GB2478082A - Identification of compounds for use in tissue repair - Google Patents
Identification of compounds for use in tissue repair Download PDFInfo
- Publication number
- GB2478082A GB2478082A GB1108815A GB201108815A GB2478082A GB 2478082 A GB2478082 A GB 2478082A GB 1108815 A GB1108815 A GB 1108815A GB 201108815 A GB201108815 A GB 201108815A GB 2478082 A GB2478082 A GB 2478082A
- Authority
- GB
- United Kingdom
- Prior art keywords
- integrin
- beta
- tissue
- allosteric
- cell
- 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.)
- Granted
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 152
- 230000017423 tissue regeneration Effects 0.000 title claims abstract description 59
- 108010044426 integrins Proteins 0.000 claims abstract description 346
- 102000006495 integrins Human genes 0.000 claims abstract description 345
- 108010022222 Integrin beta1 Proteins 0.000 claims abstract description 306
- 102000012355 Integrin beta1 Human genes 0.000 claims abstract description 301
- 210000004027 cell Anatomy 0.000 claims abstract description 148
- 238000009739 binding Methods 0.000 claims abstract description 132
- 230000027455 binding Effects 0.000 claims abstract description 130
- 230000000694 effects Effects 0.000 claims abstract description 99
- 101001033280 Homo sapiens Cytokine receptor common subunit beta Proteins 0.000 claims abstract description 68
- 102000055647 human CSF2RB Human genes 0.000 claims abstract description 68
- 102000007469 Actins Human genes 0.000 claims abstract description 39
- 108010085238 Actins Proteins 0.000 claims abstract description 38
- 238000003556 assay Methods 0.000 claims abstract description 38
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 32
- 230000005764 inhibitory process Effects 0.000 claims abstract description 31
- 102000003727 Caveolin 1 Human genes 0.000 claims abstract description 17
- 108090000026 Caveolin 1 Proteins 0.000 claims abstract description 17
- 230000026731 phosphorylation Effects 0.000 claims abstract description 9
- 238000006366 phosphorylation reaction Methods 0.000 claims abstract description 9
- 238000012423 maintenance Methods 0.000 claims abstract description 6
- 230000001629 suppression Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 228
- 210000001519 tissue Anatomy 0.000 claims description 81
- 210000004072 lung Anatomy 0.000 claims description 58
- 125000000539 amino acid group Chemical group 0.000 claims description 51
- 150000007523 nucleic acids Chemical class 0.000 claims description 43
- 108020004707 nucleic acids Proteins 0.000 claims description 41
- 102000039446 nucleic acids Human genes 0.000 claims description 41
- 108090000623 proteins and genes Proteins 0.000 claims description 37
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 37
- 239000012634 fragment Substances 0.000 claims description 35
- 210000004379 membrane Anatomy 0.000 claims description 35
- 239000012528 membrane Substances 0.000 claims description 35
- 102000004169 proteins and genes Human genes 0.000 claims description 28
- 230000008439 repair process Effects 0.000 claims description 28
- 206010014561 Emphysema Diseases 0.000 claims description 22
- 102000011971 Sphingomyelin Phosphodiesterase Human genes 0.000 claims description 18
- 108010061312 Sphingomyelin Phosphodiesterase Proteins 0.000 claims description 18
- 230000001413 cellular effect Effects 0.000 claims description 18
- 230000004770 neurodegeneration Effects 0.000 claims description 18
- 210000000170 cell membrane Anatomy 0.000 claims description 17
- 208000015122 neurodegenerative disease Diseases 0.000 claims description 17
- 206010003246 arthritis Diseases 0.000 claims description 16
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 claims description 15
- 208000024827 Alzheimer disease Diseases 0.000 claims description 14
- 150000003384 small molecules Chemical class 0.000 claims description 12
- 210000000845 cartilage Anatomy 0.000 claims description 11
- 230000003436 cytoskeletal effect Effects 0.000 claims description 11
- 238000011161 development Methods 0.000 claims description 11
- 230000018109 developmental process Effects 0.000 claims description 11
- 239000000816 peptidomimetic Substances 0.000 claims description 10
- 230000010355 oscillation Effects 0.000 claims description 8
- 230000019439 energy homeostasis Effects 0.000 claims description 6
- 230000001976 improved effect Effects 0.000 claims description 6
- 208000018737 Parkinson disease Diseases 0.000 claims description 5
- 210000000988 bone and bone Anatomy 0.000 claims description 5
- 230000007423 decrease Effects 0.000 claims description 5
- 210000000748 cardiovascular system Anatomy 0.000 claims description 4
- 210000000653 nervous system Anatomy 0.000 claims description 4
- 238000004064 recycling Methods 0.000 claims description 4
- 210000005228 liver tissue Anatomy 0.000 claims 2
- 210000005084 renal tissue Anatomy 0.000 claims 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 abstract description 50
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 abstract description 50
- 210000002744 extracellular matrix Anatomy 0.000 abstract description 49
- 230000004913 activation Effects 0.000 abstract description 39
- 230000001404 mediated effect Effects 0.000 abstract description 27
- 102000011727 Caspases Human genes 0.000 abstract description 20
- 108010076667 Caspases Proteins 0.000 abstract description 20
- 230000006907 apoptotic process Effects 0.000 abstract description 17
- 230000009467 reduction Effects 0.000 abstract description 17
- 102100036597 Basement membrane-specific heparan sulfate proteoglycan core protein Human genes 0.000 abstract description 8
- 108010049224 perlecan Proteins 0.000 abstract description 8
- 102000005353 Tissue Inhibitor of Metalloproteinase-1 Human genes 0.000 abstract description 6
- 108010031374 Tissue Inhibitor of Metalloproteinase-1 Proteins 0.000 abstract description 6
- 230000003281 allosteric effect Effects 0.000 description 143
- 239000005557 antagonist Substances 0.000 description 101
- 238000011282 treatment Methods 0.000 description 82
- 108090000765 processed proteins & peptides Proteins 0.000 description 66
- 229940125516 allosteric modulator Drugs 0.000 description 62
- 125000003275 alpha amino acid group Chemical group 0.000 description 62
- 241000282414 Homo sapiens Species 0.000 description 57
- 230000006378 damage Effects 0.000 description 53
- 208000014674 injury Diseases 0.000 description 46
- 108010067372 Pancreatic elastase Proteins 0.000 description 45
- 102000016387 Pancreatic elastase Human genes 0.000 description 45
- 208000027418 Wounds and injury Diseases 0.000 description 45
- 239000003446 ligand Substances 0.000 description 31
- 102000004196 processed proteins & peptides Human genes 0.000 description 31
- 230000008859 change Effects 0.000 description 29
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 29
- 230000006870 function Effects 0.000 description 27
- 235000018102 proteins Nutrition 0.000 description 25
- 238000002474 experimental method Methods 0.000 description 24
- 230000001419 dependent effect Effects 0.000 description 23
- 201000010099 disease Diseases 0.000 description 23
- 229920001184 polypeptide Polymers 0.000 description 23
- 230000008860 allosteric change Effects 0.000 description 22
- 235000001014 amino acid Nutrition 0.000 description 22
- 241001465754 Metazoa Species 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 21
- 150000001413 amino acids Chemical class 0.000 description 21
- 239000000203 mixture Substances 0.000 description 20
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 19
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 19
- 238000001727 in vivo Methods 0.000 description 19
- 241000699670 Mus sp. Species 0.000 description 18
- 238000000338 in vitro Methods 0.000 description 18
- 230000030833 cell death Effects 0.000 description 17
- 210000001612 chondrocyte Anatomy 0.000 description 17
- 206010071155 Autoimmune arthritis Diseases 0.000 description 16
- 238000003501 co-culture Methods 0.000 description 16
- 230000002401 inhibitory effect Effects 0.000 description 16
- 230000001225 therapeutic effect Effects 0.000 description 16
- 230000000451 tissue damage Effects 0.000 description 16
- 238000013459 approach Methods 0.000 description 15
- 150000002632 lipids Chemical class 0.000 description 15
- 239000003981 vehicle Substances 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 14
- 238000011321 prophylaxis Methods 0.000 description 14
- 238000011069 regeneration method Methods 0.000 description 14
- 239000000126 substance Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 14
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 13
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 13
- 102000005962 receptors Human genes 0.000 description 13
- 108020003175 receptors Proteins 0.000 description 13
- 231100000827 tissue damage Toxicity 0.000 description 13
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 12
- 108060003951 Immunoglobulin Proteins 0.000 description 12
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 12
- 229940106189 ceramide Drugs 0.000 description 12
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 12
- 239000003814 drug Substances 0.000 description 12
- -1 for example Chemical class 0.000 description 12
- 102000018358 immunoglobulin Human genes 0.000 description 12
- 201000006417 multiple sclerosis Diseases 0.000 description 12
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 12
- 230000004044 response Effects 0.000 description 12
- 208000024172 Cardiovascular disease Diseases 0.000 description 11
- 230000004071 biological effect Effects 0.000 description 11
- 230000015556 catabolic process Effects 0.000 description 11
- 230000005779 cell damage Effects 0.000 description 10
- 239000003795 chemical substances by application Substances 0.000 description 10
- 238000005259 measurement Methods 0.000 description 10
- 239000003068 molecular probe Substances 0.000 description 10
- 201000008482 osteoarthritis Diseases 0.000 description 10
- 102000047934 Caspase-3/7 Human genes 0.000 description 9
- 108700037887 Caspase-3/7 Proteins 0.000 description 9
- 108091006146 Channels Proteins 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 9
- 238000007792 addition Methods 0.000 description 9
- 150000001768 cations Chemical class 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 9
- 210000002950 fibroblast Anatomy 0.000 description 9
- JHDGGIDITFLRJY-UHFFFAOYSA-N laurdan Chemical compound C1=C(N(C)C)C=CC2=CC(C(=O)CCCCCCCCCCC)=CC=C21 JHDGGIDITFLRJY-UHFFFAOYSA-N 0.000 description 9
- 108090000397 Caspase 3 Proteins 0.000 description 8
- 102100029855 Caspase-3 Human genes 0.000 description 8
- 108010035532 Collagen Proteins 0.000 description 8
- 102000008186 Collagen Human genes 0.000 description 8
- 102100027893 Homeobox protein Nkx-2.1 Human genes 0.000 description 8
- 101710114425 Homeobox protein Nkx-2.1 Proteins 0.000 description 8
- 108010067787 Proteoglycans Proteins 0.000 description 8
- 102000016611 Proteoglycans Human genes 0.000 description 8
- 101710088547 Thyroid transcription factor 1 Proteins 0.000 description 8
- 101710159262 Transcription termination factor 1 Proteins 0.000 description 8
- 230000004075 alteration Effects 0.000 description 8
- 230000033115 angiogenesis Effects 0.000 description 8
- 230000000903 blocking effect Effects 0.000 description 8
- 229920001436 collagen Polymers 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 230000008929 regeneration Effects 0.000 description 8
- 230000002441 reversible effect Effects 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 108010067306 Fibronectins Proteins 0.000 description 7
- 102000016359 Fibronectins Human genes 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 7
- 206010061218 Inflammation Diseases 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 125000003636 chemical group Chemical group 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 230000004054 inflammatory process Effects 0.000 description 7
- 230000003447 ipsilateral effect Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 108091033319 polynucleotide Proteins 0.000 description 7
- 102000040430 polynucleotide Human genes 0.000 description 7
- 239000002157 polynucleotide Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 6
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 6
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 6
- 108091023037 Aptamer Proteins 0.000 description 6
- 208000023275 Autoimmune disease Diseases 0.000 description 6
- 206010006458 Bronchitis chronic Diseases 0.000 description 6
- 108090000567 Caspase 7 Proteins 0.000 description 6
- 102100038902 Caspase-7 Human genes 0.000 description 6
- 102000012422 Collagen Type I Human genes 0.000 description 6
- 108010022452 Collagen Type I Proteins 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 6
- 208000004454 Hyperalgesia Diseases 0.000 description 6
- 208000035154 Hyperesthesia Diseases 0.000 description 6
- 208000004852 Lung Injury Diseases 0.000 description 6
- 208000012902 Nervous system disease Diseases 0.000 description 6
- 208000025966 Neurological disease Diseases 0.000 description 6
- 108010038807 Oligopeptides Proteins 0.000 description 6
- 102000015636 Oligopeptides Human genes 0.000 description 6
- 206010069363 Traumatic lung injury Diseases 0.000 description 6
- 239000013543 active substance Substances 0.000 description 6
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 206010006451 bronchitis Diseases 0.000 description 6
- 150000001720 carbohydrates Chemical class 0.000 description 6
- 235000014633 carbohydrates Nutrition 0.000 description 6
- 208000007451 chronic bronchitis Diseases 0.000 description 6
- 208000010877 cognitive disease Diseases 0.000 description 6
- 238000012790 confirmation Methods 0.000 description 6
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 6
- 210000004292 cytoskeleton Anatomy 0.000 description 6
- 230000001086 cytosolic effect Effects 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 108020001507 fusion proteins Proteins 0.000 description 6
- 102000037865 fusion proteins Human genes 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 210000001503 joint Anatomy 0.000 description 6
- 231100000515 lung injury Toxicity 0.000 description 6
- 208000027061 mild cognitive impairment Diseases 0.000 description 6
- 210000001258 synovial membrane Anatomy 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- GUEHESDOJBMSSE-UHFFFAOYSA-M (2-aminophenyl)mercury(1+);acetate Chemical compound CC(=O)O[Hg]C1=CC=CC=C1N GUEHESDOJBMSSE-UHFFFAOYSA-M 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- OFNXOACBUMGOPC-HZYVHMACSA-N 5'-hydroxystreptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](CO)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O OFNXOACBUMGOPC-HZYVHMACSA-N 0.000 description 5
- 239000012099 Alexa Fluor family Substances 0.000 description 5
- 108700021041 Disintegrin Proteins 0.000 description 5
- 102100025304 Integrin beta-1 Human genes 0.000 description 5
- 102000005741 Metalloproteases Human genes 0.000 description 5
- 108010006035 Metalloproteases Proteins 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 201000004681 Psoriasis Diseases 0.000 description 5
- 108010081750 Reticulin Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 208000015114 central nervous system disease Diseases 0.000 description 5
- 208000037976 chronic inflammation Diseases 0.000 description 5
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- OFNXOACBUMGOPC-UHFFFAOYSA-N hydroxystreptomycin Natural products CNC1C(O)C(O)C(CO)OC1OC1C(C=O)(O)C(CO)OC1OC1C(N=C(N)N)C(O)C(N=C(N)N)C(O)C1O OFNXOACBUMGOPC-UHFFFAOYSA-N 0.000 description 5
- OKPOKMCPHKVCPP-UHFFFAOYSA-N isoorientaline Natural products C1=C(O)C(OC)=CC(CC2C3=CC(OC)=C(O)C=C3CCN2C)=C1 OKPOKMCPHKVCPP-UHFFFAOYSA-N 0.000 description 5
- 210000000629 knee joint Anatomy 0.000 description 5
- 210000002414 leg Anatomy 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 229910021645 metal ion Inorganic materials 0.000 description 5
- 230000003278 mimic effect Effects 0.000 description 5
- 238000002823 phage display Methods 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 238000003752 polymerase chain reaction Methods 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 150000004804 polysaccharides Chemical class 0.000 description 5
- 238000002203 pretreatment Methods 0.000 description 5
- 230000000069 prophylactic effect Effects 0.000 description 5
- 230000008707 rearrangement Effects 0.000 description 5
- 238000009877 rendering Methods 0.000 description 5
- JTQHYPFKHZLTSH-UHFFFAOYSA-N reticulin Natural products COC1CC(OC2C(CO)OC(OC3C(O)CC(OC4C(C)OC(CC4OC)OC5CCC6(C)C7CCC8(C)C(CCC8(O)C7CC=C6C5)C(C)O)OC3C)C(O)C2OC)OC(C)C1O JTQHYPFKHZLTSH-UHFFFAOYSA-N 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 238000012800 visualization Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 4
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 4
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 208000009386 Experimental Arthritis Diseases 0.000 description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 4
- 241001111421 Pannus Species 0.000 description 4
- KPKZJLCSROULON-QKGLWVMZSA-N Phalloidin Chemical compound N1C(=O)[C@@H]([C@@H](O)C)NC(=O)[C@H](C)NC(=O)[C@H](C[C@@](C)(O)CO)NC(=O)[C@H](C2)NC(=O)[C@H](C)NC(=O)[C@@H]3C[C@H](O)CN3C(=O)[C@@H]1CSC1=C2C2=CC=CC=C2N1 KPKZJLCSROULON-QKGLWVMZSA-N 0.000 description 4
- 108010029485 Protein Isoforms Proteins 0.000 description 4
- 102000001708 Protein Isoforms Human genes 0.000 description 4
- 208000006011 Stroke Diseases 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 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 4
- 238000004220 aggregation Methods 0.000 description 4
- 235000010443 alginic acid Nutrition 0.000 description 4
- 229920000615 alginic acid Polymers 0.000 description 4
- 230000001640 apoptogenic effect Effects 0.000 description 4
- 235000003704 aspartic acid Nutrition 0.000 description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 229910052791 calcium Inorganic materials 0.000 description 4
- 244000309466 calf Species 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 229940072221 immunoglobulins Drugs 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 230000033001 locomotion Effects 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000013508 migration Methods 0.000 description 4
- 230000002438 mitochondrial effect Effects 0.000 description 4
- 230000000750 progressive effect Effects 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000009870 specific binding Effects 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 206010065040 AIDS dementia complex Diseases 0.000 description 3
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical group NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 3
- 102000000844 Cell Surface Receptors Human genes 0.000 description 3
- 108010001857 Cell Surface Receptors Proteins 0.000 description 3
- 108010077544 Chromatin Proteins 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 3
- 208000020406 Creutzfeldt Jacob disease Diseases 0.000 description 3
- 208000010859 Creutzfeldt-Jakob disease Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010019196 Head injury Diseases 0.000 description 3
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 description 3
- 208000023105 Huntington disease Diseases 0.000 description 3
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 3
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 3
- 102000004890 Interleukin-8 Human genes 0.000 description 3
- 108090001007 Interleukin-8 Proteins 0.000 description 3
- 206010023203 Joint destruction Diseases 0.000 description 3
- 208000003456 Juvenile Arthritis Diseases 0.000 description 3
- 206010059176 Juvenile idiopathic arthritis Diseases 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 208000024777 Prion disease Diseases 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 3
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108010031318 Vitronectin Proteins 0.000 description 3
- 102100035140 Vitronectin Human genes 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 239000000783 alginic acid Substances 0.000 description 3
- 229960001126 alginic acid Drugs 0.000 description 3
- 150000004781 alginic acids Chemical class 0.000 description 3
- 230000009042 allosteric modification Effects 0.000 description 3
- 210000002821 alveolar epithelial cell Anatomy 0.000 description 3
- 230000025164 anoikis Effects 0.000 description 3
- 230000008485 antagonism Effects 0.000 description 3
- 210000001188 articular cartilage Anatomy 0.000 description 3
- 230000001363 autoimmune Effects 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 210000003483 chromatin Anatomy 0.000 description 3
- 208000019069 chronic childhood arthritis Diseases 0.000 description 3
- 238000001218 confocal laser scanning microscopy Methods 0.000 description 3
- 238000004624 confocal microscopy Methods 0.000 description 3
- 210000003618 cortical neuron Anatomy 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- 210000005220 cytoplasmic tail Anatomy 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000007850 degeneration Effects 0.000 description 3
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 206010014599 encephalitis Diseases 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 210000001650 focal adhesion Anatomy 0.000 description 3
- 230000005714 functional activity Effects 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 230000003284 homeostatic effect Effects 0.000 description 3
- 230000013632 homeostatic process Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 230000004941 influx Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 201000002215 juvenile rheumatoid arthritis Diseases 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 239000002773 nucleotide Chemical group 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 210000004940 nucleus Anatomy 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- 230000000241 respiratory effect Effects 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 230000003019 stabilising effect Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 208000037816 tissue injury Diseases 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 230000014616 translation Effects 0.000 description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 3
- 241001515965 unidentified phage Species 0.000 description 3
- YZOUYRAONFXZSI-SBHWVFSVSA-N (1S,3R,5R,6R,8R,10R,11R,13R,15R,16R,18R,20R,21R,23R,25R,26R,28R,30R,31S,33R,35R,36R,37S,38R,39S,40R,41S,42R,43S,44R,45S,46R,47S,48R,49S)-5,10,15,20,25,30,35-heptakis(hydroxymethyl)-37,39,40,41,42,43,44,45,46,47,48,49-dodecamethoxy-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.23,6.28,11.213,16.218,21.223,26.228,31]nonatetracontane-36,38-diol Chemical compound O([C@@H]([C@H]([C@@H]1OC)OC)O[C@H]2[C@@H](O)[C@@H]([C@@H](O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3O)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O[C@@H]3[C@@H](CO)O[C@@H]([C@H]([C@@H]3OC)OC)O3)O[C@@H]2CO)OC)[C@H](CO)[C@H]1O[C@@H]1[C@@H](OC)[C@H](OC)[C@H]3[C@@H](CO)O1 YZOUYRAONFXZSI-SBHWVFSVSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 208000037259 Amyloid Plaque Diseases 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 102100023995 Beta-nerve growth factor Human genes 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 206010053567 Coagulopathies Diseases 0.000 description 2
- 206010055665 Corneal neovascularisation Diseases 0.000 description 2
- 229920000858 Cyclodextrin Polymers 0.000 description 2
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 2
- 206010012689 Diabetic retinopathy Diseases 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 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 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 208000010412 Glaucoma Diseases 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 101001009079 Homo sapiens Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 102000018297 Immunoglobulin subtype Human genes 0.000 description 2
- 108050007411 Immunoglobulin subtype Proteins 0.000 description 2
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 208000008589 Obesity Diseases 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 239000005662 Paraffin oil Substances 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 108010009711 Phalloidine Proteins 0.000 description 2
- 241000276498 Pollachius virens Species 0.000 description 2
- 102100027391 Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 Human genes 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- 102100024550 Sphingomyelin phosphodiesterase 2 Human genes 0.000 description 2
- QHNORJFCVHUPNH-UHFFFAOYSA-L To-Pro-3 Chemical compound [I-].[I-].S1C2=CC=CC=C2[N+](C)=C1C=CC=C1C2=CC=CC=C2N(CCC[N+](C)(C)C)C=C1 QHNORJFCVHUPNH-UHFFFAOYSA-L 0.000 description 2
- 206010064390 Tumour invasion Diseases 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 238000002441 X-ray diffraction Methods 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000005856 abnormality Effects 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 102000019997 adhesion receptor Human genes 0.000 description 2
- 108010013985 adhesion receptor Proteins 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 238000004873 anchoring Methods 0.000 description 2
- 238000003782 apoptosis assay Methods 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 230000023555 blood coagulation Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 238000000423 cell based assay Methods 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 230000007248 cellular mechanism Effects 0.000 description 2
- 210000003850 cellular structure Anatomy 0.000 description 2
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000010205 computational analysis Methods 0.000 description 2
- 201000000159 corneal neovascularization Diseases 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000006735 deficit Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 208000037765 diseases and disorders Diseases 0.000 description 2
- 230000007783 downstream signaling Effects 0.000 description 2
- 238000003708 edge detection Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000011536 extraction buffer Substances 0.000 description 2
- 210000003414 extremity Anatomy 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 230000009760 functional impairment Effects 0.000 description 2
- 230000009454 functional inhibition Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 201000011066 hemangioma Diseases 0.000 description 2
- 239000000833 heterodimer Substances 0.000 description 2
- 235000003642 hunger Nutrition 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 208000018937 joint inflammation Diseases 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 210000005265 lung cell Anatomy 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 229910052748 manganese Inorganic materials 0.000 description 2
- 239000011572 manganese Substances 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000002284 membrane microdomain Anatomy 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 210000003470 mitochondria Anatomy 0.000 description 2
- 210000001700 mitochondrial membrane Anatomy 0.000 description 2
- 230000004660 morphological change Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 201000003142 neovascular glaucoma Diseases 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 235000020824 obesity Nutrition 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 238000012753 partial hepatectomy Methods 0.000 description 2
- 210000000426 patellar ligament Anatomy 0.000 description 2
- 229940124531 pharmaceutical excipient Drugs 0.000 description 2
- 230000000704 physical effect Effects 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 208000005069 pulmonary fibrosis Diseases 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000012306 spectroscopic technique Methods 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 210000003518 stress fiber Anatomy 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000036962 time dependent Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 1
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 1
- 102000001049 Amyloid Human genes 0.000 description 1
- 108010094108 Amyloid Proteins 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 206010051728 Bone erosion Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 229940123169 Caspase inhibitor Drugs 0.000 description 1
- 102000005600 Cathepsins Human genes 0.000 description 1
- 108010084457 Cathepsins Proteins 0.000 description 1
- 102000004201 Ceramidases Human genes 0.000 description 1
- 108090000751 Ceramidases Proteins 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 101001013520 Coccidioides posadasii (strain C735) Neutral protease 2 homolog MEP7 Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 229940123907 Disease modifying antirheumatic drug Drugs 0.000 description 1
- 101800001224 Disintegrin Proteins 0.000 description 1
- 101000844746 Drosophila melanogaster Drosomycin Proteins 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 241000724791 Filamentous phage Species 0.000 description 1
- 102000016621 Focal Adhesion Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108010067715 Focal Adhesion Protein-Tyrosine Kinases Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 102100024025 Heparanase Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 101100118545 Holotrichia diomphalia EGF-like gene Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 description 1
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 1
- 101001013150 Homo sapiens Interstitial collagenase Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 238000012313 Kruskal-Wallis test Methods 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 229940124761 MMP inhibitor Drugs 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- YALRCXHVQYBSJC-UHFFFAOYSA-N Mammea A/AB Chemical compound C12=C(O)C(C(=O)C(C)CC)=C(O)C(CC=C(C)C)=C2OC(=O)C=C1C1=CC=CC=C1 YALRCXHVQYBSJC-UHFFFAOYSA-N 0.000 description 1
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 description 1
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 1
- 101710170181 Metalloproteinase inhibitor Proteins 0.000 description 1
- 102000002151 Microfilament Proteins Human genes 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 229910020700 Na3VO4 Inorganic materials 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 244000203593 Piper nigrum Species 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108010003541 Platelet Activating Factor Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 101710193132 Pre-hexon-linking protein VIII Proteins 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 102100021651 SUN domain-containing ossification factor Human genes 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000004133 Sodium thiosulphate Substances 0.000 description 1
- 101710201923 Sphingomyelin phosphodiesterase 2 Proteins 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102000006463 Talin Human genes 0.000 description 1
- 108010083809 Talin Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108010046075 Thymosin Proteins 0.000 description 1
- 102000007501 Thymosin Human genes 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 1
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 108091000387 actin binding proteins Proteins 0.000 description 1
- 108091009126 actinin binding proteins Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 206010069351 acute lung injury Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 210000002588 alveolar type II cell Anatomy 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000006933 amyloid-beta aggregation Effects 0.000 description 1
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 1
- 230000003941 amyloidogenesis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- POJOORKDYOPQLS-UHFFFAOYSA-L barium(2+) 5-chloro-2-[(2-hydroxynaphthalen-1-yl)diazenyl]-4-methylbenzenesulfonate Chemical compound [Ba+2].C1=C(Cl)C(C)=CC(N=NC=2C3=CC=CC=C3C=CC=2O)=C1S([O-])(=O)=O.C1=C(Cl)C(C)=CC(N=NC=2C3=CC=CC=C3C=CC=2O)=C1S([O-])(=O)=O POJOORKDYOPQLS-UHFFFAOYSA-L 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000009460 calcium influx Effects 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000009787 cardiac fibrosis Effects 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000017455 cell-cell adhesion Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000005786 degenerative changes Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 239000002988 disease modifying antirheumatic drug Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 230000000547 effect on apoptosis Effects 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000002172 effect on chromatin Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000008508 epithelial proliferation Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000027948 extracellular matrix binding Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 239000012909 foetal bovine serum Substances 0.000 description 1
- 210000005224 forefinger Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- QPJBWNIQKHGLAU-IQZHVAEDSA-N ganglioside GM1 Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](NC(=O)CCCCCCCCCCCCCCCCC)[C@H](O)\C=C\CCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](CO)O1 QPJBWNIQKHGLAU-IQZHVAEDSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- BCQZXOMGPXTTIC-UHFFFAOYSA-N halothane Chemical compound FC(F)(F)C(Cl)Br BCQZXOMGPXTTIC-UHFFFAOYSA-N 0.000 description 1
- 229960003132 halothane Drugs 0.000 description 1
- 230000004217 heart function Effects 0.000 description 1
- 108010037536 heparanase Proteins 0.000 description 1
- 230000000917 hyperalgesic effect Effects 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000010569 immunofluorescence imaging Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000012212 insulator Substances 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 210000002570 interstitial cell Anatomy 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- RUTXIHLAWFEWGM-UHFFFAOYSA-H iron(3+) sulfate Chemical compound [Fe+3].[Fe+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O RUTXIHLAWFEWGM-UHFFFAOYSA-H 0.000 description 1
- 208000012947 ischemia reperfusion injury Diseases 0.000 description 1
- 210000005067 joint tissue Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 150000002611 lead compounds Chemical class 0.000 description 1
- 238000012917 library technology Methods 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000003475 metalloproteinase inhibitor Substances 0.000 description 1
- 229940126170 metalloproteinase inhibitor Drugs 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000012775 microarray technology Methods 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000006676 mitochondrial damage Effects 0.000 description 1
- 230000004898 mitochondrial function Effects 0.000 description 1
- 230000008965 mitochondrial swelling Effects 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000037230 mobility Effects 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000007491 morphometric analysis Methods 0.000 description 1
- 238000013425 morphometry Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000006654 negative regulation of apoptotic process Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 208000004296 neuralgia Diseases 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 1
- 208000021722 neuropathic pain Diseases 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 108040005466 neutral sphingomyelin phosphodiesterase activity proteins Proteins 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229960003379 pancuronium bromide Drugs 0.000 description 1
- NPIJXCQZLFKBMV-YTGGZNJNSA-L pancuronium bromide Chemical compound [Br-].[Br-].C[N+]1([C@@H]2[C@@H](OC(C)=O)C[C@@H]3CC[C@H]4[C@@H]5C[C@@H]([C@@H]([C@]5(CC[C@@H]4[C@@]3(C)C2)C)OC(=O)C)[N+]2(C)CCCCC2)CCCCC1 NPIJXCQZLFKBMV-YTGGZNJNSA-L 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 210000004417 patella Anatomy 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000008024 pharmaceutical diluent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- YHHSONZFOIEMCP-UHFFFAOYSA-O phosphocholine Chemical compound C[N+](C)(C)CCOP(O)(O)=O YHHSONZFOIEMCP-UHFFFAOYSA-O 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000013105 post hoc analysis Methods 0.000 description 1
- 238000010149 post-hoc-test Methods 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 210000002243 primary neuron Anatomy 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 210000003456 pulmonary alveoli Anatomy 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009703 regulation of cell differentiation Effects 0.000 description 1
- 230000004202 respiratory function Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 239000000565 sealant Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 230000008409 synovial inflammation Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 210000003813 thumb Anatomy 0.000 description 1
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 1
- 230000005919 time-dependent effect Effects 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 239000003656 tris buffered saline Substances 0.000 description 1
- IHIXIJGXTJIKRB-UHFFFAOYSA-N trisodium vanadate Chemical compound [Na+].[Na+].[Na+].[O-][V]([O-])([O-])=O IHIXIJGXTJIKRB-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000013042 tunel staining Methods 0.000 description 1
- 238000007492 two-way ANOVA Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000011870 unpaired t-test Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 229960005356 urokinase Drugs 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 239000002821 viper venom Substances 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
- C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/74—Inducing cell proliferation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
An assay for identifying compounds for use in tissue repair comprising the steps of providing a candidate compound which has binding specificity to human beta 1 integrin, bringing the candidate compound into contact with human beta 1 integrin and determining the presence or absence of modulation of the beta 1 integrin or the derivative thereof into an intermediate affinity state conformation by the candidate compound; wherein the presence of modulation of the beta 1 integrin or the derivative thereof into the intermediate affinity state conformation is indicative of utility of that compound in tissue repair. Modulation may be detected by various means such as remodelling of the extracellular matrix, inhibition of caspase activation, a reduction in apoptosis, suppression of F-actin aggregate formation, maintenance of beta 1 integrin expression levels, inhibition of caveolin-1 phosphorylation, increase in perlecan, increase in inhibtors of metalloproteinase-1 (TIMP-1) and also enhancement of adhesion-mediated activity of the cell.
Description
COMPOUNDS AND METHODS FOR THE MODULATION OF INTEGRIN
FUNCTION TO MEDIATE TISSUE REPAIR
Field of the Invention
The present invention provides compounds and methods for the selective modulation of the ligand binding function of an integrin, in particular betal integrin. More specifically, there is provided a method for mediating tissue repair following insult and injury by allosterically modulating beta 1 integrin.
The invention further provides compounds which act as allosteric antagonists of beta 1 integrin and which modulate betal integrin function in order to mediate tissue repair. The invention further extends to methods for the treatment of tissue damage associated with disease conditions such as chronic obstructive pulmonary disease, arthritis, and neurodegenerative conditions.
Background to the Invention
Integrins constitute a family of widespread heterodimeric cell surface adhesion receptors comprised of non-covalently associated alpha (a) and beta (13) subunits. To date, 18 different alpha subunits and 8 different beta subunits have been described in vertebrates, these fomling 24 distinct heterodimers. Of these heterodimers, 12 contain the betal (131, beta 1, beta-i, CD29) subunit.
To date, the precise structure of beta 1 integrin has not been identified.
However, a prediction of the X-ray crystalline structure of beta I has been produced, and is shown in Figure 17. The alpha and beta subunit of all integrins fold forming an extracellular headpiece which is connected to the membrane by a structure which can be likened to "2 supporting legs', followed by a short transmembrane domain and cytoplasmic tail. The headpiece of the integrin heterodimer is composed of a beta propeller domain of the alpha subunit which closely interacts with the A domain of the beta subunit (the A domain also being known as the I-like domain of the beta subunit).
The affinity state of an integrin is regulated by the conformation of the headpiece. Rearrangement of the headpiece can be initiated by intrinsic ligands and by the binding of specific adaptors to the cytoplasmic domain.
The activation of beta 1 integrin is regulated in a cell-type dependent manner, and plays an important role in modulating cell functions. The avidity of an integrin is increased by the transition to an active conformational state. The specific conformational state of an integrin receptor can have a fundamental effect on integrin function. As such, in many instances, integrin conformation is more important than integrin expression levels when considering the physiological and pathological remodelling of tissue.
Integrin antagonists fall into three classes; direct inhibitors of ligand binding to the I domain of the alpha chain, allosteric inhibitors of the I domain of the alpha chain and allosteric antagonists of alpha chain I beta I-like domains (also known as the A domain).
Functional modification of betal integrin using antibodies is known. For example, International Patent Application Number WO 05/037313 discloses the use of compounds which modulate beta I integrin in order to mediate an increase in anabolism of the extracellular matrix. Such compounds are identified as being of use in tissue regeneration.
Further approaches to the modulation of beta 1 integrin functional activity focus on the modulation of beta 1 integrin by means of activation or blocking of adhesion to a substrate under a defined set of conditions.
Modulation of betal integrin in such a way has a number of limitations.
Firstly, modulation is based on the understanding that integrins can exist in inactive, active and active and occupied states. Secondly, functional modulation was often achieved via different domains of the integrin. This may entail different downstream intracellular signalling, and therefore even if the effect on adhesion is similar, the functional end outcome can be different as each region appears to possess a different function. Further, betal integrin is known to exist in four different splice variants, with the resulting differences in the cytoplasmic domain implicating different downstream signalling.
Following extensive experimentation, the inventors have now surprisingly identified that compounds which interact with beta 1 integrin and function as antagonists of allosteric modulation have utility in tissue repair. The inventors have identified that such allosteric antagonists can function to promote tissue repair, this being characterised by the physical and/or mechanical restoration of damaged or diseased tissues through the growth of new cellular structures. Further, the inventors have further recognised that direct allosteric modulation of beta 1 integrin with a compound which functions as an allosteric antagonist can result in the reversal of functional and structural changes associated with tissue repair.
Summary of the Invention
According to the first aspect of the present invention there is provided a method of modulating one or more of the biological activities of beta 1 integrin in a beta 1 integrin expressing cell or tissue in order to mediate cellular or tissue repair, comprising: -contacting the cell or tissue with an allosteric antagonist of beta 1 integrin, in an amount sufficient to reduce one or more biological activities of beta 1 integrin in the cell or tissue which causes in tissue damage or cell death.
In certain embodiments, the reduction of one or more of the biological activities of beta 1 integrin may relate to one of more of; inhibiting cell death, modulating cellular differentiation, modulating cellular mechanisms, such as inhibiting caspase activation, F-actin aggregate formation, causing fluctuations in cellular AlP levels, and increasing cellular impedance.
Typically the reduction of said one or more biological activities of beta 1 integrin relates to the conformation of the beta 1 integrin being modulated such that it assumes an intermediate affinity state.
In certain embodiments of this aspect of the invention, the allosteric modulator is an allosteric antagonist which, when bound to an epitope present on beta 1 integrin, and in particular, the extracellular domain of beta 1 integrin, causes a conformational change of beta 1 integrin such that the extracellular domain of beta 1 integrin assumes a conformation defined as the intermediate affinity state. In this "intermediate conformatory state", the extracellular domain of beta 1 integrin assumes an extended conformation, however, the headpiece assumes a closed conformation.
The "intermediate affinity state" differs from the conformation of an inactive integrin where the extracellular domain assumes a low affinity bent confirmation, wherein the integrin is conformationally bent, and the headpiece is spaced approximately 5 nm from the cell membrane upon which the integrin is expressed.
The intermediate affinity state also differs from the conformation of an activated beta 1 integrin molecule wherein the integrin assumes a high affinity extended form which has an open headpiece configuration.
The change in integrin conformation is mediated by a conformational change in the extracellular domain of beta 1 integrin which is mediated by the opening or closing of the hinge angle between the A domain and the hybrid domain of beta 1 integrin. The opening of this hinge is indicative of a conformational switch to a high binding affinity. Inactive beta 1 integrin has bent conformation, wherein the headpiece is spaced approximately 5 nm from the cell membrane upon which the integrin is expressed.
nm. In the activated, extended conformation, the headpiece projects from the membrane at a distance of between 20 to 25 nm. Accordingly, when in the intermediate affinity state conformation, where the extracellular domain of the integrin is extended, but the headpiece is in a closed conformation, the headpiece can be between about 6 nm to about 19 nm from the cell membrane.
The inventors have identified that when beta 1 integrin is bound by a ligand, the integrin assumes a high affinity extended conformational form.
However, tissue damage and injury occurs when beta 1 integrin is in this form. The inventors have therefore surprisingly identified that modulatory compounds which alter the allostery of the beta 1 integrin, and which in particular act as allosteric antagonists to prevent the integrin assuming an active, extended form, and which instead cause the beta I integrin to assume a conformation associated with the integrin being in a intermediate affinity state, can reverse functional and structural effects which result in tissue injury. In particular, the functional and structural reversal of tissue injury which are observed by the inventors following allosteric antagonism of beta 1 integrin mediate tissue repair, this being defined as the physical or mechanical restoration of damaged or diseased tissues by growth of healthy new cells.
Accordingly, the provision of compounds which bind to beta 1 integrin at a site distinct from the ligand binding site, and which mediate an allosteric modulation of beta 1 integrin such that it assume the intermediate affinity conformatory state, has utility in methods for tissue repair, wound healing and tissue regeneration.
In particular, the inventors have identified that when beta 1 integrin is in the activated, extended conformation, which, for example, results when beta 1 integrin is bound by a ligand, tissue damage and injury can occur.
Accordingly, the present inventors provided herewith methods and compounds which allosterically modulate beta 1 integrin such that it reverts to the intermediate affinity conformation, a conformatory state which has been identified by the inventors as mediating tissue repair.
The inventors have further identified that binding of beta 1 integrin by divalent cations such as, for example, calcium, manganese and rnagnesiurrl, can result in beta 1 integrin assuming high affinity extended conformation. The inventors have identified that such divalent cations bind to the extracellular domain of beta 1 integrin at sites termed MIDAS (metal ion dependent adhesion sites), and ADMIDAS (adjacent to metal ion dependent adhesion sites).
In particular, the inventors have identified potential MIDAS sites as being present in the human beta 1 integrin amino acid sequence a residue 150 (aspartic acid), 152 (serine), 154 (serine), 249 (glutamic acid), 279 (aspartic acid). Further, ADMIDAS sites are predicted to be present at residues 157 (aspartic acid), 158 (aspartic acid), and 362 (alanine) of the amino acid sequence of human beta 1 integrin.
The inventors have identified that the binding of divalent cations to these MIDAS and ADMIDAS sites result in a conformational activation of the beta 1 integrin into an extended, high affinity conformation. In order to prevent the binding of such divalent cations to beta 1 integrin, the inventors have identified that binding beta 1 integrin with an allosteric antagonist which further prevents divalent cation binding to the MIDAS and ADMIDAS sites can have utility in the methods of the present invention, as the integrin will not assume the activated, extended conformation.
As defined herein, the term "allosteric modulator", "allosteric antagonist" or "allosteric modulator compound" relates to a compound which can allosterically modulate betal integrin in order to cause the beta 1 integrin to assume an intermediate affinity conformation, wherein the extracellular domain and headpiece of beta 1 integrin is neither fully bent, nor fully extended. Allosteric modulation refers to a control mechanism which alters protein behaviour wherein the binding of an allosteric modulator occurs at a binding site distinct from the active site, this resulting in a conformational change which influences protein function.
As defined herein, the term "modulates" relates to a change in the conformation or functional activity of beta I integrin.
The allosteric antagonist of the invention mediates a conformational change to beta 1 integrin such that it assumes a conformation defined as the intermediate affinity state. As defined herein, the term "intermediate affinity state" describes the conformation of the extracellular domain of betal integrin when under normal homeostatic conditions. When in this state, betal integrin is in an intermediate affinity binding state.
The term "normal homeostatic conditions" as used herein in relation to the conformational arrangement of beta 1 integrin, relates to the structural conformation of the extracellular domain assumed by betal integrin which is in a conformational equilibrium between the accepted high and low affinity binding conformational states which betal integrin can assume. In the intermediate state, betal integrin is not in a completely inactive state, however "swing" out of the hybrid domain, a process required for beta 1 integrin to assume a high affinity conformation, is prevented. Further, the intermediate affinity state can be further characterised in that it maintains extracellular binding.
Without wishing to be bound by theory, the inventors predict that the selective allosteric modulation of betal integrin such that the extracellular domain assumes an intermediate affinity binding conformation can result in a reduction in caspase activation and cell death, and thus can confer significant associated benefits in terms of tissue repair and regeneration.
This intermediate affinity conformation state is equivalent to the conformation assumed by the integrin when it is under normal homeostatic conditions. Selective allosteric modulation of the betal integrin, in accordance with the present invention, can induce the reversal of structural and functional changes resulting from cellular insult and injury.
Such changes are important in preventing ECM degeneration and in allowing tissue repair and regeneration. The inventors have identified that complete inhibition or blocking of betal integrin results in cell death as the cell becomes detached from the ECM. The modulation effected by the methodology and compositions of the present invention prevents detachment of a cell from the ECM, this permitting cell repair, due to the maintenance of ECM anchoring. Accordingly, the identification by the inventors that the targeted binding of the hybrid domain of the beta 1 integrin can modulate betal integrin allostery, this causing a marked observed therapeutic benefit in terms of the prevention of cell death following tissue insult or injury, is significant in that it promotes cell survival and permits cell/tissue repair and regeneration.
The allosteric modulation of the betal integrin can be characterised and/or monitored in terms of the effect on downstream signalling and function mediated by and through beta 1 integrin. Accordingly, allosteric modulation of the betal integrin such that it assumes an intermediate affinity conformational state may be further co-characterised by the occurrence of at least one of the following which serve to reduce, prevent or revert cellular damage which may be associated with ECM degradation, cellular injury or death and disease progression: (i) remodelling of the extracellular matrix (ECM), (ii) inhibition of caspase activation, and in particular caspase 3 and caspase 7 and an associated reduction in apoptosis, (iii) suppression of F-actin aggregate formation, this in turn preventing an increase in cytoskeletal tension (iv) improved cellular energy homeostasis resulting from a reversion to normal sinusoidal oscillation of ATP levels, and associated improved levels of ATP recycling within a cell, (v) maintenance of betal integrin expression levels, (vi) inhibition of caveolin-1 phosphorylation, this being an indicator of an alteration of the cytomechanics of the cell and/or the degradation of the ECM and a reduction in tethering forces, which can result in clustering or aggregation of receptors, and (vii) inhibition of cell membrane physicochemical changes. (increased stiffness as a result of increased sphingomyelinase activity leading to increased ceramide content and raft formation.
Allosteric modulation of betal integrin in accordance with the present invention may be further characterised in that an increase in perlecan results, as well as there being an initial increase in tissue inhibitors of nietalloproteinase-1 (TIMP-1) and a subsequent increase in pro-nietalloproteinase-9.
In certain embodiments, the allosteric modulator compound can bind to betal integrin when betal integrin assumes any structural conformation, that is, the modulator compound can bind betal integrin irrespective of whether betal integrin assumes a low affinity (bent) or high affinity (extended) conformational state.
In certain embodiments, the allosteric modulator is selected from the group comprising of, but not limited to a: protein, peptide, peptidomimetic, nucleic acid, polynucleotide, polysaccharide, oligopeptide, carbohydrate, lipid, small molecule compound, aptamer, and naturally occurring compound such as a plant derived compound. Typically the allosteric modulator has binding specificity for beta 1 integrin and functions as an allosteric antagonist.
In certain embodiments, where the allosteric modulator is a protein, the protein is an antibody. An "antibody" is an immunoglobulin, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide, protein or peptide having a binding domain that is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced.
Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses and fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd, and a bi-specific antibody.
In certain embodiments, the allosteric modulator is an antibody selected from, but not limited to, the group comprising: a polyclonal antibody, a rnonoclonal, a humanized antibody, a chimeric antibody or a synthesized antibody. The antibody may be of an isotype selected from the group consisting of IgG, IgA, 1gM, and IgE.
In certain embodiments, the antibody may bind to an epitope on beta 1 integrin with a dissociation constant (Kd) selected from the group of from about 107M to about 1011M.
In certain further embodiments, where the allosteric modulator is a protein, the protein is a fusion protein, for example a fusion protein comprising an Fc domain derived from an antibody, said Fc domain being conjoined, by a linker, or otherwise, to a protein fragment which has binding specificity for beta 1 integrin, and which acts as an allosteric modulator.
In certain embodiments, the allosteric modulator binds to at least one epitope present on the extracellular domain of human beta 1 integrin. In certain embodiments, the epitope which is bound by the beta 1 integrin allosteric rriodulator is distinct to the ligand binding epitope of beta 1 integrin.
In certain embodiments the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 and 87 of the mature amino acid sequence of human beta I integrin as defined in SEQ ID NO:1. Amino acid resides 82 (threonine) and 87 (lysine) are determined when the leader signal peptide is deducted from the defined amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
In certain embodiments the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 to 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
In certain embodiments the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TAEKLK (SEQ ID NO:2) of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
In certain embodiments the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 179 to 184 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
In certain embodiments the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TPAKLR (SEQ ID NO:3) of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
In certain embodiments the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within amino acid residues 207 to 218 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
In certain embodiments the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within the hybrid domain comprising residues 60 to 139 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
In certain embodiments the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 360 to 461 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
The allosteric modulators may also be defined as beta 1 integrin binding compounds. A binding compound is a specific binding agent and refers to a natural or non-natural molecule that specifically binds to a target, in particular an epitope present on beta 1 integrin. Examples of suitable binding compounds for use in the present invention include, but are not limited to, proteins, peptides, peptidomimetics, nucleic acids, carbohydrates, aptamers, lipids, and small molecule compounds.
The term "specifically binds" or "binding specificity" refers to the ability of the binding compound to bind to a target epitope present on beta 1 integrin with a greater affinity than it binds to a non-target epitope. In certain embodiments, specific binding refers to binding to a target with an affinity that is at least 10, 50, 100, 250, 500, or 1000 times greater than the affinity for a non-target. In certain embodiments, affinity is determined by an affinity [LISA assay. In certain embodiments, affinity is determined by a BlAcore assay. In certain embodiments, affinity is determined by a kinetic method. In certain embodiments, affinity is determined by an equilibrium/solution method.
In certain embodiments, the binding compound binds to at least one epitope present on epitope, wherein binding to this epitope results in the modulation of the allostery of beta 1 integrin. An "epitope" refers to a portion of beta 1 integrin which is capable of being recognised by and bound by a binding compound such as a small molecule or antibody by the binding region of said small molecule or antibody. Epitopes generally consist of chemically active surface groups and have specific three dimensional structural characteristics as well as specific charge characteristics. Typically, the binding compound antagonises the allostery of beta 1 integrin and as such binds to an epitope known as an inhibiting or inhibitory allosteric epitope. An "inhibiting" or "inhibitory" allosteric epitope means an epitope, that when bound by a binding compound, such as a small molecule or an antibody, results in the loss of allostery, and in particular of the ability of beta 1 integrin to assume an extended, activate conformation. An epitope present on beta 1 integrin, and which is bound by the binding compounds in order to antagonise beta 1 integrin allosteric function, may comprise 5 or more amino acid residues. In certain embodiments, the binding compounds recognise an epitope comprised of at least 5 continuous amino acid residues which is present from amino acid residues 60 to 139 and 360 to 461 of the amino acid sequence of the hybrid domain of the extracellular domain of human beta 1 integrin as defined herein in SEQ ID NO:1.
In certain embodiments, the betal integrin may be any member of the betal integrin family which comprises; betal integrin bound to al, a2, a3, a7, a9, a4, a5, a8, and av to form a heterodirner. The amino acid sequence of the hybrid domain of beta 1 integrin is identical irrespective of the particular beta 1 isoform.
In one embodiment the allosteric modulator binds to beta 1 integrin and induces a conformational change from a high affinity conformational state, wherein the extracellular domain is in an extended form with an open headpiece to an intermediate state, where the extracellular domain of beta 1 integrin is extended, but wherein the headpiece is in a closed conformation.
In an alternative embodiment, the allosteric modulator may bind betal integrin and induce a conformational change from an inactive state where the extracellular domain is in a bent conformation to an intermediate state.
The allosteric modulation of betal integrin, when bound by the allosteric modulator compound of the invention, therefore causes a modulation of the special or positional relationship of a cell to a substrate, wherein anchoring of a cell to the ECM is maintained. Such as relationship is important as adhesion of cells to their surrounding extracellular matrix (ECM) in-vivo regulates their morphology, proliferation, migration, survival and differentiation. In vitro, the interactions of cells with ECM molecules such as fibronectin result in cell attachment, spreading and the assembly of focal adhesions and actin stress fibres. The method of this aspect of the present invention may further modulate at least one of; cell attachment, cell spreading, migration, or the formation of a focal adhesion.
The intracellular response of a cell to the ECM may also be modulated.
Without being bound by theory, the inventors predict that binding beta 1 integrin with at least one allosteric modulator compound serves to induce a conformational change in the structure of betal integrin which causes beta 1 integrin to assume an intermediate affinity state. This intermediate affinity state can be differentiated from the low and high affinity states which betal integrin is known to assume following inactivation and insult/injury respectively. Allosteric modulation of betal integrin which induces the assumption of an intermediate affinity state has been unexpectedly shown by the inventors to result in the prevention of ECM degradation and cell death following tissue insult or injury, with this observed cell survival allowing cell repair and regeneration processes to occur. Accordingly, cells and tissue which would have formerly have died through a process of programmed cell death, can be maintained in living form and associated repair can occur.
In particular, allosteric modulation of betal integrin has been identified by the inventors as being a potentially important therapeutic approach for use in tissue repair following injury or following the onset or development of autoimmune disease such as an autoimmune arthritis, such as rheumatoid arthritis or a lung condition, such as COPD, emphysema or chronic bronchitis. Pathogenesis observed following such tissue damage causes a change in the mechanical tethering of the cells to the ECM, with this in turn can change cell mechanosensing, this leading to a depletion in ATP levels due to the cessation of energy homeostasis resulting from sinusoidal oscillation of ATP levels within a cell. It has been shown herein for the first time that allosteric modulation, which causes betal integrin to assume an intermediate affinity state can result in a modification of cellular attachment to the underlying ECM, a reduction in mechanical stress and an increase in energy homeostasis, this further resulting in a reduction in detachment-induced apoptosis (anoikis).
Accordingly, the methods and modulatory compounds of the present invention which induce selective modulation of beta 1 integrin can prevent cell death which can result from tissue insult, injury and disease. This reduction in cell death allows cells to survive, this allowing them to undergo repair and regeneration.
In one embodiment the allosteric modulator is the monoclonal antibody JB1a. The JB1a monoclonal antibody is obtainable as a commercial clone JB1a from Chemicon (Chemicon Europe Ltd, Hampshire, UK, and Millipore, catalogue number MAB-1965). The JB1a monoclonal antibody may also be referred to as the J1O monoclonal antibody.
The JB1a monoclonal antibody (JB1A mouse anti-CD29 (Integrin beta-i subunit) IgG monoclonal antibody, unconjugated, clone JB1a, Millipore, Catalogue number MAB-i 965) which is derived from the JBia hybridoma cell line binds to an epitope between amino acid residues 82-87 of the betai integrin hybrid domain, irrespective of the conformational state of the receptor. This epitope may be defined as amino acid resides 62-67 when the 20 amino acid signal peptide leader sequence is removed from the defined human beta 1 integrin amino acid sequence.
The inventors have identified that the compositions and methods of the present invention have utility in the treatment of a number of conditions where tissue insult or injury occurs. These conditions include, but are not limited to, autoimmune arthritis, COPD, including emphysema and chronic bronchitis, cardiovascular disease and neurodegenerative disease.
Accordingly, a yet further aspect of the invention provides a method for the prophylaxis and/or treatment of autoimmune arthritis, the method comprising the steps of: -providing a therapeutically effective amount an allosteric antagonist compound, wherein said allosteric modulator binds to betai integrin and mediates an allosteric change thereof such that the conformation of betai integrin assumes the intermediate affinity state, and -administering the same to a subject in need of such treatment.
In certain embodiments, the autoimmune arthritis is at least one condition is selected from the group comprising, but not limited to: rheumatoid arthritis, inflammatory arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis. In further embodiments, the autoimmune arthritis may be collagen-induced arthritis.
In certain embodiments, where the autoimmune arthritis is rheumatoid arthritis, the beta 1 integrin allosteric modulator is brought into contact will a cell of the synovium, such the cells of the synovial membrane, for example, the intima or subintima.
Accordingly, a yet further aspect of the invention provides for the prophylaxis and/or treatment of chronic obstructive pulmonary disease (COPD), the method comprising the steps of: -providing a therapeutically effective amount an allosteric antagonist compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state, and -administering the same to a subject in need of such treatment.
In certain embodiments the chronic obstructive pulmonary disease (COPD) indication is selected from the group comprising, but not limited to: emphysema or chronic bronchitis.
Accordingly, a yet further aspect of the invention provides for the prophylaxis and/or treatment of a neurodegenerative disease, the method comprising the steps of: -providing a therapeutically effective amount an allosteric antagonist compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of betal integrin assumes the intermediate affinity state, and -administering the same to a subject in need of such treatment.
In certain embodiments the neurodegenerative disease is selected from the group comprising, but not limited to: Alzheimer's disease (AD), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.
Accordingly, a yet further aspect of the invention provides for the prophylaxis and/or treatment of tissue damage associated with a cardiovascular disease, the method comprising the steps of: -providing a therapeutically effective amount an allosteric antagonist compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state, and -administering the same to a subject in need of such treatment.
In certain embodiments, the cardiovascular disease is selected from the group comprising, but not limited to: stroke, ischemia, ischemia repertusion and heart attack.
The invention further extends to a binding fragment derived from the JB1 a nionoclonal antibody, or an antibody which has binding specificity for an epitope present on beta 1 integrin, wherein binding of the antibody mediates allosteric modulation such that the beta 1 integrin assumes an intermediate conformation. For example, the allosteric antagonist may be a Fab fragment, scFV or fusion protein which is derived from, and maintains the binding specificity of, the JBIa monoclonal antibody.
Furthermore, in certain embodiments, the allosteric antagonist of beta 1 integrin may be an aptamer which has binding specificity for beta 1 integrin, and which causes allosteric antagonism of the beta 1 integrin receptor. Techniques for the selection of suitable aptamers will be well known to the person skilled in the art, for example, using SELEX technology.
Accordingly the invention extends to a method of identifying and isolating nucleic acid ligands which have binding specificity for beta 1 integrin and which modulate the function of beta 1 integrin by acting as an allosteric antagonist, the method comprising the steps of: (a) providing a candidate mixture of nucleic acids (b) contacting beta 1 integrin protein with the candidate nucleic acid mixture (C) selecting nucleic acids which have an increased affinity to beta 1 integrin protein relative to the other candidate nucleic acids, (d) amplifying the selected nucleic acids in order to provide at least one nucleic acid with affinity for beta 1 integrin protein, and (e) selecting at least one nucleic acid therefrom which has a high affinity and specificity for beta 1 integrin protein.
As used herein, reference to "JB1a" include antibodies and binding fragment derived therefrom which have variable light, variable heavy or CRD (corriplementarity detemlining) sequences which show substantial homology with JB1a and which can be characterised in that they exhibit substantially identical binding specificity to that defined herein in relation to JB1 a. In certain embodiments, the degree of homology between the amino acid resides which comprise the JB1a monoclonal antibody complementarity determining regions (CDR5) and the CDRs of other antibodies, or related binding fragments will be at least 60%, more preferably 70%, further preferably 80%, even more preferably 90% or most preferably at least 95% identical.
The invention further extends to immunoglobulins which have binding specificity for betal integrin, wherein binding of said immunoglobulin mediates a conformatory change which causes beta 1 integrin to assume a conformation representative of an intermediate affinity state, and in particular where this binding is allosteric and effected at the hybrid domain of beta 1 integrin, most preferably epitope between amino acid residues 82-87 of the human beta 1 integrin hybrid domain.
In further embodiments, the allosteric modulator compound has binding specificity for the hybrid domain of betal integrin. The amino acid sequence of the hybrid domain is identical irrespective of the isoforrn of any beta 1 integrin family member presently identified. In a further, specific embodiment, the allosteric modulator has binding specificity for the domain of betal integrin corresponding to residues 82-87 of the mature betal integrin. These residues have the sequence as defined in SEQ ID NO:1, namely TAEKLK (SEQ ID NO:2) (Threonine-Alanine-Glutamic Acid-Lysine-Leucine-Lysine), and can be further characterised in that they bind to this epitope irrespective of the overall conformation assumed by betal integrin.
For example, such allosteric modulator compounds may be small molecules (low molecular weight), or non-antibody modulating agents which are distinct from oligopeptide fragments of integrin ligands (e.g., ECM proteins, such as fibrinogen and fibronectin) and cyclic derivatives of these fragments.
More specifically, such allosteric modulator compounds may be derived from a fibronectin protein, for example the CBD portion of fibronectin, or the RGD sequence of the CBD; a vitronectin, or an analogue or an integrin binding portion of vitronectin; a laminin, or an analogue or an integrin binding portion of a laminin; a collagen, or an analogue or an integrin binding portion of a collagen; a polypeptide other than an ECM molecule which binds to a betal chain of integrin, e.g., a CBD-binding portion of integrin, e.g., a polypeptide selected for binding in, for example, a phage display or 2 hybrid assay; a small molecule, e.g., a small molecule capable of binding a betal chain of integrin, such as a CBD-binding portion of integrin. In a preferred embodiment, the method of this aspect of the invention comprises administering a nucleic acid (polynucleotide) which encodes one of the above-described agents.
In further embodiments, the allosteric modulator compound of the invention may be a disintegrin, or a variant or analogue thereof.
Disintegrins are a family of naturally-occurring cysteine-rich peptides originally isolated from viper venom, but also found on cells and elsewhere, many of which contain the sequence Arg Gly Asp (RGD) as an integrin recognition site. Disintegrins are defined by their specific amino acid sequences and three-dimensional structures. "Variants" of disintegrins are disintegrins engineered to have one or more amino acids added, deleted or replaced. "Analogues" are non-peptide mimetics of disintegrins or their variants.
A further aspect of the invention provides a method for mediating tissue repair, the method comprising the step of administering to an individual in need of such treatment, a therapeutically effective amount of a compound which is an allosteric modulator of beta I integrin, wherein the binding of the allosteric modulator to betal integrin induces the conformation of the betal integrin to assume an intermediate affinity state.
As used herein, the term tissue repair' means the physical or mechanical restoration or growth of damaged or diseased tissue by the repair or regeneration of tissue or cells following injury, insult, damage or trauma or following the onset of autoimmune disease or a disorder mediated by the immune system.
In one embodiment of this aspect of the invention, the method further comprises the step of administering at least one compound which inhibits matrix metalloproteinase (MMP) activity.
The method of this aspect of the invention may be used for the prophylaxis and/or treatment of any condition wherein extracellular matrix breakdown occurs. Such conditions may include; tumour invasion, infectious diseases, chronic inflammatory diseases such as rheumatoid arthritis or osteoarthritis, joint destruction in rheumatoid arthritis and osteoarthritis, periodontotitis and angiogenesis-dependent diseases such as corneal neovascularization, diabetic retinopathy, neovascular glaucoma, haemangioma, psoriasis, scieroderma and solid tumours, a neurological disease or disease of the central nervous system such as Alzheimer's disease, angiogenesis-dependent diseases, multiple sclerosis.
A yet further aspect of the invention provides a method for the prophylaxis and/or treatment of a condition which causes degeneration of the extracellular matrix, the method comprising the step of administering a therapeutically effective amount of an allosteric modulatory compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of beta I integrin assumes the intermediate affinity state to an individual in need of such treatment.
In one embodiment of this aspect of the invention, the method further comprises the step of administering at least one compound which inhibits matrix metalloproteinase (MMP) activity.
As herein defined, a "condition which causes degradation of the extracellular matrix" is a condition which causes or results from degenerative changes or break down to the extracellular matrix or to the cells of the extracellular matrix. Such degradation can result from the onset and progression of disease, such as infectious diseases and autoimmune or immune-mediated diseases. Further such conditions may include tumour.
In particular embodiments, the degeneration of the extracellular matrix or cells thereof comprises joint tissues, including articular cartilage or bone, intra-articular ligaments and tendons, wherein degradation leads to a loss of function. Such conditions may result from pathologies such as; tumours, chronic inflammatory diseases such as rheumatoid arthritis or osteoarthritis, a neurological disease or disease of the central nervous system such as Alzheirner's disease, angiogenesis-dependent diseases, multiple sclerosis, and ischerriia.
The methods of the present invention may have further utility in the treatment and/or prophylaxis of; methods of treating inflammation; methods of treating and preventing viral infections; a method of treating shock; a method of treating arthritis; a method of suppressing immune responses; a method of treating an autoimmune disease; a method of inhibiting undesirable integrin-mediated cell-cell fusion; a method of inhibiting the formation of lesions; a method of treating psoriasis; a method of treating atherosclerosis; a method of treating diseases or conditions involving a plurality of integrin-dependent etiopathogenetic mechanisms; and a method of inhibiting the transfer of genetic material.
In addition, this aspect of the present invention further extends to a method of treating a tissue by contacting the tissue with an allosteric modulator compound according to the present invention. Such treatment improving the cellular structure of the tissue for subsequent use, as compared to tissue which is not treated with such a modulator compound.
In particular, tissue which is to be transplanted into a recipient may be treated with an allosteric modulator compound according to the present invention, in order to increase the viability of the tissue.
In one embodiment of this aspect of the invention, the method further comprises the step of administering at least one compound which inhibits matrix metalloproteinase (MMP) activity to a subject in need of such treatment in order to mediate tissue repair.
In one embodiment the subject is a mammal. In a further embodiment the mammal is a human.
In one embodiment, the condition may include any condition where ECM breakdown or cell death occurs, for example such conditions may include; tumours, chronic inflammatory diseases such as rheumatoid arthritis or osteoarthritis, a neurological disease or disease of the central nervous system such as Alzheimer's disease, angiogenesis-dependent diseases, multiple sclerosis..
In a further embodiment the condition may be chronic obstructive pulmonary disease (COPD), chronic bronchitis or emphysema.
A yet further aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an allosteric modulatory compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state along with a pharmaceutically acceptable diluent, excipient or carrier.
In one embodiment of this aspect of the invention, the pharmaceutical composition may further comprise at least one compound which inhibits matrix metalloproteinase (MMP) activity.
Assays The present invention further extends to assay methods which can be used to identify further compounds which serve to allosterically modulate betal integrin such that it assumes an intermediate affinity state.
Novel compounds identified using the assays of the invention as being allosteric modulators of betal integrin which mediate an allosteric change such that the conformation of beta 1 integrin assumes the intermediate affinity state form a further independent aspect of the invention.
Accordingly, a yet further aspect of the present invention provides an assay method for the determination of an allosteric modulator compound, wherein said allosteric modulator binds betal integrin and mediates an allosteric change such that the conformation of beta I integrin assumes the intermediate affinity state, said method comprising the steps of: -providing an assay comprising beta I integrin or a derivative thereof; -activating betal integrin such that it assumes a conformation representative of a high affinity state, -contacting a the activated beta 1 integein with a candidate modulating agent under suitable conditions; and -detecting a change of the conformation of beta 1 integrin to an intermediate affinity state, wherein modulation of the allostery of the beta 1 integrin from the high affinity conformational state to the intermediate affinity conformational state indicates that the candidate compound is an allosteric modulator of beta 1 integrin which has utility in mediating tissue repair.
In one embodiment the derivative of betal integrin may comprise an amino acid sequence comprising the hybrid domain of betal integrin, or a peptide fragment comprising at least residues 82-87 of the betal integrin hybrid domain having the amino acid sequence of SEQ ID NO:1.
In one embodiment, the contacting step is followed by an incubating step wherein the mixture obtained in the contacting step is incubated for a time period and under conditions suitable to allow interaction between the components and/or the test substance.
Further methods for identifying an allosteric modulator compound can be performed, for example, using a cell free assay. In a preferred embodiment, a compound can be identified using a cell based assay.
These methods include identifying a compound based on its ability to allosterically modulate betal integrin this being characterised by enhancement of an adhesion-mediated activity of the cell. Examples of such adhesion mediated activities include: promoting focal adhesion assembly, promoting actin stress fibre formation; promoting an adhesion-mediated signalling pathway, e.g., a Rho dependent signalling pathway; promoting the interaction, e.g., binding, with a component of the extracellular matrix, e.g., fibronectin, vitronectin, a laminin, a collagen. In addition, the method includes identifying a compound on the basis of its ability to affect cell survival.
It is preferred that the assay be a cellular assay which may comprise cultured cells of primary or transformed cell lines, said cell lines being the result of the transfection of a cell line with an expression vector comprising the sequence or a substantial portion of the sequence a betal integrin polynucleotide sequence.
A yet further aspect of the present invention provides novel compounds identified from the assay methods described herein which act as allosteric modulator compounds of betal integrin in that they can bind to betal integrin and mediate an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state.
Such compounds can be used in tissue repair in any tissue, for example tissue of the lung, skin, liver, kidney, nervous system, cartilage, bone and cardiovascular system. The compounds could have further utility in methods for the treatment of any condition where degradation of the ECM occurs, or where cell damage or cell death occurs, for example autoimmune conditions or infectious disease conditions and in particular in the treatment of conditions such as inflammation; viral infections shock; arthritis; suppressing immune responses; autoimmune disease; inhibiting undesirable integrin-mediated cell-cell fusion; inhibiting the formation of lesions; treating psoriasis; treating atherosclerosis; treating diseases or conditions involving a plurality of integrin-dependent etiopathogenetic mechanisms; and neurological diseases.
The present invention further provides assay methods for determining compounds which may be used to mediate tissue repair by virtue of their ability to act as allosteric modulator compounds, wherein said allosteric modulators binds to betal integrin and mediates an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state. As used herein, an "assay system" encompasses all the components required for performing and analysing results of an assay that detects and/or measures a particular event or events.
A variety of assays are available to detect the activity of compounds such as antibodies, peptides and chemicals which have specific binding activity to betal integrin examples of which are described herein. However, the precise format of the assay(s) of the invention may be varied by those skilled in the art using routine skill and knowledge. Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening potential candidate compounds to identify their ability to allosterically modulate betal integrin such that the conformation of beta 1 integrin assumes the intermediate affinity state.
Betal integrin allosteric modulating activity may be assessed in the assays of the invention using any suitable means. For example, the effect of the agent on MMP levels or balance, and / or the effect on apoptosis and apoptotic pathways. Exemplary assays are western blotting analyses and ELISA based assays for MMPs protein in both active and inactive forms, proteoglycans synthesis using western analyses and ELISA based assays, cell adhesion based assays, apoptosis assays using in-situ labelling, immunohistochemistry, immunofluorescence imaging and gel analyses.
In the assays of the invention, derivatives of beta 1 integrin may be used.
Such derivatives may comprise one or more fragments of beta 1 may be used, for example the hybrid domain, or alternatively one or more binding sites of beta 1 integrin may be used, for example the binding site corresponding to amino acid residues 82 to 87 of the mature betal integrin molecule.
Alternatively, the derivative may comprise a betal integrin mimetic. The skilled person is well aware of how to design such a mimetic. Briefly, a template molecule is selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in-vivo, while retaining the biological activity of the betal integrin.
The mimetic found by this approach can then be used in assays of the invention in place of beta 1 integrin to ascertain whether the candidate compounds can allosterically modulate the betal integrin mimetic to identify whether said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the confirmation of beta 1 integrin assumes the intermediate affinity state.
The inventors have further recognised the utility of the beta 1 integrin allosteric modulatory compounds of the present invention in uses for the treatment of tissue repair in any tissue, for example tissue of the lung, skin, liver, kidney, nervous system, cartilage, bone and cardiovascular system and further in relation to increasing the lifespan of a cell, and treating and! or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing as well as diseases or disorders.
Accordingly, a yet further aspect of the present invention provides for the use of an allosteric modulator of betal integrin which modulates the betal integrin into an intermediate affinity state in the preparation of a med icament for increasing the lifespan of a cell, and treating and! or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, cardiovascular disease, blood clotting disorders, inflammation, cancer, and/or flushing as well as diseases or disorders such as COPD or emphysema.
A yet further aspect of the present invention provides for the use of an allosteric modulator of betal integrin which modulates the betal integrin into an intermediate affinity state in the preparation for the treatment of an autoimmune disease. The autoimmune disease may be rheumatoid arthritis, or psoriasis, or multiple sclerosis.
A yet further aspect of the present invention provides the use of an allosteric modulator of betal integrin which modulates the betal integrin into an intermediate affinity state in the preparation of a med icament for mediating tissue repair and regeneration.
A yet further aspect of the present invention provides the use of an allosteric modulator of betal integrin which modulates betal integrin into an intermediate affinity state for the inhibition of caspase activation and/or apoptosis. In one embodiment the caspase is caspase 3. In a further embodiment the caspase is caspase 7.
A yet further aspect of the present invention provides the use of an allosteric modulator of betal integrin which modulates the betal integrin into an intermediate affinity state for the inhibition of caveolin-1 phosphorylation.
A yet further aspect of the invention provides a method for the inhibition of apoptosis within a cell, said effect being mediated by inhibiting or decreasing the production of at least one of caspase 3 and caspase 7, the method comprising the step of administering a therapeutically effective amount of an allosteric modulator compound which binds to betal integrin and mediates an allosteric change such that the conformation of betal integrin assumes the intermediate affinity state to an individual in need of such treatment.
A yet further aspect of the present invention provides for the use of an allosteric modulator of betal integrin which modulates the betal integrin into an intermediate affinity state and a compound which inhibits matrix metalloproteinase activity in the preparation of a combined medicament for the prophylaxis and/or the treatment of pathologies selected from the group comprising; tumours, chronic inflammatory diseases such as rheumatoid arthritis or osteoarthritis, a cardiovascular disease, a neurological disease or disease of the central nervous system such as Alzheimer's disease, angiogenesis-dependent diseases, multiple sclerosis or conditions requiring the inhibition of matrix metalloproteinase (MMP) activity.
A yet further aspect of the present invention provides a pharmaceutical composition for the prophylaxis and/or the treatment of pathologies selected from the group comprising; tumours, chronic inflammatory diseases such as rheumatoid arthritis or osteoarthritis, a neurological disease or disease of the central nervous system such as Alzheimer's disease, angiogenesis-dependent diseases, multiple sclerosis or conditions requiring the inhibition of matrix metalloproteinase (MMP) activity comprising an allosteric modulator of betal integrin which modulates the betal integrin into an intermediate affinity state and a compound which inhibits matrix metalloproteinase activity along with at least one suitable pharmaceutical excipient.
Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the
art in the field of the present invention.
Throughout the specification, unless the context demands otherwise, the terms comprise' or include', or variations such as comprises' or comprising', includes' or including' will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
As used herein, terms such as "a", "an" and "the" include singular and plural referents unless the context clearly demands otherwise. Thus, for example, reference to "an active agent" or "a pharmacologically active agent" includes a single active agent as well as two or more different active agents in combination, while references to "a carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.
The term "or" is used herein to mean, and is used interchangeably with, the term "and/or", unless context clearly indicates otherwise.
As used herein, the terms "about" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically within 10%, and more typically, within 5% of a given value or range of values. In the context of a range value for an amino acid or nucleotide sequence, the term "about" includes a range that differs by 1, 2, 3, 4 or 5 residues or nucleotides at one or both end points.
For example, the phrase "about amino acids 9 to 22" of a sequence can include amino acid sequences, such as 7 to 23 and 11 to 20 of the amino acid sequence specified.
The nomenclature used to describe the polypeptide constituents of the fusion protein of the present invention follows the conventional practice wherein the amino group (N) is presented to the left and the carboxy group to the right of each amino acid residue.
The expression "amino acid" as used herein is intended to include both natural and synthetic amino acids, and both D and L amino acids. A synthetic amino acid also encompasses chemically modified amino acids, including, but not limited to salts, and amino acid derivatives such as am ides. Amino acids present within the polypeptides of the present invention can be modified by rriethylation, amidation, acetylation or substitution with other chemical groups which can change the circulating half life without adversely affecting their biological activity.
The terms "peptide", "polypeptide" and "protein" are used herein interchangeably to describe a series of at least two amino acids covalently linked by peptide bonds or modified peptide bonds such as isosteres. No limitation is placed on the maximum number of amino acids which may comprise a peptide or protein. Furthermore, the term polypeptide extends to fragments, analogues and derivatives of a peptide, wherein said fragment, analogue or derivative retains the same biological functional activity as the peptide from which the fragment, derivative or analogue is derived Furthermore the term "fusion protein" as used herein can also be taken to mean a fusion polypeptide, fusion peptide or the like, or may also be referred to as an immunoconjugate. The term "fusion protein" refers to a molecule in which two or more subunit molecules, typically polypeptides, are covalently or non-covalently linked.
As used herein, the term "therapeutically effective amount" means the amount of beta 1 integrin allosteric modulator which is required to mediate a conformational change of beta 1 integrin in order to mediate tissue repair. The tissue repair may be used to treat a cancerous condition, an autoimmune condition, such as autoimmune arthritis, a cardiovascular disease, or a neurodegenerative disease or at least one symptom thereof.
As used herein, the term "prophylactically effective amount" relates to the amount of a composition which is required to prevent the initial onset, progression or recurrence of a tissue injury in a disease condition such as a cancerous condition, an autoirrimune condition, such as autoimmune arthritis, a cardiovascular disease, or a neurodegenerative disease or at least one symptom thereof, said tissue damage, insult or injury being mediated by beta 1 integrin assuming an extended conformation, in a subject following the administration of the compounds of the present invention.
As used herein, the term "subject" refers to an animal, preferably a mammal and in particular a human. In a particular embodiment, the subject is a mammal, in particular a human. The term "subject" is interchangeable with the term "patient" as used herein.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
Other features and advantages of the invention will be apparent from the
description, drawings and from the claims.
Brief Description of Drawings
The present invention will now be described with reference to the following examples which are provided for the purpose of illustration and are not intended to be construed as being limiting on the present invention, and further, with reference to the figures.
Figure 1 shows the effect of porcine pancreatic elastase (PPE) on respiratory function in mice and its reversal using the anti-betal integrin antibody JB1 a. Figure 1 (a) shows the effect of PPE on mean respiratory pressure-volume curves in mice 35 days (35d) after instillation and its reversal by JB1a (JB) (Vehicle=Veh). Figure 1(b) shows the reversal of PPE-induced increase in the constant k by JB1 a treatment at different time points post injury. JB1 a treatment once or twice following PPE-induced injury reversed the time-dependent effect on peak respiratory pressure from the pressure-volume curves (Figure 1 (c)) and the quasi-static elastase derived from a. (Figure 1(d)). n=5-6 in 35d (35 day) groups and n=1O in 21d (21 day) groups, Figure 2 shows the effect of PPE on lung structure and its reversal by JB1a treatment. Figure 2(a) shows haematoxylin and eosin staining of lung sections from veh, PPE and JB instilled mice from 21d (21 day) and 35d (35 day) groups. Figure 2(b) shows mean linear intercept measurements from the 21d and 35d groups.
Automated stereological estimates of the number of (Figure 2(c)) contiguous airspaces, (Figure 2(d)) septal junctions, and (Figure 2(e)) septal ends from the 21 day group (n=5), Figure 3 shows the effect of allosteric modulation of betal integrin GATA-6 and TTF-1 expression after PPE-induced. Figure 3(a) shows 3D reconstructions of PPE-injured lung in the 21 day group of lung parenchyma (blue, Column I), and the expression patterns of GATA-6 (yellow) and TTF-1 (red, Column II). Column III shows an overlay of parenchyma and expression patterns of GATA-6 and TTF-1. Column IV shows a detail of (Column I) at higher magnification. Column V shows a detail of (Column II) at higher magnification. Figure 3(b) shows TUNEL staining of lung tissue sections from 21d (21 day) and 35d (35 day) groups demonstrating the effect of JB1 a treatment after PPE-induced lung injury. Figure 3(c) shows the quantification of TUNEL positive cells following PPE-induced injury and JB1 a treatment. n=5-6 per group, Figure 4 shows the effects of PPE-induced injury and JB1 a treatment on caspase levels (Figure 4(a)), F-actin (Figure 4(b)) and ATP levels (Figure 4(c)) in-vitro using human lung co-culture during with mechanical stretch for 6 hours. The effects of PPE-induced injury alone and in the presence of JB1a treatment on betal integrin (Figure 4(d)), caveolin-1 Figure 4(e)) and phosphorylated caveolin-1 levels (Figure 4(f)) in membrane fractions.
Representative blots from n4. Loading controlled by total amount of protein before gradient separation, Figure 5 shows the effect of allosteric modulation of betal integrin using JB1a on metalloproteinases and tissue inhibitors of metalloproteinases (MMP5) in primary human lung alveolar/mesenchymal co-cultures. Figure 5(a) shows the effect of JB1a on MMP1 levels and activity at two different time points when added alone and in the presence of APMA, cycloheximide (CXH), neutralising antibodies against MMPs 7 and 9 and broad spectrum MMP inhibitor. Figure 5(b) demonstrates the effects JB1a on tissue inhibitor of metalloproteinase-1 (TIMP1). Figure 5(c) shows the effect of JB1 a on the levels of inactive MMP9 (pro-MMP9). The experiment was repeated 3 times using cells derived from different patients, Figure 6 shows automated image processing of the septal junctions, septal ends, and contiguous airspaces in the pulmonary acinus in the 21 day group. Figure 6 (a)-(c) shows RGB images from JB (Figure 6(a)), PPE (Figure 6(b)), and veh lungs (Figure 6(c)) were pre-processed with morphological filters (Figures 6(d)-(f)) to aid automated edge detection and to remove noise. In Figure 6(g)-(i) a 200mm x 200mm frame was superimposed over preprocessed images. Only events completely inside the frame or intersecting the two inclusion edges (South-West boundaries) are considered and any events intersecting the exclusion lines (North-East boundaries) are not sampled. The number of contiguous airspaces (Figure 6(g)-(i)), septal branches (Figure (j)-(l), white dots), and free septal ends (m-o, white dots) was determined in reticulin-stained sections of lung, Figure 7 shows images from time-lapse movies showing the effect of vehicle (Figure 7(a)) elastase ((Figure 7(b), 0.6 U/mI) on F-actin (blue) and caspase 3/7 activation (red) in vitro using human lung co-culture during mechanical stretch and its inhibition by JB1a (Figure 7(c), I pg/mI). Syto 16-green. A figure of the selected frames from the recording is provided), Figure 8 shows a 3D reconstruction of images of human lung co-culture after injury using elastase (b, 0.6 U/mI) demonstrating the formation of F-actin (blue) and caspase 3/7 activation (red).
(Ganglioside GM1 for the cell membrane-green) and its inhibition by JB1a (c, I pg/mI). Vehicle (a), Figure 9 shows 3D reconstruction of images of in-vivo mouse lung after injury using elastase (b) (0.2UIg body weight) demonstrating the formation of F-actin (green) after 35 days and its inhibition by JB1a (c)(3mgfkg on days 21 and 28). (a: vehicle instilled animals).
Each figure is from a separate animal, Figure 10(a) shows the effect of elastase-induced injury (0.3UIml) on sphingomyelinase activity in human lung co-culture during mechanical stretch at 2-12% amplitude at different time points and its inhibition by JB1a (2ug/mI) (n=3-4). Figure 10(b) shows the level of sphingomyelinase activity and kinetics in in-vivo mouse lung after injury using elastase (0.2U/g body weight) demonstrating the an increase in the level of sphingomylinase after 35 days and its inhibition by JBIa (3mg/kg on days 21 and 28) (n=5), Figure 11 shows the effect of elastase-induced injury (0.6U/mI) on raft formation using laurdan in human lung co-culture and its inhibition by JB1 a (2ug/ml). The intensity of laurdan emission at 488nm indicates increase in lipid rafts after elastase-induced injury.
Methyl-beta-cyclodextrin was used as a control as it depletes cholesterol thus reducing the intensity of laurdan at 488nm, Figure 12 shows the effect of elastase-induced injury (0.3U/ml) on electrical impedance of human lung co-culture and its inhibition by JB1 a (2ug/ml). The experiment was repeated 3 times and each was done in duplicate. Each well had 8 sampling electrodes. The figure is one representative experiment, Figure 13 shows the effects of Freund's complete adjuvant (FCA) on the function and structure of joints in mice and its reversal by JB1 a treatment. Figure 13 (a) shows the effect of two doses of JB1a on FCA-induced ipsilateral joint dimension increase. Figure 14(b) shows the effect of two doses of JB1a on the contralateral joint dimension after FCA intra-articular injection. Figure 13 (c) shows the effects of JB1a on FCA-induced hyperalgesia. Figure 13(d) shows the effects of JB1a on the reversal of abnormal histological scores of the ipsilateral joints induced by FCA. Figure 13(e) shows the haematoxylin and eosin staining of median sections from Vehicle (Veh), FCA, JB1 a, isotype and sham treated mice. Figure 13(f) shows alcian blue staining of the ipsilateral articular joint demonstrating the reversal effects of JB1 a on FCA-induced glycosaminoglycan loss and metachromasia. Figure 13(g) shows a graph of the quantification of the effect of JB1 a on FCA-induced chondrocytes cell death, Figure 14 shows the effects of IL-i beta-induced injury and JB1a treatment on caspase activity, ATP levels and IL-8 concentration.
Figure 14(a) shows a bar chart indicating that JB1a inhibits IL-ibeta-induced caspase 3/7 activation (n=9). Data from all time points for a given treatment were pooled as the effect of time was not significant. Figure 14(b) shows a graph illustrating IL-i beta (10 ng/ml) effects on ATP post 3 hours IL-i beta-induced injury and the effect of JBi a on ILl beta-induced ATP changes (each curve represents n=2, each done in triplicates). c. IL-i beta effect on 1L8 levels and its partial reversal by JBia (n=3, each done in duplicates), Figure 15(a) shows the effect of beta-amyloid fibrils on primary cortical neurons. Beta amyloid fibrils induced caspase activation (arrowhead) and raft formation (arrow). Both effects were inhibited by JB1a. Figure 15(b) shows JB1a inhibition of amyloid fibril effects were accompanied by morphological changes, Figure 16 shows SEQ ID NO:i, this being the amino acid sequence of human beta 1 integrin (Genbank Accession Number P05556 (Integrin beta-i precursor (Fibronectin receptor subunit beta) Integrin VLA-4 subunit beta, CD29 antigen)), and Figure 17 shows the predicted crystalline structure of human beta 1 integrin.
Detailed Description of the Invention
The present invention relates to compounds and methods for use in allosterically modulating beta I integrin in order to mediate tissue repair.
The amino acid sequence of human beta 1 integrin is provide in Figure 16 as SEQ ID NO:1. The 798 amino acid sequence of beta 1 integrin is available from the Genbank database of sequences, under Genbank Accession Number P05556 ((Integrin beta-i precursor (Fibronectin receptor subunit beta) Integrin VLA-4 subunit beta, CD29 antigen).
The key region involved in ligand recognition and binding is the top face of the A domain. This domain contains a metal ion dependent adhesion site (MIDAS). The A domain of beta integrin is linked to the immunoglobulin-like hybrid domain. The hybrid domain acts as a lever, wherein the angle between the A domain and hybrid domain controls beta 1 integrin receptor conformation and, accordingly, ligand binding affinity. The "legs" of the integrin are composed of multiple globular domains known as EGF-repeats (epidermal like growth factor repeats), which incorporate two knee-like joints referred to as genu. The EGF-repeats are linked to cytoplasmic tail domains via a single transmembrance pass.
Ligand binding has been shown to induce the opening (or an increase) in the angle between the A and hybrid domains of beta 1 integrin in the integrin headpiece. The opening of this hinge is indicative of the switch to a high binding affinity. A further site which is of importance in affinity regulation is the metal ion dependent adhesion site (MIDAS) within the A domain. A MIDAS site is also thought to be present in the alpha chains containing the I domain. Integrin-ligand interactions have been shown to be affected by the binding of divalent cations, such as magnesium, manganese and calcium at these MIDAS sites. For example, the binding of calcium is shown to have an inhibitory effect on beta 1 integrin by stabilising the low-affinity conformation of beta I integrin.
Integrins are known to undergo global structural rearrangement upon activation, with this structural rearrangement resulting in the exposure of ligand binding sites. The overall strength of cellular adhesiveness or avidity is governed by affinity and valency. Valency relates to the density of the receptor and its ligand on the cell surface, as well as the special and geometric arrangement and movement.
Integrins can be summarised as having three main possible structural conformations of the extracellular domain. These are (i) a low affinity, bent conformation, (ii) an intermediate affinity state which is characterised by an extended conformation, with a bent headpiece, and (iii) a high affinity, extended form, wherein ligand binding induces the headpiece to extend in an open conformation.
The function of beta 1 integrin has been studied using a genetic approach.
These studies have shown that beta 1 integrin has an effect on cell survival and proliferation, as well as remodelling of the extra-cellular matrix. These effects are both cell type and development stage type dependent.
Inactive integrins are conformationally bent and the headpiece is approximately 5nm away from the membrane. Upon activation, integrins undergo a switchblade-like extension, this resulting in the integrin transfomling from a bent state to an extended state. This extended state increases the accessibility of the headpiece to interact with ligands. For example, the binding of fibronectin to the alpha5betal integrin was shown to induce an open conformation, by means of an 80 degree increase in the angle of the hybrid domain of the beta subunit relative to the I-like (A) domain.
The separation of the alpha and beta subunit legs is a critical step in inside-out integrin activation, which results in transformation of the extended conformation and allowing headpiece-ligand engagement.
Disrupting the integrin heterodimeric transmembrane helix-helix interface activates ligand binding mainly by increasing the monomeric affinity for ligand, and not the receptor valency (i.e. clustering). This separation is initiated by the heterodimer intersubunit dissociation between their transmembrane and cytoplasmic domains. Activation and opening of the beta 1 integrin MIDAS can result from the swing-out of the beta subunit hybrid domain, pulling on the C-terminal alpha helix of the beta 1 domain in a manner similar to a bell rope. At this level of extension, the integrin headpiece projects at 20 to 25 nm above the plasma membrane, making the receptor ligand competent. Both the EGF-like and hybrid domains are key in transducing the cytoplasmic tail rearrangements into separation and straightening of the integrin ectodomain. This is because when the hybrid domain undergoes a sing-out motion through inside-out signalling, it causes a partial opening of the beta 1 integrin I-like domain, and ligand binding occurs leading to outside-in activation.
Inside-out activation of integrins entails changes in the conformation initiated at the cytoplasmic clasp and transmitted to the headpiece MIDAS domain via the leg domains. Outside-in activation is a term which describes the activation occurring when ligand binding to the headpiece domain induces changes in the headpiece as well as in the orientation of the leg domains and the cytoplasmic regions. High-affinity states can be artificially induced by chemically locking extended and open integrin conformations or by stabilising these states with monoclonal antibodies prior to binding.
There are multiple cation binding sites present in both the alpha and beta integrin subunits. Divalent cations have been shown to induce a conformational relaxation in the integrin, this resulting in exposure of ligand binding sites. These sites lie near the interface between the alpha beta-propeller and the beta subunit A (I-like) domain.
One such site lies in the beta subunit A domain and is known as the metal ion dependent adhesion site (MIDAS). Another cation binding site is known as the ADMIDAS (adjacent to the MIDAS). The ADMIDAS is important in stabilising the active conformation of the integrin, since mutations in this site inhibit integrin activation and conformational changes are markedly increased. The predicted position of the MIDAS and ADMIDAS sites in the crystal structure of beta 1 integrin is shown in Figure 17.
The extracellular matrix (ECM) interacts with integrins and modifies their function. In turn, integrins are key regulators of the ECM. Their main functions are cell-ECM and cell-cell adhesion. One hallmark feature is ability of integrins to process signals bidirectionally across the cell membrane. Additionally, integrins are mechanotransducers, converting mechanical signals to biochemical changes. These properties give integrins key roles in cell growth, differentiation, migration, and survival.
Integrins in general, including the betal subfamily, are known to exhibit global structural rearrangement and exposure of ligand binding sites upon activation. The overall strength of cellular adhesiveness or avidity is governed by affinity and valency; the latter governed by the density of the receptor and its ligand on the cell surface as well as the spatial and geometric arrangement and movement of the integrin.
Heterodimers of betal integrin bind collagens (al, a2), laminins (al, a2, a3, a7, a9) and fibronectin (a3, cx4, a5, a8, av). Furthermore, beta 1 integrin can also bind rnetalloproteinases (MMP5) such as MMP2 and MMP9 and affect their activation state.
Several pathological processes involve tissue remodelling and degradation including; tumour invasion, joint destruction in rheumatoid arthritis and osteoarthritis, periodontotitis and angiogenesis-dependent diseases such as corneal neovascularization, diabetic retinopathy, neovascular glaucoma, haemangioma, psoriasis, scieroderma and solid tumours. Basement membrane and interstitial connective tissue make up the ECM, which is composed of collagen, proteoglycans and adhesion glycoproteins. ECM provides a physical support to cells and tissues and cell-ECM interactions regulate cell growth, differentiation and migration.
ECM degrading enzymes include matrix metalloproteases (MMP5), urokinase, tissue plasminogen activator, cathepsins, trypsins and heparanases. The integrin-mediated function of adhesion is important for a variety of physiological and pathological responses. The extent of adhesion is controlled by integrin signalling.
Antibodies An "antibody" is an imrnunoglobulin, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide, protein or peptide having a binding domain that is, or is homologous to, an antibody binding domain, particularly that of the JB1 a rnonoclonal antibody. Such binding compounds can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses and fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd, and a bi-specific antibody. So called "domain antibodies" may also be produced, as can binding fragments based on or derived from Camelid or shark antibodies.
As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering any binding member or compound having a binding domain with the required binding specificity as defined hereinbefore. The antibody may be a monoclonal antibody, or a fragment, derivative, functional equivalent or homologue thereof. The term includes any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in European Patent Application Publication Number EP 0,120,694 and European Patent Application Publication Number EP 0,125,023.
The constant region of the antibody may be of any suitable immunoglobulin subtype, however it is preferred that the antibody subtype is IgGi. However, in alternative embodiments, the subtype of the antibody may be of the class IgA, 1gM, IgD and IgE where a human immunoglobulin molecule is used. Such an antibody may further belong to any sub class e.g. IgGi, IgG2a, 2b, IgG3 and IgG4. In further embodiments, the constant region may be derived from an immunoglobulin subtype from a non-human source such as any other animal, in particular a mouse.
Fragments of a whole antibody can perform the function of antigen binding. Examples of such binding fragments are; a Fab fragment comprising of the VL, VH, CL and CH1 antibody domains; an Fv fragment consisting of the VL and VH domains of a single antibody; a F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; a single chain Fv molecule (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; or a bi-specific antibody, which may be multivalent or multispecific fragments constructed by gene fusion.
A fragment of an antibody or of a polypeptide for use in the present invention, for example, a fragment of the JB1 a antibody, generally means a stretch of amino acid residues of at least 5 to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids, more preferably at least about 20 to 30 or more contiguous amino acids and most preferably at least about 30 to 40 or more consecutive amino acids. A preferred group of fragments are those which include all or part of the CDR regions of monoclonal antibody JB1a.
A "derivative" of such an antibody or polypeptide, or of a fragment of a JB1 a antibody means an antibody or polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion and/or substitution of one or more amino acids, preferably while providing a peptide having interferon alpha binding activity. Preferably such derivatives involve the insertion, addition, deletion and/or substitution of 25 or fewer amino acids, more preferably of 15 or fewer, even more preferably of 10 or fewer, more preferably still of 4 or fewer and most preferably of 1 or 2 amino acids only.
The term "antibody" includes antibodies which have been "humanised".
Methods for making humanised antibodies are known in the art. Methods are described, for example, in Winter, U.S. Patent No. 5,225,539. A humanised antibody may be a modified antibody having the hypervariable region of a monoclonal antibody such as JB1 a and the constant region of a human antibody. Thus the binding member may comprise a human constant region.
The variable region other than the hypervariable region may also be derived from the variable region of a human antibody and/or may also be derived from a monoclonal antibody such as JB1a. In such case, the entire variable region may be derived from murine monoclonal antibody JB1a and the antibody is said to be chimerised. Methods for making chimerised antibodies are known in the art. Such methods include, for example, those described in U.S. patents by Boss (Celltech) and by Cabilly (Genentech). See U.S. Patent Nos. 4,816,397 and 4,816,567, respectively.
It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDR5), of an antibody to the constant regions, or constant regions plus framework regions, of a different irnmunoglobulin. See, for instance, EP-A-1 84,187, GB 2,1 88,638A or EP-A-239,400. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
Production of Antibodies Certain methodologies for producing antibodies which have an affinity and binding specificity for beta 1 integrin are described hereinbefore.
The antibodies provided by the present invention may be provided by a number of techniques. For example, a combinatorial screening technique such as a phage display-based biopanning assay may be used to in order to identify amino acid sequences which have binding specificity to the binding epitopes of the invention. Such phage display biopanning techniques involve the use of phage display libraries, which are utilised in methods which identify suitable epitope binding ligands in a procedure which mimics immune selection, through the display of antibody binding fragments on the surface of filamentous bacteria. Phage with specific binding activity are selected. The selected phage can thereafter be used in the production of chimeric, CDR-grafted, humanised or human antibodies.
In further embodiments, the antibody is a monoclonal antibody may be produced using any suitable method which produces antibody molecules by continuous cell lines in culture. Suitable methods will be well known to the person skilled in the art and include, for example, the method of Kohler and Milstein (Kohler et al. Nature, 256, 495-497. 1975), Chimeric antibodies or CDR-grafted antibodies are further provided within the scope of the present invention. In further embodiments, the antibodies of the invention may be produced by the expression of recombinant DNA in host cell.
In further embodiments, humanized antibodies are also provided.
Humanized antibodies may be produced by the method of Winter as described in US Patent No 5,585,089.
In further certain embodiments, the monoclonal antibodies may be human antibodies, produced using transgenic animals, for example, transgenic mice, which have been genetically modified to delete or suppress the expression of endogenous murine immunoglobulin genes, with loci encoding for human heavy and light chains being expressed in preference, this resulting in the production of fully human antibodies.
The antibodies or antibody fragments of and for use in the present invention may also be generated wholly or partly by chemical synthesis.
The antibodies can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available and are well known by the person skilled in the art. Further, they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry.
Another convenient way of producing antibodies or antibody fragments suitable for use in the present invention is to express nucleic acid encoding them, by use of nucleic acid in an expression system.
Nucleic acid for use in accordance with the present invention may comprise DNA or RNA and may be wholly or partially synthetic. In certain aspects, nucleic acid for use in the invention encodes antibodies or antibody fragments for use in the invention as defined above. The skilled person will be able to determine substitutions, deletions and/or additions to such nucleic acids which will still provide an antibody or antibody fragment of the present invention.
Nucleic acid sequences encoding antibodies or antibody fragments for use with the present invention can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook et al. (1989), and Ausubel et al, (1992)), given the nucleic acid sequences and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences. DNA encoding antibody fragments may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preferences in the host cells used to express the nucleic acid.
The nucleic acid may be comprised as constructs in the form of a plasmid, vector, transcription or expression cassette which comprises at least one nucleic acid as described above. The construct may be comprised within a recombinant host cell which comprises one or more constructs as above.
Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression the antibody or antibody fragments may be isolated and/or purified using any suitable technique, then used as appropriate.
Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, insect and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse myeloma cells. A common, preferred bacterial host is E. co/i. The expression of antibodies and antibody fragments in prokaryotic cells such as E. co/i is well established in the art. Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a binding member.
General techniques for the production of antibodies are well known to the person skilled in the field, with such methods being discussed in, for example, Kohler and Milstein (1975) Nature 256: 495-497; US Patent No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, the contents of which are incorporated herein by reference.
Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example, EP 0,623,679 and EP 0,368,684, which are incorporated herein by reference.
In preferred embodiments of the invention, recombinant nucleic acids comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies are employed. By definition such nucleic acids comprise coding single stranded nucleic acids, double stranded nucleic acids consisting of said coding nucleic acids and of complementary nucleic acids thereto, or these complementary (single stranded) nucleic acids themselves.
Furthermore, nucleic acids encoding a heavy chain variable domain and/or a light chain variable domain of antibodies can be enzymatically or chemically synthesised nucleic acids having the authentic sequence coding for a naturally-occurring heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof.
Recombinant DNA technology may be used to improve the antibodies of the invention. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity within, for example, a transgenic organism such as a pig, may be minimised, by altering the antibodies by CDR grafting in a technique analogous to hurnanising antibodies.
Examples of such techniques are described in EP 0,239,400 to Winter.
In order to reduce immunogenicity within a recipient, the invention may employ recombinant nucleic acids comprising an insert coding for a heavy chain variable domain of an antibody fused to a human constant domain.
Likewise the invention concerns recombinant DNA5 comprising an insert coding for a light chain variable domain of an antibody fused to a human constant domain K or A. Antibodies may moreover be generated by mutagenesis of antibody genes to produce 5 artificial repertoires of antibodies. This technique allows the preparation of antibody libraries. Antibody libraries are also available commercially. Hence, the present invention advantageously employs artificial repertoires of immunoglobulins, preferably artificial scFv repertoires, as an immunoglobulin source in order to identify binding molecules which have specificity for the epitope of the present invention.
Antibody selection systems Immunoglobulins which are able to bind to the epitope of the present invention and which accordingly may be used in the methods of the invention can be identified using any technique known to the skilled person. Such immunoglobulins may be conveniently isolated from libraries comprising artificial repertoires of imrnunoglobulin polypeptides.
A "repertoire" refers to a set of molecules generated by random, semi-random or directed variation of one or more template molecules, at the nucleic acid level, in order to provide a multiplicity of binding specificities.
Methods for generating repertoires are well characterised in the art.
Any library selection system may be used in conjunction with the invention. Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques.
Such systems, in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage, have proven useful for creating libraries of antibody fragments (and the nucleotide sequences that encode them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen. The nucleotide sequences encoding the VH and VL regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E. co/i and as a result the resultant antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage coat proteins (e.
g.,pIII or pVIII). Alternatively, antibody fragments are displayed externally on lambda phage capsids (phage bodies). An advantage of phage-based display systems is that, because they are biological systems, selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells. Furthermore, since the nucleotide sequence that encodes the polypeptide library member is contained on a phage or phagernid vector, sequencing, expression and subsequent genetic manipulation is relatively straight forward.
Methods for the construction of bacteriophage antibody display libraries and lambda phage expression libraries are well known in the art (for example, McCafferty et al. (1990) Nature 348 552-554. One particularly advantageous approach has been the use of scFv phage-libraries (see for example Huston et al., 1988, Proc. NatI. Acad. Sci USA).
An alternative to the use of phage or other cloned libraries is to use nucleic acid, preferably RNA, derived from the B cells of an animal which has been immunised with the selected target, e.g. beta 1 integrin.
Isolation of V-region and C-region mRNA permits antibody fragments, such as Fab or Fv, to be expressed intracellularly. Briefly, RNA is isolated from the B cells of an irnmunised animal, for example from the spleen of an immunised mouse, and PCR primers used to amplify VH and VL cDNA selectively from the RNA pool. The VH and VL sequences thus obtained are joined to make scFv antibodies. PCR primer sequences may be based on published VH and VL sequences.
Peptidomimetics Whilst numerous strategies to improve the pharmaceutical properties of peptides found to exert biological effects are known in the art including, for example, amide bond replacements, incorporation of non-peptide moieties, peptide small molecule conjugates or backbone cyclisation, the optimisation of pharmacological properties for particular peptides still presents those involved in the optimisation of such pharmaceutical agents with considerable challenges.
Peptides of and for use in the present invention may be modified such that they comprise amide bond replacement, incorporation of non peptide moieties, or backbone cyclisation. Suitably if cysteine is present the thiol of this residue is capped to prevent damage of the free sulphate group.
Suitably a peptide of and for use in the present invention may be modified from the natural sequence to protect the peptides from protease attack.
Suitably a peptide of and for use in the present invention may be further modified using at least one of C and I or N-terminal capping, and I or cysteine residue capping. Suitably a peptide of and for use in the present invention may be capped at the N terminal residue with an acetyl group.
Suitably a peptide of and for use in the present invention may be capped at the C terminal with an amide group. Suitably the thiol groups of cysteines are capped with acetamido methyl groups.
Pharmaceutical Compositions As described above, the present invention compounds which allosterically modulate betal integrin such that is assumes an intermediate affinity state. Pharmaceutical compositions for use in accordance with the present invention may comprise, in addition to active ingredient (i.e. an allosteric modulator of beta 1 integrin which induces the beta 1 integrin to assume an intermediate affinity state), a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be, for example, oral, intravenous, intranasal or via oral or nasal inhalation. Dose
The modulatory compounds of the present invention are preferably administered to an individual in a "therapeutically effective amount", this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the condition being treated.
Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners, physicians or other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. The optimal dose can be determined by physicians based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered and the route of administration.
Small Molecules In various further aspects, the present invention relates to screening and assay methods for use in identifying compounds which act as allosteric modulators of beta 1 integrin activity. Certain further aspects extend to the compounds identified thereby, wherein said binding compounds have affinity and binding specificity for the epitope of the invention.
A substance identified an allosteric modulator of beta 1 integrin may be a peptide or may be non-peptide in nature, for example a peptidomimetic as described hereinbefore. However, non-peptide "small molecules" are often preferred for many in-vivo pharmaceutical uses. Accordingly, a mimetic or mimic of a compound which acts as an allosteric modulator of beta 1 integrin for use in the present invention may be designed for pharmaceutical uses.
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise, or where it is unsuitable for a particular method of administration. For example, peptides are not well suited as active agents for oral compositions and administration as they are degraded by proteases present in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.
There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, for example by substituting each amino acid residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore".
Once the pharmacophore has been determined, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can also be used in this modelling process.
In a variant of this approach, the three-dimensional structure of beta 1 integrin extracellular domain, or a compound which binds thereto, and which acts as an allosteric rriodulator is modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of the design of the mimetic.
A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in-vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in-vivo or clinical testing.
In certain embodiments, the mimetic binding compound may be a natural or synthetic chemical compound used in drug screening programmes.
Extracts of plants which contain several characterised or uncharacterised components may also be used.
A candidate binding compound which has affinity and binding specificity to beta 1 integrin may be isolated and/or purified, manufactured and/or used to modulate beta 1 integrin allosteric activity.
In yet further aspects, the invention extends to the use of combinatorial library technology (Schultz, JS (1996) Biotechnol. Frog. 12:729-743) which provides an efficient way of testing a potentially vast number of different substances for ability their ability to bind to an epitope or to modulate the activity of a ligand which binds to an epitope. Prior to, or as well as, being screened for modulation of activity, test substances may be screened for ability to interact with the polypeptide, e.g. in a yeast two-hybrid system (which requires that both the polypeptide and the test substance can be expressed in yeast from encoding nucleic acid). This may be used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide.
The amount of test substance or compound which may be added to an assay of the invention will normally be determined by trail and error depending upon the type of compound used. Typically, from about 0.01 to nM concentrations of inhibitor compound may be used, for example from 0.1 to 10 nM. Greater concentrations may be used when a peptide is the test substance.
Combination medicaments The present invention extends to combinational therapies wherein compositions or methods relates to the administration of an allosteric modulator of beta 1 integrin according to the invention, which is administered to a subject in combination with at least one further therapeutic compound which serves to, for example, suppress the immune response which mediates tissue damage or injury.
Typically the primary and secondary therapeutic compositions are given contemporaneously. In certain embodiments, the primary therapeutic composition (i.e. the beta 1 integrin allosteric modulator) and the secondary therapeutic compounds are administered simultaneously. In certain further embodiments, they are administered sequentially.
Treatment I Therapy The term treatment' is used herein to refer to any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment).
Treatment may include curative, alleviation or prophylactic effects.
More specifically, reference herein to "therapeutic" and "prophylactic" treatment is to be considered in its broadest context. The term "therapeutic" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition.
Accordingly, therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylactic" may be considered as reducing the severity or the onset of a particular condition. "Therapeutic" may also reduce the severity of an existing condition.
Administration The allosteric modulatory compound of the present invention, such as an antibody or protein may be administered alone but will preferably be administered as a pharmaceutical composition, which will generally comprise a suitable pharmaceutical excipient, diluent or carrier selected depending on the intended route of administration. The allosteric modulator may be administered to a patient in need of treatment via any
suitable route.
Routes of administration may include; parenteral administration (including subcutaneous, intramuscular, intravenous, by means of, for example a drip patch). Further suitable routes of administration include (but are not limited to) oral, rectal, nasal, topical (including buccal and sublingual), infusion, vaginal, intradermal, intraperitoneally, intracranially, intrathecal and epidural administration or administration via oral or nasal inhalation, by means of, for example a nebuliser or inhaler, or by an implant.
The composition may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of sustained release carriers include semi-permeable polymer matrices in the form of shared articles, e.g. suppositories or microcapsules.
Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Lippincott Williams & Wilkins; 20th edition (December 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, H.C. et al. 7th Edition ISBN 0- 683305-72-7 the entire disclosures of which is herein incorporated by reference.
Mimetics A substance identified as an allosteric modulator of betal integrin may be peptide or non-peptide in nature. Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly is a peptide) may be designed for pharmaceutical uses. The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesise of where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.
There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore".
Once the pharmacophore has been found, its structure is modelled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used I this modelling process.
In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the rnirrietic is easy to synthesise, is likely to be pharrriacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the led compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
Mimetics of substances identified as having ability to modulate the confirmation of betal integrin are included within the scope of the present invention. A polypeptide, peptide or substance which can modulate the confirmation of beta 1 integrin according to the present invention may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment. Such a kit may include instructions for use.
EXAMPLES
Example I -Allosteric modulation of betal integrin function reverses functional impairment and lung iniury in a mouse model of emphysema Chronic Obstructive Pulmonary Disease (COPD) is characterised by poorly reversible and progressive airflow limitation associated with chronic bronchitis and emphysema. There are no effective treatments for emphysema to halt, slow or reverse the disease.
There is also limited information on how the lung is remodelled in emphysema. The extracellular matrix (ECM) interacts with integrins and modifies their function. In turn, integrins are key regulators of the ECM.
Integrins bind metalloproteinases (MMPs), these being key enzymes in ECM homeostasis, and thus affects their activation state.
Excision of beta 1 integrin gene in cardiac myocytes resulted in postnatal cardiac fibrosis, depressed cardiac function and increased myocardial glucose metabolism leading to spontaneous heart failure. Conditional deletion of betal integrin in the intestinal epithelium of mice causes a significant increase in epithelial proliferation without affecting cell adhesion.
Although transgenic approaches have highlighted the crucial role for betal integrin, they do not elucidate more subtle functions, such as those controlled by avidity regulation.
Materials and methods PPE-induced air space enlarqement model in mice Female C57/BL6 mice (6-8 weeks old) were instilled intra-tracheally with porcine pancreatic elastase as detailed before. At day 14 or 21, mice were treated intra-tracheally with the anti-integrin antibody JB1 a at 3mg/kg in sterile PBS. The dose chosen is equivalent to the dose of clinically used antibodies against a4131 integrin (Miller DH, et al. 2003). Control group was instilled initially with PBS and at day 14 or 21 with PBS. For the group treated at day 14, the animals were terminated at day 21 as follows: The animals were anaesthetised using sodium pentobarbitone (45mg/kg), paralysed using pancuronium bromide (0.8mg/kg) and tracheostomised and ventilated using a small animal ventilator (Flexivent, SCIREQ, Montreal) at 8m1/kg and a rate of 150 breaths/minute and positive end expiratory pressures (PEEP) of 3.5cm H20.
The pressure-volume curve was obtained during inflation and deflation in a stepwise manner by applying volume perturbation incrementally during 16 seconds. The pressure signal was recorded and the pressure-volume (P-V) curve calculated from the plateau of each step. The constant K was obtained using the Salazar-Knowles equation and reflects the curvature of the upper portion of the deflation P-V curve. Quasi-static elastance reflects the static elastic recoil pressure of the lungs at a given lung volume. It was obtained by calculating the slope of the linear part of P-V curve.
After the measurements, the animals were sacrificed and bronchoalveolar lavage collected. The bronchoalveolar lavage was centrifuged at 2000 rpm for 10 minutes and the supernatants lyophilised and analysed for rnetalloproteinase-12 (clone M19, polyclonal goat IgG, Santa Cruz) by Western blotting.
H istochem istry The lungs were removed and formalin-fixed at a pressure of 25cm H20, paraffin-embedded and sectioned at 4 pm thickness. Sagittal sections were used from each animal for histological and immunohistochemical assessment of damage, and morphometric analysis (mean linear intercept, Lm). Images from 10 fields per section were digitised using Image-Pro plus (version 5.1) and micropublisher 3.3 RTV camera connected to a Zeiss axioskope RTM with 10 x objective. The field size was 0.83 pm x 0.63 pm. Mean linear intercept was calculated from each field (horizontal and vertical) by dividing the length of the line by the number of alveolar intercepts.
Gordon and Sweet's Reticulin stain was performed on 3pm deparaffinised sections as a method of visualising collagen distribution in both injured and vehicle-treated lung. Briefly, sections were oxidised with 0.5% potassium permanganate, bleached with 1 % oxalic acid, and treated with 2.5% arrirrionium iron (III) sulphate and a 5% silver nitrate/i.5% sodium hydroxide solution prior to reduction with 10% unbuffered formaldehyde.
Visualisation of reticulin fibres was accomplished via treatment with 0.1% sodium chloroaurate and 3% sodium thiosulphate solutions. Sections were counterstained with neutral red prior to mounting.
Imacie Processinci and Stereolociical Analysis Colour images of 3pm reticulin-stained sections were subjected to automated image processing methods developed in-house using Image Pro Plus (Media Cybernetics). Briefly, a greyscale image representing the green channel (which shows the highest differentiation from background) was obtained by splitting the RGB channels. Sobel edge detection operators, dilation-erosion and erosion-dilation algorithms were used to enhance the image and to remove salt and pepper' noise. An area of interest measuring 200pm x 200 jim was overlaid over a digitally-captured image of a reticulin-stained section and only events completely inside the frame or intersecting the two inclusion edges of the frame's left and lower aspects (South-West) were included. Events intersecting the right and upper aspects (North-East) of the frame were excluded, as were blood vessels other than the capillaries of the alveolus. 2D objects that were counted using this method included alveolar septal junctions, free septal ends, and contiguous airspaces. A total of 15 non-overlapping frames were included per section as this was sufficient to achieve a running mean. The mean number of events on sections obtained from vehicle-treated (n=5), PPE-treated (n=5), and JB1 a-treated (n=5) mice were scored as a function of area (counts per 200 jim x 200 jim field of view).
Immunoh istochem istry 3pm paraffin sections of lung were deparaffinised, rehydrated, and washed in water. For TTF-1 (clone 8G7G311, DAKO) immunohistochemistry, a microwave antigen retrieval method was used (3 x 5 minute 700W microwave treatments, in 10mM citrate buffer, pH 6.0).
GATA-6 (H-92 rabbit polyclonal IgG, Santa Cruz) immunohistochemistry required proteinase K pretreatment (2Opg/ml, 20 minutes, 37°C).
Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in water. Endogenous biotin was blocked with Avid in/Biotin Blocking Kit (Vector). Sections were blocked with either M.O.M IgG Blocking Reagent (Vector) or DAKO Protein Block Serum-Free (used for rabbit polyclonal antibodies) prior to incubation with primary antibody (diluted 1:50) overnight at 4°C. The following day, sections were washed 5 times in TBST (Tris-Buffered Saline pH 7.6, with 0.1% Tween 20) prior to incubation with biotinylated secondary antibody (30 minutes) and DAKO StreptABComplex/HRP (20 minutes). Immunohistochemical staining was visualised using DAB chromogen, a haematoxylin counterstain was used for sections other than those used to generate 3D reconstructions.
Sections were washed, dehydrated and mounted in DPX. Negative controls omitted the primary antibody step.
3D Reconstructions consecutive 3pm sections were used to generate 3D reconstructions of tissue parenchyma. Odd (1, 3, 5...) sections were labelled with GATA-6 whilst even (2, 4, 6...) sections were labelled with TTF-1. Digitally captured images were obtained with a QCapture Pro imaging system and a brightfield microscope (Zeiss Axioskop RTM, 20x objective lens) and were patched into larger images (3x3), which were interleaved to form their original order in an image stack prior to alignment using Adobe Photoshop RTM 7.0.1. TIFF images were cropped, resized (6000x6000 pixels) and image stacks were imported into Amira 4.0 for isosurtace rendering, which was accomplished at threshold values sufficient to detect either immunohistochemical label or tissue parenchyma. Isosurfaces were coloured either red (TTF-1), blue (parenchyma) or yellow (GATA-6) for enhanced visualisation.
Apoptosis measurement Terminal Deoxyribonucleotidyl Transferase (TdT)-Mediated dUTP Nick End Labelling (TUNEL) was assessed in sections using the Red ApopTagTM Kit (Chemicon). Data for the quantification of positively stained apoptotic nuclei was acquired using the x40 oil objective of a Zeiss 510 Axiovert RTM confocal microscope system (Carl Zeiss Ltd, Welwyn Gardens City, Herts, UK). The stage-tiling utility was employed for the collection of 4x4 tiled images, equivalent to a total area of 0.921mm x 0.921mm, imaged from a lung section of -8mm x 8mm (two tiles each from right and left lobes). Images of mainly alveolar tissue were constructed. The images were then converted to 8-bits grey scale and ImageJ was used to count total number of cells. TUNEL positive cells were counted manually.
F-actin staining in 35dav mouse lungs Separate experiments were performed where lung tissue was fixed using fixed volume of 4% paraformaldehyde for 1 hour at 37oC. The tissue was then immersed in 40% sucrose in PBS overnight at 4oC. The lung tissue blocks were embedded into OCT and frozen. Cryostat sections (4 microns) were stained using Alexa-fluor RTM phalloidin and TO-PRO3 (Molecular Probes). Images were acquired using Zeiss Axioskope RTM using a x63 oil achroplan objective and analysed using LSM 5 Image software.
Caspase 3/7 activity measurements using human mesenchyme and epithelial cell co-culture in vitro Adult human lung fibroblasts (CCD-8Lu) were seeded onto collagen I coated BioFlex RTM 6 well plates at 0.5 X 106/well. The following day, NCI-H441 were seeded on top of the fibroblasts at the same density. NCI-H441 cells possess alveolar type II cell characteristics. Cells were starved with media containing 0.1 % FCS. The plates were subjected to stretching at 0- 5%, 0-10% or 2-10% sinusoidal stretch at 1 Hz for 6 hours. Control plates on plastic or bioflex RTM plates without stretch were also included. PPE was added at 0.06 or 0.3U/ml alone or in combination with JBIa (1 pg/mI) and ZVAD-fmk at 10 or 20 pM. At the end of the 6 hour period, the media was aspirated and caspase 3 activity assayed using Caspase-GIoTM 3/7 (Promega) according to the manufacturers' instructions. Separate experiments were performed at up to 6 hours of stretching where the cells were fixed with 4% paraformaldehyde and stained using Alexa-fluor RTM phalloidin and TO-PRO3 (Molecular Probes). Images were aquired using Zeiss Axioskope RTM using a x63 water achroplan objective and analysed using LSM 5 Image software.
In a separate set of experiments lung fibroblasts and epithelial cells were seeded onto 96 multi-well plates as described above. The cells were starved in media containing 0.1% FCS then in DMEM-glucose-free with 0.1% FCS for 45 minutes before treating with (i) PPE at O.3U/ml alone or (ii) PPE preceded by JB1a (1 jig/mI). At the end of the experiment, AlP levels were measured using a bioluminescent ATP kit (Perkin Elmer).
Human lunç exilant culture and human lung cell isolation Human lung tissue specimens were obtained (and approved by NHS ethics committee and with consent) and cultured as either 20-30 mg explant strips or cells. Alveolar epithelial cells were isolated as described before (Elbert et. al., 1999 and Murphy et al., 1999). The cells were then plated onto Transwells of 0.3um pore size (Sigma) and maintained in culture using 1:1 DMEM/F12:Small airway growth media (Cambrex BioScience Wokingharn Ltd.) containing 1% foetal calf serum. The remaining tissue was treated with DMEM containing 40% foetal calf serum to inactivate the digestive enzymes and then washed using HEPES buffer.
To isolate fibrobiasts and smooth muscle cell populations, the tissue was then incubated in DMEM containing 1mg/mi collagenase, 0.5% trypsin and 200U/g DNAsI and maintained at 5% in an 002 incubator. The cell suspension was washed as above and cells seeded on muitiweil culture plates and maintained in DMEM containing 10% foetal calf serum.
Cultures were subjected to serum starving overnight in a medium containing 0.1 % foetal calf serum. Some coilagenase digested plated cells were co-cultured with isolated alveolar epithelial cells Transwells at the time of commencing the starvation.
Functional modifying antibody against betal integrin (Chemicon, clone JB1a, IgG1; also commercially available as JB1A mouse anti-CD29 (Integrin beta-i subunit) IgG monoclonal antibody, unconjugated, clone JBia, Millipore, Catalogue number MABi965) was added to the cultures at a concentration of i.44 and 0.48 pg/mI. The betai integrin stimulatory antibody clone TS2Ii 6 (IgGi) was also added at 0.9pg/mI for i hour to demonstrate the specificity of the JB1 a action. After antibody addition to the cells in culture, the medium was aspirated and the cell layer rinsed twice with ice-cold PBS. The media was aspirated and preserved after the addition of protease inhibitors at 80°C. In additional experiments, the effect of protein synthesis inhibition on betai integrin mediated proteoglycan increase was tested by pretreating the human lung derived cells with 25pM cycloheximide. The effect of non-specific activation of rnetalloproteinases on betai integrin mediated proteoglycan increase was tested by pretreating the human lung derived cells with 0.5M APMA (aminophenylmercuric acetate). To investigate the involvement of selected rrietalloproteinases in initiating the response observed with beta 1 integrin, specific neutralising antibodies for metalloproteinase-7 (i:i 000, goat IgG, R&D systems) and metalloproteinase-9 (i:1000 of clone 6-6B, mouse IgGi, Oncogene Research Products) were used. A broad spectrum inhibitor of metalloproteinases was also used at 2.3nM (metalloproteinase inhibitor III, Calbiochem).
SDS-PAGE was also used to separate the denatured proteoglycan and proteins from the tissue concentrated tissue culture supernatants.
Perlecan antibody immunoreactive to non-degraded forms of perlecan was used (7B5, mouse IgGi, Zymed Laboratories). Metalloproteinase-1 (clone 41-1 E5, IgG2a against amino acids 332-350 of human metalloproteinase- 1), inactive metalloproteinase-9 (clone 7-1 10, mouse IgGi) and TIMP1 (clone 7-6C1, mouse IgGi) antibodies were all derived from Oncogene Research Products.
Time-lapse studies Cells were cultured as described in methods at 50,000 cell/membrane onto collagen I Bioflex RTM membranes using silicone gaskets of 10mm diameter. Cells were starved with media containing 0.1% FCS and Syto 16 (Molecular Probes). The media was removed and Alexa-Fluor RTM 647 labelled G-actin (lOOpg/membrane) from rabbit was loaded using Influx (Molecular Probes). The cells were loaded with PhiPhiLux-G2D2 for visualisation of caspase activation (Oncoimmune). The membrane was then mounted onto the StageFlexer (FlexCell), placed on the stage of an upright Leica-TCS-NT confocal microscope system (Leica Microsystems GmbH, Heidelberg, Germany) and subjected to 2-10% cyclic stretch at 1 Hz for up to 6 hours. Images were collected simultaneously from 3 channels at 1 minute intervals, using the xlO lens. The resulting time-lapse movies were collated and analysed with Imaris RTM software (Bitplane AG, Switzerland). At various time points during the study, the membrane was held static while serial optical sections were acquired, the three fluorescent channels supplemented by the collection of the
brightfield channel image.
Three-dimensional confocal microscopy NCI-H441 cells and human lung fibroblasts were cultured as described above onto collagen I-coated glass cover slips at 20,000 cells within an area of 5mm in diameter. The media was removed and Alexa-Fluor RTM 647 labelled G-actin (3Opg/coverslip) from rabbit was loaded using Influx (Molecular Probes). The cells were loaded with PhiPhiLux-G2D2 for visualisation of caspase activation (Oncoimmune) and FL-ganglioside 1 (GM1, Molecular probes) to visualise the plasma membrane. Images were collected through 4 separate channels (GM 1: A = 488,caspase A = 568, actin: A = 647 nm and brightfield) using x63 water lens and Zeiss LSM51O CLSM microscope. The resulting images were analysed with Imaris RTM software (Bitplane AG, Switzerland). Three-dimensional images were reconstructed.
Caveolin-1 extraction and analyses NCI-H441 cells and human lung fibroblasts were cultured as described above. Media was changed prior to the experiment to 0.1% FCS containing media. Vehicle, PPE (0.3UIml) or PPE in the presence of JB1a (1 pg/mI) containing media was added onto the cells for 2 hours. Cells were extracted using lysis buffer composed of 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 1 mM Na3VO4 containing 1% Triton RTM X-100 and protease inhibitors (Roche) for 1 hour at 4°C and sheared repeatedly using 22G needle on ice. Total protein was quantified using Bradford's assay method (BioRad). The extracts (1 ml) were mixed with equal volume of 85% w/v sucrose in extraction buffer without Triton RTM X-1 00 and layered with 2m1 of 35% w/v sucrose in buffer and then 1 ml of 5% w/v sucrose in buffer.
The gradients were prepared at 4°C. The gradients were then centrifuged at 38,000 rpm for 16 hours at 4°C. Fractions were collected from the top (0.4 ml fractions) and separated onto 13% SDS-PAGE and transferred onto nitrocellulose membranes then probed for 131 integrin using JB1a (Chemicon), caveolin-1 (BD Biosciences), phosphorylated caveolin-1 (Cell Biosciences) and talin (Sigma-Aldrich), and developed with HRP-conjugated secondary antibodies using the ECL-plus chemiluminescence system (Amersham Biosciences).
Shinqomyelinase activity measurements in vivo and in vitro Adult human lung fibroblasts (CCD-8Lu) were seeded onto collagen I coated BioFlex RTM 6 well plates at 0.5 X 106/well. The following day, Nd-H441 were seeded on top of the fibroblasts at the same density. Cells were starved with media containing 0.1% FCS. The plates were subjected to stretching at 2-10% sinusoidal stretch at 1 Hz for 5, 10, 15 and 30 minutes. PPE was added at 0.3U/ml alone or in combination with JB1a (2pg/ml). At the end of stimulation, the media was aspirated and sphingomyelinase was were extracted using 200u1 buffer composed of 20mM HEPES, 1mM sodium fluoride, 0.1mM sodium molybdate, EDTA-free protease inhibitors (Roche) and 0.2% Triton RTM x-100 (pH 7.4).
Sphingomylinase activity was assayed using the amplex RTM red assay (Molecular Probes) according to manufacturer's instructions.
In separate in vivo experiments, mouse lung tissue (35 day group) was frozen using liquid nitrogen. The tissue was then sliced at 4Oum thick section using a cryostat and extracted using the above mentioned sphingomyelinase buffer for 1 hour. Sphingomyelinase activity was assayed over time using the amplex RTM red assay (Molecular Probes).
The activity was calculated according to wet tissue weight.
Lipid raft formation in vitro In a separate set of experiments lung fibroblasts and epithelial cells were seeded onto collagen I-coated glass coverslips at 20,000 cells within an area of 5mm in diameter. The media was removed and the dye laurdan was added. The lipophilic probe, 2-dimethylamino-6-lauroyl-naphthalene or Laurdan (Molecular Weight 353.54,lOOmg or 0.28mmoles, Aldrich-Sigma), was prepared in DMSO (Sigma) to give a 6 mM stock solution (dissolve 100mg in 47u1).lul of Laurdan stock solution was added per coverslip in the dark at 37°C, and incubated for 15 minutes.
Laurdan fluorescence was excited with a titanium-sapphire laser. Laurdan intensity images were recorded simultaneously with emission in the range of 400-460 nm and 470-530 nm for the two channels, respectively. The relative sensitivity of the two channels was verified by exposing control cells to 1 mg/mI methyl-beta-cyclodextrin for 1 hour to deplete lipid rafts Images were collected using x63 water lens and Zeiss LSM51O CLSM microscope. The resulting images were analysed with Imaris RTM software (Bitplane AG, Switzerland). Three-dimensional images were reconstructed.
The excitation generalized polarization (GPex) spectra were derived from the following equation.
where L40 and 1490 are the intensities at each excitation wavelength, from 320 to 410 nm, using a fixed emission of 440 and 490 nm, respectively.
Electric Cell-Substrate Impedance Sensing (ECIS) Measurments Epithelial-mesenchymal human lung cell cultures were grown on collagen I pre-coated ECIS-arrays (model 8W1OE, Applied BioPhysics, Tray, NY), which consist of 8 wells with 10 electrodes deposited on the bottom of each well (Applied-Biophysics, Inc. Tray, NY). ECIS-measurements were performed via the ECISTM Model 1600 (Applied BioPhysics, Tray, NY).
Both data acquisition and processing were performed using the ECIS Data Analysis Software supplied by Applied Biophysics (Tray, NY).
Media (DMEM/F12: MEM at 1:1 containing 0.1 foetal bovine serum) was added and a baseline was established for 2 hours. The media was then changed to one containing: PBS, porcine pancreatic elastase (0.3U/ml), JB1a (2ug/ml) or a combination of elastase and JB1a monoclonal antibody. ECIS measurements were carried out for at least 4 hours at 37 °C and 5% CO2 at 30 Hz and 1 recording/minute.
ECIS instrumentation The sensing area within a single well of the 8-well ECIS arrays consists of active working electrodes in parallel that are distributed over the bottom of the well, and a counter electrode much bigger in surface area. A non-invasive alternating current (< 1 pA) is applied to the electrodes which are electrically connected via the electrolytes of the cell culture medium above the cells. The ECIS device reads the associated voltage drop across the system (in-phase with the current) and determines the electrical resistance of the cell-covered electrodes. Previous studies have indicated that the resistance measured is most sensitive to monitor the establishment of cell-cell junctions as a function of time.
Experimental Protocols and Results The experimentation first considered whether beta 1 integrin allosteric modulation altered the ECM. This experimentation focused on perlecan because of its known role in basement membrane integrity.
In a co-culture of primary human alveolar epithelial cells with lung interstitial cells, the anti-betal integrin antibody JB1a caused an increase in perlecan (Figure 5(a)); a change that persisted for 24 hours and was partially sensitive to pre-treatment with cycloheximide and the non-specific MMPs activator aminophenylmercuric acetate (APMA). Treatment with neutralising anti-MMPs 7 and 9 antibodies failed to produce a similar increase in perlecan expression. The changes in perlecan in response to JB1a were accompanied by an increase in tissue inhibitors of metalloproteinase-1 (TIMP1) initially (Figure 5(b)) and pro-MMP-9 subsequently (Figure 5(c)).
The results therefore indicate that JB1 a-induced changes in proteoglycan are not solely due to alteration in the MMPITIMP balance. Similar responses in human lung explants and the alveolar cell line, NCI-H441, were also obtained and were specific to JB1a treatment (data not shown).
As controls, it was shown that the anti-beta 1 integrin antibody TS2I1 6, which binds the 207-218 amino acid sequence, and 6S6 clone, which binds a discontinuous unmapped epitope, had no effect on proteoglycans.
In order to investigate whether allosteric modulation of betal integrin was of therapeutic value in a model of emphysema, mice were treated with the anti betal integrin monoclonal antibody, JB1a, or vehicle, either once on day 14(21 daygroup, 21d)oron days 21 and 28 (3sdaygroup, 35d)after elastase instillation and the achievement of maximal air space enlargement on the basis of a pilot time course study and previous reports. Diseases such as emphysema, where remodelling is a key feature were assessed. Both JB1a and 6S6 clones bind betal integrin in mouse tissues (U. Cavallaro, et al., Nat.Cell Biol. 3, 650-657 (2001)). An additional group receiving elastase and the isotype control IgGi, MOPC21, was performed and showed no reversal of elastase-induced structural damage (data not shown). The clone 6S6 did not induce repair in vivo (data not shown).
A marked leftward shift in the respiratory pressure-volume curve (PV) in response to elastase was observed, and there was a progressive increase in the PV shape exponential constant, k, particularly in the 35 day group (figure 1 (a)-(b)). This shift was reversed by JB1 a treatment (figure 1(a)- (b), for 35 day data, 21 day data not shown). Furthermore, the effect of elastase treatment on the peak pressures and quasi-static elastance derived from the PV curves were significantly reversed by JB1a treatment (figures 1(c) and 1(d)).
Lung morphometry demonstrated that JB1a treatment also reversed elastase-induced air space enlargement (Figures 2(a) and 2(b)). The explanation for the discrepancy between the functional and structural differences of the 21 day and 35 day treatment groups reflects the presence of inflammation, noted histologically only in the 21 day group.
The functional reversal was accompanied by reversal in elastase-induced damage to elastic fibres (data not shown).
To study the effect of JB1 a on regeneration of lung and alveolar septation, we used image analysis of histological sections from the 21 day group to determine the number of contiguous airspaces, septal junctions and septal ends (Figures 2(c)-(e), Figure 6 for method). Elastase pre-treatment decreased the number of contiguous airspaces and was reversible by JB1a treatment (Figure 2(c)). In addition, elastase pre-treatment caused a decrease in the number of septal junctions which was partially reversed by JB1 a (Figure 2(d)). No change was seen in the number of septal ends (Figure 2(e)). Using 3D irriage reconstructions of expression pattern of TTF-1 and GATA-6 in 2lday group animals, two transcription factors which control septation 13;14, we found that GATA-6 expression increased following elastase pre-treatment (Figure 3(a)). Treatment with JB1 a induced increased numbers of TTF-1 expressing cells, and attenuated the GATA-6 upregulation associated with elastase-injury.
Allosteric modulation of beta 1 integrin affects both mitosis and cell death.
Since cell death has been implicated in COPD progression, the prevalence of TUNEL positive cells in mouse lung at days 21 and 35 following injury with or without JB1 a treatment was evaluated. There was a significant increase in TUNEL positive cells in the lung both at 21 days and 35 days post-elastase instillation: this was significantly reduced by subsequent treatment with JB1 a (Figures 3(b) and (c)).
Elastase can induce apoptosis of epithelial cells secondary to altered attachment (anoikis), accompanied by alteration in mitochondrial permeability and caspase activation. In a co-culture of adult human lung fibroblasts overlayered with NCI-H441 cells, under cyclic mechanical stimulation, we showed that elastase treatment caused a significant increase in caspase 3 and 7 activity. Addition of JB1a to the cultures inhibited elastase-induced caspase activation in a similar fashion to the broad spectrum caspase inhibitor, ZVAD-fmk (Figure 4(a)). Furthermore, it was found that elastase treatment increased F-actin aggregates, changes inhibited by both JB1a and ZVAD-fmk (Figure 4(b)). In time-lapse studies 3D reconstructions, it was demonstrated that the increase in F-actin formation during the course of elastase-induced injury which was followed by activation of caspase 3 and 7 (Figures 7(a)-(c) and 8). These findings are supportive of the notion that cytomechanics determine cell fate and effect repair.
Formation of F-actin from monomeric G-actin is energy dependent and under ATP depletion conditions, there is a net conversion of monomeric G-actin to polymeric F-actin. We measured ATP levels in co-cultures in response to elastase and JB1a. Elastase reduced the levels of ATP, a response inhibited byJBla (Figure 4(c)).
Betal integrin-mediated adhesion has been shown to regulate cholesterol- rich membrane microdomain internalisation mediated by phospho- caveolin-1. In co-cultures, elastase significantly reduced cell membrane-associated beta 1 integrin levels and induced phosphorylation of caveolin-1 within two hours; changes inhibited by JB1a (Figure 4(d)).
It has previously been shown that the administration of beta4 thymosin, an actin-sequestering protein, promoted cardiac repair after ischemia-reperfusion injury. Changes in actin dynamics also affect mitochondrial function and the release of reactive oxygen species eliciting death signals.
Additionally, gene disruption of caveolin-1, which is known to be involved in integrin clustering and activation, results in pulmonary fibrosis and impairment in liver regeneration after partial hepatectomy which was reversible by treatment with glucose, indicating the probable importance of energy preservation.
In conclusion, it is suggested that elastase-induced progressive damage is partly due to anoikis resulting from an increase in cytoskeletal tension caused by increased actin polymerisation, rendering the cell rigid and susceptible to physiological forces. Without being bound by theory, the inventors predicts that the allosteric modulation could effect alteration in the receptor focal density leading to changes in cytoskeletal organisation and cell membrane composition hence affecting cell behaviour and fate.
Conclusions
The present experimentation has identified a novel cellular mechanism for mediating repair in cases of lung injury caused by emphysema. The surprising advances in understanding made by the inventors in relation to the role of betal integrin in emphysema and how altering its allostery facilitates structural and functional repair have elucidated a novel mechanism of cellular injury in emphysema to which a therapeutic approach for treatment can be applied.
Significant components of the mechanism of action of beta 1 integrin-med iated repair include the following: A. The role of the actin cytoskeleton in betal integrin mediated repair in lung emphysema and in particular (i) the in-vitro findings which show an alteration in actin polyrnerisation, and (ii) the observation that changes in receptor movement can cause membrane disruption thus wounding the cell membrane. Integrin engagement in addition to calcium influx can cause a wide range of responses one of which is initiation of actin polymerisation to form a "sealant" to be filled later with membrane rnicrodomains. It has been reported that the induction of calcium oscillations rather than the net concentration protects from injury in adult tissue; a feature present in the developing tissues. The present experimentation has shown a similar phenomenon in ATP oscillation in two independent injury models. This part identifies the role of allosteric changes in betal integrin and its effects on cellular mechanics and cytoskeletal alterations in repair after injury in the lung.
B. The role of receptor clustering and/or altered diffusibility in injury and betal integrin repair. Recently, betal integrin-rriediated adhesion was shown to regulate cholesterol-rich membrane microdomain internalisation mediated by phospho-caveolin-1. Gene disruption of caveolin-1 in mice resulted in pulmonary fibrosis as well as cytoskeletal changes and inhibition of focal adhesion kinases in-vitro. It has also been shown that in these mice there is impairment in liver regeneration after partial hepatectomy which was reversible by treatment with glucose.
Experimentation was conducted in which epithelial-mesnchymal co-cultures were exposed to vehicle, elastase alone or in the presence of allosteric modifying anti-betal integrin antibody. Elastase induced caveolin-1 phosphorylation and this effect was inhibited by allosteric modulation of beta 1 integrin. Furthermore, elastase resulted in a significant reduction in membrane associated betal integrin levels in two hours. Association of beta 1 integrin with lipid-rich domains 3 hours after the addition of elastase was not detected. However, the presence of phospho-caveolin-1 in the heavy fractions of extracts from the elastase- injured cultures was detected. The addition of allosteric modifying anti-betal integrin antibody inhibited the elastase-induced changes.
Sphingomyelin is a ubiquitouse lipid of cell membranes with the plasma membrane containing 70-90% of total cellular content. Sphingomyelin is hydrolysed by sphingomyelinases (SMase) into ceramide and phosphocholine. Ceramide is degraded into sphingosine and fatty acid by ceramidases. To date there are five SMase isoforms; alkaline SMase, lysosomal acidic SMase (aSMase), secreted Zn2-dependent acidic SMase, membrane-bound Mg2-dependent SMase (nSMase), and cytosolic Mg2-independent SMase. The SMase isoforms differ in their subcellular localization, tissue specificity, and enzymatic properties. The role of aSMase and ceramide synthesis in platelet-activating factor, endotoxin or acid instillation induced acute lung injury.
Ceramide is a lipid "second messenger" molecules involved in the regulation of cell differentiation and TNF and Fas-induced apoptosis as well as stress-induced apoptosis. The alteration in composition of lipid raft transforms into a gel-like state and thus alters its viscosity and diffusion properties rendering it more rigid. Changing the tether in lipid rafts leads to further change in ceramide content and fusion of ceramide microdornains to form rigid ceram ide-enriched membrane platforms.
Changes in the activity of SMase could also be altered in the mitochondria leading to increase ceramide synthesis. This could cause changes in the membrane permeability which could effect a change in mitochondrial membrane potential, volume and leakage of molecules such as cytochrome C. Time-lapse confocal microscopy studies of human epithelial/mesenchymal co-cultures using mitochondrial dyes and the membrane potential indicator dye JC-1 were performed. It was observed that elastase affected mitochondrial membrane potential and caused mitochondrial swelling. Additional experiments using fluorescent mitochondrial dye revealed signs of swelling in response to elastase injury which was reversed by treatment with the anti-betal integrin antibody, JB1a. This demonstrates that betal integrin allosteric modulation induced repair in lung injury as a result of inhibiting membrane disruption due to clustering of cell surface receptors and/or altered membrane composition (due to increase ceramide) rendering it biophysically rigid.
C. The effect of changes in the actin cytoskeleton in emphysematous injury on transcription and translation as determined by (i) the presence of polymerised cytoskeleton scaffolding can affect the half-life of intracellular molecules such as mRNA. Reports have linked alteration in cytoskeleton-associated mRNA pool secondary to differentiation or integrin binding. An investigation of the downstream effectors of beta 1 integrin in allosteric modification-induced repair in the lung is in preparation. The potential for such experiments are the identification of effectors which could provide additional specific targets in repair and regeneration.
The effect of beta I integrin-induced repair after injury using therapeutic antibodies in vitro and in vivo on cytoskeleton-associated mRNA done using microarray technology (Affymetrix). Human co-cultures and in vivo lung tissue subjected to injury alone or injury and repair via betal integrin allosteric modification then subjected to selective extraction buffers (Cytoskeleton, Inc.) followed by RNA extraction as detailed before (A.
Brock, S. Huang, D. E. Ingber, BMCCell Biol. 4,6(2003)). All procedures will include the appropriate controls. The RNA will be reverse transcribed and labelled before hybrid ising it on microarrays. RT-PCR of identifiable genes will be done subsequently for confirmation. These experiments will identify effector gene translation downstream of betal integrin induced repair after injury.
This will be further determined by (ii) the effect betal integrin-mediated repair effect on nuclear actin and its effect on chromatin. Actin plays a direct role in gene transcription. It has been found to associate with components of ATP-dependent chromatin remodelling complexes, RNP particles, and the three RNA polymerases in the cell nucleus. Various actin subtypes have been reported to be in the nucleus such as 3-actin monomeric, oligomeric, and filamentous). Experiment will focus on determining the effect betal integrin mediated repair after elastase-induced injury in vitro using human and mouse co-cultures on the association of nuclear actin with chromatin. This will be done using promoter chromatin immunoprecipitation-on-chip (ChIP-on-chip). The immunoprecipitation will be conducted using antibodies against actin and actin-binding proteins (Abcarn). Further experiments using the wild type and mutant beta 1 integrin expressing cells will be conducted. This will demonstrate further effector genes downstream of beta 1 integrin and their involvement in repair.
In vitro experiments using epithelial-mesenchymal cultures to measure the activity of neutral sphingomyelinase have demonstrated that there was a transient increase in activity in the presence of elastase which was blocked by JB1a (Figure 10). Furthermore, there was an increase in lipid rafts in the presence of elastase; an effect reversed by JB1 a (Figure 11).
Changes in the activity of SMase could also be altered in the mitochondria leading to increase ceramide synthesis. This could cause changes in the membrane permeability.
ECIS core technology, which is based on a technique of measuring the change in impedance of a small electrode to AC current flow, was used.
Cells are attached and spread into an 8 individual wells (with 10 identical electrodes in each well), and then the current flows in the spaces under and between the cells, as the cell membrane are normal insulators. The base of the device has an array of gold film electrodes that connect to the ECIS electronics to each of the 8 wells. Firstly, pre-treatment was performed as a baseline for 2 hours prior to treatment to ensure that the drop in impedance induced by the change of media was recoverable.
Elastase was then added, this resulting in an initial surge in mechanical impedance above control levels followed by time-dependent decrease at a rate faster than control and JB1 a treated-elastase injured cells. Both effects were blocked by the JB1a monoclonal antibody (Figure 12).
LiThese observations demonstrates that beta allosteric modulation induced repair in lung injury as a result of inhibiting membrane disruption due to clustering of cell surface receptors andlor altered membrane composition (due to increase ceramide) rendering it biophysically rigid.
Summary
The results of this example confirm that beta integrin is an effective target for tissue repair and regeneration in emphysema. The results show that allosteric modulation of betal integrin using a single dose of monoclonal antibody, induced functional and structural reversal of elastase-induced lung injury in vivo, and found that similar matrix remodelling changes occurred in human lung tissue. The results further identified inhibition of elastase-induced caspase activation, F-actin aggregate formation and changes in cellular ATP levels. This was accompanied by maintenance of betal integrin levels and inhibition of caveolin-1 phosphorylation. We propose that allosteric modulation of betal integrin-mediated niechanosensing prevents cell death associated with lung injury and progressive emphysema, thus allowing cells to survive and for repair and regeneration to ensue.
Example 2 -Functional modulation of betal integrin via the hybrid domain reverses articular cartilage iniury and functional impairment in a mouse model of arthritis Rheumatoid arthritis (RA) affects 0.8-1 % of the population in developed countries. It is characterised by synovial proliferation and infiltration of inflammatory cells. This occurs in conjunction with angiogenesis leading to the formation of inflammatory pannus which contributes to bone erosion and cartilage thinning. The pathogenesis of RA has not been clearly defined. However, studies suggest that CD4+ T cell-mediated autoimmune responses play a critical role in the pathogenesis of RA. In addition, interferon gamma producing Thi cells are though to be pivotal in the development of autoimmune arthritis in both humans and animal models.
Current disease-modifying anti-rheumatic drug therapies have been shown to slow joint destruction in only a subpopulation of patients. Novel approaches have focused on specific modulation of the inflammatory response by targeting cytokines and their receptors. More recently, PI3Ky inhibitors have been reported to suppress joint inflammation and damage in a mouse model of RA. To date there has been no treatment targeting chondrocytes.
Articular cartilage is avascular tissue and is devoid of nerves and lymphatics. Cartilage extracellular matrix (ECM) homeostasis is dependent upon the interaction between chondrocytes and their surrounding ECM via cell surface adhesion receptors such as integrins.
Normal chondrocytes express integrins a1131, Q5131, aVI35, v133, and a313i.
In-vitro studies have demonstrated the importance of the interaction of 131 integrin with ECM during mesenchymal condensation and chondrocytes differentiation. The administration of anti betal integrin antibodies to mouse limb mesenchynial buds has been previously shown to inhibit the formation of cartilage nodules. Furthermore, betal integrin inhibition in cartilage explants and also specific deletion of the betal integrin gene in mice in-vivo has been shown to cause a reduction in growth and collagen X deposition, accompanied by abnormal chondrocytes size, disorganised actin and increased apoptosis.
Without being bound by theory, the inventors hypothesise that in injury, beta integrin activation may occur as a result of fragmentation of the ECM.
This might lead to cytoskeletal abnormalities, mitochondrial changes, altered ATP homeostasis and increased susceptibility to apoptosis. Failure of removal of apoptotic debris could act as a proinflammatory stimulus. In this setting integrin disengagement could be of therapeutic value.
Materials and methods Induction of arthritis in-vivo Animals were injected with either Freund's Complete Adjuvant (FCA, lOOpg in 20 p1) from Mycobacterium tuberculosis in paraffin oil (Sigma) or vehicle (heavy liquid paraffin oil; controls). Briefly, injections were carried out by transiently anaesthetising the animal (3% halothane in oxygen), a small incision was made over the stifle joint to allow visual identification of the patella tendon then using a 30-gauge needle mounted on a 50-p1 Hamilton syringe, FCA, vehicle or JB1 a (20pl) was injected under the patella tendon and directly into the synovial space of the stifle joint. The animals were treated with JB1 a (mouse anti-human IgGi, Chemicon), isotype control (IgGi, MOPC21, Sigma) or vehicle twice at weekly intervals starting at day 7 post FCA or injection (days 7 and 14). Sham animals were also included.
Assessment of arthritis Animals were weighed, the diameter across the joint (left and right), just below the level of the patella, was measured weekly according to previously described method. Measurements were taken once a week prior to the injection on that day. Subjective measures were used to correlate an increased hyperalgesia score with an applied pressure to the joint induced by the injection of FCA and its reversal by JB1a. The scale was 0 (normal, high pressure applied) to 3 (very hyperalgesic, very low pressure applied). Testing involved squeezing the joint between thumb and forefinger and determining the amount of pressure required for the animal to withdraw the limb (normal end-point) or vocalise (rare).
Histoiatholoqy Animals were killed (002) on day 21 and the left (injected, ipsilateral) and right (control, contralateral) stifle joints surgically removed for histology.
Tissues were fixed for 48 hours in 10% neutral buffered formalin then decalcified using 15% EDTA (pH 7) in 10% neutral buffered formalin for 2 weeks. Samples were embedded in paraffin wax then cut (8 pm sections) and stained haematoxylin/eosin and alcian blue (pH 1.0)/brilliant red.
Images from the haematoxylin/eosin and alcian blue stained sections were digitised using Image-Pro plus (version 5.1) and micropublisher 3.3 RTV camera connected to a Zeiss axioskope RTM with 5x objective (haematoxylin I eosin) and 20x objective (Alcian blue). The severity of the synovial inflammation, joint damage, metachromasia and pannus tissue formation were arbitrarily and subjectively scored on an analogue scale (0 no abnormality detected; 1 = very slight to maximum; and 3 = very marked), as previously described.
Apoptosis measurement Terminal Deoxyribonucleotidyl Transferase (TdT)-Mediated dUTP Nick End Labelling (TUNEL) was assessed in sections using the Red ApopTagTM Kit (Chemicon). Data for the quantification of positively stained apoptotic nuclei was acquired using the x40 oil objective of a Zeiss 510 Axiovert RTM confocal microscope system (Carl Zeiss Ltd, Welwyn Gardens City, Herts, UK). Images of cartilage mainly tissue were constructed. The images were then converted to 8-bits grey scale and ImageJ (NIH) was used to count total number of cells. TUNEL positive cells were counted manually.
Human articular chondrocytes in-vitro Primary normal adult human chondrocytes derived from the knee joint were cultured according to supplier's instructions. Cells were cultured for 2 weeks in 1.2% alginic acid (Keltone RTM LV) in nornial saline (3 weeks for bead experiments) polymerised using 102mM CaCl2. Cells were dispersed on the day of experiment from the alginate using 55mM sodium citrate and allowed to attach onto plastic for 6 hours before being starved with DMEM/F12 media containing 0.1% FCS for 1 hour. IL-i 13 (carrier-free, R&D) was added at I Ong/mI alone or in combination with JBI a (I pg/mI) and the anti-betal integrin antibody, 6S6 (1 pg/mI). At the end of the 3, 6, 12 or 24 hour period, the media was aspirated and preserved and caspase 3 activity assayed using Caspase-GIoTM 3/7 (Promega) according to the manufacturers' instructions.
In a separate set of experiments chondrocytes were dispersed and seeded onto 96 multi-well plates as described above. The cells were starved in media containing 0.1% FCS then in DMEM-glucose-free with 0.1% FCS before treating with (i) IL-i 13 (lOng/mi) alone,(ii) IL-113 at lOng/mi and JB1a (1 pg/mI) (iii) JB1a (1 pg/mI) alone or (iv) control. At the third hour, ATP levels were measured minute-by-minute using a bioluminescent ATP kit (Perkin Elmer).
Data analysis and statistics Data were collected and analysed using Microsoft Excel, GraphPad Prism and SPSS. One way and multi-way ANOVA followed by unpaired t-tests were used to analyse differences between the means of normally distributed groups. When the sample size for each group was too small or the data was not normally distributed then the non-parametric Man n-Whitney U-test was used. Where significance was not achieved in two-way ANOVA for the effect of time, data were pooled for a given treatment from across all time points. For repeated measure values, a repeated measure ANOVA was used followed by post-hoc test. The medians of two or more groups of non-parametric data were analysed with a Kruskal-Wallis test, and post-hoc analysis was done using Dunn's multiple comparison. A P-value of less than 0.05 was considered significant, and where possible, the actual P-value was quoted to show proximity to the 0.05 limit. All values are presented as means or scatter.
Experimental Protocol and Results Preliminary work performed by the inventors demonstrated the ability of specific inhibitory anti-beta 1 integrin antibodies to induce an increase in ECM proteoglycans in human chondrocytes (data not shown).
To evaluate whether the modulation of betal integrin function facilitated repair, a modified unilateral model of arthritis in mice induced by intra-articular administration of Freund's complete adjuvant (FCA, 1 OOug) was studied (Gauldie,S.D., et al. J. Neurosci. Methods 139, 281-291 (2004)).
This model resembles chronic rheumatoid arthritis but does not cause marked significant systemic effects or mobility and body weight changes.
Mice injected with FCA showed a significant increase in their ipsilateral joint diameter and hyperalgesia score on days 7, 14 and 21 compared to either vehicle -treated or sham animals (Figure 12(a)). In mice injected with FCA followed by intra-articular injection of 60 mg of JB1 a on days 7 and 14, the increase in the ipsilateral joint diameter and hyperalgesia score was abolished on days 14 and 21. The contralateral joint diameter and the hyperalgesia scores showed no significant change (Figure 12(b),(c)).
Histologically, mice injected with FCA showed joint inflammation with cellular infiltrate, synovial proliferation, pannus formation and cartilage erosion (Figure 12(d),(e),(f)). The JB1a treated group showed a significant amelioration in histological markers of damage; with a marked reduction in synovial thickness and cellular infiltrate. This was accompanied by the reversal of cartilage thinning and reduced pannus formation. No adverse effects were seen after the treatment with JB1a. This indicates that the reduction in hyperalgesia scores is not simply the result of previously described blockade of neuropathic pain by functional inhibition of beta I integrin.
The prevalence of TUNEL positive cells in mouse joints following injury and treatment was then investigated. The experiments were performed in order to ascertain whether chondrocyte apoptosis and failure of removal of apoptotic bodies by phagocytosis may itself provoke further inflammation and autoimmunity. The results showed that background prevalence of apoptosis was low, and there was a significant increase in TUNEL positive cells in the ipsilateral joints: this was significantly reduced by treatment with JB1a monoclonal antibody(Figure 12(g)).
To investigate the role of betal integrin in chondrocyte injury, primary human chondrocytes derived from the knee joint were cultured in 1.2% alginic acid for 2 weeks, then dispersed and allowed to attach to plastic before study.
Figure 13(a) demonstrates that IL-i beta (lOng/mi) causes a significant increase in caspase 3/7 activity. Treatment with the anti-betal integrin antibody (1.5pgIml), JB1 a, a monoclonal antibody with binding specificity to an epitope present in the hybrid domain, alone had no effect on caspase 3/7 activity ((JB1A mouse anti-CD29 (Integrin beta-i subunit) IgG nionoclonal antibody, unconjugated, clone JBIa, Millipore, Catalogue number MAB-i 965)). However, when IL-i beta was added with JB1 a, caspase 3/7 activation was abolished.
Similar responses were obtained in experiments conducted using primary human chondrocytes cultured in alginic acid beads for 3 weeks (data not shown). The anti-betal integrin antibody 6S6 (1 pg/mI) which is also an adhesion blocking antibody, caused activation of caspase 3/7 activity. The epitope for this antibody is discontinuous but has not been mapped. In addition to blocking adhesion, it has been described to induce homotypic aggregation.
A further antibody, JBS5 directed against a51 (alpha5betai) integrin also causes clustering and induces inflammation 17. We found that IL-i3 (iL-ibeta) stimulated the secretion of IL-8 in a time-dependent manner (Figure 13(b)). This response was reduced by 50% in the presence of JB1a.
Interestingly, although the 6S6 clone had no effect on its own, when added with IL-i beta, it amplified the IL-i beta induced IL-8 secretion at both 3 and 6 hours.
Changes in ATP affect both cytoskeletal dynamics particularly actin polymerisation. ATP and actin have also been described to affect niitochondrial membrane potential and cell survival. It was demonstrated that the addition of IL-i beta (1 Ong/mI) to chondrocytes in vitro, reduced ATP concentration in extended time-course studies and abrogated its sinusoidal oscillation at earlier time points (Figure 13(c)). When IL-ibeta was added with JB1a, the oscillation of ATP levels was restored although the mean concentration was reduced. The significance of this is unknown.
The present example used the JB1a monoclonal antibody which is thought to bind to betal integrin at an epitope defined by the amino acid sequence 82-87 of the hybrid domain. The epitope is non-conformation dependent.
Although this clone is defined as an adhesion blocking antibody, the nature of the functional modification remains obscure. This definition does not take into account the multiple states of integrins, their multifunctional domains and splice variants. From our studies modulation of function via differing epitopes entailed a different outcome. The therapeutic effect was achieved via modulation of the hybrid domain.
JB1 a epitope convey an allosteric modulation of betal integrin which stabilises the receptor in the physiological low affinity state of the receptor by preventing the swing out of the domain. Although the resulting conformation maintains extracellular matrix binding, both the association and dissociation constants are partially affected raising the possibility that JB1a is a partial antagonist.
This experiment shows that in articular chondrocytes, betal integrin has a pivotal role in energy homeostasis and cell survival. Without wishing to be bound by theory, the inventors hypothesise that upon local injury, degradation of the extracellular matrix results in "aggregation" of cell surface receptors; one of which is betal integrin. Macro-or micro-aggregation could initiate an outside-inside signalling and cytoskeletal engagement. This in turn alters the cellular mechanical properties by increasing F-actin formation, decreasing ATP recycling, and cell surface tethering forces; all of which are known to contribute to mitochondrial and cellular damage. Taken together with the accompanying findings in emphysema, this provides new and unexpected insights into the principles of cellular injury and disease progression, leading to the identification of novel targets for therapeutics.
Summary
The results of Example 2 illustrate that direct modulation of betal integrin using the rrionoclonal antibody JB1a causes functional and structural amelioration of adjuvant-induced arthritis in vivo. It was further shown that such changes are associated with a reduction in chondrocyte apoptosis and restitution of normal cellular ATP fluctuation.
Example 3 -Functional modulation of betal inteqrin inhibits beta amyloid fibril-induced cell death The main pathological hallmarks of Alzheimer's disease are the formation of extracellular amyloid plaques, intracellular neurofibrillary tangles, and neurodegeneration. Amyloid plaques are mainly composed of tangles of beta amyloid peptide which is believed to play a causal role in Alzheimer's disease.
It has been demonstrated that amyloid beta deposition and neurotoxicity in human cortical primary neurons is mediated through alpha2betal and alphaVbetal integrins. It was further claime that blocking betal integrin inhibits amyloid fibril formation via functional inhibition of adhesion via betal integrin. Although the beta 1 integrin binding epitope of the JB1a rnonoclonal antibody maps to the hybrid region of beta 1 integrin, there is a possibility that it may recognise another sequence in the A domain almost sandwiched between the MIDAS and the ADMIDAS (Figure 17).
Whether the real epitope could be discontinuous and/or combinatorial, remains unclear. Indeed the hybrid domain has been implicated in allosteric alteration and targeting residues within this region induced a low affinity physiological conformation.
In cultured primary human cortical neurons, 1-42 beta amyloid induced caspase 3/7 activation and an increase in GM1 staining indicative of raft formation (Figure 15). Allosteric modulation of betal integrin using the JB1a rrionoclonal antibody ((JB1A mouse anti-CD29 (Integrin beta-i subunit) IgG monoclonal antibody, unconjugated, clone JB1a, Millipore, Catalogue number MAB-1 965) conferred protection against beta amyloid-induced apoptosis. This study further indicated that there was a marked morphological change in the presence of JB1 a.
Experimental Methods Primary human cortical neuron cultures: The HCN2 cell line was obtained from the American Type Culture Collection (ATCC). HCN2 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells were maintained at 37°C in 5% Co2.
For differentiation experiments, cells were cultured on collagen-coated glass coverslips. Differentiation of these neuronal cells was induced by adding fresh medium containing 25 ng/ml beta-nerve growth factor (NGF), 20 nM phorbol-1 2-myristate-1 3-acetate (PMA) and 0.5 mM 3-isobutyl-1-rnethylxanthine (IBMX). When the neurons differentiated and developed neuritic processes, the effect of amyloid was examined as detailed below.
Three-dimensional time-lapse con focal microscopy The media was removed and Alexa-Fluor RTM 647 labelled G-actin (3Opg/coverslip) from rabbit was loaded using Influx (Molecular Probes).
The cells were loaded with PhiPhiLux-G2D2 for visual isation of caspase activation (1/50 dilution, Oncoimmune) and FL-ganglioside 1(1/100 dilution) GM1, Molecular probes) to visualise the plasma membrane rafts.
The cells were treated with vehicle, lOuM amyloid-beta fibrils (1-42) (American Peptide Co., Sunnyvale, CA), or arnyloid fibrils and JB1a (2ug/ml). Images were collected every 30 through 4 separate channels (emissions: GM1: A = 488,caspase A = 568, actin: A = 647 and brightfield) using x63 water lens and Zeiss LSM51 0 CLSM microscope. The resulting images were analysed with Irnaris RTM software (Bitplane AG, Switzerland).
Three-dimensional images were reconstructed.
All documents referred to in this specification are herein incorporated by reference. Various modifications and variations to the described embodiments of the inventions will be apparent to those skilled in the art without departing from the scope of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
The present application is a divisional application of UK Patent Application No. 0916943.4 which in turn is derived from PCT Application No. PCTIGB2008IO5O1 31. The following numbered clauses, which correspond to the claims of the PCT application as filed, form part of the present disclosure and in particular form further aspects of the invention, whether or not they appear in the present claims.
1. A method of modulating one or more of the biological activities of beta 1 integrin in a beta 1 integrin expressing cell or tissue in order to mediate cellular or tissue repair, comprising: -contacting the cell or tissue with an allosteric antagonist of beta 1 integrin, in an amount sufficient to modulate one or more biological activities of beta 1 integrin in the cell or tissue which results in tissue damage or cell death.
2. The method of clause 1, wherein the beta 1 integrin expressing cell or tissue is a cell or tissue of the synovium.
3. The method of clause 1, wherein the beta 1 integrin expressing cell or tissue is a neuronal cell or tissue.
4. The method of clause 1, wherein the beta 1 integrin expressing cell or tissue is a cell or tissue of the lung.
5. The method as referred to in any preceding clause, wherein the contacting step occurs in a cell lysate, a reconstituted system or cells in culture.
6. The method as referred to in any preceding clause, wherein the contacting step occurs on cells present in a subject.
7. The method of clause 6, wherein the subject is a human patient having, or at risk of having tissue damage to any of the cells of the lung, synovium or central nervous system.
8. The method of clause 1, wherein the beta 1 integrin is human or niurine beta 1 integrin.
9. The method of clause 1, wherein the beta 1 integrin allosteric antagonist binds to human beta 1 integrin.
10. The method as referred to in any preceding clause, wherein the beta 1 integrin allosteric antagonist is selected from the group consisting of a: protein, peptide, peptidomimetic, nucleic acid, polynucleotide, polysaccharide, oligopeptide, carbohydrate, lipid, small molecule compound, aptamer, and naturally occurring compounds such as a plant derived compound.
11. The method as referred to in clause 10, wherein the beta I integrin allosteric antagonist is an antibody molecule or a fragment thereof which has binding specificity to beta 1 integrin.
12. The method of clause 11, wherein the antibody molecule is a human, humanized, chimeric, or camelid antibody.
13. The method of clause 11, wherein the antibody molecule is an in vitro generated antibody to beta 1 integrin.
14. The method of clause 11, wherein the antibody molecule comprises two complete heavy chains, and two complete light chains, or an antigen-bind ing fragment thereof.
15. The method of clause 10, wherein the beta 1 integrin allosteric antagonist binds to the extracellular domain of human beta 1 integrin.
16. The method of clause 11, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 and 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
17. The method of clause 11, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 to 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
18. The method of clause 11, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TAEKLK of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
19. The method of clause 11, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 179 to 184 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
20. The method of clause 11, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TPAKLR of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
21. The method of clause 11, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within amino acid residues 207 to 218 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
22. The method of clause 11, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within the hybrid domain comprising residues 60 to 139 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
23. The method of clause 11, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 360 to 461 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
24. A method for mediating tissue repair, the method comprising the steps of: -providing a therapeutically effective amount of an agent which acts as an allosteric antagonist of beta I integrin, wherein the binding of the allosteric antagonist to betal integrin induces the conformation of the betal integrin to assume an intermediate affinity state, and -administering the same to a subject in need of such treatment.
25. A method as referred to in clause 24 wherein the tissue is a tissue of the lung, synovium or a neuronal tissue.
26. A method as referred to in clause 24 or clause 25 wherein the agent is selected from the group consisting of a: protein, peptide, peptidomimetics, nucleic acid, polynucleotide, polysaccharide, oligopeptide, carbohydrate, lipid, small molecule compound, and a naturally occurring compound.
27. The method as referred to in clause 26, wherein the beta 1 integrin allosteric antagonist is an antibody molecule or a fragment thereof which has binding specificity to beta 1 integrin.
28. The method of clause 27, wherein the antibody molecule is a human, humanized, chimeric, orcamelid antibody.
29. The method of clause 27, wherein the antibody molecule is an in vitro generated antibody to beta 1 integrin.
30. The method of clause 27, wherein the antibody molecule comprises two complete heavy chains, and two complete light chains, or an antigen-bind ing fragment thereof.
31. The method of clause 27, wherein the beta 1 integrin allosteric antagonist binds to the extracellular domain of human beta I integrin.
32. The method of clause 26, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 and 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
33. The method of clause 26, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 to 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
34. The method of clause 26, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TAEKLK of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
35. The method of clause 26, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 179 to 184 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
36. The method of clause 26, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TPAKLR of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
37. The method of clause 26, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within amino acid residues 207 to 218 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
38. The method of clause 26, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within the hybrid domain comprising residues 60 to 139 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
39. The method of clause 26, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 360 to 461 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
40. A method for modulating the conformational allostery of betal integrin such that it assumes an intermediate affinity state conformation wherein the extracellular headpiece is neither bent such that the headpiece is at a distance of around 5 nm from the membrane of the cell on which the beta 1 integrin is provided, or wherein the headpiece is in an extended conformation wherein the headpiece projects at a distance of greater than 25 nm from the membrane of the cell on which the beta 1 integrin is provided, the method comprising the step of exposing betal integrin to an effective amount of an allosteric antagonist compound, wherein said allosteric antagonist binds to beta 1 integrin and mediates an allosteric change thereof such that the confirmation of beta 1 integrin assumes the intermediate affinity state conformation.
41. A rriethod as referred to in clause 40 wherein the allosteric antagonist further mediates at least one of: (i) remodelling of the extracellular matrix (ECM), in particular a reduction in cytoskeletal stiffness, (ii) inhibition of caspase activation, and in particular caspase 3 and caspase 7 and an associated reduction in apoptosis, (iii) suppression of F-actin aggregate formation, and a decrease in cytoskeletal tension (iv) improved cellular energy homeostasis resulting from a reversion to sinusoidal oscillation of ATP levels, and associated improved levels of ATP recycling within a cell, (v) maintenance of beat 1 integrin expression levels, (vi) inhibition of caveolin-1 phosphorylation and hence receptor clustering, and (vii) inhibition of cell membrane physicochemical changes.
42. A method as referred to in clause 40 wherein the tissue is a tissue of the lung, synovium or a neuronal tissue.
43. A method as referred to in clause 40 wherein the allosteric antagonist is selected from the group consisting of a: protein, peptide, peptidomimetics, nucleic acid, polynucleotide, polysaccharide, oligopeptide, carbohydrate, lipid, small molecule compound, and a naturally occurring compound.
44. The method as referred to in clause 40, wherein the beta 1 integrin allosteric antagonist is an antibody molecule or a fragment thereof which has binding specificity to beta 1 integrin.
45. The method of clause 40, wherein the antibody molecule is a human, humanized, chimeric, or camelid antibody.
46. The method of clause 40, wherein the antibody molecule is an in vitro generated antibody to beta 1 integrin.
47. The method of clause 40, wherein the antibody molecule comprises two complete heavy chains, and two complete light chains, or an antigen-bind ing fragment thereof.
48. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to the extracellular domain of human beta 1 integrin.
49. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 and 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
50. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 to 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
51. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TAEKLK of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
52. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 179 to 184 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
53. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TPAKLR of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
54. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within amino acid residues 207 to 218 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
55. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within the hybrid domain comprising residues 60 to 139 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
56. The method of clause 40, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 360 to 461 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
57. A method for the prophylaxis and/or treatment of autoimmune arthritis, the method comprising the steps of: -providing a therapeutically effective amount an allosteric antagonist compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state, and -administering the same to a subject in need of such treatment.
58. A method as referred to in clause 57 wherein the autoimmune arthritis is at least one condition selected from the group comprising: rheumatoid arthritis, inflammatory arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis. In further embodiments, the autoimmune arthritis may be collagen-induced arthritis.
59. A method for the prophylaxis and/or treatment of chronic obstructive pulmonary disease (COPD), the method comprising the steps of: -providing a therapeutically effective amount an allosteric antagonist compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of betal integrin assumes the intermediate affinity state, and -administering the same to a subject in need of such treatment.
60. A method as referred to in clause 59 wherein the chronic obstructive pulmonary disease (COPD) indication is emphysema or chronic bronchitis.
61. A method for the prophylaxis and/or treatment of a neurodegenerative disease, the method comprising the steps of: -providing a therapeutically effective amount an allosteric antagonist compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state, and -administering the same to a subject in need of such treatment.
62. A method as referred to in clause 61 wherein the neurodegenerative disease is selected from the group comprising: Alzheimer's disease (AD), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.
63. A method for the prophylaxis and/or treatment of tissue damage associated with cardiovascular disease, the method comprising the steps of: -providing a therapeutically effective amount an allosteric antagonist compound, wherein said allosteric modulator binds to betal integrin and mediates an allosteric change thereof such that the conformation of betal integrin assumes the intermediate affinity state, and -administering the same to a subject in need of such treatment.
64. A method as referred to in any one of clauses 57, 59, 61 or 63, wherein the allosteric antagonist is selected from the group consisting of a: a: protein, peptide, peptidomimetic, nucleic acid, polynucleotide, polysaccharide, oligopeptide, carbohydrate, lipid, small molecule compound, aptamer, and naturally occurring compounds such as a plant derived compound.
65. A method as referred to in any one of clauses 57, 59, 61 or 63, wherein the beta 1 integrin allosteric antagonist is an antibody molecule or a fragment thereof which has binding specificity to beta 1 integrin.
66. A method as referred to in clause 65, wherein the antibody molecule is a human, humanized, chimeric, or camelid antibody.
67. A method as referred to in clause 65, wherein the antibody molecule is an in vitro generated antibody to beta 1 integrin.
68. A method as referred to in clause 65, wherein the antibody molecule comprises two complete heavy chains, and two complete light chains, or an antigen-binding fragment thereof.
69. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to the extracellular domain of human beta I integrin.
70. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 and 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
71. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 82 to 87 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
72. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TAEKLK of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
73. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 179 to 184 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
74. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues TPAKLR of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
75. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta I integrin present within amino acid residues 207 to 218 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
76. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin present within the hybrid domain comprising residues 60 to 139 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
77. A method as referred to in clause 65, wherein the beta 1 integrin allosteric antagonist binds to an epitope on beta 1 integrin comprising amino acid residues 360 to 461 of the mature amino acid sequence of human beta 1 integrin as defined in SEQ ID NO:1.
78. A pharmaceutical composition comprising a therapeutically effective amount of compound which acts as an allosteric antagonist of beta 1 integrin, said antagonism of beta 1 integrin mediating an allosteric change in the conformation of betal integrin such that it assumes an intermediate affinity state conformation, along with at least one pharmaceutically acceptable diluent, excipient or carrier.
79. A composition as referred to in clause 78 wherein the allosteric modulator binds to the hybrid domain of betal integrin, the hybrid domain comprising amino acid residues 60 to 139 and 360 to 461 of the amino acid sequence of human beta 1 integrin as defined in the SEQ ID NO:1.
80. An assay method for the identification of a compound which acts as an allosteric antagonist of beta 1 integrin, wherein said allosteric antagonist binds to the extracellular domain of betal integrin and mediates an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state conformation, said method comprising the steps of: -providing an assay comprising betal integrin or a derivative thereof; -activating betal integrin such that it assumes a conformation representative of a high affinity state; -contacting the beta 1 integrin with a candidate modulatory compound under suitable conditions; and -detecting a change of the conformation of betal integrin to an intermediate affinity state, wherein an allosteric change of the beta 1 integrin into an intermediate affinity state indicates that the candidate compound acts as an allosteric antagonist of beta 1 integrin.
81. A method as referred to in clause 80 wherein the contacting step is followed by an incubating step wherein the mixture obtained in the contacting step is incubated for a time period and under conditions suitable to allow interaction between the components and/or the test substance.
82. A method as referred to in clause 80 or 81 wherein the intermediate affinity state conformation of beta 1 integrin is characterised in that the extracellular headpiece of beta 1 integrin is neither bent such that the headpiece is at a distance of around 5 nm from the membrane of the cell on which the beta 1 integrin is provided, or wherein the headpiece is in an extended conformation wherein the headpiece projects at a distance of greater than 25 nm from the membrane of the cell on which the beta 1 integrin is provided.
83. An assay for identifying compounds which function as allosteric antagonists of betal integrin, wherein the allosteric antagonist binds to beta 1 integrin and mediates an allosteric change thereof such that the conformation of beta 1 integrin assumes the intermediate affinity state conformation, said assay comprising the steps of: -providing a candidate compound, -bringing the candidate compound into contact with betal integrin or a derivative thereof, -determining the presence or absence of modulation of beta 1 integrin wherein the conformation of beta 1 integrin assumes the intermediate affinity state activity by the candidate compound, wherein modulation of betal integrin into the intermediate affinity state is indicative of utility of that compound in tissue repair.
84. A method as referred to in clause 83 wherein the intermediate affinity state conformation of beta 1 integrin is characterised in that the extracellular headpiece of beta 1 integrin is neither bent such that the headpiece is at a distance of around 5 nm from the membrane of the cell on which the beta 1 integrin is provided, or wherein the headpiece is in an extended conformation wherein the headpiece projects at a distance of greater than 25 nm from the membrane of the cell on which the beta 1 integrin is provided.
85. Use of a compound which acts as an allosteric antagonist of betal integrin, wherein binding of said antagonist to beta 1 integrin mediates a confirmatory change of betal integrin into an intermediate affinity state conformation in the preparation of a med icament for the treatment of a disease or condition which is characterised by cellular or tissue damage or death.
86. Use of a compound which acts as an allosteric antagonist of beta I integrin, wherein binding of said antagonist to beta 1 integrin mediates a confirmatory change of betal integrin into an intermediate affinity state conformation in the preparation of a medicament for the treatment of autoimmune arthritis.
87. Use as referred to in clause 86 wherein the autoimmune arthritis is selected from the group consisting of: rheumatoid arthritis, inflammatory arthritis, juvenile rheumatoid arthritis, osteoarthritis and psoriatic arthritis.
In further embodiments, the autoimmune arthritis may be collagen-induced arthritis.
88. Use of a compound which acts as an allosteric antagonist of betal integrin, wherein binding of said antagonist to beta 1 integrin mediates a confirmatory change of beta 1 integrin into an intermediate affinity state conformation in the preparation of a med icament for the treatment of tissue damage which results from cardiovascular disease.
89. Use of a compound which acts as an allosteric antagonist of betal integrin, wherein binding of said antagonist to beta 1 integrin mediates a confirmatory change of betal integrin into an intermediate affinity state conformation in the preparation of a medicament for the treatment of a neurodegenerative disease.
90. Use of a compound as referred to in clause 89 wherein the neurodegenerative disease is selected from the group consisting of: Alzheimer's disease (AD), mild cognitive impairment (MCI), multiple sclerosis (MS), Parkinson's disease, Amyotrophic lateral sclerosis (ALS), Huntington's disease, prion diseases such as CJD, AIDS-related dementia, encephalitis, stroke and head trauma.
91. Use as referred to in any one of clauses 85 to 90 wherein the intermediate affinity state conformation of beta 1 integrin is characterised in that the extracellular headpiece of beta 1 integrin is neither bent such that the headpiece is at a distance of around 5 nm from the membrane of the cell on which the beta 1 integrin is provided, or wherein the headpiece is in an extended conformation wherein the headpiece projects at a distance of greater than 25 nm from the membrane of the cell on which the beta 1 integrin is provided.
92. Use of an allosteric antagonistic of betal integrin which modulates the betal integrin into an intermediate affinity state in the preparation of a med icament for mediating tissue repair and regeneration.
93. Use of an allosteric antagonist of betal integrin which modulates the betal integrin into an intermediate affinity state in the preparation of a rnedicament which mediates an inhibition of caspase activation and/or apoptosis.
94. Use of an allosteric modulator of betal integrin which modulates the betal integrin into an intermediate affinity state in the preparation of a niedicament which mediates an inhibition of caveolin-1 phosphorylation.
95. An allosteric antagonist of beta 1 integrin for use in treating tissue or cellular damage caused by cardiovascular disease.
96. An allosteric antagonist of beta 1 integrin for use in treating tissue or cellular damage caused by autoimmune arthritis.
97. An allosteric antagonist of beta 1 integrin for use in treating tissue or cellular damage caused by emphysema.
Claims (17)
- Claims 1. An assay for identifying compounds for use in tissue repair, said assay comprising the steps of: -providing a candidate compound which has binding specificity to human beta 1 integrin; -bringing the candidate compound into contact with human beta 1 integrin or a derivative thereof; and -determining the presence or absence of modulation of the beta 1 integrin or the derivative thereof into an intermediate affinity state conformation by the candidate compound; wherein the presence of modulation of the beta 1 integrin or the derivative thereof into the intermediate affinity state conformation is indicative of utility of that compound in tissue repair.
- 2. The method as claimed in claim 1 wherein the intermediate affinity state conformation of beta 1 integrin is characterised in that the extracellular headpiece of beta 1 integrin is in an extended closed conformation.
- 3. The method as claimed in claim 1 or 2 wherein the intermediate affinity state conformation of beta 1 integrin is characterised in that the extracellular headpiece of beta 1 integrin is neither bent wherein the headpiece is at a distance of around 5 nm from the membrane of the cell on which the beta I integrin is provided nor in an open extended conformation wherein the headpiece has an open configuration and projects at a distance of greater than 25 nm from the membrane of the cell on which the beta 1 integrin is provided.
- 4. The method as claimed in any one of claims 1 to 3 wherein the candidate compound binds to the extracellular domain of human beta 1 integrin.
- 5. The method as claimed in any one of claims 1 to 4 wherein the candidate compound binds to an epitope on beta 1 integrin comprising amino acid residues 82 and 87 of human beta 1 integrin as defined in SEQ ID NO:1.
- 6. The method as claimed in claim 5 wherein the candidate compound binds to an epitope on beta 1 integrin comprising amino acid residues 82 to 87 of human beta 1 integrin as defined in SEQ ID NO:1.
- 7. The method as claimed in any one of claims 1 to 6 wherein the candidate compound is selected from the group consisting of a protein, a peptidomimetic, a nucleic acid and a small molecule.
- 8. The method as claimed in any one of claims 1 to 6 wherein the candidate compound is an antibody or a fragment thereof.
- 9. The method as claimed in any one of claims 1 to 8 wherein the tissue repair is repair of a tissue selected from the group consisting of lung tissue, skin tissue, liver tissue, kidney tissue, tissue of the nervous system, cartilage, bone and tissue of the cardiovascular system.
- 10. The method as claimed in any one of claims 1 to 8 wherein the tissue repair is tissue repair following the onset or development of arthritis.
- 11. The method as claimed in claim 10 wherein the arthritis is rheumatoid arthritis.
- 12. The method as claimed in any one of claims ito 8 wherein the tissue repair is tissue repair following the onset or development of neurodegenerative diseases.
- 13. The method as claimed in claim 12 wherein the neurodegenerative disease is Alzheimer's disease.
- 14 The method as claimed in claim 12 wherein the neurodegenerative disease is Parkinson's disease.
- 15. The method as claimed in any one of claims 1 to 8 wherein the tissue repair is tissue repair following the onset or development of chronic obstructive pulmonary disease.
- 16. The method as claimed in claim 15 wherein the chronic obstructive pulmonary disease is emphysema.Amendments to the claims have been filed as follows: 1. An assay for identifying compounds for use in tissue repair, said assay comprising the steps of: -providing a candidate compound which has binding specificity to human beta 1 integrin; -bringing the candidate compound into contact with human beta 1 integrin or a derivative thereof; and -determining the presence or absence of modulation of the beta 1 integrin or the derivative thereof into an intermediate affinity state conformation by the candidate compound; wherein modulation of the beta 1 integrin or the derivative thereof into the intermediate affinity state conformation is monitored in terms of at least one of: (I) suppression of F-actin aggregate formation and a decrease in cytoskeletal tension; (ii) improved cellular energy homeostasis resulting from a reversion to sinusoidal oscillation of ATP levels and associated improved levels of ATP recycling within a cell; (iii) maintenance of beta 1 integrin expression levels; (iv) inhibition of caveolin-1 phosphorylation; and (v) inhibition of cell membrane physicochemical changes; wherein the presence of modulation of the beta 1 integrin or the derivative thereof into the intermediate affinity state conformation is indicative of utility of that compound in tissue repair.2. The method as claimed in claim 1 wherein the intermediate affinity state conformation of beta 1 integrin is characterised in that the extracellular headpiece of beta 1 integrin is in an extended closed conformation.3. The method as claimed in claim 1 or 2 wherein the intermediate affinity state conformation of beta 1 integrin is characterised in that the extracellular headpiece of beta 1 integrin is neither bent wherein the headpiece is at a distance of around 5 nm from the membrane of the cell on which the beta 1 integrin is provided nor in an open extended conformation wherein the headpiece has an open configuration and projects at a distance of greater than 25 nm from the membrane of the cell on which the beta 1 integrin is provided.4. The method as claimed in any one of claims 1 to 3 wherein the candidate compound binds to the extracellular domain of human beta 1 integrin.5. The method as claimed in any one of claims 1 to 4 wherein the candidate compound binds to an epitope on beta 1 integrin within amino acid residues 82 and 87 of human beta 1 integrin as defined in SEQ ID NO:1.6. The method as claimed in claim 5 wherein the candidate compound binds to an epitope on beta 1 integrin comprising amino acid residues 82 to 87 of human beta 1 integrin as defined in SEQ ID NO:1.7. The method as claimed in any one of claims I to 6 wherein the candidate compound is selected from the group consisting of a protein, a peptidomimetic, a nucleic acid and a small molecule.8. The method as claimed in any one of claims 1 to 6 wherein the candidate compound is an antibody or a fragment thereof.9. The method as claimed in any one of claims 1 to 8 wherein the tissue repair is repair of a tissue selected from the group consisting of lung tissue, skin tissue, liver tissue, kidney tissue, tissue of the nervous system, cartilage, bone and tissue of the cardiovascular system.10. The method as claimed in any one of claims 1 to 8 wherein the tissue repair is tissue repair following the onset or development of arthritis.11. The method as claimed in claim 10 wherein the arthritis is rheumatoid arthritis.12. The method as claimed in any one of claims ito 8 wherein the tissue repair is tissue repair following the onset or development of neurodegenerative diseases.13. The method as claimed in claim 12 wherein the neurodegenerative disease is Alzheimer's disease.14 The method as claimed in claim 12 wherein the neurodegenerative disease is Parkinson's disease.15. The method as claimed in any one of claims 1 to 8 wherein the tissue repair is tissue repair following the onset or development of chronic obstructive pulmonary disease.16. The method as claimed in claim 15 wherein the chronic obstructive pulmonary disease is emphysema.
- 17. The method as claimed in claim 1 wherein the inhibition of cell membrane physicochemical changes is as a result of increased sphingomyelinase activity. Co
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB0703652.8A GB0703652D0 (en) | 2007-02-26 | 2007-02-26 | Method for the allosteric modulation of beta1 integrin |
GB0916943A GB2460009B (en) | 2007-02-26 | 2008-02-26 | Compounds and methods for the modulation of beta-1 integrin function to mediatetissue repair |
Publications (3)
Publication Number | Publication Date |
---|---|
GB201108815D0 GB201108815D0 (en) | 2011-07-06 |
GB2478082A true GB2478082A (en) | 2011-08-24 |
GB2478082B GB2478082B (en) | 2011-11-30 |
Family
ID=44315456
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
GB1108815A Active GB2478082B (en) | 2007-02-26 | 2008-02-26 | Compounds and methods for the modulation of beta-1 integrin function to mediate tissue repair |
Country Status (1)
Country | Link |
---|---|
GB (1) | GB2478082B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013038196A1 (en) * | 2011-09-17 | 2013-03-21 | Rehab Aljamal-Naylor | Method for reversing or inhibiting replicative senescence |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005037313A2 (en) * | 2003-10-17 | 2005-04-28 | University Court Of The University Of Edinburgh | Tissue repair by modulation of beta-1 integrin biological function |
-
2008
- 2008-02-26 GB GB1108815A patent/GB2478082B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005037313A2 (en) * | 2003-10-17 | 2005-04-28 | University Court Of The University Of Edinburgh | Tissue repair by modulation of beta-1 integrin biological function |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013038196A1 (en) * | 2011-09-17 | 2013-03-21 | Rehab Aljamal-Naylor | Method for reversing or inhibiting replicative senescence |
Also Published As
Publication number | Publication date |
---|---|
GB2478082B (en) | 2011-11-30 |
GB201108815D0 (en) | 2011-07-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20140322222A1 (en) | Compounds and methods for the modulation of beta-1 integrin function to mediate tissue repair | |
Liu et al. | Human junction adhesion molecule regulates tight junction resealing in epithelia | |
EP2839843B1 (en) | VLA-1 antagonist for use in treating stroke | |
US8865645B2 (en) | Method of treating lung fibrosis using ST2 polypeptide | |
EA016193B1 (en) | Antibodies directed against amyloid-beta peptide and methods using same | |
KR101429783B1 (en) | Antihuman α9 integrin antibody and use of the same | |
KR20220162819A (en) | Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (csfir) | |
US20030026803A1 (en) | Compositions for inhibiting macrophage activity | |
WO2010030813A2 (en) | Methods for inhibiting ocular angiogenesis | |
JP2019081796A (en) | Anti-tlr4 antibodies and methods of use thereof | |
WO2006088890A2 (en) | Treating stroke | |
CN106046158A (en) | Antibody against human prostaglandin e2 receptor ep4 | |
JP2004519215A (en) | Claudin polypeptide | |
TW201121570A (en) | A method of promoting dendritic spine density | |
UA126908C2 (en) | Methods for reducing proteinuria in a human subject suffering from immunoglobulin a nephropathy | |
US9403908B2 (en) | Method for altering hematopoietic progenitor cell adhesion, differentiation, and migration | |
MXPA01012388A (en) | A blocking monoclonal antibody to vla-1 and its use for the treatment of inflammatory disorders. | |
KR20100123857A (en) | Anti-adam-15 antibodies and utilization of the same | |
CA2494870A1 (en) | Methods for inhibiting angiogenesis | |
US20050074823A1 (en) | Selective inhibition of toll-like receptor-2 | |
TWI609692B (en) | Novel stip1 polypeptides and uses thereof | |
GB2478082A (en) | Identification of compounds for use in tissue repair | |
JP5037134B2 (en) | Tissue repair | |
US20100211325A1 (en) | Laminin binding site | |
Geiger | Roles of Transient Receptor Potential (TRP) cation channels in primary pulmonary fibroblasts |