＰ８活性の測定；および
ii）ＡＱＰ８発現細胞株におけるＡＱＰ８の転写または翻訳の測定。
【００２４】
被検化合物のＡＱＰ８の活性に対する影響を決定するために用いられるアッセイは、ＡＱＰ８仲介水輸送の測定により達成することができる。アクアポリン仲介水輸送は、一般にはアクアポリンのｃＲＮＡを発現するアフリカツメガエル卵母細胞での浸透膨潤アッセイを用いて測定される。最初に記載されたように（Zhang et la. 1990）、定量的画像分析を用い、２００から０〜１００mosmol／ｋｇＨ2Ｏへの細胞外溶液の浸透圧の急激な低下に対する反応で卵細胞膨潤のタイムコースを推定する。アクアポリンをトランスフェクトした哺乳類細胞における水輸送もまた多様な生物物理学的アプローチによって調べられ、該アプローチには以下が含まれる：液流停止光散乱（stopped-flow light scattering）（ Ma et al. 1993）、総内部反射蛍光顕微鏡法（total internal reflection fluorescence microscopy）（Farinas et al. 1995）、レーザーインターフェロメトリー（laser interferometry）（Farinas and Verkman 1996）、およびフーリエ光学暗視野顕微鏡法／位相差顕微鏡法（Farinas et al. 1997）。液流停止光散乱はまた、精製アクアポリンタンパク質を含む酵母由来の小胞（Laize et al. 1995）および再構成プロテオリポソーム（Van Hoek and Verkman, 1992）におけるアクアポリン仲介水輸送のアッセイにも用いられた。
【００２５】
被検化合物のＡＱＰ８の転写または翻訳に対する影響を決定するために用いられるアッセイは以下を基にすることができる：
ｉ）例えばノザンブロット分析または定量的リアルタイムＰＣＲを用いる生成ＡＱＰ８ｍＲＮＡの量の測定；
ii）例えばウェスタンブロット分析または免疫化学的分析（例えばＥＬＩＳＡ）を用いる生成ＡＱＰ８タンパク質の量の測定；または
iii）ＡＱＰ８発現細胞でのＡＱＰ８活性の上記に述べたような測定。
【００２６】
アッセイで用いられる細胞はＡＱＰ８を天然に発現している細胞でも、またリコンビナントＡＱＰ８を発現するトランスフェクト細胞でもよい。好ましくは該ＡＱＰ８はヒトリコンビナントＡＱＰ８である。
【００２７】
ＡＱＰ８は多様な宿主、例えば細菌、植物細胞、昆虫細胞、真菌細胞並びにヒトおよび動物細胞で発現させることができる。真核リコンビナント宿主細胞が特に好ましい。例として、酵母、哺乳類細胞（ヒト、ウシ、ブタ、サルおよびげっ歯類起源の細胞株を含む）、および昆虫細胞（ショウジョウバエおよびカイコに由来する細胞株を含む）が含まれる。市販されている使用可能な哺乳類種由来の細胞株には、Ｌ細胞Ｌ−Ｍ（ＴＫ−）（ATCC CCL 1.3）、Ｌ細胞Ｌ−Ｍ（ATCC CCL 1.2）、ＨＥＫ２９３（ATCC CRL 1573）、Ｒａｊｉ（ATCC CCL 86）、ＣＶ−１（ATCC CCL 70）、ＣＯＳ−１（ATCC CRL 1650）、ＣＯＳ−７（ATCC CRL 1651）、ＣＨＯ−Ｋ１（ATCC CCL 61）、３Ｔ３（ATCC CCL 92）、ＮＩＨ／３Ｔ３（ATCC CRL 1658）、ＨｅＬａ（ATCC CCL 2）、Ｃ１２７Ｉ（ATCC CRL 1616）、ＢＳ−Ｃ−１（ATCC CCL 26）およびＭＲＣ−５（ATCC CCL 171）が含まれる。
【００２８】
以下を含む多数の方法のいずれかを用い、ＡＱＰ８をコードする核酸を含む発現ベクターを宿主細胞に導入し、本発明のポリペプチドを発現させることができる：リン酸カルシウムによる形質転換、ＤＥＡＥ−デキストランによる形質転換、陽イオン脂質仲介リポフェクチン、電気穿孔または感染。
【００２９】
トランスフェクトした宿主細胞を増殖させ、例えば制限希釈によってクローニングし、さらにリコンビナントＡＱＰ８の発現レベルを決定するために分析する。ＡＱＰ８を発現する形質転換宿主細胞の同定は、抗体との免疫学的反応性および／または本明細書に述べるアッセイによる生物学的活性の検出を含むいくつかの手段によって達成できる。
リコンビナントヒトＡＱＰ１またはＡＱＰ２は酵母内で発現され、分泌小胞内に局在することが判明した。ＡＱＰ１およびＡＱＰ２を含む分泌小胞は、高い水分透過性および水流に対する低い活性化エネルギーを示し、機能的ＡＱＰ１およびＡＱＰ２の発現を示唆した（Coury et al. 1998）。酵母での発現およびそれに続く単離酵母小胞の水透過性の測定を用いて、ＡＱＰ２変異体の機能が調べられた（Shinbo et al. 1999）。
【００３０】
遺伝子発現の転写の調節は転写因子の結合を支配し、それによって遺伝子の転写を仲介するプロモーター中の特定のＤＮＡエレメントによって仲介される。真核生物の転写因子は以下の２つの主な群に分けることができる。ｉ）転写開始部に近位のプロモーター配列と相互作用し、それによってＲＮＡポリメラーゼIIが補充されたとき転写を開始させる基礎転写因子、および ii）特定の遠位プロモーターエレメントに結合し、それによって基礎転写機構と接触したときに転写を仲介する転写因子。真核生物における基礎的な生理学的プロセスは、細胞がその環境とコミュニケーションをもち、シグナリング分子（例えばホルモンおよび増殖因子）を介して細胞外刺激に対して反応することができるということである。そのようなシグナリングのための最終事象は、特定の遠位プロモーターエレメントと転写因子との結合であり、これは、例えば遺伝子発現のアップレギュレーションまたは組織特異的遺伝子の発現をもたらす。それらプロモーターエレメントの調節的な役割のために、プロモーターエレメントは治療薬剤スクリーニングのための仮定的標的である。ヒトＡＱＰ８プロモーター配列（本出願に開示）は、ＡＱＰ８の転写を選択的に調節する治療薬剤のスクリーニングを可能にする。
【００３１】
適当な宿主細胞は、ＡＱＰ８を発現することが判明している細胞、またはＡＱＰ８の転写に影響を与えることができる転写因子を発現することが判明している細胞である。好ましくは、特定の転写因子をコードするＤＮＡをトランスフェクトされた宿主細胞を用いて、特定の転写因子とＡＱＰ８プロモーターとの相互作用を調べることができる。
【００３２】
被検化合物のＡＱＰ８の転写に対する効果を判定するために用いられるアッセイはまた、レポーター遺伝子系を用いるＡＱＰ８プロモーターの活性の測定を基準にすることができる。該レポーター遺伝子系はＡＱＰ８プロモーターを構成する核酸分子またはそのフラグメントを含む発現系で、この発現系はさらにレポーター遺伝子を含む。これらプロモーターおよびレポーター遺伝子は、レポーター遺伝子の発現がＡＱＰ８プロモーターによって調節されるように配置されている。生成されるレポータータンパク質の量は、ＡＱＰ８プロモーターの活性の指標として用いられる。
【００３３】
好ましくはＡＱＰ８プロモーターはヒトＡＱＰ８プロモーター、好ましくは配列番号：１のヌクレオチド配列１−１９５６、またはそのフラグメントである。
レポーター遺伝子系の構築に用いることができる適当なレポーター遺伝子は、例えばホタルのルシフェラーゼ遺伝子、細菌のクロラムフェニコールアセチルトランスフェラーゼ（ＣＡＴ）遺伝子、β−ガラクトシダーゼ（β−ＧＡＬ）遺伝子、および緑色蛍光タンパク質（ＧＦＰ）である。
【００３４】
本発明の別の特徴にしたがえば、以下の工程を含む医薬組成物の製造方法が提供される：
ｉ）本明細書に記載される方法にしたがってＩＢＤの治療に有用な化合物を特定し；さらに
ii）該化合物または医薬的に許容できるその塩を医薬的に許容できる賦形剤または希釈剤と混合する。
【００３５】
本発明のさらに別の特徴では診断方法が提供される。該方法は、患者から得られたＤＮＡサンプルで染色体１６の配列またはその部分をＩＢＤに対する感受性の判定のために分析することを含み、さらにＢＡＣ ５０４Ｎ１９（ＥＭＢＬ ＡＦ２６５３４０）、ＢＡＣ Ａ−２４９Ｂ１０（ＥＭＢＬ ＡＣ００２２８８）またはＢＡＣ ＣＴＤ−２５４７Ｇ２３（ＥＭＢＬ ＡＣ００８７４１）に一致する染色体フラグメントに存在する多型マーカーの対立遺伝子の有無を判定することを含む。好ましくは、該方法は、患者から得られたＤＮＡサンプル中のＡＱＰ８遺伝子またはその一部分の配列をＩＢＤに対する感受性の判定のために分析することを含み、さらに患者のＡＱＰ８遺伝子に存在する多型マーカーの対立遺伝子の有無を判定することを含む。
【００３６】
本発明のまた別の特徴では、ＡＱＰ８タンパク質上の自己免疫エピトープに対する免疫反応を調節する方法が提供される。好ましくは、本方法は免疫学的耐性を誘発する方法を含む。免疫学的耐性は、例えば自己抗原と結合させたV. cholera由来のコレラ毒素（ＣＴ）のＢサブユニットまたは大腸菌（E. coli）由来の易熱性毒素（ＬＴ）を用いた経口免疫によって誘発することができる（Simmons et al. 2001）。自己免疫反応は多数の炎症性疾患、例えば慢性関節リウマチ、膵炎および多発性硬化症の原因であることが判明している。
【００３７】
本発明のまた別の特徴では、ＡＱＰ８タンパク質に対して向けられる自己抗体の測定を含むＩＢＤの診断方法が提供される。
【００３８】
略語
アクアポリン：ＡＱＰ
アクアポリン−８：ＡＱＰ８
罹患兄弟ペア：ＡＳＰ
細菌性人工染色体：ＢＡＣ
クロラムフェニコールアセチルトランスフェラーゼ：ＣＡＴ
クローン病：ＣＤ
センチモルガン：ＣＭ
コレラ毒素：ＣＴ
グリコサミノグリカン：ＧＡＧ
緑色蛍光タンパク質：ＧＦＰ
炎症性腸疾患：ＩＢＤ
易熱性毒素：ＬＴ
腎性尿崩症：ＮＤＩ
オンラインヒトメンデル遺伝：ＯＭＩＭ
ペディグリー不平衡検査（Pedigree disequilibrium test）：ＰＤＴ
配列付加部位：ＳＴＳ
伝達不平衡検査：ＴＤＴ
潰瘍性大腸炎：ＵＣ
【００３９】
以下の非限定的実施例を用いて本発明をこれから説明する。図１は、該実施例の、問題の染色体領域マップを示す。
アンプリタック（AMPLITAQ（登録商標））（Perkin-Elmer Cetusから入手できる）が耐熱性ＤＮＡポリメラーゼの供給源として用いられる。
以下の文献に記載されている方法のいずれかに由来する一般的な分子生物学的方法にしたがうことができる：“ Molecular Cloning-A Laboratory Manual", Second Edition, Sambrook, Fritsch and Maniatis ( Cold Spring Harbor Laboratory, 1989) または“Current Protocols in Molecular Biology Vol.1-3, F.M. Asubel, R. Brent and R.E. Kingston (eds), John Wiley発行, 1998。
【実施例１】
【００４０】
本発明者らは、ＳＴＳマーカーおよび未だ公表されていない単一ヌクレオチド多型による高密度遺伝子型決定を用い、続いて標準的な手順の連鎖不平衡検査（Terwillinger, 1995）にしたがって、ＩＢＤ１に関する感受性遺伝子を含む領域の範囲を物理的距離で約７Ｍｂと限定した。ＳＴＳマーカー、Ｄ１６Ｓ４０１およびＤ１６Ｓ４０９とそれぞれ隣接する領域全体に及ぶＴＤＴ検査によって（合計３２マーカー（１３のＳＴＳおよび１９のＳＮＰ）を用いた）最初の連鎖データが確認され、さらに２つの部位における連鎖のゆがみが明らかにされた。本発明者らのデータは、ＩＢＤ１に対する感受性遺伝子が、Ｄ１６Ｓ４０１およびＤ１６Ｓ４０９に隣接する領域におそらく位置することを示した。
【００４１】
１ . １ 家族確認および表現型：
患者は以下の施設のＩＢＤプログラムから募集された：Charite University Hospital (Berlin)およびthe Ist Department of Medicine at the Christian-Albrechts-University of Kiel。ＩＢＤと診断された２人以上の罹患兄弟を有する小児をサンプルとし、２５０を越える罹患兄弟ペアーが本分析に含まれた。書面によるインフォームドコンセントを全ての研究参加者から得た。募集プロトコルは院内の監査委員会で承認された。ＩＢＤの診断およびＣＤおよびＵＣにおける分類は、Lennard-Jones（1989）およびPodolsky（1991）が記載した標準的な診断基準によって判定した。患者は１人または２人以上の研究責任者が直接診察した。また別には詳細な病歴および全ての診断過程の結果を記載した２つの記録書を各患者について入手し、研究責任者が概覧した。静脈血サンプルを罹患兄弟および可能な場合にはその両親から得た。
【００４２】
１ . ２ 遺伝子型決定：
ピュアージーンシステム（Puregene System）（Gentra Systems）を用いて全血サンプルからゲノムＤＮＡを調製した。個々のＤＮＡサンプルを９６ウェルのマイクロタイタープレートに並べ、個々のマーカーアンプリコンを用いてＰＣＲによる増幅を実施した。マイクロサテライトマーカーを文献（Hall and Nanthakumar (1997)）に記載されたように蛍光法で遺伝子型決定した。データは、ＡＢＩ３７７自動配列決定装置を用いて収集し、データ分析はＧＥＮＥＳＣＡＮ（バージョン２.１）およびＧＥＮＯＴＹＰＥＲ（バージョン１.１.１）を用いて実施した。対立遺伝子分析および個々のアレルコーリングは、Hall and Nanthakumar (1997)およびIdury and Cardon (1997)が記載したように実施した。ＳＮＰはＡＢＩ３７００自動配列決定装置、ｃＤＮＡ選別および変異検出方法を用いゲノム配列決定によって特定した。情報量の多いＳＮＰの遺伝子型決定は、タックマン（TAQMAN（登録商標））技術（PE Biosystems）を用いるリアルタイムＰＣＲによって実施した。ＰＣＲ反応はＡＢＩ９７００で実施し、蛍光の結果はＡＢＩ７７００シークェンス−ディテクターシングルポイント測定を用いて判定した。対立遺伝子分析および個々の対立遺伝子コーリングは、Hall and Nanthakumar (1997)の記載にしたがって実施した。遺伝子マップは自動マッピングプログラムＭＵＬＴＩＭＡＰ（バージョン２．０、Matise et al. 1994）を用いて構築した。得られたマーカー間の距離（Ｋｏｓａｍｂｉマップアルゴリズムによって決定）はセンチモルガン（ｃＭ）で示される。
【００４３】
１ . ３ 遺伝解析：
遺伝解析は前述の２つの標準的診断カテゴリー、ＣＤおよびＵＣを使用して実施した。第三のカテゴリー、ＡＬＬはＣＤ／ＣＤ、ＵＣ／ＵＣおよびＣＤ／ＵＣ（混合ＡＳＰ）を含み、したがってＩＢＤを分析用単一表現型として表す。各マーカーの対立遺伝子頻度は全ての個体についてのコホート遺伝子型決定データから計算した。関連の統計はＡＮＡＬＹＺＥソフトウェアパッケージのＴＤＴＬＩＫＥプログラムによって計算した（Terwilliger 1995）。検査する対立遺伝子は１０人以上に観察された伝達に限定される。プログラムアルゴリズムは多数の対立遺伝子の検査について修正されたｐ値を提供する。
【００４４】
Ｄ１６Ｓ４０１およびＤ１６Ｓ４０９間の領域のマーカー（１ＳＴＳ，３ＳＮＰ）に関する配列情報
ＩＢＤの２００罹患兄弟ペア（ＡＳＰ）の１６ｐ上の陽性マーカーに対するＴＤＴの結果
ａ）ＴＤＴＬＩＫＥ（Terwilliger, 1995）
【表１】
ｂ）ＰＤＴ（Martin et al. 2000）
【表２】
【００４７】
１ . ４ 生物情報科学：
染色体１６に由来する３つのヒトＢＡＣ、マーカーＤ１６Ｓ３０６８を含むＲＰ１１−４５１Ｎ２０（ＥＭＢＬ ＡＣ０６０７８５）、５０４Ｎ１９（ＥＭＢＬ ＡＦ２６５３４０）、Ａ−２４９Ｂ１０（ＥＭＢＬ ＡＣ００２２８８）を、ＢＡＣ ＣＴＤ−２５４７Ｇ２３（ＥＭＢＬ ＡＣ００８７４１）（部分的にＢＡＣ Ａ−２４９Ｂ１０とオーバーラップする）と同様に潜在的疾患関連遺伝子について解析した。ＡＱＰ８ｃＤＮＡと一致する配列がＢＡＣ ＣＴＤ−２５４７Ｇ２３（ＥＭＢＬ ＡＣ００８７４１）で見出された。
このＢＡＣの配列（ＥＭＢＬで登録番号ＡＣ００８７４１として入手できる）（３４の順不同のフラグメントを含む）をさらに分析し、１つのフラグメントはＡＱＰ８遺伝子（１．９ｋｂのプロモーター領域を含む）を含むことが判明した。
【００４８】
１ . ５ ＡＱＰ８遺伝子のＳＮＰの特定：
ＡＱＰ８遺伝子のプロモーター領域とともにエキソンに隣接するＤＮＡ配列を基にしてＰＣＲプライマーを構築する。ＰＣＲ増幅の後で、２３人のＩＢＤ罹患個体および２０人の正常コントロールから得られたゲノムＤＮＡサンプル由来のエキソンおよびプロモーター領域の配列を決定する。１０％を越える個体に存在する配列の変異体を特定し、上記で述べたタックマン（登録商標）技術を用いてコホートの残りのＳＮＰの遺伝子型決定のために選択する。
【００４９】
１ . ６ 患者のＩＢＤ感受性を診断する方法：
患者からＤＮＡサンプルを得る。ＩＢＤに対する感受性増加に関連する１つまたは２つ以上の多型マーカーの対立遺伝子の有無を、例えばタックマン（登録商標）技術を用いて判定する。
【００５０】
１ . ７ ＡＱＰ８アッセイ：
ＡＱＰ８活性は、リコンビナントＡＱＰ８を発現する酵母に由来する酵母の分泌小胞の水の透過性として測定される。
酵母株およびプラスミドの構築：ＡＱＰ８（配列番号：１）をコードするｃＤＮＡを酵母発現ベクターｐＹＥＳ２（Invitrogen, San Diego, CA）で標準的技術（Sambrook et al. 1989）によってクローニングする。酵母のＳＹ１株（Potenza et al. 1992）を、例えばバイオラド（Bio-Rad）遺伝子パルサー（設定は１．５ｋＶ、２００ｏｈｍで２５μＦ）を用いて電気穿孔によって形質転換する。形質転換した酵母を増殖させ、さらに、ウラシルを欠き、炭素源としてラフィノースを含有する限定的培地で維持する。酵母を栄養豊富な酵母抽出物／ペプトン（ＹＥＰ）−ガラクトース培地（０．５％酵母抽出物（ｗ／ｖ）、１.０％バクトペプトン（ｗ／ｖ）および２.０％ガラクトース（ｗ／ｖ））に２５℃で２〜４時間移してタンパク質発現を開始させ、続いて３７℃に一晩切り換えて分泌小胞を蓄積させる。コントロール実験は、バックグラウンド株またはＡＱＰ８挿入物を欠くｐＹＥＳ２ベクターで形質転換した酵母から調製した小胞を用いて実施する。
【００５１】
イムノブロッティング：サンプルを６０−８０℃で５分インキュベートし、続いてドデシル硫酸ナトリウム（ＳＤＳ）−ポリアクリルアミドゲル電気泳動によって分離する。該電気泳動では、４−２０％連続グラディエントのトリス（ヒドロキシメチル）アミノメタン（Tris）−Ｃｌ−グリシンレディゲル（Ready Gel）または１２％ＳＤＳ−ポリアクリルアミドスラブを用い、さらにニトロセルロース（Bio-Rad）に移す。リン酸緩衝食塩水（ｐＨ７．２５）中の１％粉乳および３％トゥイーン２０から成るブロット緩衝液でブロットをブロックする。抗ＡＱＰ８抗体とともにブロットを一晩４℃でインキュベートし、続いて例えば強化化学発光法（NENまたはAmersham）によって可視化する。
【００５２】
小胞の調製：１００ｍＭトリス−Ｃｌ（ｐＨ９.４）中の１０ｍＭジチオスレイトール（ＤＴＴ）で酵母を処理し、細菌で発現させたリコンビナントリチカーゼにより細胞壁を消化することによってスフェロプラストを作製する。細胞質膜をコンカナバリンＡで架橋して、その密度を分泌小胞の密度より高くする。スフェロプラストを５,６−カルボキシフルオレセイン（ＣＦ）（７.５ｍｇ／ｍＬ）を含む溶解緩衝液（０.８Ｍソルビトール、１０ｍＭトリエタノールアミン、１ｍＭのＥＤＴＡ、ｐＨ７.２）で溶解し、未溶解細胞およびコンカナバリン架橋細胞質膜をソーバルＧＳＡローターで１１０００ｒｐｍ（２０,０００ｇ）１０分、４℃でペレットにする。上清から小胞をペレットにし、さらにソーバルＴＨ−６４１スィングバケットローターで１時間４℃で遠心する（２９０００ｒｐｍ、１４４０００ｇ）ことによって洗浄し小胞外ＣＦを除去する。
【００５３】
水の透過性：溶解中にＣＦをロードされた酵母小胞の完全性は、その蛍光の強さを分光蛍光測定器（例えばＳＬＭ−ＡＭＩＮＣＯ ＳＰＦ−５００Ｃ）で最初に測定することによって実証される。続いて小胞外ＣＦを抗ＣＦ抗体の添加によってクエンチし、浸透圧上昇剤（osmoficant）としてシュクロースを用いた高浸透圧溶液の連続添加によって小胞を収縮させる。酵母小胞を液流停止（stopped-flow）装置（例えばApplied Photophysics SF7mv）にロードする。小胞に対して急激に浸透圧を二倍すると小胞は縮み、ＣＦはセルフクエンチする。浸透圧膜の水の透過性は、酵母小胞の初期縮み速度から計算される。
【００５４】
前記アッセイは、縮みおよびＣＦのセルフクエンチによる蛍光の減少を惹起させるようにデザインされる。蛍光の減少を測定し、容積の減少に対して修正を実施する。蛍光は、４９０±１ｎｍの入射光および５１０ｎｍを越える波長で放出される光を測定するカットオンフィルターを用いて測定される。
【００５５】
参考文献
Agre P et al. 1998. The aquaporins, blueprints for cellular plumbing systems. J. Biol. Chem. 273,14659-14662.
Bai C et al. 1999. Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice. J. Clin. Invest. 103,555-561.
Brant SR. 1998. American families with Crohn's diseas have strong evidence for linkage to chromosome 16 but not chromosome 12. Gastroenterlogy 115: 1056-1061.
Cho JH et al. 1998. Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: evidence for epistasis between 1p and IBD1. Proc. Nat. Acad. Sci. 95: 7502-7507.
Cho JH et al. 2000. Linkage and linkage disequilibrium in chromosome band 1p36 in American Chaldeans with inflammatory bowel disease. Hum. Molec. Genet. 9: 1425-1432.
Coury LA et al. 1998. Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles. Am J Physiol. Renal Physiol. Vol. 274, F34-F42.
Curran ME et al. 1998. Genetic analysis of inflammatory bowel disease in a large European cohort supports linkage to chromosomes 12 and 16. Gastroenterology 115: 1066-1071．
Deen PM et al. 1994. Requirement of human renal water channel aquaporin 2 for vasopressindependent concentration of urine. Science 264,92-95.
Duerr RH et al. 1998. Linkage and association between inflammatory bowel disease and a locus on chromosome 12. Am. J. Hum. Genet. 63: 95-100.
Echevarria M and Ilundain AA. 1998. Aquaporins. J Physiol Biochem 54: 107-18.
Farinas J et al. 1995. Cell volume measured in adherent cells by total internal reflection microfluorimetry: application to permeability in cells transfected with water channel homologs. Biophys. J. 68: 1613-1620.
Farinas J and Verkman AS. 1996. Measurement of cell volume and water permeability in epithelial cell layers by interferometry. Biophys. J. 71: 3511-3522.
Farinas J et al. 1997. Plasma membrane water permeability of cultured cells and epithelia measured by light microscopy with spatial filtering. J. Gen. Physiol. 110:283-296.
Fischer H et al. 2001. Differential expression of Aquaporin 8 in human colonic epithelial cells and colorectal tumors. BMC Physiol. 1 (1): 1.
Frokiaer J et al. 1996. Bilateral ureteral obstruction downregulates expression of vasopressinsensitive AQP-2 water channel in rat kidney. Am J Physiol 270: F657-68.
Fujita N et al. 1995. Role of water channel AQP-CD in water retention in SIADH and cirrhotic rats. Am. J. Physiol., 269:F926-F931..
Garcia F et al. 2001. The water channel Aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cAMP. J. Biol. Chem. 276, 12147-12152.
Hall J and Nanthakumar E. 1997. Automated flourescent geno-typing. In: Boyle AL (ed) Current protocols in human genetics. Vol 2. John Wiley & Sons, pp 2.8.1-2.8.19.
Hampe J et al.1999a. A genomewide analysis provides evidence for novel linkages in Inflammatory Bowel Disease in a large European cohort. Am. J. Hum. Genet. 64:808-816.1.
Hampe J et al.1999b. Linkage of inflammatory bowel disease to human chromosome 6p. Am. J. Hum. Genet. 65,1647-1655.
Hugot JP et al. 1996 Mapping of a susceptibility locus for Crohn's disease on chromosome 16. Nature 379,821-823.
Idury RM and Cardon LR. 1997. A simple method for automated allele binning in microsatellite markers. Genome Res 7:1104-1109.
King LS et al. 2000. Aquaporins in health and disease. Mol Med Today 6: 2 60-5.
Koyama Y et al. 1997. Molecular cloning of a new aquaporin from rat pancreas and liver. J. Biol. Chem. 272,30329-30333.
Koyama N et al. 1998. Cloning and functional expression of human aquaporin8 cDNA and analysis of its gene. Genomics 54: 169-72.
Koyama Y et al. 1999. Expression and localization of aquaporins in rat gastrointestinal tract. American Journal of Physiology. 276 (3 Pt 1): C621-7.
Laize V et al. 1995. Functional expression of the human CHIP28 water channel in a yeast secretory mutant. FEBS Lett. 373: 269-274.
Lennard-Jones JE. 1989. Classification of inflammatory bowel disease. Scand J Gastroenterol Suppl 170: 2-6.
Ma T et al. 1993. Localization and functional analysis of CHIP28k water channels in stably transfected CHO cells. J. Biol. Chem. 268: 22756-22764.
Ma T et al. 1997a. Cloning af a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver and heart. Biochem. Biophys. Res. Commun. 240, 324-328.
Ma T et al. 1997b. Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4. J. Clin. Invest., 100: 957-962.
Ma T et al. 1998. Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels. J. Biol. Chem. 273,4296-4299.
Ma T and Verkman AS. 1999. Aquaporin water channels in gastrointestinal physiology. J Physiol 517: 317-26.
Ma T et al. 1999. Defective secretion of saliva in transgenic mice lacking aquaporin-5 channels. J Biol Chem 274: 20071-20074.
Ma T et al. 2000. Nephrogenic diabetes insipidus in mice lacking aquaporin-3 water channels. PNAS 97,4386-4391.
Marples D et al. 1995a. Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla. J. Clin. Invest, 95: 1838-1845.
Marples D et al. 1995b. Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex. J. Clin. Invest., 97: 1960-1968.
Martin ER. 2000. A test for linkage and association in general pedigrees: the pedigree disequilibrium test. Am J Hum Genet 67: 146-154.
Mathai JC et al. 1996. Functional analysis of aquaporin-1 deficient red cells. J. Biol. Chem. 271,1309-1313.
Matise TC et al. 1994. Automated con-struction of genetic linkage maps using an expert system (MultiMap): a human genome linkage map. Nat Genet 6: 384-390.
Nielsen S et al. 1997. Congestive heart failure in rats is associated with increased expression and targeting of aquaporin-2 water channel in collecting duct. Proc Natl Acad Sci U S A. 13 ; 94 (10): 5450-5.
Ohara M et al. 1998. Upregulation of aquaporin-2 water channel expression in pregnant rats. J. Clin. Invest.,101 : 1076-1083.
Parkes M et al. 2000. The IBD2 locus shows linkage heterogeneity between ulcerative colitis and Crohn disease. Am. J. Hum. Genet. 67: 1605-1610.
Podolsky DK. 1991. Inflammatory bowel disease. N Engl J Med 325:928-937.
Potenza M et al. 1992. SEC6 encodes an 85 kD soluble protein required for exocytosis in yeast. Yeast 8: 549-558.
Preston GM et al. 1994. Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels. Science 265,1585-1587.
Sambrook J et al. 1989. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shinbo I et al. 1999. Functional analysis of aquaporin-2 mutants associated with nephrogenic diabetes insipidus by yeast expression. Am J Physiol. Renal Physiol. 277,734-741.
Simmons CP et al. 2001. Immunomodulation using bacterial enterotoxins. Scand J Immunol 53 (3),218-226.
Steinfeld S et al. 2001. Abnormal Distribution of Aquaporin-5 Water Channel Protein in Salivary Glands from Sjogren's Syndrome Patients. Lab Invest 81:143-148.
Terwilliger J. 1995. A powerful likelihood method for the analysis of linkage disequilibrium between trait loci and one or more polymorphic marker loci. Am J Hum Genet 56: 777-787.
Van Hoek AN and Verkman AS. 1992. Functional reconstitution of the isolated erythrocyte water channel CHIP28. J. Biol. Chem. 267:18267-18269.
Verkman AS and Mitra AK. 2000. Structure and function of aquaporin water channels. Am J Physiol. Renal. Physiol. 278, F13-28.
Wang KS et al. 2000. Colon water transport in transgenic mice lackin aquaporin-4 water channels. Am J Physiol. Gastrointest. Liver physiol. 279, G463-G470.
Xu DL et al. 1997. Upregulation of aquaporin-2 water channel expression in chronic heart failure rat. J. Clin. Invest., 99: 1500-1505.
Yasui M et al. 1999. Rapid gating and anion permeability of an intracellular aqua-porin. Nature 402, 184-187.
Zeidel ML et al. 1992. Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein. Biochemistry 31: 7436-7440.
Zhang R et al. 1990. Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes. J. Biol. Chem. 265: 15375-15378.
【図面の簡単な説明】
【００５６】
【図１】染色体領域マップを示す。【Technical field】
[0001]
The present invention relates to a method for identifying a compound useful for treating IBD, a method for producing a pharmaceutical composition, a diagnostic method for measuring susceptibility to IBD, a method for diagnosing IBD or a predisposition thereof, and regulating the activity or amount of AQP8. Provided for the manufacture of a medicament for the treatment of IBD.
[Background Art]
[0002]
IBD is characterized by chronic recurrent intestinal inflammation of the gastrointestinal tract. IBD affects about 1 in 1000 people in Western countries and has an average age of onset in early adulthood. To date, the etiology of the disease is unknown. Based on clinical and histopathological characteristics, IBD is divided into two main subtypes: Crohn's disease (CD) (“On Line Mendelian Inheritance in Man”) OMIM 266600. And ulcerative colitis (UC) (OMIM 191390). Although the cause of IBD is unknown, the familial outbreak of the disease and increased concordance in identical twins indicate strong genetic susceptibility. Estimates of the risk for intrabrotherhood (λs) range from 10 to 50, suggesting that genetic factors play an important role in IBD predisposition. Epidemiological data suggests that IBD is a multifactorial disorder caused by many susceptibility genes as well as environmental factors. In the present invention, the term IBD is intended to include IBD in addition to Crohn's disease and ulcerative colitis.
[0003]
Earlier genome-wide linkage analyzes revealed a number of susceptibility loci for IBD, such as IBD1 (OMIM 266600) (Hugot et al. 1996; Brant et al. 1998; Curran et al. 1998; Hampe et al. 1999a), IBD2 ( OMIM 601458) (Duerr et al. 1998; Parkes et al. 2000), IBD3 (OMIM 604519) (Hampe et al. 1999b), and IBD7 (OMIM 605225) (Cho et al. 1998; Cho et al. 2000) Was done. The IBD1 locus is located in the pericentromere region of human chromosome 16 and maps to the 16p12-q13 region.
[0004]
AQP8 belongs to the group of water channel proteins (aquaporins). Aquaporins (AQPs) are members of the major intrinsic superfamily that make up essential membrane proteins and function as special channels that facilitate cell membrane water passage in animals, plants and bacteria. So far, ten AQP homologs, numbered 0-9, have been cloned in mammals. They are widely distributed and more than one AQP can be present in the same cell (Echevarria and Ilundain, 1998). Several human AQP genes have been cloned and many disorders have been found to be associated with dysfunction of these proteins (Echevarria and Ilundain, 1998).
[0005]
First, the cDNA encoding mouse AQP8 was cloned by Ma et al. (1997a). Northern blot analysis showed expression in a number of tissues, including placenta, colon, liver, heart, pancreas, lung, kidney, testis, spleen, stomach and brain.
[0006]
The cDNA encoding human AQP8 was cloned in 1998 and shown to be important for cell fluid transport (Koyama et al. 1998). Expression of the gene was shown in human pancreatic and colonic tissues by Northern blot (Koyama et al. 1998).
[0007]
Koyama et al. (1999) show that in rats AQP8 is expressed in absorbable columnar cells of the jejunum, proximal and distal colon, and that AQP8 uptakes water in the gastrointestinal tract. And / or have a role in emissions. In mice, AQP3, AQP4 and AQP8 are expressed on the colonic epithelial surface, suggesting a joint role for these aquaporins in fluid transport (Ma and Verkman 1999).
[0008]
The expression, non-cellular localization and regulation of AQP8 in rat hepatocytes was examined in more detail by Garcia et al. (2001). More recently, the expression of AQP8 in the human colon has been demonstrated by Fischer et al. (2001) and has been proposed to be a marker for normally proliferating colon epithelial cells.
[0009]
Aquaporins are small, membrane-wide, highly hydrophobic intrinsic membrane proteins (monomer size about 30 kDa) that are expressed on the cytoplasmic membrane of many cell types required for fluid transport. The basic features of the structure of aquaporins were revealed using mutagenesis, epitope addition, and spectroscopy and freeze-fracturing electron microscopy. Aquaporins appear to assemble as homotetramers in the membrane. In this homotetramer, each monomer (consisting of six membrane spanning α-helical domains with the amino and carboxy termini facing the cytoplasm) contains distinct water pores.
[0010]
The structure and function of aquaporins have been reviewed by Verkman and Mitra (2000) and King et al. (2000).
Humans with a rare Colton-null phenotype have a deficiency in AQP1, but do not show a clear clinical phenotype (Preston et al 1994) and only reduced urine concentration (Mathai et al.). al. 1996). In contrast, AQP1 knockout mice showed marked urinary dysfunction (Ma et al. 1998). Similarly, AQP3 knockout mice develop renal diabetes insipidus, which indicates severe dysfunction of the urinary concentration mechanism (Ma et al. 2000).
[0011]
Mutations in AQP2 cause a rare hereditary diabetes insipidus with a dramatic clinical phenotype (NDI) (Deen et al. 1994). NDI is a disease of which etiology is due to renal resistance to vasopressin, the clinical feature of which is the excretion of large amounts of dilute urine. This also indicates that the expression and / or functional state of aquaporin under certain circumstances is under the influence of hormones. Patients with autosomal recessive NDI with a mutation in the AQP2 gene provide the first clear example that aquaporin may be the rate-limiting factor for water transport. Acquired NDI is more common than genotype and has a variety of causes. Lithium (Marples et al. 1995a), bilateral ureteral obstruction (Fr [phi] kiaer 1996) and chronic hypokalemia (Marples et al. 1995b), which are responsible for known NDI, all have significant AQP2 expression in animals. It causes a decrease and at the same time reduces the ability to concentrate urine. On the other side of the water imbalance spectrum, increased AQP2 expression is associated with congestive heart failure (Nielsen et al. 1997; Xu et al. 1997), cirrhosis (Fujita et al. 1995) and pregnancy (Ohara et al. 1998).
[0012]
Disruption of the AQP4 gene in mice resulted in moderate urine concentration dysfunction (Ma et al. 1997b), but had little or no effect on colonic drainage or fecal dehydration (Ma et al. 1997b). Wang et al. 2000).
[0013]
Lung fluid transport in AQP1 and AQP4 knockout mice was examined by Bai et al. (1999). The absorption rate of isotonic alveolar fluid was not significantly different in AQP1 knockout mice compared to wild-type mice, despite a 10-fold reduction in air-capillary water permeability. There was little effect of the deficiency of the airway water channel AQP4.
[0014]
Ma et al. (1999) reported a decrease in salivary secretion in transgenic mice lacking AQP5, suggesting a physiological relevance of AQP5 in salivary gland function. Steinfeld et al. (2001) report the localization of AQP5 in human salivary glands and show an abnormal distribution of AQP5 in salivary glands of patients with Sjogren's syndrome, which is the cause of decreased salivary secretion in patients with Sjogren's syndrome. Will.
[0015]
Currently, two functional groups of mammalian aquaporins are recognized (Agre et al. 1998). The first group (including AQP0, AQP1, AQP2, AQP4 and AQP5) is only permeable to water as classically defined. The second group (including AQP3, AQP7 and AQP9) has strong permeability to water but also penetrates glycerol and other small molecules. The structural explanation and physiological relevance of these differences are unknown. The sequence of AQP6 is similar to the water-selective group, but recent surprising observations of gated anion conductance indicate that functional differences are more complex than previously thought. (Yasui et al. 1999). The sequence of AQP8 is intermediate between the water-selective and glycerol-permeable groups, and a functional definition has been reserved (Koyama et al. 1997). Thus, aquaporins (including AQP8) will play an important role in gate control in the intestines of water and possibly other small molecules in the intestine and other tissues, which are thought to have high nearby cell permeability.
[0016]
The present invention is based on the discovery of a genetic predisposition to IBD associated with a genetic marker located in a region on chromosome 16 very close to the location of the AQP8 gene. The use of a compound capable of modulating the activity or amount of AQP8 has been identified as a novel therapeutic concept for treating IBD according to the present invention. Sequence analysis of the AQP8 gene will provide a novel diagnostic method of susceptibility to IBD.
DISCLOSURE OF THE INVENTION
[0017]
According to one aspect of the present invention, there is provided the use of a compound capable of modulating the activity or amount of AQP8 for the treatment of IBD. Modulation of the amount of AQP8 by a compound can be achieved, for example, by altering gene expression levels or message stability. Modulation of AQP8 activity by a compound can be achieved, for example, by binding the compound to an AQP8 protein. In certain embodiments, modulation of AQP8 comprises a compound capable of decreasing the activity or amount of AQP8. In another embodiment, modulation of AQP8 comprises a compound capable of increasing the activity or amount of AQP8.
[0018]
An example of a compound that can modulate the activity of AQP8 is an antibody. Antibodies can be prepared using any suitable method. For example, specific antibodies can be prepared using purified polypeptides. The term "antibody" refers to polyclonal antibodies, monoclonal antibodies and various types of antibody constructs, such as F (ab ')Two, Fab and single-chain Fv. About 10 antibodies7M-1Greater than or equal to KaIs defined as specifically binding when it binds to an allelic variant of SLC10A2. Binding affinity can be determined by conventional techniques (eg, Scatchard et al., Ann. N.Y. Acad. Sci.,51: 660 (1949)).
[0019]
Polyclonal antibodies can be readily produced from a variety of sources (eg, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats) using methods well known in the art. Generally, the antigen is administered to the host animal, typically by intraperitoneal injection. The immunogenicity of an antigen can be increased by using an adjuvant (eg, Freund's complete or incomplete adjuvant). After the booster immunization, a small serum sample is collected and tested for reactivity to the antigen. Examples of various assays useful for such measurements include those described in the following literature (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988), Methods such as, for example, cross immunoelectrophoresis (CIEP), radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot assays and sandwich assays (see US Pat. Nos. 4,376,110 and 4,486,530). included.
[0020]
Monoclonal antibodies can be readily produced using well-known methods. See, for example, the methods described in the following references: U.S. Patents RE32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn and Bechtol (eds.), (1980).
[0021]
The monoclonal antibody of the present invention can be prepared using other techniques such as the technique described by Alting-Mees et al. (“Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas”, Strategies in Molecular Biology).Three1-9 (1990), which is hereby incorporated by reference). Similarly, binding partners can be constructed using recombinant DNA technology to incorporate the variable region of a gene encoding a specific binding antibody. Such techniques are described by Larrick et al. (Biotechnology,7: 394 (1989)).
Once isolated and purified, the antibody can be used to detect the presence of the antigen by established assay protocols (see, eg, “A Practical Guide to ELISA”, DM Kemeny, Pergamon Press). , Oxford, England).
[0022]
According to another aspect of the present invention there is provided the use of a compound capable of modulating the activity or amount of AQP8 in the manufacture of a medicament for the treatment of IBD.
[0023]
In accordance with yet another aspect of the present invention, there is provided a method of identifying a compound that is potentially useful for the treatment of IBD, the method comprising: determining the compound's ability to modulate the activity or amount of AQP8. Assaying. Preferably, the assay is selected from:
i) measuring AQP8 activity using a cell line expressing AQP8 or using purified AQP8 protein; and
ii) Measurement of AQP8 transcription or translation in AQP8 expressing cell lines.
[0024]
The assay used to determine the effect of a test compound on AQP8 activity can be accomplished by measuring AQP8-mediated water transport. Aquaporin-mediated water transport is generally measured using an osmotic swelling assay in Xenopus oocytes expressing aquaporin cRNA. As described initially (Zhang et la. 1990), using quantitative image analysis, 200 to 0-100 mosmol / kg HTwoThe time course of egg cell swelling is estimated in response to a sudden drop in the osmotic pressure of the extracellular solution to O. Water transport in mammalian cells transfected with aquaporins has also been investigated by a variety of biophysical approaches, including: stopped-flow light scattering (Ma et al. 1993). ), Total internal reflection fluorescence microscopy (Farinas et al. 1995), laser interferometry (Farinas and Verkman 1996), and Fourier optical dark-field microscopy / phase contrast microscopy. (Farinas et al. 1997). Flow-stop light scattering was also used to assay aquaporin-mediated water transport in yeast-derived vesicles containing purified aquaporin protein (Laize et al. 1995) and reconstituted proteoliposomes (Van Hoek and Verkman, 1992). .
[0025]
Assays used to determine the effect of a test compound on AQP8 transcription or translation can be based on:
i) determination of the amount of generated AQP8 mRNA using, for example, Northern blot analysis or quantitative real-time PCR;
ii) determination of the amount of produced AQP8 protein, for example using Western blot analysis or immunochemical analysis (eg, ELISA); or
iii) Measurement of AQP8 activity in AQP8 expressing cells as described above.
[0026]
Cells used in the assay can be cells that naturally express AQP8 or transfected cells that express recombinant AQP8. Preferably, the AQP8 is a human recombinant AQP8.
[0027]
AQP8 can be expressed in a variety of hosts, such as bacteria, plant cells, insect cells, fungal cells, and human and animal cells. Eukaryotic recombinant host cells are particularly preferred. Examples include yeast, mammalian cells (including cell lines of human, bovine, porcine, monkey and rodent origin), and insect cells (including cell lines derived from Drosophila and silkworm). Commercially available cell lines derived from mammalian species include L-cell LM (TK-) (ATCC CCL 1.3), L-cell LM (ATCC CCL 1.2), HEK293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH / 3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
[0028]
An expression vector containing a nucleic acid encoding AQP8 can be introduced into a host cell to express a polypeptide of the invention using any of a number of methods, including: transformation with calcium phosphate, transformation with DEAE-dextran. Conversion, cationic lipid-mediated lipofectin, electroporation or infection.
[0029]
The transfected host cells are grown, cloned, eg, by limiting dilution, and analyzed to determine the expression level of recombinant AQP8. Identification of transformed host cells expressing AQP8 can be accomplished by several means, including detection of immunological reactivity with the antibody and / or biological activity by the assays described herein.
Recombinant human AQP1 or AQP2 was expressed in yeast and found to be localized in secretory vesicles. Secretory vesicles containing AQP1 and AQP2 exhibited high water permeability and low activation energy for water flow, suggesting the expression of functional AQP1 and AQP2 (Coury et al. 1998). The function of AQP2 variants was examined using expression in yeast and subsequent measurement of water permeability of isolated yeast vesicles (Shinbo et al. 1999).
[0030]
Regulation of transcription of gene expression governs the binding of transcription factors and is thus mediated by specific DNA elements in the promoter that mediate transcription of the gene. Eukaryotic transcription factors can be divided into two main groups: i) a basal transcription factor that interacts with a promoter sequence proximal to the transcription initiation site, thereby initiating transcription when RNA polymerase II is recruited; and ii) binds to a particular distal promoter element and thereby A transcription factor that mediates transcription when in contact with the transcription machinery. The fundamental physiological process in eukaryotes is that cells can communicate with their environment and respond to extracellular stimuli via signaling molecules (eg, hormones and growth factors). The final event for such signaling is the binding of certain distal promoter elements to transcription factors, which results in, for example, up-regulation of gene expression or expression of tissue-specific genes. Because of their regulatory role, promoter elements are putative targets for therapeutic drug screening. The human AQP8 promoter sequence (disclosed in the present application) allows for the screening of therapeutic agents that selectively regulate AQP8 transcription.
[0031]
Suitable host cells are cells that are known to express AQP8, or cells that are known to express transcription factors that can affect the transcription of AQP8. Preferably, the interaction between a specific transcription factor and the AQP8 promoter can be examined using a host cell transfected with a DNA encoding a specific transcription factor.
[0032]
Assays used to determine the effect of a test compound on AQP8 transcription can also be based on measuring the activity of the AQP8 promoter using a reporter gene system. The reporter gene system is an expression system containing a nucleic acid molecule constituting the AQP8 promoter or a fragment thereof, and the expression system further contains a reporter gene. These promoter and reporter gene are arranged such that the expression of the reporter gene is regulated by the AQP8 promoter. The amount of reporter protein produced is used as an indicator of AQP8 promoter activity.
[0033]
Preferably the AQP8 promoter is a human AQP8 promoter, preferably the nucleotide sequence 1-1956 of SEQ ID NO: 1, or a fragment thereof.
Suitable reporter genes that can be used to construct the reporter gene system include, for example, firefly luciferase gene, bacterial chloramphenicol acetyltransferase (CAT) gene, β-galactosidase (β-GAL) gene, and green fluorescent protein ( GFP).
[0034]
According to another aspect of the present invention, there is provided a method of making a pharmaceutical composition comprising the steps of:
i) identifying a compound useful for treating IBD according to the methods described herein;
ii) mixing the compound or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient or diluent.
[0035]
In yet another aspect of the invention, a diagnostic method is provided. The method comprises analyzing a sequence of chromosome 16 or a portion thereof in a DNA sample obtained from the patient for determination of susceptibility to IBD, and further comprising BAC 504N19 (EMBL AF265340), BAC A-249B10 (EMBL AC002288). Alternatively, the method includes determining the presence or absence of an allele of a polymorphic marker present in a chromosome fragment corresponding to BAC CTD-2547G23 (EMBL AC008741). Preferably, the method comprises analyzing the sequence of the AQP8 gene or a portion thereof in a DNA sample obtained from the patient for determination of susceptibility to IBD, and further comprising the steps of: Determining the presence or absence of the allele.
[0036]
In another aspect of the invention, there is provided a method of modulating an immune response to an autoimmune epitope on an AQP8 protein. Preferably, the method comprises a method of inducing immunological resistance. Immunological resistance is induced, for example, by oral immunization with the B subunit of cholera toxin (CT) from V. cholera or a thermolabile toxin (LT) from E. coli conjugated to a self antigen. (Simmons et al. 2001). Autoimmune reactions have been found to be responsible for a number of inflammatory diseases, such as rheumatoid arthritis, pancreatitis and multiple sclerosis.
[0037]
In another aspect of the invention, there is provided a method of diagnosing IBD, comprising measuring an autoantibody directed against the AQP8 protein.
[0038]
Abbreviation
Aquaporin: AQP
Aquaporin-8: AQP8
Affected brother pair: ASP
Bacterial artificial chromosome: BAC
Chloramphenicol acetyltransferase: CAT
Crohn's disease: CD
Centi Morgan: CM
Cholera toxin: CT
Glycosaminoglycan: GAG
Green fluorescent protein: GFP
Inflammatory Bowel Disease: IBD
Heat-labile toxin: LT
Renal diabetes insipidus: NDI
Online Human Mendelian Genetics: OMIM
Pedigree disequilibrium test: PDT
Sequence addition site: STS
Transmission imbalance test: TDT
Ulcerative colitis: UC
[0039]
The invention will now be described by way of the following non-limiting examples. FIG. 1 shows the chromosome region map of interest in this example.
AMPLITAQ® (available from Perkin-Elmer Cetus) is used as a source of thermostable DNA polymerase.
General molecular biology methods can be followed from any of the methods described in the following references: "Molecular Cloning-A Laboratory Manual", Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989) or “Current Protocols in Molecular Biology Vol. 1-3, FM Asubel, R. Brent and RE Kingston (eds), published by John Wiley, 1998.
Embodiment 1
[0040]
We used high-density genotyping with STS markers and an unpublished single nucleotide polymorphism, followed by sensitivity to IBD1 according to standard procedures of linkage disequilibrium testing (Terwillinger, 1995). The range of the region containing the gene was limited to a physical distance of about 7 Mb. TDT testing across the area adjacent to the STS markers, D16S401 and D16S409, respectively, confirms the first linkage data (using a total of 32 markers (13 STSs and 19 SNPs)), plus linkage distortion at two sites. Was revealed. Our data indicated that the susceptibility gene for IBD1 is probably located in a region adjacent to D16S401 and D16S409.
[0041]
1 . 1. Family identification and phenotype:
Patients were recruited from the IBD program at the following institutions: Charite University Hospital (Berlin) and the Ist Department of Medicine at the Christian-Albrechts-University of Kiel. Children with two or more affected siblings diagnosed with IBD were sampled and over 250 affected sibling pairs were included in the analysis. Written informed consent was obtained from all study participants. The recruitment protocol was approved by the in-house audit committee. Diagnosis of IBD and classification on CD and UC was determined by standard diagnostic criteria described by Lennard-Jones (1989) and Podolsky (1991). Patients were consulted directly by one or more principal investigators. Alternatively, two records containing detailed medical history and results of all diagnostic procedures were obtained for each patient and reviewed by the principal investigator. Venous blood samples were obtained from affected siblings and, where possible, their parents.
[0042]
1 . 2. Genotyping:
Genomic DNA was prepared from whole blood samples using the Puregene System (Gentra Systems). Individual DNA samples were aligned in a 96-well microtiter plate and PCR amplification was performed using individual marker amplicons. Microsatellite markers were genotyped by fluorescence as described in the literature (Hall and Nanthakumar (1997)). Data was collected using an ABI 377 automated sequencer and data analysis was performed using GENESCAN (version 2.1) and GENOTTYPER (version 1.1.1). Allele analysis and individual allele calling were performed as described by Hall and Nanthakumar (1997) and Idury and Cardon (1997). SNPs were identified by genomic sequencing using the ABI 3700 automatic sequencer, cDNA selection and mutation detection methods. Genotyping of informative SNPs was performed by real-time PCR using TAQMAN technology (PE Biosystems). PCR reactions were performed on an ABI 9700 and fluorescence results were determined using ABI 7700 sequence-detector single point measurements. Allele analysis and individual allele calling were performed as described by Hall and Nanthakumar (1997). Genetic maps were constructed using the automatic mapping program MULTIMAP (version 2.0, Matise et al. 1994). The resulting distance between markers (determined by the Kosambi map algorithm) is given in centiMorgans (cM).
[0043]
1 . 3 Genetic analysis:
Genetic analysis was performed using the two standard diagnostic categories described above, CD and UC. The third category, ALL, includes CD / CD, UC / UC and CD / UC (mixed ASP), thus representing IBD as a single phenotype for analysis. Allele frequencies for each marker were calculated from cohort genotyping data for all individuals. Relevant statistics were calculated by the TDTLIKE program of the ANALYZE software package (Terwilliger 1995). Alleles tested are limited to transmissions observed in more than 10 individuals. The program algorithm provides a modified p-value for multiple allele testing.
[0044]
Sequence information on markers (1STS, 3SNP) in the region between D16S401 and D16S409
TDT results for positive markers on 16p of 200 affected sib pairs (ASP) of IBD
a) TDTLIKE (Terwilliger, 1995)
[Table 1]
b) PDT (Martin et al. 2000)
[Table 2]
[0047]
1 . 4. Bioinformatics:
Three human BACs derived from chromosome 16; RP11-451N20 (EMBL AC060785), 504N19 (EMBL AF265340), and A-249B10 (EMBL AC002288) containing marker D16S3068, BAC CTD-2547G23 (EMBL AC008741) (partially BAC A-249B10) and analyzed for potential disease-related genes. A sequence consistent with the AQP8 cDNA was found in BAC CTD-2547G23 (EMBL AC008741).
Further analysis of the sequence of this BAC (available under EMBL under accession number AC008741), including 34 unordered fragments, revealed that one fragment contained the AQP8 gene, including the 1.9 kb promoter region. .
[0048]
1 . 5 Identification of ANP8 gene SNP:
PCR primers are constructed based on the DNA sequence adjacent to the exon together with the promoter region of the AQP8 gene. After PCR amplification, the sequences of exon and promoter regions from genomic DNA samples obtained from 23 IBD affected individuals and 20 normal controls are determined. Sequence variants present in more than 10% of the individuals are identified and selected for genotyping the remaining SNPs of the cohort using the Taqman® technique described above.
[0049]
1 . 6. Method for diagnosing IBD susceptibility in a patient:
Obtain a DNA sample from the patient. The presence or absence of an allele of one or more polymorphic markers associated with increased susceptibility to IBD is determined, for example, using the Tackman® technique.
[0050]
1 . 7 AQP8 assay:
AQP8 activity is measured as the water permeability of secretory vesicles of yeast derived from yeast expressing recombinant AQP8.
Construction of yeast strains and plasmids: The cDNA encoding AQP8 (SEQ ID NO: 1) is cloned into the yeast expression vector pYES2 (Invitrogen, San Diego, CA) by standard techniques (Sambrook et al. 1989). The yeast strain SY1 (Potenza et al. 1992) is transformed by electroporation using, for example, a Bio-Rad gene pulser (setting at 1.5 kV, 25 μF at 200 ohm). The transformed yeast is grown and further maintained in a defined medium lacking uracil and containing raffinose as a carbon source. The yeast was converted to a nutrient-rich yeast extract / peptone (YEP) -galactose medium (0.5% yeast extract (w / v), 1.0% bactopeptone (w / v) and 2.0% galactose (w / v). Transfer to v)) at 25 ° C for 2-4 hours to initiate protein expression, followed by switching to 37 ° C overnight to accumulate secretory vesicles. Control experiments are performed with vesicles prepared from yeast transformed with the background strain or the pYES2 vector lacking the AQP8 insert.
[0051]
Immunoblotting: Incubate the samples at 60-80 ° C for 5 minutes, followed by separation by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis. In the electrophoresis, a 4-20% continuous gradient of Tris (hydroxymethyl) aminomethane (Tris) -Cl-glycine ready gel (Ready Gel) or 12% SDS-polyacrylamide slab was used, and nitrocellulose (Bio-Rad) was used. ). Blots are blocked with blot buffer consisting of 1% milk powder and 3% Tween 20 in phosphate buffered saline (pH 7.25). Blots are incubated overnight at 4 ° C. with anti-AQP8 antibody and subsequently visualized, for example, by enhanced chemiluminescence (NEN or Amersham).
[0052]
Preparation of vesiclesSpheroplasts are made by treating yeast with 10 mM dithiothreitol (DTT) in 100 mM Tris-Cl (pH 9.4) and digesting the cell wall with bacterially expressed recombinant triticase. The cytoplasmic membrane is cross-linked with concanavalin A to increase its density above that of secretory vesicles. Spheroplasts were dissolved in a lysis buffer (0.8 M sorbitol, 10 mM triethanolamine, 1 mM EDTA, pH 7.2) containing 5,6-carboxyfluorescein (CF) (7.5 mg / mL), and were not dissolved The cells and the concanavalin crosslinked cytoplasmic membrane are pelleted at 11000 rpm (20,000 g) for 10 minutes at 4 ° C. with a Soval GSA rotor. The vesicles are pelleted from the supernatant and further washed by centrifugation (29000 rpm, 144000 g) at 4 ° C. for 1 hour in a Soval TH-641 swinging bucket rotor to remove extra-vesicular CF.
[0053]
Water permeability: The integrity of the yeast vesicles loaded with CF during lysis is demonstrated by first measuring its fluorescence intensity on a spectrofluorometer (eg SLM-AMINCO SPF-500C). Subsequently, extravesicular CF is quenched by the addition of an anti-CF antibody, and the vesicles are contracted by continuous addition of a hyperosmolar solution using sucrose as an osmoficant. The yeast vesicles are loaded into a stopped-flow device (eg, Applied Photophysics SF7mv). When the osmotic pressure is rapidly doubled for the vesicles, the vesicles shrink and the CF self-quenches. The water permeability of the osmotic membrane is calculated from the initial shrinkage rate of the yeast vesicles.
[0054]
The assay is designed to cause a reduction in fluorescence due to shrinkage and self-quenching of CF. Measure the decrease in fluorescence and make corrections for the decrease in volume. Fluorescence is measured using a cut-on filter that measures incident light at 490 ± 1 nm and light emitted at wavelengths above 510 nm.
[0055]
References
Agre P et al. 1998. The aquaporins, blueprints for cellular plumbing systems. J. Biol. Chem. 273,14659-14662.
Bai C et al. 1999. Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice. J. Clin. Invest. 103,555-561.
Brant SR. 1998.American families with Crohn's diseas have strong evidence for linkage to chromosome 16 but not chromosome 12.Gastroenterlogy 115: 1056-1061.
Cho JH et al. 1998.Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: evidence for epistasis between 1p and IBD1. Proc. Nat.Acad. Sci. 95: 7502-7507.
Cho JH et al. 2000.Linkage and linkage disequilibrium in chromosome band 1p36 in American Chaldeans with inflammatory bowel disease.Hum.Molec.Genet.9: 1425-1432.
Coury LA et al. 1998.Reconstitution of water channel function of aquaporins 1 and 2 by expression in yeast secretory vesicles. Am J Physiol. Renal Physiol. Vol. 274, F34-F42.
Curran ME et al. 1998. Genetic analysis of inflammatory bowel disease in a large European cohort supports linkage to chromosomes 12 and 16. Gastroenterology 115: 1066-1071.
Deen PM et al. 1994.Requirement of human renal water channel aquaporin 2 for vasopressindependent concentration of urine. Science 264,92-95.
Duerr RH et al. 1998. Linkage and association between inflammatory bowel disease and a locus on chromosome 12. Am. J. Hum. Genet. 63: 95-100.
Echevarria M and Ilundain AA. 1998. Aquaporins. J Physiol Biochem 54: 107-18.
Farinas J et al. 1995.Cell volume measured in adherent cells by total internal reflection microfluorimetry: application to permeability in cells transfected with water channel homologs.Biophys. J. 68: 1613-1620.
Farinas J and Verkman AS. 1996.Measurement of cell volume and water permeability in epithelial cell layers by interferometry.Biophys. J. 71: 3511-3522.
Farinas J et al. 1997. Plasma membrane water permeability of cultured cells and epithelia measured by light microscopy with spatial filtering.J. Gen. Physiol. 110: 283-296.
Fischer H et al. 2001. Differential expression of Aquaporin 8 in human colonic epithelial cells and colorectal tumors. BMC Physiol. 1 (1): 1.
Frokiaer J et al. 1996.Bilateral ureteral obstruction downregulates expression of vasopressinsensitive AQP-2 water channel in rat kidney.Am J Physiol 270: F657-68.
Fujita N et al. 1995. Role of water channel AQP-CD in water retention in SIADH and cirrhotic rats. Am. J. Physiol., 269: F926-F931 ..
Garcia F et al. 2001.The water channel Aquaporin-8 is mainly intracellular in rat hepatocytes, and its plasma membrane insertion is stimulated by cAMP. J. Biol. Chem. 276, 12147-12152.
Hall J and Nanthakumar E. 1997.Automated flourescent geno-typing.In: Boyle AL (ed) Current protocols in human genetics.Vol 2. John Wiley & Sons, pp 2.8.1-2.8.19.
Hampe J et al. 1999a.A genomewide analysis provides evidence for novel linkages in Inflammatory Bowel Disease in a large European cohort. Am. J. Hum. Genet. 64: 808-816.1.
Hampe J et al. 1999b.Linkage of inflammatory bowel disease to human chromosome 6p.Am.J.Hum.Genet. 65,1647-1655.
Hugot JP et al. 1996 Mapping of a susceptibility locus for Crohn's disease on chromosome 16.Nature 379,821-823.
Idury RM and Cardon LR. 1997.A simple method for automated allele binning in microsatellite markers. Genome Res 7: 1104-1109.
King LS et al. 2000. Aquaporins in health and disease.Mol Med Today 6: 2 60-5.
Koyama Y et al. 1997.Molecular cloning of a new aquaporin from rat pancreas and liver.J. Biol. Chem. 272, 30329-30333.
Koyama N et al. 1998. Cloning and functional expression of human aquaporin8 cDNA and analysis of its gene.Genomics 54: 169-72.
Koyama Y et al. 1999.Expression and localization of aquaporins in rat gastrointestinal tract. American Journal of Physiology. 276 (3 Pt 1): C621-7.
Laize V et al. 1995. Functional expression of the human CHIP28 water channel in a yeast secretory mutant.FEBS Lett. 373: 269-274.
Lennard-Jones JE. 1989.Classification of inflammatory bowel disease.Scand J Gastroenterol Suppl 170: 2-6.
Ma T et al. 1993. Localization and functional analysis of CHIP28k water channels in stably transfected CHO cells.J. Biol. Chem. 268: 22756-22764.
Ma T et al. 1997a.Cloning af a novel water and urea-permeable aquaporin from mouse expressed strongly in colon, placenta, liver and heart.Biochem.Biophys.Res.Commun. 240, 324-328.
Ma T et al. 1997b.Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4.J. Clin. Invest., 100: 957-962.
Ma T et al. 1998.Severely impaired urinary concentrating ability in transgenic mice lacking aquaporin-1 water channels.J. Biol. Chem. 273,4296-4299.
Ma T and Verkman AS. 1999. Aquaporin water channels in gastrointestinal physiology. J Physiol 517: 317-26.
Ma T et al. 1999.Defective secretion of saliva in transgenic mice lacking aquaporin-5 channels.J Biol Chem 274: 20071-20074.
Ma T et al. 2000.Nephrogenic diabetes insipidus in mice lacking aquaporin-3 water channels.PNAS 97,4386-4391.
Marples D et al. 1995a.Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla.J. Clin. Invest, 95: 1838-1845.
Marples D et al. 1995b.Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex.J. Clin. Invest., 97: 1960-1968.
Martin ER. 2000.A test for linkage and association in general pedigrees: the pedigree disequilibrium test.Am J Hum Genet 67: 146-154.
Mathai JC et al. 1996. Functional analysis of aquaporin-1 deficient red cells. J. Biol. Chem. 271,1309-1313.
Matise TC et al. 1994.Automated con-struction of genetic linkage maps using an expert system (MultiMap): a human genome linkage map.Nat Genet 6: 384-390.
Nielsen S et al. 1997.Congestive heart failure in rats is associated with increased expression and targeting of aquaporin-2 water channel in collecting duct.Proc Natl Acad Sci U S A. 13; 94 (10): 5450-5.
Ohara M et al. 1998.Upregulation of aquaporin-2 water channel expression in pregnant rats.J. Clin. Invest., 101: 1076-1083.
Parkes M et al. 2000.The IBD2 locus shows linkage heterogeneity between ulcerative colitis and Crohn disease.Am. J. Hum.Genet. 67: 1605-1610.
Podolsky DK. 1991. Inflammatory bowel disease. N Engl J Med 325: 928-937.
Potenza M et al. 1992.SEC6 encodes an 85 kD soluble protein required for exocytosis in yeast.Yeast 8: 549-558.
Preston GM et al. 1994.Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels.Science 265,1585-1587.
Sambrook J et al. 1989. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shinbo I et al. 1999.Functional analysis of aquaporin-2 mutants associated with nephrogenic diabetes insipidus by yeast expression.Am J Physiol. Renal Physiol. 277,734-741.
Simmons CP et al. 2001. Immunomodulation using bacterial enterotoxins. Scand J Immunol 53 (3), 218-226.
Steinfeld S et al. 2001.Abnormal Distribution of Aquaporin-5 Water Channel Protein in Salivary Glands from Sjogren's Syndrome Patients.Lab Invest 81: 143-148.
Terwilliger J. 1995.A powerful likelihood method for the analysis of linkage disequilibrium between trait loci and one or more polymorphic marker loci.Am J Hum Genet 56: 777-787.
Van Hoek AN and Verkman AS. 1992. Functional reconstitution of the isolated erythrocyte water channel CHIP28. J. Biol. Chem. 267: 18267-18269.
Verkman AS and Mitra AK. 2000. Structure and function of aquaporin water channels. Am J Physiol. Renal. Physiol. 278, F13-28.
Wang KS et al. 2000. Colon water transport in transgenic mice lackin aquaporin-4 water channels.Am J Physiol. Gastrointest. Liver physiol. 279, G463-G470.
Xu DL et al. 1997.Upregulation of aquaporin-2 water channel expression in chronic heart failure rat.J. Clin. Invest., 99: 1500-1505.
Yasui M et al. 1999.Rapid gating and anion permeability of an intracellular aqua-porin.Nature 402, 184-187.
Zeidel ML et al. 1992.Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein.Biochemistry 31: 7436-7440.
Zhang R et al. 1990. Expression of mRNA coding for kidney and red cell water channels in Xenopus oocytes. J. Biol. Chem. 265: 15375-15378.
[Brief description of the drawings]
[0056]
FIG. 1 shows a chromosome region map.
Claims (11)
ｉ）ＡＱＰ８を発現する細胞株を用いるか、または精製ＡＱＰ８タンパク質を用いるＡＱＰ８活性の測定；および
ii）ＡＱＰ８発現細胞株におけるＡＱＰ８の転写または翻訳の測定
から選択される請求項１に記載の方法。Assay
i) measuring AQP8 activity using a cell line expressing AQP8 or using purified AQP8 protein; and
ii) The method of claim 1, wherein the method is selected from measuring AQP8 transcription or translation in an AQP8 expressing cell line.
ｉ）ＡＱＰ８ｍＲＮＡの量の測定；
ii）生成されたＡＱＰ８タンパク質の量の測定；または
iii）ＡＱＰ８発現細胞におけるＡＱＰ８活性の測定
のいずれかによって測定される請求項２に記載の方法。Measurement of the transcription or translation of AQP8
i) measuring the amount of AQP8 mRNA;
ii) measuring the amount of AQP8 protein produced; or
3. The method according to claim 2, wherein the method is measured by any one of measurement of AQP8 activity in AQP8-expressing cells.
ｉ）請求項１〜７のいずれか一項に記載された方法にしたがってＩＢＤの治療に有用な化合物を特定し；そして
ii）化合物または医薬的に許容できるその塩を医薬的に許容できる賦形剤または希釈剤と混合する
ことを含む医薬組成物の製造方法。The following steps:
i) identifying a compound useful for treating IBD according to the method of any one of claims 1-7; and
ii) A process for preparing a pharmaceutical composition comprising mixing a compound or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable excipient or diluent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB0111639.1A GB0111639D0 (en) | 2001-05-12 | 2001-05-12 | Methods |
| PCT/GB2002/002134 WO2002093176A2 (en) | 2001-05-12 | 2002-05-08 | Methods for identifying compounds for treatment of ibd |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2004534531A true JP2004534531A (en) | 2004-11-18 |
Family
ID=9914520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2002589805A Pending JP2004534531A (en) | 2001-05-12 | 2002-05-08 | Method |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP1397683A2 (en) |
| JP (1) | JP2004534531A (en) |
| AU (1) | AU2002255159A1 (en) |
| GB (1) | GB0111639D0 (en) |
| WO (1) | WO2002093176A2 (en) |
-
2001
- 2001-05-12 GB GBGB0111639.1A patent/GB0111639D0/en not_active Ceased
-
2002
- 2002-05-08 WO PCT/GB2002/002134 patent/WO2002093176A2/en active Application Filing
- 2002-05-08 EP EP02724467A patent/EP1397683A2/en not_active Withdrawn
- 2002-05-08 JP JP2002589805A patent/JP2004534531A/en active Pending
- 2002-05-08 AU AU2002255159A patent/AU2002255159A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU2002255159A1 (en) | 2002-11-25 |
| WO2002093176A2 (en) | 2002-11-21 |
| WO2002093176A3 (en) | 2003-10-16 |
| GB0111639D0 (en) | 2001-07-04 |
| EP1397683A2 (en) | 2004-03-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Paulusma et al. | A mutation in the human canalicular multispecific organic anion transporter gene causes the Dubin‐Johnson syndrome | |
| US5239060A (en) | Muscular dystrophy protein, dystrophin | |
| US5621091A (en) | Probes for and nucleic acid encoding the muscular dystrophy protein, dystrophin | |
| JP3449419B2 (en) | Long QT syndrome gene encoding KVLQT1 and its association with minK | |
| US7229816B2 (en) | Sitosterolemia susceptibility gene (SSG) polypeptides | |
| US6893842B2 (en) | T-type calcium channel variants; compositions thereof; and uses | |
| US7041448B2 (en) | Gene encoding a new TRP channel is mutated in mucolipidosis IV | |
| JP4903136B2 (en) | Hepoxylin and ICHTHYIN modulator for the treatment of skin diseases | |
| JP2004534531A (en) | Method | |
| WO2008039445A9 (en) | Polymorphisms in the human xbp-1 gene are associated with inflammatory bowel disease | |
| US20030013087A1 (en) | Novel mutations in the freac3 gene for diagnosis and prognosis of glaucoma and anterior segment dysgenesis | |
| EP1506228B1 (en) | Role of p62 in aging-related disease | |
| Berne et al. | Identification and genetic control of the Mt-3 and Mt-4 allotypes of rabbit serum α2-macroglobulin | |
| US6821750B2 (en) | ABCG8 vectors, host cells, and method of making | |
| WO1999048905A1 (en) | Methods for identifying a risk to ucp2 and ucp3 gene variant-related afflictions and compositions thereof | |
| Tanaka et al. | Gene responsible for dificient activity of the β subunit of C8, the eighth component of complement, is located on mouse chromosome 4 | |
| WO2004072282A9 (en) | Nephropathy-associated gene | |
| US20030054418A1 (en) | Gene and sequence variation associated with cancer | |
| JP2001508291A (en) | Methods for diagnosis and treatment of pathological conditions caused by insufficient ion transport | |
| US20050096258A1 (en) | Methods and compositions for treating gastrointestinal tract mucin production associated disease conditions | |
| JP4960951B2 (en) | Use of MGC4504 | |
| US20030064372A1 (en) | Gene and sequence variation associated with lipid disorder | |
| AU9679998A (en) | Methods to modulate blood pressure and diagnose bartter's syndrome type iii | |
| US20050277618A1 (en) | Methods | |
| JPWO2003083110A1 (en) | Novel galactose transferase, peptide thereof and nucleic acid encoding the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20050114 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080415 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20080930 |