WO2008028692A2 - Method for screening drug candidates for inflammatory diseases mediated by lps-inducible cc chemokine receptor mechanism - Google Patents
Method for screening drug candidates for inflammatory diseases mediated by lps-inducible cc chemokine receptor mechanism Download PDFInfo
- Publication number
- WO2008028692A2 WO2008028692A2 PCT/EP2007/008509 EP2007008509W WO2008028692A2 WO 2008028692 A2 WO2008028692 A2 WO 2008028692A2 EP 2007008509 W EP2007008509 W EP 2007008509W WO 2008028692 A2 WO2008028692 A2 WO 2008028692A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ccrl2
- seq
- mammal
- degree
- ccr
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 70
- 229940000406 drug candidate Drugs 0.000 title claims abstract description 59
- 238000012216 screening Methods 0.000 title claims abstract description 25
- 208000027866 inflammatory disease Diseases 0.000 title claims abstract description 23
- 230000001404 mediated effect Effects 0.000 title claims abstract description 15
- 108091008927 CC chemokine receptors Proteins 0.000 title claims abstract description 13
- 102000005674 CCR Receptors Human genes 0.000 title claims abstract description 13
- 230000007246 mechanism Effects 0.000 title description 6
- 102100025074 C-C chemokine receptor-like 2 Human genes 0.000 claims abstract description 97
- 101000934394 Homo sapiens C-C chemokine receptor-like 2 Proteins 0.000 claims abstract description 92
- 230000005764 inhibitory process Effects 0.000 claims abstract description 50
- 206010039073 rheumatoid arthritis Diseases 0.000 claims abstract description 38
- 238000002360 preparation method Methods 0.000 claims abstract description 37
- 239000003112 inhibitor Substances 0.000 claims abstract description 20
- 230000012292 cell migration Effects 0.000 claims abstract description 17
- 201000010099 disease Diseases 0.000 claims abstract description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 17
- 230000000172 allergic effect Effects 0.000 claims abstract description 15
- 208000010668 atopic eczema Diseases 0.000 claims abstract description 15
- 238000000338 in vitro Methods 0.000 claims abstract description 14
- 238000011282 treatment Methods 0.000 claims abstract description 13
- 208000006673 asthma Diseases 0.000 claims abstract description 12
- 241000699670 Mus sp. Species 0.000 claims description 90
- 210000004443 dendritic cell Anatomy 0.000 claims description 69
- 210000004027 cell Anatomy 0.000 claims description 52
- 241000124008 Mammalia Species 0.000 claims description 46
- 108010058846 Ovalbumin Proteins 0.000 claims description 26
- 229940092253 ovalbumin Drugs 0.000 claims description 26
- 239000012634 fragment Substances 0.000 claims description 24
- 108091006082 receptor inhibitors Proteins 0.000 claims description 24
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 15
- 102000000503 Collagen Type II Human genes 0.000 claims description 13
- 108010041390 Collagen Type II Proteins 0.000 claims description 13
- 239000002975 chemoattractant Substances 0.000 claims description 12
- 210000004969 inflammatory cell Anatomy 0.000 claims description 11
- 102000004127 Cytokines Human genes 0.000 claims description 10
- 108090000695 Cytokines Proteins 0.000 claims description 10
- 230000035605 chemotaxis Effects 0.000 claims description 10
- 238000001727 in vivo Methods 0.000 claims description 10
- 230000001939 inductive effect Effects 0.000 claims description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 230000000692 anti-sense effect Effects 0.000 claims description 9
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 9
- 208000024891 symptom Diseases 0.000 claims description 9
- 238000003556 assay Methods 0.000 claims description 8
- 230000028709 inflammatory response Effects 0.000 claims description 8
- 102000008186 Collagen Human genes 0.000 claims description 7
- 108010035532 Collagen Proteins 0.000 claims description 7
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 claims description 7
- 229920001436 collagen Polymers 0.000 claims description 7
- 238000000684 flow cytometry Methods 0.000 claims description 7
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 claims description 6
- 238000011156 evaluation Methods 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 claims description 5
- 101000934396 Mus musculus C-C chemokine receptor-like 2 Proteins 0.000 claims description 5
- 230000006041 cell recruitment Effects 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 5
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 claims description 4
- 230000000007 visual effect Effects 0.000 claims description 4
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 claims description 3
- 201000009961 allergic asthma Diseases 0.000 claims description 3
- 230000001960 triggered effect Effects 0.000 claims description 3
- 101150035842 CCRL2 gene Proteins 0.000 claims description 2
- 208000035473 Communicable disease Diseases 0.000 claims description 2
- 206010039085 Rhinitis allergic Diseases 0.000 claims description 2
- 201000010105 allergic rhinitis Diseases 0.000 claims description 2
- 230000001413 cellular effect Effects 0.000 claims 1
- 102000043803 human CCL19 Human genes 0.000 claims 1
- 102000043711 human CCL21 Human genes 0.000 claims 1
- 102000043525 human CXCL12 Human genes 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 11
- 229940079593 drug Drugs 0.000 abstract description 10
- 238000012750 in vivo screening Methods 0.000 abstract description 7
- 238000002474 experimental method Methods 0.000 description 25
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 19
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 19
- 239000000203 mixture Substances 0.000 description 17
- 241001529936 Murinae Species 0.000 description 15
- 208000009386 Experimental Arthritis Diseases 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 206010003246 arthritis Diseases 0.000 description 13
- 230000005012 migration Effects 0.000 description 13
- 238000013508 migration Methods 0.000 description 13
- 239000000443 aerosol Substances 0.000 description 12
- 239000002158 endotoxin Substances 0.000 description 12
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 11
- 102000005962 receptors Human genes 0.000 description 11
- 108020003175 receptors Proteins 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 101100273674 Mus musculus Ccrl2 gene Proteins 0.000 description 10
- 239000012530 fluid Substances 0.000 description 10
- 210000000265 leukocyte Anatomy 0.000 description 10
- 229920006008 lipopolysaccharide Polymers 0.000 description 10
- 239000013543 active substance Substances 0.000 description 9
- 239000003446 ligand Substances 0.000 description 9
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 210000003491 skin Anatomy 0.000 description 8
- 102000019034 Chemokines Human genes 0.000 description 7
- 108010012236 Chemokines Proteins 0.000 description 7
- 206010061218 Inflammation Diseases 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 108091034117 Oligonucleotide Proteins 0.000 description 7
- 230000004054 inflammatory process Effects 0.000 description 7
- 210000001165 lymph node Anatomy 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 239000013566 allergen Substances 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 230000006698 induction Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 239000012071 phase Substances 0.000 description 6
- 230000008961 swelling Effects 0.000 description 6
- 239000003826 tablet Substances 0.000 description 6
- 208000037883 airway inflammation Diseases 0.000 description 5
- 239000006071 cream Substances 0.000 description 5
- 230000002950 deficient Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 102000009410 Chemokine receptor Human genes 0.000 description 4
- 108050000299 Chemokine receptor Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 4
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 4
- 108090000176 Interleukin-13 Proteins 0.000 description 4
- 108090000978 Interleukin-4 Proteins 0.000 description 4
- 108010002616 Interleukin-5 Proteins 0.000 description 4
- 108010004729 Phycoerythrin Proteins 0.000 description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 4
- 102100040247 Tumor necrosis factor Human genes 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 210000001185 bone marrow Anatomy 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 238000012377 drug delivery Methods 0.000 description 4
- 239000003995 emulsifying agent Substances 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 210000003979 eosinophil Anatomy 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 210000005174 lung dendritic cell Anatomy 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 230000035800 maturation Effects 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102100036846 C-C motif chemokine 21 Human genes 0.000 description 3
- 239000004821 Contact adhesive Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 3
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 3
- 239000012981 Hank's balanced salt solution Substances 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 208000002200 Respiratory Hypersensitivity Diseases 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 239000004480 active ingredient Substances 0.000 description 3
- 230000010085 airway hyperresponsiveness Effects 0.000 description 3
- 210000002798 bone marrow cell Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000002757 inflammatory effect Effects 0.000 description 3
- 210000005265 lung cell Anatomy 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 210000004498 neuroglial cell Anatomy 0.000 description 3
- 239000002674 ointment Substances 0.000 description 3
- 230000002685 pulmonary effect Effects 0.000 description 3
- 230000007115 recruitment Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 2
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 description 2
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 2
- 241000239218 Limulus Species 0.000 description 2
- 101000746372 Mus musculus Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 2
- 239000004264 Petrolatum Substances 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 229920005372 Plexiglas® Polymers 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 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 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000000853 adhesive Substances 0.000 description 2
- 230000001070 adhesive effect Effects 0.000 description 2
- 208000037884 allergic airway inflammation Diseases 0.000 description 2
- 208000028004 allergic respiratory disease Diseases 0.000 description 2
- 239000005557 antagonist Substances 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 208000037976 chronic inflammation Diseases 0.000 description 2
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 210000003414 extremity Anatomy 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- 210000005210 lymphoid organ Anatomy 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 230000010534 mechanism of action Effects 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 2
- GLDOVTGHNKAZLK-UHFFFAOYSA-N octadecan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 235000019271 petrolatum Nutrition 0.000 description 2
- 229940066842 petrolatum Drugs 0.000 description 2
- 239000006187 pill Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- -1 polyethylenes Polymers 0.000 description 2
- 239000004926 polymethyl methacrylate Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000033300 receptor internalization Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000005212 secondary lymphoid organ Anatomy 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- XWTYSIMOBUGWOL-UHFFFAOYSA-N (+-)-Terbutaline Chemical compound CC(C)(C)NCC(O)C1=CC(O)=CC(O)=C1 XWTYSIMOBUGWOL-UHFFFAOYSA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100036305 C-C chemokine receptor type 8 Human genes 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108010040471 CC Chemokines Proteins 0.000 description 1
- 102000001902 CC Chemokines Human genes 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229940123150 Chelating agent Drugs 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010008165 Etanercept Proteins 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 101710162577 Fms-related tyrosine kinase 3 ligand protein Proteins 0.000 description 1
- 102100020715 Fms-related tyrosine kinase 3 ligand protein Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 239000004705 High-molecular-weight polyethylene Substances 0.000 description 1
- 101000716063 Homo sapiens C-C chemokine receptor type 8 Proteins 0.000 description 1
- 101000932480 Homo sapiens Fms-related tyrosine kinase 3 ligand Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 206010060820 Joint injury Diseases 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 208000008771 Lymphadenopathy Diseases 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102100032341 PCNA-interacting partner Human genes 0.000 description 1
- 101710196737 PCNA-interacting partner Proteins 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 244000303512 Peucedanum ostruthium Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920002367 Polyisobutene Polymers 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 102000018594 Tumour necrosis factor Human genes 0.000 description 1
- 108050007852 Tumour necrosis factor Proteins 0.000 description 1
- 206010047139 Vasoconstriction Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- SMYKVLBUSSNXMV-UHFFFAOYSA-K aluminum;trihydroxide;hydrate Chemical compound O.[OH-].[OH-].[OH-].[Al+3] SMYKVLBUSSNXMV-UHFFFAOYSA-K 0.000 description 1
- 239000002280 amphoteric surfactant Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- 230000003266 anti-allergic effect Effects 0.000 description 1
- 230000001088 anti-asthma Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000000924 antiasthmatic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 230000002917 arthritic effect Effects 0.000 description 1
- NBMKJKDGKREAPL-DVTGEIKXSA-N beclomethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(Cl)[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O NBMKJKDGKREAPL-DVTGEIKXSA-N 0.000 description 1
- 229940092705 beclomethasone Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 102000014974 beta2-adrenergic receptor activity proteins Human genes 0.000 description 1
- 108040006828 beta2-adrenergic receptor activity proteins Proteins 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 239000012578 cell culture reagent Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229960000541 cetyl alcohol Drugs 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- IMZMKUWMOSJXDT-UHFFFAOYSA-N cromoglycic acid Chemical compound O1C(C(O)=O)=CC(=O)C2=C1C=CC=C2OCC(O)COC1=CC=CC2=C1C(=O)C=C(C(O)=O)O2 IMZMKUWMOSJXDT-UHFFFAOYSA-N 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 229960000403 etanercept Drugs 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 244000144993 groups of animals Species 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000007762 localization of cell Effects 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 208000018555 lymphatic system disease Diseases 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000002433 mononuclear leukocyte Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000003843 mucus production Effects 0.000 description 1
- GOQYKNQRPGWPLP-UHFFFAOYSA-N n-heptadecyl alcohol Natural products CCCCCCCCCCCCCCCCCO GOQYKNQRPGWPLP-UHFFFAOYSA-N 0.000 description 1
- BXWNKGSJHAJOGX-UHFFFAOYSA-N n-hexadecyl alcohol Natural products CCCCCCCCCCCCCCCCO BXWNKGSJHAJOGX-UHFFFAOYSA-N 0.000 description 1
- 210000004296 naive t lymphocyte Anatomy 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 231100000344 non-irritating Toxicity 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000003883 ointment base Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000003209 petroleum derivative Substances 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 229960002052 salbutamol Drugs 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- 150000004760 silicates Chemical class 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
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000005065 subchondral bone plate Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- NCEXYHBECQHGNR-QZQOTICOSA-N sulfasalazine Chemical compound C1=C(O)C(C(=O)O)=CC(\N=N\C=2C=CC(=CC=2)S(=O)(=O)NC=2N=CC=CC=2)=C1 NCEXYHBECQHGNR-QZQOTICOSA-N 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 201000004595 synovitis Diseases 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960000195 terbutaline Drugs 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- CWERGRDVMFNCDR-UHFFFAOYSA-M thioglycolate(1-) Chemical compound [O-]C(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-M 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000002303 tibia Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000012049 topical pharmaceutical composition Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013271 transdermal drug delivery Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 230000025033 vasoconstriction Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
- C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
-
- 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
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0368—Animal model for inflammation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
Definitions
- the present invention relates to a screening method for identifying novel drugs for inflammatory diseases mediated by LPS-inducible CC chemokine receptor mechanism.
- the diseases are particularly allergic airways diseases such as asthma or rheumatoid arthritis .
- Allergic asthma is a chronic inflammatory disease of the airways triggered by inhalation of environmental allergens and characterized clinically by a reversible obstruction in airflow resulting from edema, mucus production and an exaggerated vasoconstriction of the bronchi to non-specific stimuli known as airway hyperresponsiveness (AHR) (1) .
- the asthma response in its first acute phase is associated with the activation of mast cells by IgE, and is followed by a second, more sustained and dangerous phase, in which an accumulation of inflammatory leukocytes, in particular eosinophils and T cells, takes place.
- chemokines are recruited to the lung by the generation of chemokine gradients secreted by resident as well as infiltrating lung cells. Extensive studies have demonstrated a central role for chemokines in orchestrating leukocyte accumulation in the asthmatic response (2, 3) .
- Dendritic cells are a unique group of mononuclear leukocytes that play a regulatory role in the immune system. They are specialized for antigen capture and presentation to naive T lymphocytes and therefore are critical for the initiation of the immune responses. Recent reports suggest that the immune outcome is associated with distinct DC subpopulations (4, 5) .
- a hallmark of asthma is the abnormal expansion of T helper type 2 (Th2) cells. This event is crucial for the disease since Th2 cells produce key cytokines, including IL-4, IL-5 and IL-13, that in turn activate parenchymal and resident lung cells that release mediators, including chemokines, that lead to airway inflammation (1) .
- RA Rheumatoid arthritis
- CIA Collagen-induced arthritis
- CII is the major constituent protein of cartilage in diarthrodial joints, the predominant site of inflammation in RA.
- pathogenesis of CIA is in many ways similar to that of RA as both RA and CIA are characterized by an intense synovitis accompanied by erosions of cartilage and subchondral bone by a pannus-like tissue (20) .
- Susceptibility to CIA was considered to be MHC class-linked (H-2 q and H- 2 r ) (21), however, recently Campbell et al. (22,23) modified the immunization procedure and showed that clinically and histologically similar CIA may be induced in C57BL/6 (B6) mice.
- the CIA model has proven equally useful to investigate inflammatory joint injury and led to the development of novel TNF- based therapies for human RA (24,25).
- LPS-inducible CC chemokine related gene is an orphan chemokine receptor originally identified in the mouse macrophage cell line RAW 264.7 (9) .
- L-CCR expression at the mRNA level has been described in murine macrophages (9) and in glial cells stimulated with LPS (10) .
- L-CCR shares the highest degree of homology with the putative orphan human chemokine receptor HCR (11) .
- HCR is largely distributed across leukocyte populations and, interestingly, it is upregulated in mature DC (12) .
- LPS-inducible CC chemokine receptor is functionally involved in several diseases, among which allergic airways diseases such as asthma and rheumatoid arthritis. Due to the high degree of homology and similarity between L-CCR and HCR, it has been found that it is possible to identify new inhibitors of CCRL2 receptor
- CCRL2 is the name of the LPS-inducible CC chemokine receptor in all the species
- an object of the present invention to provide an in vitro method for screening drug candidates for inflammatory diseases, the said method comprising a step of putting into contact a L- CCR-expressing cell preparation with a drug candidate for CCRL2 inhibition and then evaluating the degree of L-CCR-expressing cell migration against a control.
- L-CCR is induced in mouse DCs during maturation. Therefore, mice deficient for L-CCR (L-CCR " ' " ) were generated and studied in an established model of allergen-induced airway inflammation in which DC plays a prominent role (13) . The results of these studies show that OVA- sensitized L-CCR " ' " mice had significantly reduced number of total leukocytes, eosinophils and lymphocytes/mononuclear cells in the airways, thus proving an important role of this receptor to elicit an hyperreactive response and therefore asthma.
- an in vivo model for inducing rheumatoid arthritis in a mammal has been provided, as will be described below. Therefore, it is another object of the present invention to provide an in vivo method for screening drug candidates for rheumatoid arthritis, the said method comprising a step of treating a mammal genetically expressing L-CCR receptor with an amount of a drug candidate for CCRL2 inhibition and then evaluating the mammal response in a model of induced rheumatoid arthritis against a control.
- a further object of the present invention is a cell preparation generated from a LCCR-depleted mammal, to be used as a control tool in the method of the present invention.
- a still further object of the present invention are monoclonal antibodies to LCCR receptor, to be used as a triggering tool in the in vitro method of the present invention.
- Another object of the present invention are inhibitors of CCRL2 receptor, in particular of HCR receptor, obtained by the screening method of the invention, as well as a method of treatment of an inflammatory disease mediated by LPS-inducible CC chemokine receptor, particularly an allergic airways inflammation such as asthma or rheumatoid arthritis, by using such inhibitors.
- the term "inhibitor” means a molecule that prevents CCRL2 receptor from engaging in or producing an action on the body of the patient to be treated, by blocking CCRL2 receptor, replacing CCRL2 receptor in binding to a chemoattractant or preventing CCRL2 receptor expression in an inflammatory cell.
- a CCRL2 inhibitor may be a molecule that binds to CCRL2 receptor and antagonize its function or a molecule that mimics CCRL2 receptor and prevents it to elicit an inflammatory response by reacting with specific cytokines on CCRL2 behalf.
- FIG. 1 shows experimental data on the induction of L-CCR in maturing mouse dendritic cells
- Figure 2 is a schematic view of the method used to generate L-CCR deficient mice
- Figure 3 depicts the role of L-CCR in the model of OVA-induced airways inflammation
- Figure 4 shows the trafficking of lung dendritic cells in L-CCR-depleted mice upon OVA antigen challenge
- FIG. 5 shows the role of L-CCR in CCR7-mediated dendritic cells migration
- Figure 6 shows the chemokine and chemoattractant molecules tested for chemotaxis and receptor internalization on L 1.2 and CHO L-CCR transfected cells
- Figures 7a, 7b show sequence listings of inhibitors of CCRL2 receptor
- Figure 8 depicts the role of L-CCR in the model of induced rheumatoid arthritis
- Figure 9 shows a diagram reporting the paw swelling in LCCR-depleted mice against WT mice
- Figure 10 shows a diagram reporting weight ans cell number of limph nodes in LCCR-depleted and WT mice.
- the present invention relates to an in vitro method for screening drug candidates for inflammatory diseases mediated by LPS-inducible CC chemokine receptor, the said method comprising a step of putting into contact a CCRL2-expressing cell preparation with a drug candidate for CCRL2 inhibition and then evaluating the degree of CCRL2-expressing cell migration against a control.
- the cell preparation of the invention is obtained preferably from mice, i.e. a L-CCR-expressing cell preparation. More preferably, the said cells are dendritic cells.
- the method of the invention comprises a chemotaxis assay as depicted below, wherein the dendritic cell migration toward a chemoattractant is evaluated.
- the said chemoattractant is preferably a cytokine.
- Human CCLl 9 and CCL21 cytokines are preferably used as the chemoattractants in the invention method, as they are specific CCR7 ligands. However, as a control tool, CXCL12, ligand for CXCR4 but not for CCR7, is also used.
- the chemotaxis assay is performed by using a chemotaxis chamber having an upper and a lower compartment.
- the cell preparation is put together with the triggering tool, i.e. the anti-L-CCR antibody, while in the lower compartment the chemoattractant is present.
- the drug candidate is added to the upper compartment while a control experiment without the drug candidate is performed.
- the inhibition of migration of the dendritic cells from the upper to the lower compartment is evaluated, taking the control experiment as a measure of 0% inhibition.
- the dendritic cell migration is triggered by a suitable substance.
- a specific anti-L-CCR monoclonal antibody (anti-L-CCR mAb) is preferably used as a triggering tool.
- IgG2a mAb and IgG2b mAb generated from L-CCR depleted mice are used as a triggering tool.
- the said mAbs are obtained as depicted below, i.e. IgG2a mAb from clone 4.2.1 and IgG2b mAb from clone 2.5.1 of hybridomes cultured from L-CCR-depleted mice.
- a further control is used for the scope of the invention.
- the said control is a cell preparation, specifically a dendritic cell preparation, that is depleted of L-CCR receptor.
- the said cell preparation is obtained from L-CCR-depleted mice.
- the step of evaluation of the L-CCR expressing cell migration against the control is preferably accomplished by flow cytometry, as depicted below.
- the drug candidate is usually tested in a suitable form that allows good contact between the substance and the cell preparation.
- the drug candidate is administered in solution or suspension.
- the drug candidate is used in amounts or concentrations that depend on several factors, among which the molecular weight of the substance, its solubility and so on. Several concentrations of the same drug candidate can also be tested, in order to calculate its IC50.
- Dendritic cell (DC) culture DC were generated from bone marrow cells as described in Vecchi et al . J. Leukoc. Biol. 66: 489-494; 1999.
- 8- to 12-week-old WT and LCCR-KO male mice (L-CCR- depleted mice) on C57B1/6J background were used.
- CD34+ bone marrow cells from femurs and tibias were positively immunoselected using MACS microbeads coated with goat anti-rat IgG (Miltenyi Biotec
- CD34+ positive cells (2xlO 5 /mL) were cultured in RPMI1640 medium with 5% fetal calf serum, 2xlO '5 M ⁇ -mercaptoethanol, GM-CSF (40 ng/mL) , and Flt3 ligand (100 ng/mL). Cells were diluted 1:2-1:3 every 2 or 3 days. Cultured cells were collected after 8-9 days of culture. To induce maturation, DC were cultured with 20 ng/mL TNF- ⁇ for the last 24 h of culture. DCs were characterized in terms of membrane phenotype, using phycoerythrin (PE) -conjugated hamster anti-mouse CDlIc
- Rat anti-mouse CD86 was from ATCC (HB-253) . Cells were analysed by FACScan.
- Knockout mice for L-CCR were used to raise mAbs against the extracellular part of L-CCR.
- Mice were administered i.p. with 10 7 irradiated L-CCR/L1.2 cells (a mix of 3 different clones) in PBS every other week for 6 weeks. 3 days after the last challenge, spleen cells were used for polyethylene glycol-mediated fusion following conventional protocols. Supernatants were screened for Abs that could bind to L-CCR/CHO-K1 transfectants and not parental CHO. Cultures that were positive in this primary screening were then tested for binding to the L-CCR/L1.2 cells and not to parental Ll.2.
- TNF ⁇ -matured DC 1.5 x 10 6 /mL from WT and KO mice were incubated for 30 minutes at 4 0 C with graded concentrations (0, 3 and 10 ⁇ g/mL) of the purified anti- LCCR mAbs and isotype matched irrelevant mAb, then added to the upper compartment of the chemotaxis chamber and CCL19, CCL21 (specific ligands for CCR7) and CXCL12 (not binding CCR7) were added in the lower compartment. This constitutes the control experiment.
- the screened molecule is incubated with the cell suspension in the upper chamber and the inhibition of the dendritic cell migration is evaluated against the control.
- Flow cytometry mAbs used were from BD-Pharmingen and included phycoerythrin (PE) -conjugated hamster anti-mouse CDlIc
- Rat anti-mouse CD86 was from ATCC (HB-253) . Cells were analysed by FACScan.
- DC from WT mice pretreated with mAbs to LCCR displayed an increased migration in response to the CCR7 ligands CCL19 and CCL21.
- the effect was specific, since migration to CXCL12, ligand for CXCR4, was not affected as shown in fig. 5.
- An isotype matched, irrelevant mAb did not modified DC migration to CCR7 ligands.
- migration of DC from LCCR KO mice to CCR7 ligands was not modified after exposure to anti-LCCR mAbs.
- the CCRL2 inhibitors cause an inhibition of the dendritic cell migration from the upper to the lower compartment if compared to the control.
- the drug candidates that pass the screening test on L-CCR-expressing cell preparation are then tested on a further cell preparation wherein the dendritic cells are depleted from L-CCR receptor (L-CCR "7" cell preparation) , to prove that their mechanism of action is actually an L-CCR inhibitory mechanism. No inhibition of migration is expected for pure CCRL2 inhibitors. A different degree of inhibition with respect to the previous test is indicative of an impure mechanism.
- the drug candidates that confirm their mechanism of action as being an CCRL2 inhibition are then preferably passes to the in vivo screening method, as depicted below.
- Fig. 6 reports the list of the molecules tested and found not to activate LCCR, in terms of chemotaxis and receptor internalization.
- an in vivo screening method comprises inducing an allergen-type airways inflammatory response in a mammal, treating the said mammal with a drug candidate for inhibition of CCRL2 receptor and evaluating the inhibition of inflammatory cell recruitment in the BAL of said mammal against a control .
- the said mammal is a mouse.
- the allergen-type airways inflammatory response is preferably elicited by a suitable allergen.
- OVA bronchoalveolar lavage
- the drug candidates for CCRL2 inhibition are screened in such a test.
- the drug candidate is administered to the mammal in any suitable way, such as orally, intraperitoneally, i.v. or the like, before, during or after OVA challenge.
- the drug candidate is in a suitable form, such as in solid form (powder, tablet or the like) or liquid form (solution, suspension or the like) for oral administration, liquid form for intravenous injection and so on.
- a control experiment is run, wherein no drug candidate administration is made, so that a 0% inhibition is given.
- the %inhibition obtained with the drug candidate with respect to such a control is evaluated.
- an in vivo screening method comprises inducing rheumatoid arthritis in a mammal, treating the said mammal with a drug candidate for inhibition of CCRL2 receptor and evaluating the inhibition of arthritis symptoms in said mammal against a control.
- the said mammal is a mouse.
- the rheumatoid arthritis is preferably elicited by a collagen, particularly collagen type II.
- the mammals are first treated with collagen according to the protocol described below in order to induce arthritis. Then, a visual evaluation is made of the symtoms od rheumatoid arthritis, in particular paw swelling as described below, together with the assessment of the thickness of the hind paws and the count of number of cells and weight of the limph nodes.
- the pretreatment with collagen is performed in any suitable way, such as by i.d. injection at the base of the tail.
- the injection is preferablu made twice, at day 0 and day 21.
- the drug candidates for CCRL2 inhibition are screened in such tests.
- the drug candidate is administered to the mammal in any suitable way, such as orally, intraperitoneally, i.v. or the like, before, during or after OVA challenge.
- the drug candidate is in a suitable form, such as in solid form (powder, tablet or the like) or liquid form (solution, suspension or the like) for oral administration, liquid form for intravenous injection and so on.
- a control experiment is run, wherein no drug candidate administration is made, so that a 0% inhibition is given.
- the %inhibition obtained with the drug candidate with respect to such a control is evaluated.
- the screening dose of the drug candidate can vary in a wide range of doses depending on several factors, such the IC 50 calculated from the above in vitro test results or the LD 50 that can be determined for such compound. Generally, the dose of the drug candidate used in the present in vivo screening method will be below its LD 50 . It is also possible to test different doses of the drug candidate in the in vivo test, in order to determine its ED 5 O-
- L-CCR is a murine orphan chemokine receptor.
- L-CCR mRNA was barely detectable in bone marrow (BM) -derived myeloid DC, however, lipopolysaccharide (LPS) stimulation upregulated L-CCR mRNA after 30 min, reaching a peak at 2 h and decreasing afterwards (Fig. IA) .
- LPS lipopolysaccharide
- CCR7 increased its transcripts levels in the same samples, but in a delayed kinetic.
- L-CCR "; ⁇ mice developed normally to term and were fertile. No significant differences were found after histological and flow cytometric analysis of lymphoid organs (not shown) .
- L-CCR "7" mice had a normal lifespan and did not show an overt phenotype in the steady-state.
- L-CCR is required for pulmonary DC homing to mediastinal LNs
- the in vivo screening method according to the invention is performed by sensitizing the WT (wild type, thus expressing L-CCR receptor) mice with ovalbumin (OVA) , then challenging such mice with OVA to induce the hyperreactive response and counting the number of dendritic cells in the BAL of the animals, as described below. This constitutes the control experiment.
- Another group of mice is treated in the same manner, but a drug candidate for inhibition of WT (wild type, thus expressing L-CCR receptor) mice with ovalbumin (OVA) , then challenging such mice with OVA to induce the hyperreactive response and counting the number of dendritic cells in the BAL of the animals, as described below. This constitutes the control experiment.
- Another group of mice is treated in the same manner, but a drug candidate for inhibition of
- CCRL2 receptor is administered to the mice before, during or after the sensitization with OVA.
- %inhibition of the dendritic cells recruitment in the BAL is evaluated with respect to the control experiment to which 0% inhibition is assigned.
- GM-CSF Mouse granulocyte-macrophage colony-stimulating factor
- GM-CSF Human granulocyte-macrophage colony-stimulating factor
- Human Flt3 ligand was from Peprotech Inc. (Rocky Hill, NJ).
- LPS Erichia coli strain 055 :B5
- Cell culture reagents were from Gibco and Hyclone.
- Recombinant chemokines were from Peprotech (Rocky Hill, NJ) and R&D. Cytokines were endotoxin free as assessed by Limulus amoebocyte assay (BioWhittaker Inc., Walkersville, MD). All other reagents were from Sigma unless indicated otherwise .
- a gene- targeting vector was constructed in which the whole open reading frame of the gene was replaced by a Neo cassette.
- This vector was transfected into embryonic stem (ES) cells. After selection, one targeted clone was identified by Southern blotting hybridization. It was injected into C57B1/6 blastocysts to generate germ line chimeras and subsequently L-CCR +/" heterozygote mice of 129/Sv C57B1/6 mixed genetic background. L-CCR +/+ mice are littermates of
- mice DNA derived from tail biopsies was amplified by polymerase chain reaction that detected the wild type and targeted allele, respectively. L-CCR deficiency at the mRNA level was confirmed in thioglycollate-elicited neutrophils obtained from L-CCR -/- animals.
- mice Age and sex-matched L-CCR deficient mice were sensitized to ovalbumin (OVA; grade V, Sigma) with a single i.p. injection of 10 ⁇ g OVA mixed with 4 mg aluminium hydroxide gel in 0.2 ml sterile saline solution on days 1 and 11. On day 18 mice were placed in a Plexiglas chamber and exposed to a 5.0 % OVA aerosol (w/v in sterile saline solution) for 20 min. The aerosol was generated by a PARI Master Nebulizer (Sapio Life, Milan, Italy) connected to the Plexiglas chamber.
- OVA ovalbumin
- Aerosol challenge was repeated daily for 6 days and 24 hrs post last challenge mice were anesthetized with urethane (12% w/v, 400 ⁇ l/ mouse, i.p.), bled, and tracheotomized. Sera were prepared by centrifugation and snap-frozen to -20 0 C until further use. Bronco-alveolar lavages (BAL) were carried out by injection into the airways of 3 x 1 ml sterile Hank's balanced salt solution (HBSS, Gibco-BRL) . Lavage fluid was recovered after 30 sec by gentle aspiration from the airways and lavages from each were pooled. Samples were then centrifuged at 2000 rpm for 10 min at 4 0 C.
- BAL Bronco-alveolar lavages
- BAL fluid was concentrated by using Vivaspin 2 concentrators (SIGMA) , and stored at - 20 0 C for further analysis.
- Pellets were re-suspended in 1 ml HBSS and the total cell number was determined from an aliquot of the cell suspension using a disposable multi- chamber hematocytometer (FAST-read 102, Biosigma, Italy) .
- FAST-read 102 Biosigma, Italy
- an aliquot of the cell suspension was spotted onto glass slides by centrifuging in a Cytospin (5 min, 800 rpm) . Slides were then fixed and stained with Diff-Quik staining set (Dade Behring SpA, Milan, Italy) , following manufacturer' s instructions. Standard morphological criteria were used in classifying at least 400 cells per slide under light microscopy.
- cytokines IL-4, IL-5, IL-13 and IFN ⁇
- IL-4, IL-5, IL-13 and IFN ⁇ were measured in BAL fluids by standard sandwich ELISA assays by using 150 ⁇ l of 4x concentrated fluid.
- Specific capture and detecting antibodies were from Pharmingen (IL-4, and IL-5) and R&D (IL-13, IFN ⁇ ) .
- FIG. 1 Induction of L-CCR in maturing mouse dendritic cells. DC were generated in vitro from CD34 + BM precursors.
- A Total RNA was prepared from untreated immature DC (0 h) or DC treated for different times with 100 ng/ml LPS and analyzed by Northern blot for the expression of L-CCR mRNA. The same filter was probed for CCR7. Ethidium bromide staining is shown in the lower part of the panel.
- B Flow cytometry of L- CCR and CCR7 surface expression by DC stimulated with 100 ng/ml LPS.
- the graphs show the mean fluorescence intensity (MFI) of cells labeled with L-CCR and CCR7 mAbs after subtraction of the background staining obtained in DC generated from L-CCR "/" mice (for L-CCR) or isotype-control matched mAb (for CCR7) .
- MFI mean fluorescence intensity
- FIG. 1 Generation of L-CCR deficient mice .
- A Exon/intron structure of the L-CCR gene. The figure is not drawn to scale. The size of exon 1, 2 and 3 are 125, 434 and 1603 bp respectively. Black area corresponds to the coding sequence.
- B Schematic representation of WT and KO alleles of the L-CCR gene and of targeting vector. The three exons are represented by open boxes. Neomycin-resistance gene driven by a PGK promoter (Neo) is indicated by a dashed box. Restriction sites: K, Kpnl; S, Sail; H, Hindlll; B, BamHI; Sa, Sad; Sc, Seal.
- Kpnl-digested genomic DNA fragments were detected by probe.
- C Southern blot analysis of Kpnl-digested genomic DNA from transfected ES cells hybridized with L-CCR probe generates a 12 kb WT restriction fragment and a 3.5 kb homologous recombinant restriction fragment.
- D PCR analysis of genomic DNA from L-CCR +/+ (lane 1), L-CCR "7" (lane 2,3) and L-CCR +/ ⁇ (lane 4) mice.
- E RT-PCR demonstration of disrupted L-CCR transcription in L-CCR "7" mice.
- A Differential cell counts in BAL fluid.
- B ELISA evaluation of cytokine production in BAL fluid of mice sensitized and challenged with OVA. Values are expressed as mean ⁇ SEM from three different experiments with 8 to 12 mice per group. Statistical significance: *, p ⁇ 0.05; **, p ⁇ 0.01.
- FIG. 4 Trafficking of lung DC in L-CCR "7" mice upon OVA antigen challenge. Homing of DC from the airways to draining MLNs. OVA-sensitized mice were treated by intratracheal administration of 800 ⁇ g OVA- FITC in 50 ⁇ l PBS and sacrificed after 24, 48 and 72 h. The accumulation of FITC + CDlIc + DCs in the MLNs was assessed by flow cytometry. Panel A: Kinetics analysis of FITC + DC accumulation in MLNs. Results are shown as mean ⁇ SEM from 12-15 mice per group from two separated experiments. *, p ⁇ 0.05, **, p ⁇ 0.01 in comparison with WT-treated mice.
- LCCR-/- mice originally generated on the mixed background 129Sv-C57Bl/6 were backcrossed with C57B1/6 mice for 10 generations, to have the C57B1/6 background for CIA experiments.
- CFA was prepared by mixing 100 mg of heat-killed Mycobacterium tuberculosis (H37Ra; Difco Laboratories) in 20 ml of IFA (Sigma-Aldrich) (22) .
- An emulsion was formed by dissolving 2.0 mg/ml chick CII (Collagen of type II, CII; Sigma-Aldrich) overnight at 4°C in 10 mM acetic acid and combining it with an equal volume of CFA.
- CII solution and the emulsion with CFA were always freshly prepared.
- Mice were injected i.d. at the base of the tail with a total of 100 ⁇ l of emulsion containing 100 ⁇ g of CII and 250 ⁇ g of M. tuberculosis. The same injection was repeated at day 21; however, due to toughening of the skin at the base of the tail, booster injections were distal to the primary injection site.
- mice were examined two to three times per week for the initial visual appearance of arthritis after immunization. Arthritis of each individual limb was graded using the following scoring system: 0, normal; 1, apparent swelling and redness limited to individual digits; 2, swelling in more than one joint; 3, severe redness and swelling of the entire paw including digits; and 4, maximally inflamed limb with involvement of multiple joints. The maximum score per mouse was 16. Mice were scored as arthritic if more than one paw had a score >2. The thickness of the hind paws was measured using a dial gauge caliper (Mitsutoyo) . At the end of the experiment (day 60) , inguinal lymph nodes were collected, weighted, disaggregated and the number of cells per lymph node was microscopically counted.
- Mitsutoyo dial gauge caliper
- the arthritis symptoms in LCCR-/- mice were studied after immunization with CII on day 0 and a boost with CII on day 21. Mice were examined weekly after the first immunization and every 2-3 days after the boost for signs of developing arthritis. The severity of the arthritis was assessed using a visual scoring as described above.
- the LCCR-/- and WT animals developed clinical signs of arthritis with an incidence of 8 and 28 % by day 29 and of 8 and 31 % by day 36, respectively (figure 8) . The incidence of clinical symptoms was thus lower in LCCR-/- mice through all the observation period, till day 60. As an other objective parameter to evaluate the severity of disease the thickness of the hind paws was measured.
- Paw swelling was always significant lower in LCCR-/- mice than in WT mice (figure 9) . Since it has been reported that the disease-induced inflammation cause lymph nodes enlargement, at day 60 when the experiment was stopped lymph nodes were recovered and evaluated. Weight and cell numbers of nodes from LCCR-/- mice were markedly lower, highly significantly different from those from WT mice (figure 10) .
- the in vivo screening method for rheumatoid arthritis according to the invention is performed by treating the WT (wild type, thus expressing L-CCR receptor) mice with collagen, then assessing the arthritis parameters as described above. This constitutes the control experiment. Another group of mice is treated in the same manner, but a drug candidate for inhibition of CCRL2 receptor is administered to the mice before, during or after the treatment with collagen. The %inhibition of the induced rheumatoid arthritis is evaluated with respect to the control experiment to which 0% inhibition is assigned.
- a cell preparation of inflammatory cells that are depleted of CCRL2 receptor is provided.
- a cell preparation is used, as explained above, as a control tool in the screening method of the invention, in order to confirm the CCRL2 inhibitory mechanism of the drug candidates selected according to the in vitro method performed on CCRL2 expressing cells.
- the inflammatory cell preparation is a dendritic cell preparation obtained by a CCRL2-depleted mammal. More preferably, the inflammatory cell preparation is a dendritic cell preparation obtained by a L-CCR-depleted mice.
- the inflammatory cell preparation of the invention can be obtained according to well known procedures, as described above.
- a still further object of the present invention are monoclonal antibodies to CCRL2 receptor, to be used as a triggering tool in the in vitro method of the present invention.
- the said antibodies are IgG2a mAb and IgG2b mAb generated from L-CCR depleted mice.
- the said mAbs are obtained as depicted above, i.e. IgG2a mAb from clone 4.2.1 and IgG2b mAb from clone 2.5.1 of hybridomes cultured from L-CCR- depleted mice.
- CCRL2 receptor inhibitors A still further object of the present invention are CCRL2 receptor inhibitors obtainable through the screening method of the invention.
- Object of the present invention is a CCRL2 receptor inhibitor that displays a %inhibition greater than 50% in the in vitro screening test and/or a %inhibition greater than 50% in the in vivo test of the invention.
- the CCRL2 receptor inhibitors of the invention are those inhibitors that bind to a CCRL2 receptor fragment identified by the following sequences :
- MANYTLAPEDEYDVLIEGELESDEAEQCDKYDAQALSA N-Terminal or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%;
- the said fragments of sequences SEQl- SEQ8 are the following (fig. 7b) :
- the CCRL2 receptor inhibitors are CCRL2 gene antisense sequences .
- the present invention provides antisense oligomers having a sequence effective to inhibit or block the expression of the CCRL2 encoding gene or mRNA sequence.
- Antisense technology which uses specific- oligonucleotides to inhibit expression of target gene products, is developing as a therapeutic modality for human disease.
- Several selection criteria are available to contribute to the optimization of antisense oligonucleotide antagonists. For example, it is advisable to choose sequences with 50% or more GC content.
- Preferred sequences span the AUG initiation codon of the target protein, but sites in the coding region and 5'UTR may perform equally well. Such sequences are generally about 18-30 nucleotides long.
- oligomers are often found to inhibit the target to a greater extent, indicating that a preferred length is about 25 mer for the first oligonucleotides chosen as antisense reagents.
- three oligonucleotide sequences are chosen with regard to these criteria, and compared for antagonist activity to control oligonucleotide sequences, such as "reverse" oligonucleotides or those in which about every fourth base of the antisense sequence is randomized. Therefore, a preferred sequence for making antisense oligomer sequences to
- CCRL2 is a 25 mer sequence from the CCRL2mRNA sequence (human) : GAGGAGGAAACAACTTCCCGGTTGC [SEQ ID NO.17, fig. 7c]. The whole sequence of the CCRL2 encoding gene is reported in NCBI database, under ref. No. NM 003965.
- Such preferred antisense sequences are used to construct antisense oligonucleotide agents (and suitable controls) for an in vitro comparison as inhibitors of CCRL2 receptor. These in vitro data are predictive of human clinical utility using antisense agents of comparable design.
- compositions comprising a pharmaceutically active amount of CCRL2 receptor inhibitor as defined above and a suitable carrier or excipient are also provided for.
- Pharmaceutically acceptable carriers or excipients can contain one or more physiologically acceptable compound (s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent (s) .
- physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.
- CCRL2 inhibitors as identified herein are useful for parenteral, topical, oral, or local administration, such as by inhalation, aerosol or transdermally, for prophylactic and/or therapeutic treatment of inflammatory diseases mediated by LPS- inducible CC chemokine receptor, particularly allergic airways inflammations or rheumatoid arthritis.
- Administration by inhalation or aerosol is generally preferred in the case of allaergic airways inflammation, while a systemic administration form is more suitable in the case of rheumatoid arthritis.
- a pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the like.
- Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate are employed along with various disintegrants such as starch and preferably potato or tapioca starch and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
- binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
- lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes.
- compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols.
- preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols.
- lactose or milk sugar as well as high molecular weight polyethylene glycols.
- the compounds of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents such as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
- compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, etc.
- Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms.
- Various preservatives are well known and include, for example, phenol and ascorbic acid.
- pharmaceutically acceptable carrier (s) including a physiologically acceptable compound depends, for example, on the route of administration of the active agent (s) and on the particular physio-chemical characteristics of the active agent (s) .
- the excipients are preferably sterile and generally free of undesirable matter.
- concentration of active agent (s) can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages in accordance with the dosage recommendations provided above. It will be appreciated that such dosages may be varied to optimize a therapeutic regimen in a particular subject or group of subjects.
- compositions of this invention are administered to a patient suffering from an inflammatory disease mediated by LPS-inducible CC chemokine receptor, such as an allergic airways inflammatory disease like asthma or rheumatoid arthritis, in an amount sufficient to cure or at least partially modify the issue of the condition and its complications.
- An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the active agents of the formulations of this invention to effectively treat (ameliorate one or more symptoms) the patient.
- the CCRL2 inhibitor (s) is administered orally (e.g. via a tablet) or as an injectable in accordance with standard methods well known to those of skill in the art.
- the CCRL2 inhibitors may also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the active agent (s) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin.
- the drug composition is typically contained in a layer, or "reservoir,” underlying an upper backing layer.
- the term “reservoir” in this context refers to a quantity of “active ingredient (s) " that is ultimately available for delivery to the surface of the skin.
- the “reservoir” may include the active ingredient (s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art.
- the patch may contain a single reservoir, or it may contain multiple reservoirs.
- the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery.
- suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
- the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form.
- the backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility.
- the material selected for the backing layer is preferably substantially impermeable to the active agent (s) and any other materials that are present .
- the CCRL2 inhibitor (s) is administered locally via a patch (e.g. as described above) or other topical formulation.
- Other preferred formulations for topical drug delivery include, but are not limited to, ointments and creams.
- Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives.
- Creams containing the selected active agent are typically viscous liquid or semisolid emulsions, often either oil-in-water or water-in-oil .
- Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase.
- the oil phase also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant .
- the emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant.
- the specific ointment or cream base to be used is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.
- compositions for inhalation or aerosol can be prepared according to conventional methods.
- standard inhalation devices filled with the CCRL2 inhibitor composition and a propellant gas can be used.
- a solution or suspension of the active ingredient composition in a suitable physiological medium is prepared, and the such solution or suspension is administered by aerosol with conventional aerosol machines .
- a method of treatment of an inflammatory disease mediated by LPS-inducible CC chemokine receptor involves treating a mammal in need thereof with a therapeutically effective dose of a CCRL2 receptor inhibitor as previously defined, wherein the said CCRL2 receptor inhibitor is comprised in a pharmaceutical composition together with a pharmaceutically acceptable carrier or excipient, as defined above.
- the dose of the CCRL2 receptor inhibitor that is administered can vary in a wide range of doses, depending on inhibitor's activity, the disease to be treated, its seriousness, the conditions of the patient, its weight and age as well as the route of administration, as will be determined by the clinician.
- the CCRL2 receptor inhibitor can be used in doses ranging from 0.001 mg to 50 mg per kg of body weight, from 1 to 4 times a day.
- CCRL2 receptor inhibitor as defined in any of claims from 27 to 29, for the preparation of a medicament for curing or alleviating an inflammatory disease mediated by LPS-inducible CC chemokine receptor is also claimed.
- the inflammatory disease to be treated is preferably selected from an allergic airways inflammatory disease such as allergic asthma, allergic rhinitis, airways infectious diseases and COPD (Chronic Obstructive Pulmonary Disease) , or from rheumatoid arthritis .
- an allergic airways inflammatory disease such as allergic asthma, allergic rhinitis, airways infectious diseases and COPD (Chronic Obstructive Pulmonary Disease) , or from rheumatoid arthritis .
- the CCRL2 receptor inhibitor of the invention be administered in combination with another CCRL2 receptor inhibitor or with a different antiallergic or anti asthmatic drug, such as an anti-istaminic drug, a glucocorticosteroid (like budenoside or beclomethasone) , sodium chromoglycate, ⁇ 2-adrenoceptor agonists (like salbutamol or terbutaline) ; or with one or more drugs used to treat rheumatoid arthritis or its symptoms, such as Non Steroidal Antiinflammatory Drugs (NSAIDs) , methotrexate, cyclosporin, salazopyrin, hydroxychlorokin, etanercept, or corticosteroidal drugs.
- NSAIDs Non Steroidal Antiinflammatory Drugs
- the expression w administered in combination with'' means that the drugs are contained in the same pharmaceutical composition or that they are administered in different pharmaceutical compositions during the therapy of the diseases. This means that the different drugs can be administered at the same time or at different times, depending on the needs of the patient and the protocol of administration dictated by the clinician.
- mice new insights into an important disease model of rheumatoid arthritis. Eur. J. Immunol. 30: 1568-1575.
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)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Environmental Sciences (AREA)
- Veterinary Medicine (AREA)
- General Engineering & Computer Science (AREA)
- General Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to an in vitro and in vivo screening methods for identifying novel drugs for inflammatory diseases mediated by LPS-inducible CC chemokine receptor, particularly allergic airways diseases or rheumatoid arthritis. The allergic airways disease is particularly asthma. Inhibitors of CCRL2 receptor are also disclosed, as well as a method of treatment such diseases by using these inhibitors. In particular, the screening method of the invention comprises a step of putting into contact a CCRL2- expressing cell preparation with a drug candidate for CCRL2 inhibition and then evaluating the degree of CCRL2- expressing cell migration against a control.
Description
DESCRIPTION
METHOD FOR SCREENING DRUG CANDIDATES FOR INFLAMMATORY DISEASES MEDIATED BY LPS-INDUCIBLE CC CHEMOKINE
RECEPTOR MECHANISM Field of the invention
The present invention relates to a screening method for identifying novel drugs for inflammatory diseases mediated by LPS-inducible CC chemokine receptor mechanism. The diseases are particularly allergic airways diseases such as asthma or rheumatoid arthritis .
Background art
Allergic asthma is a chronic inflammatory disease of the airways triggered by inhalation of environmental allergens and characterized clinically by a reversible obstruction in airflow resulting from edema, mucus production and an exaggerated vasoconstriction of the bronchi to non-specific stimuli known as airway hyperresponsiveness (AHR) (1) . The asthma response in its first acute phase is associated with the activation of mast cells by IgE, and is followed by a second, more sustained and dangerous phase, in which an accumulation of inflammatory leukocytes, in particular eosinophils and T cells, takes place. During allergen-induced lung inflammation,
leukocytes are recruited to the lung by the generation of chemokine gradients secreted by resident as well as infiltrating lung cells. Extensive studies have demonstrated a central role for chemokines in orchestrating leukocyte accumulation in the asthmatic response (2, 3) .
Dendritic cells (DCs) are a unique group of mononuclear leukocytes that play a regulatory role in the immune system. They are specialized for antigen capture and presentation to naive T lymphocytes and therefore are critical for the initiation of the immune responses. Recent reports suggest that the immune outcome is associated with distinct DC subpopulations (4, 5) . A hallmark of asthma is the abnormal expansion of T helper type 2 (Th2) cells. This event is crucial for the disease since Th2 cells produce key cytokines, including IL-4, IL-5 and IL-13, that in turn activate parenchymal and resident lung cells that release mediators, including chemokines, that lead to airway inflammation (1) . A large number of studies on in vitro-generated in vivo-transferred and skin DCs have underlined a mandatory role for CCR7 on DC migration to lymph nodes (6-8) . CCR7 is the only chemokine receptor described so far to be induced during DC maturation. Rheumatoid arthritis (RA) is a chronic
inflammatory disease involving multiple joints and remains an autoimmune disease of unknown etiology (17) . Collagen-induced arthritis (CIA) is a model for RA that is induced in susceptible mouse strains by intradermal immunization with collagen type II (CII) emulsified in a complete adjuvant (18, 19) . The significance of this model is that CII is the major constituent protein of cartilage in diarthrodial joints, the predominant site of inflammation in RA. In addition, the pathogenesis of CIA is in many ways similar to that of RA as both RA and CIA are characterized by an intense synovitis accompanied by erosions of cartilage and subchondral bone by a pannus-like tissue (20) . Susceptibility to CIA was considered to be MHC class-linked (H-2q and H- 2r) (21), however, recently Campbell et al. (22,23) modified the immunization procedure and showed that clinically and histologically similar CIA may be induced in C57BL/6 (B6) mice. In addition to serving as a valuable tool to study immunity to CII, the CIA model has proven equally useful to investigate inflammatory joint injury and led to the development of novel TNF- based therapies for human RA (24,25).
The LPS-inducible CC chemokine related gene (L- CCR) is an orphan chemokine receptor originally identified in the mouse macrophage cell line RAW 264.7
(9) . L-CCR expression at the mRNA level has been described in murine macrophages (9) and in glial cells stimulated with LPS (10) . L-CCR shares the highest degree of homology with the putative orphan human chemokine receptor HCR (11) . HCR is largely distributed across leukocyte populations and, interestingly, it is upregulated in mature DC (12) .
Currently, there is no evidence for a functional role of L-CCR and HCR in vivo. Summary of the invention
We have now discovered that LPS-inducible CC chemokine receptor is functionally involved in several diseases, among which allergic airways diseases such as asthma and rheumatoid arthritis. Due to the high degree of homology and similarity between L-CCR and HCR, it has been found that it is possible to identify new inhibitors of CCRL2 receptor
(CCRL2 is the name of the LPS-inducible CC chemokine receptor in all the species) by screening molecules in a screening murine test for L-CCR receptor inhibition.
Therefore, it is an object of the present invention to provide an in vitro method for screening drug candidates for inflammatory diseases, the said method comprising a step of putting into contact a L- CCR-expressing cell preparation with a drug candidate
for CCRL2 inhibition and then evaluating the degree of L-CCR-expressing cell migration against a control.
We have recently discovered that L-CCR is induced in mouse DCs during maturation. Therefore, mice deficient for L-CCR (L-CCR"'") were generated and studied in an established model of allergen-induced airway inflammation in which DC plays a prominent role (13) . The results of these studies show that OVA- sensitized L-CCR"'" mice had significantly reduced number of total leukocytes, eosinophils and lymphocytes/mononuclear cells in the airways, thus proving an important role of this receptor to elicit an hyperreactive response and therefore asthma.
It is another object of the present invention to provide an in vivo method for screening drug candidates for allergic airways diseases, the said method comprising a step of treating a mammal genetically expressing L-CCR receptor with an amount of a drug candidate for CCRL2 inhibition and then evaluating the mammal response in a model of induced airways inflammation against a control.
Similarly, an in vivo model for inducing rheumatoid arthritis in a mammal has been provided, as will be described below. Therefore, it is another object of the present
invention to provide an in vivo method for screening drug candidates for rheumatoid arthritis, the said method comprising a step of treating a mammal genetically expressing L-CCR receptor with an amount of a drug candidate for CCRL2 inhibition and then evaluating the mammal response in a model of induced rheumatoid arthritis against a control.
A further object of the present invention is a cell preparation generated from a LCCR-depleted mammal, to be used as a control tool in the method of the present invention.
A still further object of the present invention are monoclonal antibodies to LCCR receptor, to be used as a triggering tool in the in vitro method of the present invention.
Another object of the present invention are inhibitors of CCRL2 receptor, in particular of HCR receptor, obtained by the screening method of the invention, as well as a method of treatment of an inflammatory disease mediated by LPS-inducible CC chemokine receptor, particularly an allergic airways inflammation such as asthma or rheumatoid arthritis, by using such inhibitors.
In the context of the present invention, the term "inhibitor" means a molecule that prevents CCRL2
receptor from engaging in or producing an action on the body of the patient to be treated, by blocking CCRL2 receptor, replacing CCRL2 receptor in binding to a chemoattractant or preventing CCRL2 receptor expression in an inflammatory cell. For example, a CCRL2 inhibitor may be a molecule that binds to CCRL2 receptor and antagonize its function or a molecule that mimics CCRL2 receptor and prevents it to elicit an inflammatory response by reacting with specific cytokines on CCRL2 behalf.
Brief description of the drawings
Figure 1 shows experimental data on the induction of L-CCR in maturing mouse dendritic cells;
Figure 2 is a schematic view of the method used to generate L-CCR deficient mice;
Figure 3 depicts the role of L-CCR in the model of OVA-induced airways inflammation;
Figure 4 shows the trafficking of lung dendritic cells in L-CCR-depleted mice upon OVA antigen challenge;
Figure 5 shows the role of L-CCR in CCR7-mediated dendritic cells migration;
Figure 6 shows the chemokine and chemoattractant molecules tested for chemotaxis and receptor internalization on L 1.2 and CHO L-CCR transfected
cells;
Figures 7a, 7b show sequence listings of inhibitors of CCRL2 receptor;
Figure 8 depicts the role of L-CCR in the model of induced rheumatoid arthritis;
Figure 9 shows a diagram reporting the paw swelling in LCCR-depleted mice against WT mice;
Figure 10 shows a diagram reporting weight ans cell number of limph nodes in LCCR-depleted and WT mice.
Detailed description of the invention
In a first aspect, the present invention relates to an in vitro method for screening drug candidates for inflammatory diseases mediated by LPS-inducible CC chemokine receptor, the said method comprising a step of putting into contact a CCRL2-expressing cell preparation with a drug candidate for CCRL2 inhibition and then evaluating the degree of CCRL2-expressing cell migration against a control.
The cell preparation of the invention is obtained preferably from mice, i.e. a L-CCR-expressing cell preparation. More preferably, the said cells are dendritic cells.
The method of the invention comprises a chemotaxis assay as depicted below, wherein the dendritic cell
migration toward a chemoattractant is evaluated. The said chemoattractant is preferably a cytokine. Human CCLl 9 and CCL21 cytokines are preferably used as the chemoattractants in the invention method, as they are specific CCR7 ligands. However, as a control tool, CXCL12, ligand for CXCR4 but not for CCR7, is also used.
The chemotaxis assay is performed by using a chemotaxis chamber having an upper and a lower compartment. In the upper compartment the cell preparation is put together with the triggering tool, i.e. the anti-L-CCR antibody, while in the lower compartment the chemoattractant is present. In the assay, the drug candidate is added to the upper compartment while a control experiment without the drug candidate is performed. The inhibition of migration of the dendritic cells from the upper to the lower compartment is evaluated, taking the control experiment as a measure of 0% inhibition.
The dendritic cell migration is triggered by a suitable substance. A specific anti-L-CCR monoclonal antibody (anti-L-CCR mAb) is preferably used as a triggering tool. Preferably, IgG2a mAb and IgG2b mAb generated from L-CCR depleted mice are used as a triggering tool. Specifically, the said mAbs are
obtained as depicted below, i.e. IgG2a mAb from clone 4.2.1 and IgG2b mAb from clone 2.5.1 of hybridomes cultured from L-CCR-depleted mice.
A further control is used for the scope of the invention. The said control is a cell preparation, specifically a dendritic cell preparation, that is depleted of L-CCR receptor. Preferably, the said cell preparation is obtained from L-CCR-depleted mice.
As a still further control for proving the involvement of L-CCR receptor in the inhibitory action of the drug candidate, an experiment is conducted as above, by using L-CCR expressing cell preparation, but using a chemoattractant that is not a ligand for CCR7, such as CXCL12.
The step of evaluation of the L-CCR expressing cell migration against the control is preferably accomplished by flow cytometry, as depicted below.
The drug candidate is usually tested in a suitable form that allows good contact between the substance and the cell preparation. Preferably, the drug candidate is administered in solution or suspension. The drug candidate is used in amounts or concentrations that depend on several factors, among which the molecular weight of the substance, its solubility and so on.
Several concentrations of the same drug candidate can also be tested, in order to calculate its IC50.
EXPERIMENTAL METHOD
Dendritic cell (DC) culture DC were generated from bone marrow cells as described in Vecchi et al . J. Leukoc. Biol. 66: 489-494; 1999. For the study, 8- to 12-week-old WT and LCCR-KO male mice (L-CCR- depleted mice) on C57B1/6J background were used. Briefly CD34+ bone marrow cells from femurs and tibias were positively immunoselected using MACS microbeads coated with goat anti-rat IgG (Miltenyi Biotec
Inc. Auburn, CA), using the rat mAb MEC14.7 to mouse CD34
(Garlanda et al. Eur. J. Cell Biol. 73: 368-377; 1997) as selecting agent. CD34+ positive cells (2xlO5/mL) were cultured in RPMI1640 medium with 5% fetal calf serum, 2xlO'5 M β-mercaptoethanol, GM-CSF (40 ng/mL) , and Flt3 ligand (100 ng/mL). Cells were diluted 1:2-1:3 every 2 or 3 days. Cultured cells were collected after 8-9 days of culture. To induce maturation, DC were cultured with 20 ng/mL TNF-α for the last 24 h of culture. DCs were characterized in terms of membrane phenotype, using phycoerythrin (PE) -conjugated hamster anti-mouse CDlIc
(IgGl, clone HL3) , FITC-conjugated rat anti-mouse MHC- class II (IgG2a, clone 2G9) and rat anti-mouse CD80 (IgG2a, clone IGlO) from BD-Pharmingen. Rat anti-mouse
CD86 was from ATCC (HB-253) . Cells were analysed by FACScan.
Generation of mAb to L-CCR
Knockout mice for L-CCR were used to raise mAbs against the extracellular part of L-CCR. Mice were administered i.p. with 107 irradiated L-CCR/L1.2 cells (a mix of 3 different clones) in PBS every other week for 6 weeks. 3 days after the last challenge, spleen cells were used for polyethylene glycol-mediated fusion following conventional protocols. Supernatants were screened for Abs that could bind to L-CCR/CHO-K1 transfectants and not parental CHO. Cultures that were positive in this primary screening were then tested for binding to the L-CCR/L1.2 cells and not to parental Ll.2. Two hybridomes were chosen and cloned based on strong binding of its supernatant to the relevant transfectants. Clone 4.2.1 secreted an IgG2a mAb and clone 2.5.1 secreted an IgG2b mAb that were purified and used in subsequent experiments . Chemotaxis assay
Cell migration was evaluated using a 48-well chemotaxis chamber (Neuroprobe, Pleasanton, CA) with polycarbonate PVP filter (5 μm pore size; Neuroprobe) as described in Sozzani et al . J. Immunol. 155: 3292-3295; 1995. Fifty microliters of cell suspensions (1.5 x
106/mL) were seeded in the upper compartment and chemoattractants were placed in the lower compartment of the chemotaxis chamber, that was then incubated at 370C for 90 min. Results are expressed as the mean number of migrated cells in five high-power fields (100 X) . Each experiment was performed in triplicate.
To test the role of LCCR engagement on DC migration, TNFα-matured DC (1.5 x 106/mL) from WT and KO mice were incubated for 30 minutes at 40C with graded concentrations (0, 3 and 10 μg/mL) of the purified anti- LCCR mAbs and isotype matched irrelevant mAb, then added to the upper compartment of the chemotaxis chamber and CCL19, CCL21 (specific ligands for CCR7) and CXCL12 (not binding CCR7) were added in the lower compartment. This constitutes the control experiment.
To screen for a CCRL2 inhibitor drug candidate, the screened molecule is incubated with the cell suspension in the upper chamber and the inhibition of the dendritic cell migration is evaluated against the control. Cytokines
Human CCLl9, CCL21, CXCL12 and Fit3 Ligand were from Peprotech Inc. (Rocky Hill, NJ). Mouse granulocyte- macrophage colony-stimulating factor (GM-CSF) was from Pro-Spec-Tany TechnoGene LTD, Rehovot, Israel) and murine TNF-α was a kind gift from Dr P Vandenabeele (Gent
University, Belgium) . Cytokines were endotoxin free as assessed by Limulus amoebocyte assay (BioWhittaker Inc., Walkersville, MD) .
Flow cytometry mAbs used were from BD-Pharmingen and included phycoerythrin (PE) -conjugated hamster anti-mouse CDlIc
(IgGl, clone HL3) , FITC-conjugated rat anti-mouse MHC- class II (IgG2a, clone 2G9) and rat anti-mouse CD80
(IgG2a, clone IGlO) . Rat anti-mouse CD86 was from ATCC (HB-253) . Cells were analysed by FACScan.
Effect of anti-LCCR pre-treatment on DC migration
DC from WT mice pretreated with mAbs to LCCR displayed an increased migration in response to the CCR7 ligands CCL19 and CCL21. The effect was specific, since migration to CXCL12, ligand for CXCR4, was not affected as shown in fig. 5. An isotype matched, irrelevant mAb did not modified DC migration to CCR7 ligands. Moreover, migration of DC from LCCR KO mice to CCR7 ligands was not modified after exposure to anti-LCCR mAbs. These data indicates that engagement of LCCR enhance CCR7-mediated
DC migration.
The CCRL2 inhibitors cause an inhibition of the dendritic cell migration from the upper to the lower compartment if compared to the control.
The drug candidates that pass the screening test
on L-CCR-expressing cell preparation are then tested on a further cell preparation wherein the dendritic cells are depleted from L-CCR receptor (L-CCR"7" cell preparation) , to prove that their mechanism of action is actually an L-CCR inhibitory mechanism. No inhibition of migration is expected for pure CCRL2 inhibitors. A different degree of inhibition with respect to the previous test is indicative of an impure mechanism. The drug candidates that confirm their mechanism of action as being an CCRL2 inhibition are then preferably passes to the in vivo screening method, as depicted below.
Fig. 6 reports the list of the molecules tested and found not to activate LCCR, in terms of chemotaxis and receptor internalization.
According to another object of the present invention, an in vivo screening method is provided, which comprises inducing an allergen-type airways inflammatory response in a mammal, treating the said mammal with a drug candidate for inhibition of CCRL2 receptor and evaluating the inhibition of inflammatory cell recruitment in the BAL of said mammal against a control .
Preferably, the said mammal is a mouse.
The allergen-type airways inflammatory response is preferably elicited by a suitable allergen. Ovalbumin
(OVA) is preferably used as an allergen, according to a well established experimental model. The mammals are first sensitized with OVA, then a challenge with OVA is performed in order to trigger the inflammatory response. The count of dendritic cells is made in the bronchoalveolar lavage (BAL) fluid from the treated animals, in order to evaluate the recruitment of such inflammatory cells upon allergic reaction. The sensitization with OVA is performed in any suitable way, such as via aerosol or intratracheal instillation.
The drug candidates for CCRL2 inhibition are screened in such a test. The drug candidate is administered to the mammal in any suitable way, such as orally, intraperitoneally, i.v. or the like, before, during or after OVA challenge. Depending on the way of administration, the drug candidate is in a suitable form, such as in solid form (powder, tablet or the like) or liquid form (solution, suspension or the like) for oral administration, liquid form for intravenous injection and so on.
A control experiment is run, wherein no drug candidate administration is made, so that a 0%
inhibition is given. The %inhibition obtained with the drug candidate with respect to such a control is evaluated.
As a further control, the drug candidates that pass the above screening test are tested in CCRL2- depleted mammals, by running the same kind of evaluation as above. This experiment helps to confirm the actual involvement of CCRL2 receptor in the drug candidate activity. As another object of the present invention, an in vivo screening method is provided, which comprises inducing rheumatoid arthritis in a mammal, treating the said mammal with a drug candidate for inhibition of CCRL2 receptor and evaluating the inhibition of arthritis symptoms in said mammal against a control.
Preferably, the said mammal is a mouse.
The rheumatoid arthritis is preferably elicited by a collagen, particularly collagen type II. The mammals are first treated with collagen according to the protocol described below in order to induce arthritis. Then, a visual evaluation is made of the symtoms od rheumatoid arthritis, in particular paw swelling as described below, together with the assessment of the thickness of the hind paws and the count of number of
cells and weight of the limph nodes.
The pretreatment with collagen is performed in any suitable way, such as by i.d. injection at the base of the tail. The injection is preferablu made twice, at day 0 and day 21.
The drug candidates for CCRL2 inhibition are screened in such tests. The drug candidate is administered to the mammal in any suitable way, such as orally, intraperitoneally, i.v. or the like, before, during or after OVA challenge. Depending on the way of administration, the drug candidate is in a suitable form, such as in solid form (powder, tablet or the like) or liquid form (solution, suspension or the like) for oral administration, liquid form for intravenous injection and so on.
A control experiment is run, wherein no drug candidate administration is made, so that a 0% inhibition is given. The %inhibition obtained with the drug candidate with respect to such a control is evaluated.
As a further control, the drug candidates that pass the above screening test are tested in CCRL2- depleted mammals, by running the same kind of evaluation as above. This experiment helps to confirm
the actual involvement of CCRL2 receptor in the drug candidate activity.
The screening dose of the drug candidate can vary in a wide range of doses depending on several factors, such the IC50 calculated from the above in vitro test results or the LD50 that can be determined for such compound. Generally, the dose of the drug candidate used in the present in vivo screening method will be below its LD50. It is also possible to test different doses of the drug candidate in the in vivo test, in order to determine its ED5O-
EXPERIMENTAL METHOD
Induction of L-CCR in maturing mouse DC. L-CCR is a murine orphan chemokine receptor. L-CCR mRNA was barely detectable in bone marrow (BM) -derived myeloid DC, however, lipopolysaccharide (LPS) stimulation upregulated L-CCR mRNA after 30 min, reaching a peak at 2 h and decreasing afterwards (Fig. IA) . As previously described (14) , CCR7 increased its transcripts levels in the same samples, but in a delayed kinetic. We generated two mAb (2.5.1 and 4.2.1) against L-CCR that recognize specifically CHO and Ll.2 cells that stably express L-CCR and use them to study the surface expression of L-CCR protein. We used both
antibodies to detect L-CCR on primary cells with similar results, and those obtained with 4.2.1 are shown. Surface L-CCR was induced already after 6 h following LPS treatment, reaching the highest levels at 12 h and returning to nearly basal levels by 40 h. In parallel, the same cells induced CCR7 at detectable levels only after 24 h of LPS incubation and its expression was further increased after 40 h treatment (Fig IB) . No constitutive expression of surface L-CCR was found on CDlIc+ DC from any lymphoid organ. Dual labeling flow cytometric analysis of subcutaneous lymph nodes, spleen, thymus, bone marrow and lung cells from naive mice showed no expression of L-CCR in any leukocyte subpopulation. Generation of L-CCR deficient mice
To directly examine the function of L-CCR in a disease we generated mice lacking the L-CCR gene by homologous recombination techniques (Fig. 2) . L-CCR";~ mice developed normally to term and were fertile. No significant differences were found after histological and flow cytometric analysis of lymphoid organs (not shown) . L-CCR"7" mice had a normal lifespan and did not show an overt phenotype in the steady-state.
Responses of WT and L-CCR"7" in the model of OVA- induced allergic airway disease
The expression of L-CCR in maturing DC prompted the analysis of the role of L-CCR in a mouse model of allergic airway inflammation in which DC are believed to play a key role. To this end, animals were sensitized or not with OVA and then challenged with aerosolized OVA, as described in Materials and Methods. During allergen-induced airway inflammation, leukocytes traffic through the lung interstitium into the airway lumen (2). Leukocyte numbers were therefore determined in both compartments. Analysis of inflammatory cell recruitment in BAL of PBS-sensitized WT and L-CCR""7" mice showed a predominance of macrophages with no differences between WT and L-CCR"7" mice (Fig. 3A). Conversely, OVA-sensitized WT and L-CCR"7~ mice showed an increase in total cell number in BAL due to increases in lymphocytes, macrophages and especially eosinophils. OVA-sensitized L-CCR"7" mice had significantly reduced numbers of total leukocytes, eosinophils and lymphocyte/mononuclear cells in the airways compared with similarly challenged WT mice (Fig. 3A) .
One possible explanation of the decreased leukocyte recruitment into the airway of L-CCR"7" mice is that their immune response, in particular their Th2 type response, is impaired. Indeed in the BAL fluid, L-
CCR deficiency resulted in decreased levels of the Th2 cytokines IL-4 and IL-5 (Fig. 3B) . IL-13 was also decreased in the BAL fluid of L-CCR"7" mice, although not significantly (Fig. 3B) . The ThI specific cytokine INFγ was unaffected by L-CCR deficiency in either BAL or lung, which suggests that there is not an enhanced induction of the local ThI response in L-CCR"7" mice
(Fig. 3B) .
Trafficking of pulmonary DC in WT versus L-CCR"7" mice: L-CCR is required for pulmonary DC homing to mediastinal LNs
Migration of pulmonary DC to MLNs is a key step in the initiation of the allergic response in the lung (13) . To study the migration of endogenous lung DC to MLN, OVA-sensitized animals were instilled intratracheally with a single injection of OVA conjugated with the fluorescent dye FITC. This treatment is known to induce lung DC maturation and mobilization (15). Mice were sacrificed 24, 48 or 72 h later and MLN suspensions were analyzed for the presence of migrating airway-derived CDlIc+FITC+ DCs. In comparison with WT animals, significant less FITC+ DC were found in MLN of L-CCR'7" mice 48 and 72 h following OVA-FITC instillation (Fig 4A) . Importantly, airway-derived FITC+ DCs in MLNs expressed L-CCR, while
FITC DCs, which correspond to resident MLN DCs did not (Fig 4B) .
The in vivo screening method according to the invention is performed by sensitizing the WT (wild type, thus expressing L-CCR receptor) mice with ovalbumin (OVA) , then challenging such mice with OVA to induce the hyperreactive response and counting the number of dendritic cells in the BAL of the animals, as described below. This constitutes the control experiment. Another group of mice is treated in the same manner, but a drug candidate for inhibition of
CCRL2 receptor is administered to the mice before, during or after the sensitization with OVA. The
%inhibition of the dendritic cells recruitment in the BAL is evaluated with respect to the control experiment to which 0% inhibition is assigned.
The same experiment is conducted on L-CCR"/" mice, for the drug candidates that proved to be inhibitory in the screening on WT mice, to ascertain whether the said drug candidates act through CCRL2 inhibition totally or in part.
Materials and Methods Cytokines and reagents
Mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was from Pro-Spec-Tany TechnoGene LTD,
Rehovot, Israel) and murine TNF-α was a kind gift from Dr P Vandenabeele (Gent University, Belgium) . Human Flt3 ligand was from Peprotech Inc. (Rocky Hill, NJ). LPS (Escherichia coli strain 055 :B5) was from Difco Laboratories (Detroit, Michigan, USA) . Cell culture reagents were from Gibco and Hyclone. Recombinant chemokines were from Peprotech (Rocky Hill, NJ) and R&D. Cytokines were endotoxin free as assessed by Limulus amoebocyte assay (BioWhittaker Inc., Walkersville, MD). All other reagents were from Sigma unless indicated otherwise .
Generation of the L-CCR -/- null mutant "knockout" mice
To produce the L-CCR";" mouse strain, a gene- targeting vector was constructed in which the whole open reading frame of the gene was replaced by a Neo cassette.
This vector was transfected into embryonic stem (ES) cells. After selection, one targeted clone was identified by Southern blotting hybridization. It was injected into C57B1/6 blastocysts to generate germ line chimeras and subsequently L-CCR+/" heterozygote mice of 129/Sv C57B1/6 mixed genetic background. L-CCR+/+ mice are littermates of
129/Sv C57B1/6 L-CCR"7". For genotyping of mice, DNA derived from tail biopsies was amplified by polymerase chain reaction that detected the wild type and targeted
allele, respectively. L-CCR deficiency at the mRNA level was confirmed in thioglycollate-elicited neutrophils obtained from L-CCR -/- animals.
Experimental protocol for induction of allergic airway inflammation
Age and sex-matched L-CCR deficient mice were sensitized to ovalbumin (OVA; grade V, Sigma) with a single i.p. injection of 10 μg OVA mixed with 4 mg aluminium hydroxide gel in 0.2 ml sterile saline solution on days 1 and 11. On day 18 mice were placed in a Plexiglas chamber and exposed to a 5.0 % OVA aerosol (w/v in sterile saline solution) for 20 min. The aerosol was generated by a PARI Master Nebulizer (Sapio Life, Milan, Italy) connected to the Plexiglas chamber. Aerosol challenge was repeated daily for 6 days and 24 hrs post last challenge mice were anesthetized with urethane (12% w/v, 400 μl/ mouse, i.p.), bled, and tracheotomized. Sera were prepared by centrifugation and snap-frozen to -200C until further use. Bronco-alveolar lavages (BAL) were carried out by injection into the airways of 3 x 1 ml sterile Hank's balanced salt solution (HBSS, Gibco-BRL) . Lavage fluid was recovered after 30 sec by gentle aspiration from the airways and lavages from each were pooled. Samples were then centrifuged at 2000 rpm for 10 min at 40C. BAL fluid was concentrated by
using Vivaspin 2 concentrators (SIGMA) , and stored at - 200C for further analysis. Pellets were re-suspended in 1 ml HBSS and the total cell number was determined from an aliquot of the cell suspension using a disposable multi- chamber hematocytometer (FAST-read 102, Biosigma, Italy) . For differential cell counts, an aliquot of the cell suspension was spotted onto glass slides by centrifuging in a Cytospin (5 min, 800 rpm) . Slides were then fixed and stained with Diff-Quik staining set (Dade Behring SpA, Milan, Italy) , following manufacturer' s instructions. Standard morphological criteria were used in classifying at least 400 cells per slide under light microscopy.
Separated groups of animals (n= 10-15) were used for the experiments. ELISA
Several cytokines (IL-4, IL-5, IL-13 and IFNγ) were measured in BAL fluids by standard sandwich ELISA assays by using 150 μl of 4x concentrated fluid. Specific capture and detecting antibodies were from Pharmingen (IL-4, and IL-5) and R&D (IL-13, IFNγ) . Detailed explanation of figures
Figure 1. Induction of L-CCR in maturing mouse dendritic cells. DC were generated in vitro from CD34+ BM precursors. (A) Total RNA was prepared from
untreated immature DC (0 h) or DC treated for different times with 100 ng/ml LPS and analyzed by Northern blot for the expression of L-CCR mRNA. The same filter was probed for CCR7. Ethidium bromide staining is shown in the lower part of the panel. (B) Flow cytometry of L- CCR and CCR7 surface expression by DC stimulated with 100 ng/ml LPS. The graphs show the mean fluorescence intensity (MFI) of cells labeled with L-CCR and CCR7 mAbs after subtraction of the background staining obtained in DC generated from L-CCR"/" mice (for L-CCR) or isotype-control matched mAb (for CCR7) .
Figure 2. Generation of L-CCR deficient mice . (A) Exon/intron structure of the L-CCR gene. The figure is not drawn to scale. The size of exon 1, 2 and 3 are 125, 434 and 1603 bp respectively. Black area corresponds to the coding sequence. (B) Schematic representation of WT and KO alleles of the L-CCR gene and of targeting vector. The three exons are represented by open boxes. Neomycin-resistance gene driven by a PGK promoter (Neo) is indicated by a dashed box. Restriction sites: K, Kpnl; S, Sail; H, Hindlll; B, BamHI; Sa, Sad; Sc, Seal. Kpnl-digested genomic DNA fragments were detected by probe. (C) Southern blot analysis of Kpnl-digested genomic DNA from transfected ES cells hybridized with L-CCR probe generates a 12 kb
WT restriction fragment and a 3.5 kb homologous recombinant restriction fragment. (D) PCR analysis of genomic DNA from L-CCR+/+ (lane 1), L-CCR"7" (lane 2,3) and L-CCR+/~ (lane 4) mice. (E) RT-PCR demonstration of disrupted L-CCR transcription in L-CCR"7" mice. Total RNA was isolated from immature as well as BM-derived DCs stimulated for 24 h with TNFα or LPS from WT and L- CCR"7" mice. No L-CCR transcripts were detected in knockout mice. Figure 3. Role of L-CCR in the model of OVA- induced airway inflammation. WT and L-CCR"7" mice were sensitized or not with OVA intraperitoneally and subsequently challenged with OVA by aerosol as described in Materials and Methods . 24 h after the last aerosol mice were sacrificed and BAL were obtained. (A) Differential cell counts in BAL fluid. (B) ELISA evaluation of cytokine production in BAL fluid of mice sensitized and challenged with OVA. Values are expressed as mean ± SEM from three different experiments with 8 to 12 mice per group. Statistical significance: *, p < 0.05; **, p < 0.01.
Figure 4. Trafficking of lung DC in L-CCR"7" mice upon OVA antigen challenge. Homing of DC from the airways to draining MLNs. OVA-sensitized mice were treated by intratracheal administration of 800 μg OVA-
FITC in 50 μl PBS and sacrificed after 24, 48 and 72 h. The accumulation of FITC+CDlIc+ DCs in the MLNs was assessed by flow cytometry. Panel A: Kinetics analysis of FITC+ DC accumulation in MLNs. Results are shown as mean ± SEM from 12-15 mice per group from two separated experiments. *, p < 0.05, **, p < 0.01 in comparison with WT-treated mice. Panel B: Surface expression of L- CCR in FITC+ and FITC" WT MLNs-DC after OVA-FITC administration (n = 4) . Values are reported as mean fluorescence intensity after correction for background staining using L-CCR";" DC.
Jn vivo experimental model of Collagen-Induced Arthritis (CIA)
Mice used in CIA experiments
LCCR-/- mice originally generated on the mixed background 129Sv-C57Bl/6 were backcrossed with C57B1/6 mice for 10 generations, to have the C57B1/6 background for CIA experiments.
Induction and assessment of arthritis
CFA was prepared by mixing 100 mg of heat-killed Mycobacterium tuberculosis (H37Ra; Difco Laboratories) in 20 ml of IFA (Sigma-Aldrich) (22) . An emulsion was formed by dissolving 2.0 mg/ml chick CII (Collagen of type II,
CII; Sigma-Aldrich) overnight at 4°C in 10 mM acetic acid and combining it with an equal volume of CFA. CII solution and the emulsion with CFA were always freshly prepared. Mice were injected i.d. at the base of the tail with a total of 100 μl of emulsion containing 100 μg of CII and 250 μg of M. tuberculosis. The same injection was repeated at day 21; however, due to toughening of the skin at the base of the tail, booster injections were distal to the primary injection site.
Clinical and histological assessment of arthritis
All mice were examined two to three times per week for the initial visual appearance of arthritis after immunization. Arthritis of each individual limb was graded using the following scoring system: 0, normal; 1, apparent swelling and redness limited to individual digits; 2, swelling in more than one joint; 3, severe redness and swelling of the entire paw including digits; and 4, maximally inflamed limb with involvement of multiple joints. The maximum score per mouse was 16. Mice were scored as arthritic if more than one paw had a score >2. The thickness of the hind paws was measured using a dial gauge caliper (Mitsutoyo) . At the end of the experiment (day 60) , inguinal lymph nodes were collected, weighted, disaggregated and the number of cells per lymph
node was microscopically counted.
Results
The arthritis symptoms in LCCR-/- mice were studied after immunization with CII on day 0 and a boost with CII on day 21. Mice were examined weekly after the first immunization and every 2-3 days after the boost for signs of developing arthritis. The severity of the arthritis was assessed using a visual scoring as described above. The LCCR-/- and WT animals developed clinical signs of arthritis with an incidence of 8 and 28 % by day 29 and of 8 and 31 % by day 36, respectively (figure 8) . The incidence of clinical symptoms was thus lower in LCCR-/- mice through all the observation period, till day 60. As an other objective parameter to evaluate the severity of disease the thickness of the hind paws was measured. Paw swelling was always significant lower in LCCR-/- mice than in WT mice (figure 9) . Since it has been reported that the disease-induced inflammation cause lymph nodes enlargement, at day 60 when the experiment was stopped lymph nodes were recovered and evaluated. Weight and cell numbers of nodes from LCCR-/- mice were markedly lower, highly significantly different from those from WT mice (figure 10) .
The in vivo screening method for rheumatoid arthritis according to the invention is performed by
treating the WT (wild type, thus expressing L-CCR receptor) mice with collagen, then assessing the arthritis parameters as described above. This constitutes the control experiment. Another group of mice is treated in the same manner, but a drug candidate for inhibition of CCRL2 receptor is administered to the mice before, during or after the treatment with collagen. The %inhibition of the induced rheumatoid arthritis is evaluated with respect to the control experiment to which 0% inhibition is assigned.
The same experiment is conducted on L-CCR"/" mice, for the drug candidates that proved to be inhibitory in the screening on WT mice, to ascertain whether the said drug candidates act through CCRL2 inhibition totally or in part.
Cell preparation
According to a further object of the present invention, a cell preparation of inflammatory cells that are depleted of CCRL2 receptor is provided. Such a cell preparation is used, as explained above, as a control tool in the screening method of the invention, in order to confirm the CCRL2 inhibitory mechanism of the drug candidates selected according to the in vitro method performed on CCRL2 expressing cells. Preferably, the inflammatory cell preparation is a
dendritic cell preparation obtained by a CCRL2-depleted mammal. More preferably, the inflammatory cell preparation is a dendritic cell preparation obtained by a L-CCR-depleted mice. The inflammatory cell preparation of the invention can be obtained according to well known procedures, as described above.
Monoclonal antibodies
A still further object of the present invention are monoclonal antibodies to CCRL2 receptor, to be used as a triggering tool in the in vitro method of the present invention.
Preferably, the said antibodies are IgG2a mAb and IgG2b mAb generated from L-CCR depleted mice. Specifically, the said mAbs are obtained as depicted above, i.e. IgG2a mAb from clone 4.2.1 and IgG2b mAb from clone 2.5.1 of hybridomes cultured from L-CCR- depleted mice.
CCRL2 receptor inhibitors A still further object of the present invention are CCRL2 receptor inhibitors obtainable through the screening method of the invention. Object of the present invention is a CCRL2 receptor inhibitor that displays a %inhibition greater than 50% in the in vitro screening test and/or a %inhibition greater than 50% in
the in vivo test of the invention.
In one embodiment, the CCRL2 receptor inhibitors of the invention are those inhibitors that bind to a CCRL2 receptor fragment identified by the following sequences :
- (SEQ ID N0.1, human, residues 1-38)
MANYTLAPEDEYDVLIEGELESDEAEQCDKYDAQALSA (N-Terminal) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%;
- (SEQ ID NO.2, human, residues 97-104) AGGDPMCK (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%; - (SEQ ID NO.3, human, residues 172-197) PQMEDQKYKCAFSRTPFLPADETFWK (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%; - (SEQ ID NO.4, human, residues 259-275) FLSTFKEHFSLSDCKSS (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%; - (SEQ ID NO.5, murine, residues 1-37)
MDNYTVAPDDEYDVLILDDYLDNSGPDQVPAPEFLSP (N-Terminal) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%; - (SEQ ID NO.6, murine, residues 96-109) TAAHGESPGNGTCK (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%; - (SEQ ID NO.7, murine, residues 176-201) PRMERQKHKCAFGKPHFLPIEAPLWK (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity- greater than 70%; - (SEQ ID NO.8, murine, residues 263-279) FLSAFQEHLSLQDEKSS (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%. In another embodiment, CCRL2 receptor inhibitors of the invention are those depicted in fig. 7a, i.e.:
(SEQ ID N0.1)
MANYTLAPEDEYDVLIEGELESDEAEQCDKYDAQALSA; - (SEQ ID NO.2) AGGDPMCK; - (SEQ ID NO.3) PQMEDQKYKCAFSRTPFLPADETFWK;
- (SEQ ID NO.4) FLSTFKEHFSLSDCKSS;
(SEQ ID NO.5)
MDNYTVAPDDEYDVLILDDYLDNSGPDQVPAPEFLSP;
- (SEQ ID NO.6) TAAHGESPGNGTCK; - (SEQ ID NO.7) PRMERQKHKCAFGKPHFLPIEAPLWK;
- (SEQ ID NO.8) FLSAFQEHLSLQDEKSS, or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70% or chemical bioisosters thereof.
Preferably, the said fragments of sequences SEQl- SEQ8 are the following (fig. 7b) :
(SEQ ID NO.9, murine, residues 3-14) NYTVAPDDEYDVLI; - (SEQ ID NO.10, human, residues 3-14) NYTLAPEDEYDVLI;
- (SEQ ID NO.11, murine, residues 176-179) PRME;
- (SEQ ID NO.12, human, residues 172-175) PQME;
- (SEQ ID NO.13, murine, residues 183-187) HKCAF; - (SEQ ID NO.14, human, residues 179-183) YKCAF;
(SEQ ID NO.15, murine, residues 263-273) FLSAFQEHLSL;
(SEQ ID NO.16, human, residues 259-269) FLSTFKEHFSL. In a further embodiment of the present invention,
the CCRL2 receptor inhibitors are CCRL2 gene antisense sequences .
The present invention provides antisense oligomers having a sequence effective to inhibit or block the expression of the CCRL2 encoding gene or mRNA sequence. Antisense technology, which uses specific- oligonucleotides to inhibit expression of target gene products, is developing as a therapeutic modality for human disease. Several selection criteria are available to contribute to the optimization of antisense oligonucleotide antagonists. For example, it is advisable to choose sequences with 50% or more GC content. Preferred sequences span the AUG initiation codon of the target protein, but sites in the coding region and 5'UTR may perform equally well. Such sequences are generally about 18-30 nucleotides long. Longer oligomers are often found to inhibit the target to a greater extent, indicating that a preferred length is about 25 mer for the first oligonucleotides chosen as antisense reagents. Typically, three oligonucleotide sequences are chosen with regard to these criteria, and compared for antagonist activity to control oligonucleotide sequences, such as "reverse" oligonucleotides or those in which about every fourth
base of the antisense sequence is randomized. Therefore, a preferred sequence for making antisense oligomer sequences to
CCRL2 is a 25 mer sequence from the CCRL2mRNA sequence (human) : GAGGAGGAAACAACTTCCCGGTTGC [SEQ ID NO.17, fig. 7c]. The whole sequence of the CCRL2 encoding gene is reported in NCBI database, under ref. No. NM 003965. Such preferred antisense sequences are used to construct antisense oligonucleotide agents (and suitable controls) for an in vitro comparison as inhibitors of CCRL2 receptor. These in vitro data are predictive of human clinical utility using antisense agents of comparable design.
Pharmaceutical compositions Pharmaceutical compositions comprising a pharmaceutically active amount of CCRL2 receptor inhibitor as defined above and a suitable carrier or excipient are also provided for.
Pharmaceutically acceptable carriers or excipients can contain one or more physiologically acceptable compound (s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent (s) . Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as
ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers. The CCRL2 inhibitors as identified herein are useful for parenteral, topical, oral, or local administration, such as by inhalation, aerosol or transdermally, for prophylactic and/or therapeutic treatment of inflammatory diseases mediated by LPS- inducible CC chemokine receptor, particularly allergic airways inflammations or rheumatoid arthritis. Administration by inhalation or aerosol is generally preferred in the case of allaergic airways inflammation, while a systemic administration form is more suitable in the case of rheumatoid arthritis.
For oral administration, a pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate are employed along with various disintegrants such as starch and preferably potato or tapioca starch and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as
magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compounds of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents such as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, etc. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the
art would appreciate that the choice of pharmaceutically acceptable carrier (s) , including a physiologically acceptable compound depends, for example, on the route of administration of the active agent (s) and on the particular physio-chemical characteristics of the active agent (s) . The excipients are preferably sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques .
The concentration of active agent (s) can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Concentrations, however, will typically be selected to provide dosages in accordance with the dosage recommendations provided above. It will be appreciated that such dosages may be varied to optimize a therapeutic regimen in a particular subject or group of subjects.
In therapeutic applications, the compositions of this invention are administered to a patient suffering from an inflammatory disease mediated by LPS-inducible CC chemokine receptor, such as an allergic airways inflammatory disease like asthma or rheumatoid
arthritis, in an amount sufficient to cure or at least partially modify the issue of the condition and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the active agents of the formulations of this invention to effectively treat (ameliorate one or more symptoms) the patient.
In certain preferred embodiments, the CCRL2 inhibitor (s) is administered orally (e.g. via a tablet) or as an injectable in accordance with standard methods well known to those of skill in the art. In other preferred embodiments, the CCRL2 inhibitors may also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal "patches" wherein the active agent (s) are typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is typically contained in a layer, or "reservoir," underlying an
upper backing layer. It will be appreciated that the term "reservoir" in this context refers to a quantity of "active ingredient (s) " that is ultimately available for delivery to the surface of the skin. Thus, for example, the "reservoir" may include the active ingredient (s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art. The patch may contain a single reservoir, or it may contain multiple reservoirs.
In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. The backing layer in these laminates, which serves as the upper surface of the device, preferably
functions as a primary structural element of the "patch" and provides the device with much of its flexibility. The material selected for the backing layer is preferably substantially impermeable to the active agent (s) and any other materials that are present .
In the treatment of allergic airways inflammatory diseases, it is contemplated that in certain embodiments the CCRL2 inhibitor (s) is administered locally via a patch (e.g. as described above) or other topical formulation. Other preferred formulations for topical drug delivery include, but are not limited to, ointments and creams. Ointments are semisolid preparations which are typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent, are typically viscous liquid or semisolid emulsions, often either oil-in-water or water-in-oil . Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant . The emulsifier in a cream formulation is
generally a nonionic, anionic, cationic or amphoteric surfactant. The specific ointment or cream base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.
Administration by inhalation or aerosol is the preferred administration route according to the present invention. Compositions for inhalation or aerosol can be prepared according to conventional methods. For example, standard inhalation devices filled with the CCRL2 inhibitor composition and a propellant gas can be used. Alternatively, a solution or suspension of the active ingredient composition in a suitable physiological medium is prepared, and the such solution or suspension is administered by aerosol with conventional aerosol machines .
The foregoing formulations and administration methods are intended to be illustrative and not limiting. It will be appreciated that, using the teaching provided herein, other suitable formulations and modes of administration can be readily devised.
Method of treatment of inflammatory diseases mediated by LPS-inducible CC chemokine receptor
A method of treatment of an inflammatory disease mediated by LPS-inducible CC chemokine receptor is also provided, wherein the said method involves treating a mammal in need thereof with a therapeutically effective dose of a CCRL2 receptor inhibitor as previously defined, wherein the said CCRL2 receptor inhibitor is comprised in a pharmaceutical composition together with a pharmaceutically acceptable carrier or excipient, as defined above. The dose of the CCRL2 receptor inhibitor that is administered can vary in a wide range of doses, depending on inhibitor's activity, the disease to be treated, its seriousness, the conditions of the patient, its weight and age as well as the route of administration, as will be determined by the clinician. Generally, the CCRL2 receptor inhibitor can be used in doses ranging from 0.001 mg to 50 mg per kg of body weight, from 1 to 4 times a day.
The use of a CCRL2 receptor inhibitor as defined in any of claims from 27 to 29, for the preparation of a medicament for curing or alleviating an inflammatory disease mediated by LPS-inducible CC chemokine receptor is also claimed.
The inflammatory disease to be treated is preferably selected from an allergic airways
inflammatory disease such as allergic asthma, allergic rhinitis, airways infectious diseases and COPD (Chronic Obstructive Pulmonary Disease) , or from rheumatoid arthritis . It is also foreseen that the CCRL2 receptor inhibitor of the invention be administered in combination with another CCRL2 receptor inhibitor or with a different antiallergic or anti asthmatic drug, such as an anti-istaminic drug, a glucocorticosteroid (like budenoside or beclomethasone) , sodium chromoglycate, β2-adrenoceptor agonists (like salbutamol or terbutaline) ; or with one or more drugs used to treat rheumatoid arthritis or its symptoms, such as Non Steroidal Antiinflammatory Drugs (NSAIDs) , methotrexate, cyclosporin, salazopyrin, hydroxychlorokin, etanercept, or corticosteroidal drugs. In the context of the present invention, the expression w administered in combination with'' means that the drugs are contained in the same pharmaceutical composition or that they are administered in different pharmaceutical compositions during the therapy of the diseases. This means that the different drugs can be administered at the same time or at different times, depending on the needs of the patient and the protocol of administration dictated by the clinician.
References
1. Wills-Karp, M. 1999. Immunologic basis of antigen-induced airway hyperresponsiveness . Annu Rev Immunol 17:255-281. 2. Goya, I., R. Villares, A. Zaballos, J.
Gutierrez, L. Kremer, J.A. Gonzalo, R. Varona, L. Carramolino, A. Serrano, P. Pallares, L.M. Criado, R. Kolbeck, M. Torres, A.J. Coyle, J. C. Gutierrez- Ramos, A. C. Martinez, and G. Marquez . 2003. Absence of CCR8 does not impair the response to ovalbumin- induced allergic airway disease. J Immunol 170:2138- 2146.
3. Bishop, B., and CM. Lloyd. 2003. CC chemokine ligand 1 promotes recruitment of eosinophils but not Th2 cells during the development of allergic airways disease. J Immunol 170:4810- 4817.
4. de Heer, H.J., H. Hammad, T. Soullie, D. Hijdra, N. Vos, M.A. Willart, H. C. Hoogsteden, and B.N. Lambrecht. 2004. Essential role of lung plasmacytoid dendritic cells in preventing asthmatic reactions to harmless inhaled antigen. J Exp Med 200:89-98.
5. Oriss, T. B., M. Ostroukhova, C. Seguin- Devaux, B. Dixon-McCarthy, D. B. Stolz, S. C. Watkins,
B. Pillemer, P. Ray, and A. Ray. 2005. Dynamics of dendritic cell phenotype and interactions with CD4+ T cells in airway inflammation and tolerance. J Immunol 174:854-863. 6. Fδrster, R., A. Schubel, D. Breitfeld, E.
Kremmer, I. Renner-Muller, E. Wolf, and M. Lipp. 1999. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23-33. 7. Gunn, M. D., S. Kyuwa, C. Tarn, T. Kakiuchi,
A. Matsuzawa, L. T. Williams, and H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J Exp Med 189:451-460. 8. Martln-Fontecha, A., S. Sebastiani, U. E.
Hopken, M. Uguccioni, M. Lipp, A. Lanzavecchia, and F. Sallusto. 2003. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J Exp Med 198:615- 621.
9. Shimada, T., M. Matsumoto, Y. Tatsumi, A. Kanamaru, and S. Akira. 1998. A novel lipopolysaccharide inducible C-C chemokine receptor related gene in murine macrophages. FEBS Lett 425:490-494.
10. Zuurman, M. W., J. Heeroma, N. Brouwer, H.W. Boddeke, and K. Biber. 2003. LPS-induced expression of a novel chemokine receptor (L-CCR) in mouse glial cells in vitro and in vivo. Glia 41:327- 336.
11. Kodelja, V., C. Muller, 0. Politz, N. Hakij, CE. Orfanos, and S. Goerdt. 1998. Alternative macrophage activation-associated CC- chemokine-1, a novel structural homologue of macrophage inflammatory protein-1 alpha with a Th2- associated expression pattern. J Immunol 160:1411- 1418.
12. Migeotte, I., J. D. Franssen, S. Goriely, F. Willems, and M. Parmentier. 2002. Distribution and regulation of expression of the putative human chemokine receptor HCR in leukocyte populations. Eur J Immunol 32:494-501.
13. Lambrecht, B.N. 2005. Dendritic cells and the regulation of the allergic immune response. Allergy 60:271-282.
14. Vecchi, A., L. Massimiliano, S. Ramponi, W. Luini, S. Bernasconi, R. Bonecchi, P. Allavena, M. Parmentier, A. Mantovani, and S. Sozzani. 1999. Differential responsiveness to constitutive vs . inducible chemokines of immature and mature mouse
dendritic cells. J Leukoc Biol 66:489-494.
15. Vermaelen, K. Y., I. Carro-Muino, B.N. Lambrecht, and R. A. Pauwels. 2001. Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes. J Exp Med 193:51-60.
16. Garlanda, C, C. Parravicini, M. Sironi, M. De Rossi, R. Wainstok de Calmanovici, F. Carozzi, F. Bussolino, F. Colotta, A. Mantovani, and A. Vecchi . 1994. Progressive growth in immunodeficient mice and host cell recruitment by mouse endothelial cells transformed by polyoma middle-sized T antigen: implications for the pathogenesis of opportunistic vascular tumors. Proc Natl Acad Sci U S A 91:7291- 7295.
17. Goldblatt, F., D. A. Isenberg. 2005. New therapies for rheumatoid arthritis. Clin. Exp. Immunol. 140: 195-204.
18. Courtenay, J. S., M. J. Dallman, A. D. Dayan, A. Martin, B. Mosedale. 1980. Immunisation against heterologous type II collagen induces arthritis in mice. Nature 283: 666-668.
19. Wooley, P. H., H. S. Luthra, J. M. Stuart, C. S. David. 1981. Type II collagen-induced arthritis in mice. I. Major histocompatibility
complex (I region) linkage and antibody correlates. J. Exp. Med. 154: 688-700.
20. Anthony, D. D., T. M. Haqqi . 1999. Collagen-induced arthritis in mice: an animal model to study the pathogenesis of rheumatoid arthritis. Clin. Exp. Rheumatol. 17: 240-244.
21. Holmdahl, R., R. Bockermann, J. Backlund, H. Yamada. 2002. The molecular pathogenesis of collagen-induced arthritis in mice—a model for rheumatoid arthritis. Ageing Res. Rev. 1: 135-147.
22. Campbell, I. K., J. A. Hamilton, I. P. Wicks. 2000. Collagen-induced arthritis in C57BL/6
(H-2b) mice: new insights into an important disease model of rheumatoid arthritis. Eur. J. Immunol. 30: 1568-1575.
23. Campbell, I. K., K. O'Donnell, K. E. Lawlor, I. P. Wicks. 2001. Severe inflammatory arthritis and lymphadenopathy in the absence of TNF. J. Clin. Invest. 107: 1519-1527. 24. Keffer, J., L. Probert, H. Cazlaris, S.
Georgopoulos, E. Kaslaris, D. Kioussis, G. Kollias.
1991. Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J. 10: 4025-4031. 25. Williams, R. 0., M. Feldmann, R. N. Maini .
1992. Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis. Proc. Natl. Acad. Sci. USA 89: 9784-9788.
Claims
1. Method for screening in vitro drug candidates for inflammatory diseases mediated by LPS-inducible CC chemokine receptor, the said method comprising a step of putting into contact a CCRL2- expressing cell preparation with a drug candidate for CCRL2 inhibition and then evaluating the degree of CCRL2-expressing cell migration against a control .
2. Method according to claim 1, wherein the said CCRL2-exρressing cell preparation is a L-CCR- expressing cell preparation from mice.
3. Method according to claim 1 or claim 2, wherein the said cell preparation is a dendritic cell preparation.
4. Method according to any of claims from 1 to 3, comprising a chemotaxis assay, wherein the cell migration toward a chemoattractant is evaluated, the said chemoattractant being a cytokine.
5. Method according to claim 4, the chemoattractant is selected from human CCL19, CCL21 and CXCL12.
6. Method according to any of claims from 1 to 5, comprising a step of triggering the CCRL2-
59
expressing cell migration.
7. Method according to claim 6, wherein the said CCRL2-expressing cell migration is triggered by a specific anti-L-CCR monoclonal antibody (anti-L- CCR mAb) selected from IgG2a mAb and IgG2b inAb generated from L-CCR depleted mice.
8. Method according to claim 7, wherein the said mAbs are IgG2a mAb from clone 4.2.1 and IgG2b mAb from clone 2.5.1 of hybridomes cultured from L- CCR-depleted mice.
9. Method according to any of claims from 1 to 8, comprising at least one control step chosen from: i) contacting a CCRL2-depleted dendritic cell preparation with the drug candidate for CCRL2 inhibition and then evaluating the degree of CCRL2-depleted cell migration, or ii) contacting a CCRL2-expressing dendritic cell preparation with the drug candidate for CCRL2 inhibition and then evaluating the degree of CCRL2-expressing cell migration towards CXCL12 as chemoattractant .
10. Method according to any of claims from 1 to 9, wherein the step of evaluation of the CCRL2- expressing cell migration against the control is preferably accomplished by flow cytometry.
11. Method according to any one of claims
60
from 1 to 10, wherein the said inflammatory disease is an allergic airways inflammatory disease .
12. Method according to any one of claims from 1 to 10, wherein the said inflammatory disease is rheumatoid arthritis.
13. Method for screening in vivo drug candidates for an allergic airways diseases, which comprises inducing an allergen-type airways inflammatory response in a mammal, treating the said mammal with a drug candidate for inhibition of CCRL2 receptor and evaluating the inhibition of inflammatory cell recruitment in the BAL of said mammal against a control .
14. Method according to claim 13, wherein the said mammal is a mouse.
15. Method according to claim 13 or claim 14, wherein the allergen-type airways inflammatory response is elicited by ovalbumin (OVA) .
16. Method according to any of claims from 13 to 15, comprising at least one control step chosen from: a) inducing an allergen-type airways inflammatory response in a mammal and evaluating the inhibition of dendritic cell recruitment in
61
the BAL of said mammal, wherein no drug candidate administration is made, so that a 0% inhibition is given, or b) inducing an allergen-type airways inflammatory response in a CCRL2-depleted mammal, treating the said mammal with a drug candidate for inhibition of CCRL2 receptor and evaluating the inhibition of inflammatory cell recruitment in the BAL of said mammal.
17. Method for screening in vivo drug candidates for an rheumatoid arthritis, which comprises inducing rheumatoid arthritis in a mammal, treating the said mammal with a drug candidate for inhibition of CCRL2 receptor and evaluating the inhibition of the symptoms of rheumatoid arthritis in said mammal against a control .
18. Method according to claim 17, wherein the said mammal is a mouse.
19. Method according to claim 17 or claim 18, wherein rheumatoid arthritis is induced by collagen, particularly by collagen type II.
20. Method according to any of claims from 17 to 19, comprising at least one control step chosen from: a) inducing rheumatoid arthritis in a mammal
62
and evaluating the inhibition of the symptoms of rheumatoid arthritis in said mammal, wherein no drug candidate administration is made, so that a 0% inhibition is given, or b) inducing rheumatoid arthritis in a CCRL2-depleted mammal, treating the said mammal with a drug candidate for inhibition of CCRL2 receptor and evaluating the inhibition of the symptoms of rheumatoid arthritis in said mammal .
21. Method according to any one of claims from 17 to 20, wherein the step of evaluating the symptoms of rheumatoid arthritis in a mammal is made by scored visual assessment and/or by assessing the thickness of hind paws of said mammal and/or by assessing the weight and the number of cells of limph nodes of said mammal.
22. Cell preparation of CCRL2-depleted inflammatory cells as a control tool.
23. Cell preparation according to claim 22, being a dendritic cell preparation obtained by a
CCRL2-depleted mammal.
24. Cell preparation according to claim 23, being a dendritic cell preparation obtained by a L-CCR-depleted mice.
25. Monoclonal antibodies to CCRL2 receptor,
63
the said monoclonal antibodies being selected from IgG2a mAb and IgG2b mAb generated from L-CCR depleted mice.
26. Monoclonal antibodies according to claim 25, wherein the said mAbs are IgG2a mAb from clone
4.2.1 and IgG2b mAb from clone 2.5.1 of hybridomes cultured from L-CCR-depleted mice.
27. CCRL2 receptor inhibitors characterised by a %inhibition greater than 50% in the in vitro screening test of any of claims 1 to 12 and/or a %inhibition greater than 50% in the in vivo test of any of claims 13 to 21.
28. CCRL2 receptor inhibitors according to claim 27, characterised in that said inhibitors can bind to a CCRL2 receptor fragment identified by the following sequences:
- (SEQ ID NO.l)
MANYTLAPEDEYDVLIEGELESDEAEQCDKYDAQALSA (N- Terminal) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%;
(SEQ ID NO.2) AGGDPMCK (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%;
64
- (SEQ ID NO.3) PQMEDQKYKCAFSRTPFLPADETFWK (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%;
(SEQ ID NO.4) FLSTFKEHFSLSDCKSS (Extracellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%; - (SEQ ID NO.5)
MDNYTVAPDDEYDVLILDDYLDNSGPDQVPAPEFLSP (N-Terminal) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%; - (SEQ ID NO.6) TAAHGESPGNGTCK (Extracellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%;
- (SEQ ID NO.7) PRMERQKHKCAFGKPHFLPIEAPLWK (Extra-cellular loop) or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%;
(SEQ ID NO.8) FLSAFQEHLSLQDEKSS (Extra- cellular loop) or fragments thereof or homologues
65
thereof having a degree of homology greater than 50% and a degree of similarity greater than 70%.
29. CCRL2 receptor inhibitors according to claim 27, selected from: - (SEQ ID N0.1)
MANYTLAPEDEYDVLIEGELESDEAEQCDKYDAQALSA;
- (SEQ ID NO.2) AGGDPMCK;
- (SEQ ID NO.3) PQMEDQKYKCAFSRTPFLPADETFWK;
- (SEQ ID NO.4) FLSTFKEHFSLSDCKSS; - (SEQ ID NO.5)
MDNYTVAPDDEYDVLILDDYLDNSGPDQVPAPEFLSP;
- (SEQ ID NO.6) TAAHGESPGNGTCK;
- (SEQ ID NO.7) PRMERQKHKCAFGKPHFLPIEAPLWK;
- (SEQ ID NO.8) FLSAFQEHLSLQDEKSS, - (SEQ ID NO.9) NYTVAPDDEYDVLI;
- (SEQ ID NO.10) NYTLAPEDEYDVLI;
- (SEQ ID NO.11) PRME;
- (SEQ ID NO.12) PQME;
- (SEQ ID NO.13) HKCAF; - (SEQ ID NO.14) YKCAF;
- (SEQ ID NO.15) FLSAFQEHLSL;
- (SEQ ID NO.16) FLSTFKEHFSL, or fragments thereof or homologues thereof having a degree of homology greater than 50% and a degree of similarity greater than 70% or chemical bioisosters
66
thereof or CCRL2 gene antisense sequences.
30. Pharmaceutical compositions comprising a therapeutically effective amount of CCRL2 receptor inhibitor as defined in any of claims from 27 to 29 and a suitable carrier or excipient.
31. Method of treatment of an allergic airways inflammatory disease, wherein the said method involves treating a mammal in need thereof with a therapeutically effective dose of a CCRL2 receptor inhibitor as defined in any of claims from 27 to 29, wherein the said CCRL2 receptor inhibitor is comprised in a pharmaceutical composition together with a pharmaceutically acceptable carrier or excipient .
32. Method of treatment according to claim 31, wherein the said allergic airways inflammatory disease is selected from allergic asthma, allergic rhinitis, airways infectious diseases and COPD (Chronic Obstructive Pulmonary Disease) .
33. Method of treatment of rheumatoid arthritis, wherein the said method involves treating a mammal in need thereof with a therapeutically effective dose of a CCRL2 receptor inhibitor as defined in any of claims from 27 to 29, wherein the said CCRL2 receptor inhibitor is comprised in a
67
pharmaceutical composition together with a pharmaceutically acceptable carrier or excipient.
34. A CCRL2 receptor inhibitor as defined in any of claims from 27 to 29, for curing or alleviating an allergic airways inflammatory disease.
35. A CCRL2 receptor inhibitor as defined in any of claims from 27 to 29, for curing or alleviating rheumatoid arthritis.
68
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP06018661.6 | 2006-09-06 | ||
EP06018661 | 2006-09-06 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2008028692A2 true WO2008028692A2 (en) | 2008-03-13 |
WO2008028692A8 WO2008028692A8 (en) | 2008-05-15 |
WO2008028692A3 WO2008028692A3 (en) | 2008-09-25 |
Family
ID=39157601
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2007/008509 WO2008028692A2 (en) | 2006-09-06 | 2007-09-06 | Method for screening drug candidates for inflammatory diseases mediated by lps-inducible cc chemokine receptor mechanism |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2008028692A2 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010019811A2 (en) * | 2008-08-15 | 2010-02-18 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for screening for modulators of ccrl2 |
US20160075737A1 (en) * | 2013-04-25 | 2016-03-17 | Carmel-Haifa University Economic Corp. | Synthetic anti-inflammatory peptides and use thereof |
US9681647B2 (en) | 2012-07-05 | 2017-06-20 | Cornell University | Mammal with an orthotopic tumor capable of metastasis, a method of making and a method of using |
CN110456074A (en) * | 2019-08-27 | 2019-11-15 | 广东菲鹏生物有限公司 | Citrulling peptide and its application, the detection reagent of rheumatoid arthritis and kit |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002057779A2 (en) * | 2001-01-18 | 2002-07-25 | Rijksuniversiteit Groningen | Cloning and expression of a new mcp receptor in glial cells |
WO2005057220A2 (en) * | 2003-12-05 | 2005-06-23 | Oxagen Limited | Agents modulating the activity of ccrl2 and methods of using the same |
-
2007
- 2007-09-06 WO PCT/EP2007/008509 patent/WO2008028692A2/en active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002057779A2 (en) * | 2001-01-18 | 2002-07-25 | Rijksuniversiteit Groningen | Cloning and expression of a new mcp receptor in glial cells |
WO2005057220A2 (en) * | 2003-12-05 | 2005-06-23 | Oxagen Limited | Agents modulating the activity of ccrl2 and methods of using the same |
Non-Patent Citations (4)
Title |
---|
BIBER KNUT ET AL: "Expression of L-CCR in HEK 293 cells reveals functional responses to CCL2, CCL5, CCL7, and CCL8." JOURNAL OF LEUKOCYTE BIOLOGY, vol. 74, no. 2, August 2003 (2003-08), pages 243-251, XP002474350 ISSN: 0741-5400 * |
GALLIGAN C L ET AL: "Up-regulated expression and activation of the orphan chemokine receptor, CCRL2, in rheumatoid arthritis" ARTHRITIS AND RHEUMATISM, LIPPINCOTT, PHILADELPHIA, US, vol. 50, no. 6, June 2004 (2004-06), pages 1806-1814, XP002349008 ISSN: 0004-3591 * |
OOSTENDORP J ET AL: "Localization and Enhanced mRNA Expression of the Orphan Chemokine Receptor L-CCR in the Lung in a Murine Model of Ovalbumin-induced Airway Inflammation" JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, HISTOCHEMICAL SOCIETY, NEW YORK, NY, US, vol. 52, no. 3, 2004, pages 401-410, XP003009323 ISSN: 0022-1554 * |
SOZZANI SILVANO: "New therapeutic strategies for the control of inflammatory and autoimmune diseases" RESEARCH PROGRAM, [Online] 2004, XP002474351 Retrieved from the Internet: URL:http://www.ricercaitaliana.it/prin/unita_op_en-2004063113_001.htm> [retrieved on 2008-03-28] * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010019811A2 (en) * | 2008-08-15 | 2010-02-18 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for screening for modulators of ccrl2 |
WO2010019811A3 (en) * | 2008-08-15 | 2010-05-14 | The Board Of Trustees Of The Leland Stanford Junior University | Methods for screening for modulators of ccrl2 |
US9681647B2 (en) | 2012-07-05 | 2017-06-20 | Cornell University | Mammal with an orthotopic tumor capable of metastasis, a method of making and a method of using |
US20160075737A1 (en) * | 2013-04-25 | 2016-03-17 | Carmel-Haifa University Economic Corp. | Synthetic anti-inflammatory peptides and use thereof |
US9850278B2 (en) * | 2013-04-25 | 2017-12-26 | Carmel-Haifa University Economic Corp. | Synthetic anti-inflammatory peptides and use thereof |
CN110456074A (en) * | 2019-08-27 | 2019-11-15 | 广东菲鹏生物有限公司 | Citrulling peptide and its application, the detection reagent of rheumatoid arthritis and kit |
CN110456074B (en) * | 2019-08-27 | 2022-06-03 | 广东菲鹏生物有限公司 | Citrullinated peptide, application thereof, rheumatoid arthritis detection reagent and kit |
Also Published As
Publication number | Publication date |
---|---|
WO2008028692A8 (en) | 2008-05-15 |
WO2008028692A3 (en) | 2008-09-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220106381A1 (en) | Cd154 peptides and methods of inhibiting cd40 interactions with cd154 | |
Hu et al. | IL-17RC is required for IL-17A–and IL-17F–dependent signaling and the pathogenesis of experimental autoimmune encephalomyelitis | |
Henderson et al. | Soluble IL-4 receptor inhibits airway inflammation following allergen challenge in a mouse model of asthma | |
KR101510065B1 (en) | Methods and compositions for the treatment of persistent infections and cancer by inhibiting the programmed cell death 1 (pd-1) pathway | |
Walsh | Advances in the immunobiology of eosinophils and their role in disease | |
Kojima et al. | Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes | |
Sabin et al. | Schistosoma mansoni egg-induced early IL-4 production is dependent upon IL-5 and eosinophils. | |
Oshikawa et al. | Expression and function of the ST2 gene in a murine model of allergic airway inflammation | |
US8187596B1 (en) | Method of treating asthma or allergy by administering an IL-33 receptor antibody | |
Ng et al. | Fibroblast‐specific IL11 signaling drives chronic inflammation in murine fibrotic lung disease | |
Mishra et al. | Resistin-like molecule-β is an allergen-induced cytokine with inflammatory and remodeling activity in the murine lung | |
JPH11507843A (en) | Soluble divalent and multivalent heterodimer analogs of proteins | |
US7196172B2 (en) | Heterodimeric four helix bundle cytokines | |
US8795656B2 (en) | Methods of treating rheumatoid arthritis by administering an anti-IL3 antibody | |
WO2008028692A2 (en) | Method for screening drug candidates for inflammatory diseases mediated by lps-inducible cc chemokine receptor mechanism | |
JP5561689B2 (en) | Screening method for therapeutic agents for allergic airway inflammation and / or airway hypersensitivity using IL-17RB positive NKT cells | |
Yang et al. | Reduction in arthritis severity and modulation of immune function in tissue factor cytoplasmic domain mutant mice | |
WO2000019988A1 (en) | NOVEL Th2-SPECIFIC MOLECULES AND USES THEREOF | |
US20190194290A1 (en) | Methods for preventing, modulating and/or reducing cardiovascular disease | |
US7919248B2 (en) | Methods for the modulation of IL-13 | |
US20020114806A1 (en) | Uses of mammalian genes and related reagents | |
WO2005014612A1 (en) | Novel splice variant of ctla-4 | |
EP2913060A1 (en) | Inhibition of the fractalkine-receptor interaction for the treatment of atopic dermatitis | |
US20030148369A1 (en) | Novel uses of mammalian CCR8 receptors and related reagents | |
US20100098689A1 (en) | 4-1 bb ligand in inflammatory diseases |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 07818589 Country of ref document: EP Kind code of ref document: A2 |
|
NENP | Non-entry into the national phase in: |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 07818589 Country of ref document: EP Kind code of ref document: A2 |