WO2000066754A1 - Dérivés de protéine c - Google Patents
Dérivés de protéine c Download PDFInfo
- Publication number
- WO2000066754A1 WO2000066754A1 PCT/US2000/008722 US0008722W WO0066754A1 WO 2000066754 A1 WO2000066754 A1 WO 2000066754A1 US 0008722 W US0008722 W US 0008722W WO 0066754 A1 WO0066754 A1 WO 0066754A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- derivative
- human
- serpins
- seq
- Prior art date
Links
- 229960000856 protein c Drugs 0.000 title claims abstract description 109
- 101800004937 Protein C Proteins 0.000 claims abstract description 116
- 101800001700 Saposin-D Proteins 0.000 claims abstract description 113
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 68
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 66
- 229920001184 polypeptide Polymers 0.000 claims abstract description 65
- 101500025568 Homo sapiens Saposin-D Proteins 0.000 claims abstract description 34
- 229940100689 human protein c Drugs 0.000 claims abstract description 34
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 27
- 208000007536 Thrombosis Diseases 0.000 claims abstract description 26
- 208000035475 disorder Diseases 0.000 claims abstract description 14
- 210000004369 blood Anatomy 0.000 claims abstract description 7
- 239000008280 blood Substances 0.000 claims abstract description 7
- 201000005665 thrombophilia Diseases 0.000 claims abstract description 7
- 230000002792 vascular Effects 0.000 claims abstract description 7
- 230000004071 biological effect Effects 0.000 claims abstract description 6
- 102400000827 Saposin-D Human genes 0.000 claims abstract 25
- 239000003001 serine protease inhibitor Substances 0.000 claims description 53
- 102000008847 Serpin Human genes 0.000 claims description 51
- 108050000761 Serpin Proteins 0.000 claims description 51
- 238000006467 substitution reaction Methods 0.000 claims description 31
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 28
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 150000001413 amino acids Chemical class 0.000 claims description 19
- 102000004169 proteins and genes Human genes 0.000 claims description 19
- 230000002779 inactivation Effects 0.000 claims description 18
- 239000003112 inhibitor Substances 0.000 claims description 16
- 239000013598 vector Substances 0.000 claims description 12
- 206010040047 Sepsis Diseases 0.000 claims description 10
- 108020004414 DNA Proteins 0.000 claims description 9
- 102000039446 nucleic acids Human genes 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 208000009190 disseminated intravascular coagulation Diseases 0.000 claims description 7
- 208000010125 myocardial infarction Diseases 0.000 claims description 7
- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
- 208000004476 Acute Coronary Syndrome Diseases 0.000 claims description 6
- 108020004511 Recombinant DNA Proteins 0.000 claims description 6
- 229940127218 antiplatelet drug Drugs 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 206010002388 Angina unstable Diseases 0.000 claims description 5
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 claims description 5
- 206010062506 Heparin-induced thrombocytopenia Diseases 0.000 claims description 5
- 201000007023 Thrombotic Thrombocytopenic Purpura Diseases 0.000 claims description 5
- 208000007814 Unstable Angina Diseases 0.000 claims description 5
- 201000004332 intermediate coronary syndrome Diseases 0.000 claims description 5
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 claims description 4
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 claims description 4
- 208000006011 Stroke Diseases 0.000 claims description 4
- 206010047249 Venous thrombosis Diseases 0.000 claims description 4
- 208000028227 Viral hemorrhagic fever Diseases 0.000 claims description 4
- 208000011341 adult acute respiratory distress syndrome Diseases 0.000 claims description 4
- 201000000028 adult respiratory distress syndrome Diseases 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 208000014674 injury Diseases 0.000 claims description 4
- 208000007056 sickle cell anemia Diseases 0.000 claims description 4
- 238000001356 surgical procedure Methods 0.000 claims description 4
- 238000002054 transplantation Methods 0.000 claims description 4
- 230000008733 trauma Effects 0.000 claims description 4
- 206010051055 Deep vein thrombosis Diseases 0.000 claims description 3
- 208000002903 Thalassemia Diseases 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 230000035699 permeability Effects 0.000 claims description 3
- 102000040430 polynucleotide Human genes 0.000 claims description 3
- 108091033319 polynucleotide Proteins 0.000 claims description 3
- 239000002157 polynucleotide Substances 0.000 claims description 3
- 201000005380 purpura fulminans Diseases 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims 6
- 208000030090 Acute Disease Diseases 0.000 claims 2
- 239000005022 packaging material Substances 0.000 claims 2
- 238000012258 culturing Methods 0.000 claims 1
- 239000003085 diluting agent Substances 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
- 102000017975 Protein C Human genes 0.000 description 80
- 235000001014 amino acid Nutrition 0.000 description 37
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 20
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 18
- 102100024078 Plasma serine protease inhibitor Human genes 0.000 description 17
- 108010001953 Protein C Inhibitor Proteins 0.000 description 17
- 230000000694 effects Effects 0.000 description 17
- 102000010911 Enzyme Precursors Human genes 0.000 description 16
- 108010062466 Enzyme Precursors Proteins 0.000 description 16
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 15
- 229940122929 Protein C inhibitor Drugs 0.000 description 15
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 14
- 102000052586 bactericidal permeability increasing protein Human genes 0.000 description 14
- 108010032816 bactericidal permeability increasing protein Proteins 0.000 description 14
- 150000001875 compounds Chemical class 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 14
- 229940024606 amino acid Drugs 0.000 description 13
- 201000010099 disease Diseases 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 230000003024 amidolytic effect Effects 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 239000012634 fragment Substances 0.000 description 10
- 108010074105 Factor Va Proteins 0.000 description 9
- -1 His Chemical compound 0.000 description 9
- 238000003776 cleavage reaction Methods 0.000 description 9
- 230000007017 scission Effects 0.000 description 9
- 230000004913 activation Effects 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 239000003146 anticoagulant agent Substances 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 239000002753 trypsin inhibitor Substances 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 229940127219 anticoagulant drug Drugs 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000001802 infusion Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 206010053567 Coagulopathies Diseases 0.000 description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 229920002684 Sepharose Polymers 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000035602 clotting Effects 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000002035 prolonged effect Effects 0.000 description 5
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- 108091026890 Coding region Proteins 0.000 description 4
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 4
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 230000003110 anti-inflammatory effect Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 239000010432 diamond Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 230000014508 negative regulation of coagulation Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 4
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 4
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 3
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 3
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 201000005660 Protein C Deficiency Diseases 0.000 description 3
- 108010022999 Serine Proteases Proteins 0.000 description 3
- 102000012479 Serine Proteases Human genes 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 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 3
- 108090000190 Thrombin Proteins 0.000 description 3
- 238000002399 angioplasty Methods 0.000 description 3
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical compound NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003114 blood coagulation factor Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 3
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 3
- 208000013746 hereditary thrombophilia due to congenital protein C deficiency Diseases 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000012931 lyophilized formulation Substances 0.000 description 3
- 231100000219 mutagenic Toxicity 0.000 description 3
- 230000003505 mutagenic effect Effects 0.000 description 3
- 230000002093 peripheral effect Effects 0.000 description 3
- 230000004481 post-translational protein modification Effects 0.000 description 3
- 230000000019 pro-fibrinolytic effect Effects 0.000 description 3
- 230000002797 proteolythic effect Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000002741 site-directed mutagenesis Methods 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 229960004072 thrombin Drugs 0.000 description 3
- 238000007631 vascular surgery Methods 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- WPVINHLVHHPBMK-ULQDDVLXSA-N (2s)-n-[(2s)-5-(diaminomethylideneamino)-1-(4-nitroanilino)-1-oxopentan-2-yl]-1-[(2s)-5-oxopyrrolidine-2-carbonyl]pyrrolidine-2-carboxamide Chemical compound C([C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(=O)NC=2C=CC(=CC=2)[N+]([O-])=O)CCN1C(=O)[C@@H]1CCC(=O)N1 WPVINHLVHHPBMK-ULQDDVLXSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 229960001138 acetylsalicylic acid Drugs 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 208000034158 bleeding Diseases 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000058 esterolytic effect Effects 0.000 description 2
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 125000001165 hydrophobic group Chemical group 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000000415 inactivating effect Effects 0.000 description 2
- 230000000937 inactivator Effects 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000000106 platelet aggregation inhibitor Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000001732 thrombotic effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 101800001401 Activation peptide Proteins 0.000 description 1
- 102400000069 Activation peptide Human genes 0.000 description 1
- 101710081722 Antitrypsin Proteins 0.000 description 1
- 206010003178 Arterial thrombosis Diseases 0.000 description 1
- 206010003211 Arteriosclerosis coronary artery Diseases 0.000 description 1
- 239000005552 B01AC04 - Clopidogrel Substances 0.000 description 1
- 239000005528 B01AC05 - Ticlopidine Substances 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- 206010011091 Coronary artery thrombosis Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000271032 Daboia russelii Species 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010058279 Factor V Leiden mutation Diseases 0.000 description 1
- 206010053172 Fatal outcomes Diseases 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 101000766306 Homo sapiens Serotransferrin Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical group C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- NPBGTPKLVJEOBE-IUCAKERBSA-N Lys-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N NPBGTPKLVJEOBE-IUCAKERBSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 241000699673 Mesocricetus auratus Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 208000010362 Protozoan Infections Diseases 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 102000012607 Thrombomodulin Human genes 0.000 description 1
- 108010079274 Thrombomodulin Proteins 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 229960000446 abciximab Drugs 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000001475 anti-trypsic effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 238000013176 antiplatelet therapy Methods 0.000 description 1
- 229960004676 antithrombotic agent Drugs 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 201000005008 bacterial sepsis Diseases 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- OIRCOABEOLEUMC-GEJPAHFPSA-N bivalirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 OIRCOABEOLEUMC-GEJPAHFPSA-N 0.000 description 1
- 108010055460 bivalirudin Proteins 0.000 description 1
- 229960001500 bivalirudin Drugs 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000036765 blood level Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000021523 carboxylation Effects 0.000 description 1
- 238000006473 carboxylation reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229960003009 clopidogrel Drugs 0.000 description 1
- GKTWGGQPFAXNFI-HNNXBMFYSA-N clopidogrel Chemical compound C1([C@H](N2CC=3C=CSC=3CC2)C(=O)OC)=CC=CC=C1Cl GKTWGGQPFAXNFI-HNNXBMFYSA-N 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 208000026758 coronary atherosclerosis Diseases 0.000 description 1
- 208000002528 coronary thrombosis Diseases 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 229960002768 dipyridamole Drugs 0.000 description 1
- IZEKFCXSFNUWAM-UHFFFAOYSA-N dipyridamole Chemical compound C=12N=C(N(CCO)CCO)N=C(N3CCCCC3)C2=NC(N(CCO)CCO)=NC=1N1CCCCC1 IZEKFCXSFNUWAM-UHFFFAOYSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 239000003527 fibrinolytic agent Substances 0.000 description 1
- 230000003480 fibrinolytic effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- UHBYWPGGCSDKFX-VKHMYHEASA-N gamma-carboxy-L-glutamic acid Chemical group OC(=O)[C@@H](N)CC(C(O)=O)C(O)=O UHBYWPGGCSDKFX-VKHMYHEASA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 238000003312 immunocapture Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000006225 natural substrate Substances 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 238000001050 pharmacotherapy Methods 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 230000031915 positive regulation of coagulation Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- XNSAINXGIQZQOO-SRVKXCTJSA-N protirelin Chemical compound NC(=O)[C@@H]1CCCN1C(=O)[C@@H](NC(=O)[C@H]1NC(=O)CC1)CC1=CN=CN1 XNSAINXGIQZQOO-SRVKXCTJSA-N 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229940107685 reopro Drugs 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000028710 ribosome assembly Effects 0.000 description 1
- 210000004708 ribosome subunit Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 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
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960005001 ticlopidine Drugs 0.000 description 1
- PHWBOXQYWZNQIN-UHFFFAOYSA-N ticlopidine Chemical compound ClC1=CC=CC=C1CN1CC(C=CS2)=C2CC1 PHWBOXQYWZNQIN-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 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
- 239000012588 trypsin Substances 0.000 description 1
- 239000002821 viper venom Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6464—Protein C (3.4.21.69)
-
- 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
- C07K14/745—Blood coagulation or fibrinolysis factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/06—Antianaemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21069—Protein C activated (3.4.21.69)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to novel polynucleotides, polypeptides encoded by them and to the use of such polynucleotides and polypeptides. More specifically, the invention relates to protein C derivatives resistant to serpin inactivation, to their production, and to pharmaceutical compositions comprising these protein C derivatives .
- Protein C is a serine protease and naturally occurring anticoagulant that plays a role in the regulation of homeostasis by inactivating Factors V a and Villa in the coagulation cascade. Human protein C is made in vivo as a single polypeptide of 461 amino acids.
- This polypeptide undergoes multiple post-translational modifications including 1) cleavage of a 42 amino acid signal sequence; 2) cleavage of lysine and arginine residues (positions 156 and 157) to make a 2-chain inactive precursor or zymogen (a 155 amino acid residue light chain attached via a disulfide bridge to a 262 amino acid residue heavy chain) ; 3) vitamin K-dependenr carboxylation of nine glutamic acid residues of the light chain, resulting in nine gamma-carboxyglutamic acid residues; and 4) carbohydrate attachment at four sites (one in the light chain and three in the heavy chain) .
- the 2-chain zymogen may be activated by removal of a dodecapeptide at the N-terminus of the heavy chain, producing activated protein C (aPC) possessing greater enzymatic activity than the 2-chain zymogen.
- aPC functions as an anti-coagulant important in protecting against thrombosis, has anti-inflammatory effects through its inhibition of cytokine generation (e.g., TNF and IL-1), and exerts profibrinolytic properties that facilitate clot lysis.
- cytokine generation e.g., TNF and IL-1
- aPC provides a mechanism for anti-coagulation, anti-inflammation, and fibrinolysis .
- Plasma- derived and recombinantly produced aPC have been shown to be effective and safe antithrombotic agents in a variety of animal models of both venous and arterial thrombosis.
- Protein C levels have also been shown to be abnormally low in the following diseases and conditions : disseminated intravascular coagulation (DIC) [Fourrier, et al . , Chest 101:816-823, 1992], sepsis [Gerson, et al . , Pediatrics 91:418-422, 1993], major trauma/major surgery [Thomas, et al., Am J Surg. 158:491-494, 1989], burns [Lo, et al . , Burns 20:186-187 (1994)], adult respiratory distress syndrome (A DS) [Hasegawa, et al .
- DI disseminated intravascular coagulation
- HIT heparin-induced thrombocytopenia
- platelet inhibition is efficacious in both prevention and treatment of thrombotic disease.
- antiplatelet agents such as aspirin
- aPC and antiplatelet agents results in a synergy that allows the reduction of the dosages of both aPC and the antiplatelet agent (s) .
- the reduction of the dosages of the agents in combination therapy in turn results in reduced side effects such as increased bleeding often observed in combination anti- coagulant/anti-platelet therapy.
- a reason for the short half-life is that blood levels of aPC are regulated by molecules known as serpins (Serine Protease Inhibitors) , which covalently bind to aPC forming an inactive serpin/aPC complex.
- serpins Serine Protease Inhibitors
- the serpin/aPC complexes are formed when aPC binds and proteolytically cleaves a reactive site loop within the serpin; upon cleavage, the serpin undergoes a conformational change irreversibly inactivating aPC.
- the serpin/aPC complex is then eliminated from the bloodstream via hepatic receptors for the serpin/aPC complex.
- aPC has a relatively short half-life compared to the zymogen; approximately 20 minutes for aPC versus approximately 10 hours for human protein C zymogen (Okajima, et al . , Thromb Haemost 63(l):48-53, 1990). It has been shown that changes to serine protease amino acid sequences at residues which interact directly with the substrate (generally within or near the active site) can alter the specificity of the serine protease, potentially providing increased specific activity towards appropriate coagulation factors, as well as increased resistance to serpins (Rezaie, J Biol Che 271 (39) : 23807-23814 , 1996; Rezaie and Esmon, Eur. J.
- an aPC polypeptide exhibiting increased resistance to serpin inactivation, while maintaining the desirable biological activities of aPC e.g., anticoagulant, fibrinolytic, and anti-inflammatory activities
- aPC e.g., anticoagulant, fibrinolytic, and anti-inflammatory activities
- PCI protein C inhibitor
- ⁇ -antitrypsin ⁇ -AT
- Both PCI and ⁇ -AT have been demonstrated to be the primary physiological inactivators of aPC in disease states such as disseminated intravascular coagulation (Scully, et al . , Thromb Haemost 69 (5) : 448-53 , 1993)
- elevated levels of ⁇ -AT have been observed in a number of disease states involving an inflammatory response (Somayajulu, et al .
- the present invention describes novel protein C derivatives. These protein C derivatives retain the important biological activity of the wild-type protein C (SEQ ID NO: 7) and have substantially longer half-lives in human blood. Therefore, these compounds provide various advantages, eg. less frequent administration and/or smaller dosages and thus a reduction in the overall cost of production of the therapy. Additionally, these compounds exhibit an advantage in disease states with significantly elevated ⁇ -AT levels such as sepsis. Importantly, the increases in protein C derivative plasma half-lives may be achieved via single amino acid substitutions, which are less likely to be immunogenic in comparison to molecules which contain multiple amino acid substitutions (U.S. Patent No. 5,358,932; Holly, et al . , Biochemistry 33:1876-1880, 1994).
- the present invention provides a protein C derivative comprising SEQ ID NO: 1 and the corresponding amino acids in SEQ ID NO: 2, wherein one or more of amino acids 194, 195,
- the invention further provides the activated form of the above-identified protein C derivatives .
- the present invention also provides recombinant DNA molecules encoding the protein C derivatives in the preceding paragraph, in particular those comprising SEQ ID NOS: 8, 9, and 10.
- Another aspect of the present invention provides protein sequences of these same protein C derivatives, particularly those comprising SEQ ID NOS: 3, 4, and 5 and the activated forms of these protein C derivatives.
- the present invention comprises methods of treating vascular occlusive disorders and hypercoagulable states including: sepsis, disseminated intravascular coagulation, purpura fulminans, major trauma, major surgery, burns, adult respiratory distress syndrome, transplantations, deep vein thrombosis, heparin- induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.
- the invention further provides treating these same diseases and conditions employing the activated form of the above- identified protein C derivatives.
- Another embodiment of the present invention is a method of treating sepsis comprising the administration to a patient in need thereof a pharmaceutically effective amount of a protein C derivative of this invention in combination with bacterial permeability increasing protein.
- the present invention comprises methods of treating acute coronary syndromes such as myocardial infarction and unstable angina.
- the present invention further comprises methods of treating thrombotic disorders.
- thrombotic disorders include, but are not limited to, stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery.
- the present invention also provides a pharmaceutical composition comprising a protein C derivative of this invention.
- Human protein C derivatives for the above-mentioned indications and pharmaceutical compositions are preferably selected from L194S, L194S:T254S, and L194A:T254S.
- an aspect of the invention comprises treating the diseases and conditions caused or resulting from protein C deficiency as defined herein, by inhibiting binding to inhibitor recognition sequences S2 , S3', and S4 ' of the serpins, PCI and ⁇ -AT.
- This final aspect of the invention contemplates any and all modifications to any aPC molecule resulting in inhibition of the binding to said inhibitor recognition sequences of the serpins PCI and ⁇ _-AT.
- the inhibition of binding to the specific inhibitor recognition sequences of the serpins (S2, S3', and S4 ' ) being an important contribution to this aspect of the invention.
- Figure 1 Inactivation of human aPC polypeptides during incubation with normal human plasma.
- Activated protein C levels were determined using immunocapture assay, and compared to a standard curve generated from dilutions of the purified protein in rabbit plasma; the standard curve ranged from 1 to 250 ng/mL, with the calculated values within 10% of the standard samples. Data are shown for the wild-type protein (WT, circles) , T254S (squares) , L194S (triangles) , and L194S/T254S (diamonds) . The values plotted are the mean and standard error for the three animals. For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below. Antiplatelet agent - one or more agents alone or in combination which reduces the ability of platelets to aggregate.
- Agents understood and appreciated in the art include those cited in, for example, Remington, The Science and Practice of Pharmacy, Nineteenth Edition, Vol II, pages 924-25, Mack Publishing Co., herein incorporated by reference. Such agents include but are not limited to aspirin (ASA) , clopidogrel, ReoPro® (abciximab) , dipyridamole, ticlopidine and Ilb/lIIa antagonists.
- ASA aspirin
- clopidogrel ReoPro®
- abciximab dipyridamole
- ticlopidine ticlopidine
- Ilb/lIIa antagonists e.g., aPC or activated protein C refers to recombinant aPC .
- aPC includes and is preferably recombinant human aPC although aPC may also include other species having protein C proteolytic, amidolytic, esterolytic, and biological (anti- coagulant, anti-inflammatory, or pro-fibrinolytic) activities .
- Protein C derivative (s) refers to the recombinantly produced polypeptides of this invention that differ from wild-type human protein C but when activated retain the essential properties i.e., proteolytic, amidolytic, esterolytic, and biological (anti-coagulant, anti- inflammatory, pro-fibrinolytic activities) .
- the definition of protein C derivatives as used herein also includes the activated form of the above identified protein C derivatives . Treating - describes the management and care of a patient for the purpose of combating a disease, condition, or disorder whether to eliminate the disease, condition, or disorder, or prophylactically to prevent the onset of the symptoms or complications of the disease, condition, or disorder.
- Bolus injection the injection of a drug in a defined quantity (called a bolus) over a period of time up to about
- Unit dosage form - refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient .
- Hypercoagulable states - excessive coagulability associated with disseminated intravascular coagulation, pre- thrombotic conditions, activation of coagulation, or congenital or acquired deficiency of clotting factors such as aPC.
- Zymogen - protein C zymogen refers to secreted, inactive forms, whether one chain or two chains, of protein C .
- compositions comprising: a pharmaceutically effective amount - a therapeutically efficacious amount of a pharmaceutical compound.
- the particular dose of the compound administered according to this invention will, of course, be determined by the attending physician evaluating the particular circumstances surrounding the case, including the compound administered, the particular condition being treated, the patient characteristics and similar considerations.
- Acute coronary syndromes clinical manifestations of coronary atherosclerosis complicated by coronary plaque rupture, superimposed coronary thrombosis, and jeopardized coronary blood flow resulting in coronary ischemia and/or myocardial infarction.
- the spectrum of acute coronary syndromes includes unstable angina, non-Q-wave (i.e., non- ST-segment elevation) myocardial infarction, and Q-wave (i.e., ST-segment elevation) myocardial infarction.
- Thrombotic disorders a disorder relating to, or affected with the formation or presence of a blood clot within a blood vessel. Such disorders include, but are not limited to, stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery.
- Serpin any of a group of structurally related proteins that typically are serine protease inhibitors whose inhibiting activity is conferred by an active site in a highly variable and mobile peptide loop and that include but are not limited to protein C inhibitor (PCI) and ⁇ - antitrypsin ( ⁇ ]_-AT) .
- PCI protein C inhibitor
- ⁇ ]_-AT ⁇ - antitrypsin
- Inhibitor recognition sequence S2 the 2 nd residue N-terminal to the cleavage site of PCI or ⁇ -AT.
- Inhibitor recognition sequence S3' the 3 rd residue C-terminal to the cleavage site of PCI or ⁇ -AT.
- Inhibitor recognition sequence S4 ' the 4 th residue C-terminal to the cleavage site of PCI or ⁇ -AT.
- Wild-type protein C the type of protein C that predominates in a natural population of humans in contrast to that of natural or laboratory mutant or polypeptide forms of protein C.
- Bactericidal permeability increasing protein includes naturally and recombinantly produced bactericidal permeability increasing (BPI) protein; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; biologically active variant analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides.
- BPI bactericidal permeability increasing
- the complete amino acid sequence of human BPI, as well as the nucleotide sequence of DNA encoding BPI have been elucidated by Gray, et al . , 1989, J. Biol. Chem 264:9505.
- Recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI are disclosed in U.S. Patent No. 5,198,541, herein incorporated by reference .
- the activated form of aPC or isolated human aPC polypeptides may be produced by activating recombinant human protein C zymogen or recombinant protein C derivative zymogen in vitro or by direct secretion of the activated form of protein C.
- the means by which the activation occurs is not critical and the process aspects of this invention include any and all means of activation.
- Protein C derivatives may be produced in eukaryotic cells, transgenic animals, or transgenic plants, including, for example, secretion from human kidney 293 cells as a zymogen then purified and activated by techniques known to the skilled artisan.
- the present invention provides protein C derivatives, including activated forms thereof, which have increased resistance to serpins, and consequently result in extended plasma half-lives. Specific protein C derivatives include L194S, L194S:T254S, and L194A:T254S and activated forms thereof .
- Protein C derivative L194S preferably contains a serine residue at position 194 rather than a leucine residue normally found at this position.
- amino acid substitutions at residue 194 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gin.
- the activated form of protein C derivative L194S demonstrates prolonged half-life in plasma ( Figure 1) and increased resistance to serpins, for example, ⁇ _-antitrypsin ( ⁇ -AT) , Figure 4.
- Protein C derivative L194S:T254S preferably contains a serine residue at position 194 rather than a leucine residue normally found at this position and a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residues 194 and 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr.
- the activated form of human protein C derivative L194S:T254S demonstrates a prolonged half-life in normal human plasma compared to wild- type protein C, Figure 2.
- Protein C derivative L194A:T254S preferably contains an alanine residue at position 194 rather than a leucine residue normally found at this position and a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residues 194 and 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr.
- the activated form of human protein C derivative L194A:T254S demonstrates a prolonged half-life in normal human plasma compared to wild-type protein C, Figure 2.
- Further embodiments of the present invention include protein C derivatives: L194T, L194A, A195G, L228Q, T254S, F316N, Y249E, and Y302Q, and activated forms thereof which have increased resistance to serpins.
- Protein C derivatives L194T or L194A preferably contain a threonine residue or an alanine residue at position 194 rather than a leucine residue normally found at this position.
- amino acid substitutions at residue 194 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, His, Lys, Arg, Asn, Asp, Glu, and Gin.
- Protein C derivative A195G preferably contains a glycine residue at position 195 rather than an alanine residue normally found at this position.
- amino acid substitutions at residue 195 in addition to Gly may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gin.
- the activated form of protein C derivative A195G demonstrates prolonged half-life in plasma ( Figure 1) and increased resistance to serpins, for example, a ] _-antitrypsin ( ⁇ -AT) , Figure 4.
- Protein C derivative L228Q preferably contains a glutamine residue at position 228 rather than a leucine residue normally found at this position.
- amino acid substitutions at residue 228 in addition to Gin may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, and Gly.
- Protein C derivative T254S preferably contains a serine residue at position 254 rather than a threonine residue normally found at this position. It is apparent to one with skill in the art that other amino acid substitutions at residue 254 in addition to Ser may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include Ala, Thr, His, Lys, Arg, Asn, Asp, Glu, Gin, and Gly, provided that amino acid 254 is not substituted with Thr.
- the activated form of protein C derivative T254S demonstrates prolonged half-life in plasma (Figure 1) and increased resistance to serpins, for example, ⁇ -antitrypsin ( ⁇ -AT) , Figure 4.
- Protein C derivative F316N preferably contains an asparagine residue at position 316 rather than a p enylalanine residue normally found at this position.
- amino acid substitutions at residue 316 in addition to Asn may impart increased resistance to serpins in the resulting polypeptide molecule. Examples of such amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asp, Glu, Gin, and Gly.
- Protein C derivative Y249E preferably contains a glutamic acid residue at position 249 rather than a tyrosine residue normally found at this position.
- An additional polypeptide contains an Asp at position 249 rather than a tyrosine residue.
- amino acid substitutions at residue 249 in addition to Glu and Asp may impart increased resistance to serpins in the resulting polypeptide molecule.
- amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Gin, and Gly.
- Protein C derivative Y302Q preferably contains a glutamine residue at position 302 rather than a tyrosine residue normally found at this position.
- An additional polypeptide contains a Glu at position 302 rather than a tyrosine residue.
- amino acid substitutions at residue 302 in addition to Glu or Gin may impart increased resistance to serpins in the resulting polypeptide molecule.
- amino acid substitutions include, Ser, Ala, Thr, His, Lys, Arg, Asn, Asp, and Gly.
- protein C derivatives may include proteins that represent functionally equivalent gene products.
- Such an equivalent protein C derivative may contain deletions, additions, or substitutions of amino acid residues within the amino acid sequence encoded by the protein C polypeptide gene sequences described above, but which result in a silent change, thus producing a functionally equivalent protein C derivative gene product.
- Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
- polypeptides of the present invention include polypeptides having an amino acid sequence at least identical to that of SEQ ID NOS: 3, 4, or 5, or fragments thereof with at least 90% identity to the corresponding fragment of SEQ ID NOS: 3, 4, or 5.
- all of these polypeptides retain the biological activity of human aPC.
- Preferred polypeptides are those that vary from SEQ ID NOS: 3, 4, or 5 by conservative substitutions i.e., those that substitute a residue with another of like characteristics. Typical substitutions are among Ala, Val , Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues
- the invention also provides DNA compounds for use in making the protein C derivatives. These DNA compounds comprise the coding sequence for the light chain of human protein C zymogen or protein C derivative zymogen positioned immediately adjacent to, downstream of, and in translational reading frame with the prepropeptide sequence of human protein C zymogen or protein C derivative zymogen.
- the DNA sequences preferably encode the Lys-Arg dipeptide which is processed during maturation of the protein C molecule, the activation peptide and the heavy chain of the protein C derivative .
- DNA compounds of the present invention were prepared by the use of site-directed mutagenesis to change particular positions within human protein C zymogen. The methods used for the identification of residues which form critical contacts in these particular positions are described in Example 1.
- the protein C derivatives can be made by techniques well known in the art utilizing eukaryotic cell lines, transgenic animals, or transgenic plants. Skilled artisans will readily understand that appropriate host eukaryotic cell lines include but are not limited to HepG2 , LLC-MK 2 , CHO-K1, 293, or AV12 cells, examples of which are described in U.S. Patent No. 5,681,932, herein incorporated by reference. Furthermore, examples of transgenic production of recombinant proteins are described in U.S. Patent Nos . 5,589,604 and 5,650,503, herein incorporated by reference. Skilled artisans recognize that a variety of vectors are useful in the expression of a DNA sequence of interest in a eukaryotic host cell .
- Vectors that are suitable for expression in mammalian cells include, but are not limited to: pGT-h, pGT-d; pCDNA 3.0, pCDNA 3.1, pCDNA 3.1+Zeo, and pCDNA 3.1+Hygro (Invitrogen) ; and, pIRES/Hygro, and pIRES/neo (Clonetech) .
- the preferred vector of the present invention is pIG3 as described in Example 2.
- the protein C derivatives made by any of these methods must undergo post-translational modifications such as the addition of nine gamma-carboxy-glutamates, the addition of one erythro-beta-hydroxy-Asp (beta- hydroxylation) , the addition of four Asn-linked oligosaccharides (glycosylation) and, the removal of the leader sequence (42 amino acid residues) .
- post-translational modifications such as the addition of nine gamma-carboxy-glutamates, the addition of one erythro-beta-hydroxy-Asp (beta- hydroxylation) , the addition of four Asn-linked oligosaccharides (glycosylation) and, the removal of the leader sequence (42 amino acid residues) .
- post-translational modifications such as the addition of nine gamma-carboxy-glutamates, the addition of one erythro-beta-hydroxy-Asp (beta- hydroxylation) , the addition
- Human protein C may be activated by thrombin alone, by a thrombin/thrombomodulin complex, by RW-X, a protease from Russell's Viper venom, by pancreatic trypsin or by other proteolytic enzymes.
- the recombinant protein C derivatives of the present invention are useful for the treatment of vascular occlusive disorders or hypercoagulable states associated with sepsis, disseminated intravascular coagulation, major trauma, major surgery, burns, adult respiratory distress syndrome, transplantations, deep vein thrombosis, heparin- induced thrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagic fever, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome.
- the recombinant protein C derivatives of the present invention are useful for the treatment of sepsis in combination with bacterial permeability increasing protein.
- the activated protein C derivatives of the present invention are combined with an antiplatelet agent (s) to treat or prevent various disorders, such as, thrombotic disease .
- the present invention further provides for the treatment of acute coronary syndromes comprising myocardial infarction, and unstable angina with human protein C derivatives with resistance to serpin inactivation as compared to wild-type aPC.
- the recombinant human protein C derivatives of the present invention are also useful for the treatment of thrombotic disorders such as stroke, abrupt closure following angioplasty or stent placement, and thrombosis as a result of peripheral vascular surgery.
- the protein C derivatives can be formulated according to known methods to prepare a pharmaceutical composition comprising as the active agent an aPC polypeptide and a pharmaceutically acceptable solid or carrier.
- a desired formulation would be one that is a stable lyophilized product of high purity comprising a bulking agent such as sucrose, a salt such as sodium chloride, a buffer such as sodium citrate and an activated protein C derivative.
- a preferred stable lyophilized formulation comprises: 2.5 mg/ml activated protein C polypeptide, 15 mg/ml sucrose, 20 mg/ml NaCl and a citrate buffer, said formulation having a pH of 6.0.
- An additional stable lyophilized formulation comprises: 5.0 mg/ml activated protein C polypeptide, 30 mg/ml sucrose, 38 mg/ml NaCl and a citrate buffer, said formulation having a pH of 6.0.
- the human aPC polypeptides will be administered parenterally to ensure delivery into the bloodstream in an effective form by injecting the appropriate dose as a continuous infusion for 1 to 240 hours. More preferably, the human aPC polypeptides will be administered as a continuous infusion for 1 to 192 hours. Even more preferably, the human aPC polypeptides will be administered as a continuous infusion for 1 to 144 hours. Yet even more preferably, the aPC polypeptides will be administered as a continuous infusion for 1 to 96 hours.
- the amount of human aPC polypeptide administered will be from about 0.01 ⁇ g/kg/hr to about 50 ⁇ g/kg/hr. More preferably, the amount of human aPC polypeptide administered will be about 0.1 ⁇ g/kg/hr to about 25 ⁇ g/kg/hr. Even more preferably the amount of human aPC polypeptide administered will be about 1 ⁇ g/kg/hr to about 15 ⁇ g/kg/hr. The most preferable amounts of human aPC polypeptide administered will be about 5 ⁇ g/kg/hr or about 10 ⁇ g/kg/hr.
- the human aPC polypeptide will be administered by injecting a portion (1/3 to 1/2) of the appropriate dose per hour as a bolus injection over a time from about 5 minutes to about 120 minutes, followed by continuous infusion of the appropriate dose for up to 240 hours .
- the human aPC derivatives will be administered by injecting a dose of 0.01 mg/kg/day to about 1.0 mg/kg/day, B.I.D. (2 times a day), for one to ten days. More preferably, the human aPC derivatives will be administered B.I.D. for three days.
- the human aPC polypeptides will be administered subcutaneously to ensure a slower release into the bloodstream.
- Formulation for subcutaneous preparations will be done using known methods to prepare such pharmaceutical compositions.
- An additional aspect of the invention comprises treating the diseases and conditions caused or resulting from protein C deficiency as defined herein, by inhibiting binding to inhibitor recognition sequences S2 , S3', and S4 ' of the serpins, PCI and ⁇ _-AT, as described in Example 1.
- This final aspect of the invention contemplates any and all modifications to any aPC molecule resulting in inhibition of the binding to said inhibitor recognition sequences of the serpins PCI and ⁇ -AT.
- the human aPC polypeptides described in this invention have essentially the same type of biological activity as the wild-type human aPC, with substantially longer half-lives in human blood. Therefore, these compounds will require either less frequent administration and/or smaller dosage.
- Table I depicts the sequences recognized by aPC.
- the cleavage site occurs between the two residues shown in italics. Residues occupying the specific subsites, S2, S3', and S4 ' , are underlined.
- the recognized sites in factor Va are different from the sites in either factor Villa or the inhibitors, therefore, it is possible to engineer the active site of aPC to preferentially cleave the more critical coagulant factor Va, while at the same time decrease aPC's likelihood of being inhibited by serpins.
- S2 the 2 nd residue N-terminal to the cleavage site
- S3' site the S3' site
- S4' the S2 site
- the S2 site is primarily occupied by polar residues in the factor Va sequences; unlike PCI and ⁇ -AT, which have hydrophobic residues at this position.
- the S3' site occupied by polar side chains in all of the substrate sequences, but notably, a hydrophobic side chain in the ⁇ - AT sequence.
- the S4' site is occupied by charged residues in all three factor Va sequences, but is occupied by hydrophobic residues in the factor Villa and inhibitor sequences .
- Protein C Polypeptide Construction and Production Protein C derivatives were constructed using the polymerase chain reaction (PCR) following standard methods.
- the source of the wild-type coding sequence was plasmid pLPC (Bio/Technology 5:1189-1192, 1987).
- the universal PCR primers used include: PCOOlb; 5'-
- GCGATGTCTAGAccaccATGTGGCAGCTCACAAGCCTCCTGC -3' which encodes for an Xbal restriction site (underlined) used for subcloning, a Kozak consensus sequence (lowercase) (Kozak, J Cell Biol 108 (2) .229-41, 1989), and the 5' end of the coding region for protein C: PC002E; 5'-
- CAGGGATGATCACTAAGGTGCCCAGCTCTTCTGG-3 ' which encodes for the 3' end of the coding region for human protein C, and includes a Bell restriction site (underlined) for subcloning.
- Mutagenic PCR primers include: PC194SF, 5'- CTCAAAGAAGAAGTCCGCCTGCGGGGCAGTGC-3 ' and PCI94SR, 5 ' - GCACTGCCCCGCAGGCGGACTTCTTCTTTGAG-3' which encode for a Leu (CTG) to Ser (TCC) mutation at position 194 (boldfaced type) ; PCA195GF , 5 ' -GAAGAAGCTGGGGTGCGGGGCAGTGC-3 ' , and PCA195GR, 5' -GCACTGCCCCGCACCCCAGCTTCTTC-3 ' , which encode for a Ala(GCC) to Gly(GGG) mutation; PCT254SF, 5'- GC
- the first round of PCR was used to amplify two fragments of the protein C gene; the 5' fragment was generated using PCOOlb and the antisense mutagenic primer, and the 3' fragment was generated using PC002e and the sense mutagenic primer.
- the resulting amplified products were purified by standard procedures. These fragments were combined and then used as a template for a second round of PCR using primers PCOOlb and PC002e.
- the final PCR product was digested with Xbal and Bell and subcloned into similarly digested expression vector pIG3.
- a wild-type construct was similarly generated by PCR using the two universal primers and the plasmid pLPC as the template, followed by subcloning into pIG3.
- the mutations were confirmed by DNA sequencing of both the coding and non-coding strands.
- the completed expression plasmids were designated pIG3-HPC (wild-type protein C) , pGH41 (T254S) , pGH51 (A195G) , and pGH94 (L194S) .
- the pIG3 vector was generated by the insertion of an "internal ribosome entry site" (IRES) (Jackson, et al . , Trends Biochem Sci 15 (12) : 447-83 , 1990) and green fluorescent protein (GFP) (Cormack, et al . , Gene 173:33-38, 1996) gene into the mammalian expression vector pGTD (Gerlitz,et al . , Biochem J 295(Pt l):131-40, 1993).
- IRS internal ribosome entry site
- GFP green fluorescent protein
- the GBMT promoter (Berg, Nucleic Acids Res 20(20) : 5485-6, 1992) drives expression of a bicistronic mRNA (5'- cDNA - IRES - GFP - 3'). Efficient translation of the first cistron is initiated by classical assembly of ribosome subunits on the 5' -methylated cap structure of the mRNA; while the normally inefficient translation of a second cistron is overcome by the IRES sequence which allows for internal ribosome assembly on the mRNA.
- the coupling of the cDNA and reporter on a single mRNA, translated as separate proteins, allows one to screen for the highest-producing clones on the basis of fluorescence intensity.
- the expression vector also contains an ampicillin resistance cassette for maintenance of the plasmid in E. coli , and a murine DHFR gene with appropriate expression sequences for selection and amplification purposes in mammalian tissue expression.
- the adenovirus-transformed Syrian hamster AV12-664 cell line was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 ⁇ g/mL gentamicin, 200 ⁇ g /mL Geneticin (G418) , and 10 ⁇ g /mL vitamin Kl .
- Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 ⁇ g/mL gentamicin, 200 ⁇ g /mL Geneticin (G418) , and 10 ⁇ g /mL vitamin Kl .
- G418 ⁇ g /mL
- Fspl- linearized plasmids were transfected using either the calcium phosphate method (ProFection, Gibco BRL-Life Technologies) or FuGene-6 (Boehringer Mannheim) , following the manufacturer's instructions.
- Example 3 Activation of Recombinant Protein C Complete activation of the zymogen forms of protein C and polypeptides was accomplished by incubation with thrombin-sepharose . Thrombin-sepharose was washed extensively with Buffer A. 200 ⁇ L of packed thrombin- sepharose was mixed with 250 ⁇ g of protein C in 1 mL of the same buffer and incubated at 37 °C for 4 hours with gentle shaking on a rotating platform.
- the degree of protein C activation was monitored by briefly pelleting the thrombin-sepharose, and assaying a small aliquot of the supernatant for aPC activity using the chromogenic substrate S-2366 (DiaPharma) . Following complete activation, the thrombin-sepharose was pelleted, and the supernatant collected. aPC concentration was verified by Pierce BCA assay, and the aPC was either assayed directly, or frozen in aliquots at -80°C.
- Assays were performed at 25 °C, in Buffer A containing 1 mg mL "1 BSA, 3 mM CaCl 2 , and 0.5 nM aPC . Reactions (200 ⁇ L/well) were performed in a 96 -well microtiter plate, and amidolytic activity was measured as the change in absorbance units/min at 405 nm as monitored in a ThermoMax kinetic micrometer plate reader. Kinetic constants were derived by fitting velocity data at varying substrate concentrations (16 ⁇ M to 2 mM) to the Michaelis-Menten equation.
- Example 5 Inactivation of aPC Polypeptides
- the rates of inactivation of aPC polypeptides were determined by incubating normal human plasma (Helena Labs) with 20 nM aPC (or either polypeptide) at 37°C ( Figure 1) .
- Plasma concentration was 90% (v/v) in the final reaction buffer containing 150 mM NaCl, 20 mM Tris, pH 7.4, and 1 mg mL "1 BSA. Aliquots were removed at selected times, and activity was measured as amidolytic activity using S-2366 at a final concentration of ImM. The measured half-lives are summarized in Table IV.
- heparin (10 U mL " 1 ) , which is known to cause about 100- fold stimulation in the inactivation of aPC by PCI (Heeb, et al . , J Biol Chem 263(24) .11613-11616, 1988; Espana, et al . , Thromb Res 55 (3) :369-84, 1989; Aznar, et al . , Thromb Haemost 76 (6) :983-988, 1996), was added to a similar reaction ( Figure 3) .
- ⁇ -antitrypsin ⁇ -AT
- ⁇ -AT ⁇ -antitrypsin
- reaction buffer consisting of 3 mM CaCl 2 , 150 mM NaCl, 20 mM Tris, pH 7.4, and 1 mg mL "1 BSA. Aliquots were removed at selected times, and activity was measured as amidolytic activity using S-2366 at a final concentration of ImM.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- General Engineering & Computer Science (AREA)
- Communicable Diseases (AREA)
- Diabetes (AREA)
- Oncology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Biotechnology (AREA)
- Pulmonology (AREA)
- Transplantation (AREA)
- Heart & Thoracic Surgery (AREA)
- Virology (AREA)
- Cardiology (AREA)
- Vascular Medicine (AREA)
- Dermatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
Abstract
Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR0006088-7A BR0006088A (pt) | 1999-04-30 | 2000-04-13 | Derivados de proteìna c |
IL14032600A IL140326A0 (en) | 1999-04-30 | 2000-04-13 | Protein c derivatives |
US09/719,911 US6998122B1 (en) | 1999-04-30 | 2000-04-13 | Protein C derivatives |
AU41885/00A AU4188500A (en) | 1999-04-30 | 2000-04-13 | Protein c derivatives |
HU0102444A HUP0102444A3 (en) | 1999-04-30 | 2000-04-13 | Protein c derivatives |
EP00921591A EP1090130A1 (fr) | 1999-04-30 | 2000-04-13 | Derives de proteine c |
JP2000615776A JP2002542832A (ja) | 1999-04-30 | 2000-04-13 | プロテインc誘導体 |
KR1020007015115A KR20010053345A (ko) | 1999-04-30 | 2000-04-13 | 단백질 c 유도체 |
CA002338799A CA2338799A1 (fr) | 1999-04-30 | 2000-04-13 | Derives de proteine c |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13180199P | 1999-04-30 | 1999-04-30 | |
US60/131,801 | 1999-04-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000066754A1 true WO2000066754A1 (fr) | 2000-11-09 |
Family
ID=22451086
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/008722 WO2000066754A1 (fr) | 1999-04-30 | 2000-04-13 | Dérivés de protéine c |
Country Status (11)
Country | Link |
---|---|
EP (1) | EP1090130A1 (fr) |
JP (1) | JP2002542832A (fr) |
KR (1) | KR20010053345A (fr) |
AR (1) | AR029627A1 (fr) |
AU (1) | AU4188500A (fr) |
BR (1) | BR0006088A (fr) |
CA (1) | CA2338799A1 (fr) |
HU (1) | HUP0102444A3 (fr) |
IL (1) | IL140326A0 (fr) |
PE (1) | PE20010066A1 (fr) |
WO (1) | WO2000066754A1 (fr) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6270764B1 (en) * | 1998-11-20 | 2001-08-07 | Eli Lilly And Company | Method of treating viral hemorrhagic fever with activated protein C |
WO2001057193A3 (fr) * | 2000-02-02 | 2002-02-07 | Lilly Co Eli | Derives de proteine c |
WO2002028416A1 (fr) * | 2000-09-30 | 2002-04-11 | Mochida Pharmaceutical Co., Ltd. | Traitements preventifs/remedes associes a l'anemie hemolytique |
WO2002032461A3 (fr) * | 2000-10-18 | 2002-09-26 | Maxygen Aps | Molecules de proteine c ou de type proteine c activee |
US6630138B2 (en) | 2000-02-11 | 2003-10-07 | Eli Lilly And Company | Protein C derivatives |
US6933367B2 (en) | 2000-10-18 | 2005-08-23 | Maxygen Aps | Protein C or activated protein C-like molecules |
US6998122B1 (en) | 1999-04-30 | 2006-02-14 | Eli Lilly And Company | Protein C derivatives |
WO2006044294A3 (fr) * | 2004-10-14 | 2006-09-28 | Lilly Co Eli | Analogues de la proteine humaine c |
WO2023171719A1 (fr) * | 2022-03-08 | 2023-09-14 | 学校法人自治医科大学 | Séquence de protéine c activée |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20250000042A (ko) | 2023-06-23 | 2025-01-02 | 충남대학교산학협력단 | 절단 반응성 펩타이드를 발현하는 키메라 t 세포 및 이를 포함하는 감염 질환 치료용 조성물 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991009960A1 (fr) * | 1989-12-29 | 1991-07-11 | Zymogenetics, Inc. | Proteine c hybride |
US5358932A (en) * | 1989-12-29 | 1994-10-25 | Zymogenetics, Inc. | Hybrid protein C |
WO1998020118A1 (fr) * | 1996-11-08 | 1998-05-14 | Oklahoma Medical Research Foundation | Proteine c modifiee et procedes d'utilisation correspondants |
-
2000
- 2000-04-13 IL IL14032600A patent/IL140326A0/xx unknown
- 2000-04-13 HU HU0102444A patent/HUP0102444A3/hu unknown
- 2000-04-13 WO PCT/US2000/008722 patent/WO2000066754A1/fr not_active Application Discontinuation
- 2000-04-13 CA CA002338799A patent/CA2338799A1/fr not_active Abandoned
- 2000-04-13 BR BR0006088-7A patent/BR0006088A/pt not_active Application Discontinuation
- 2000-04-13 AU AU41885/00A patent/AU4188500A/en not_active Abandoned
- 2000-04-13 KR KR1020007015115A patent/KR20010053345A/ko not_active Withdrawn
- 2000-04-13 EP EP00921591A patent/EP1090130A1/fr not_active Withdrawn
- 2000-04-13 JP JP2000615776A patent/JP2002542832A/ja not_active Withdrawn
- 2000-04-26 AR ARP000101970A patent/AR029627A1/es unknown
- 2000-04-27 PE PE2000000398A patent/PE20010066A1/es not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1991009960A1 (fr) * | 1989-12-29 | 1991-07-11 | Zymogenetics, Inc. | Proteine c hybride |
US5358932A (en) * | 1989-12-29 | 1994-10-25 | Zymogenetics, Inc. | Hybrid protein C |
WO1998020118A1 (fr) * | 1996-11-08 | 1998-05-14 | Oklahoma Medical Research Foundation | Proteine c modifiee et procedes d'utilisation correspondants |
Non-Patent Citations (2)
Title |
---|
MATHER TIMOTHY ET AL: "The 2.8 A crystal structure of Gla-domainless activated protein C.", EMBO (EUROPEAN MOLECULAR BIOLOGY ORGANIZATION) JOURNAL, vol. 15, no. 24, 1996, pages 6822 - 6831, XP002145653, ISSN: 0261-4189 * |
REZAIE ALIREZA R: "Role of residue 99 at the S2 subsite of factor Xa and activated protein C in enzyme specificity.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 39, 1996, pages 23807 - 23814, XP002145652, ISSN: 0021-9258 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6270764B1 (en) * | 1998-11-20 | 2001-08-07 | Eli Lilly And Company | Method of treating viral hemorrhagic fever with activated protein C |
US6998122B1 (en) | 1999-04-30 | 2006-02-14 | Eli Lilly And Company | Protein C derivatives |
WO2001057193A3 (fr) * | 2000-02-02 | 2002-02-07 | Lilly Co Eli | Derives de proteine c |
US6841371B2 (en) | 2000-02-02 | 2005-01-11 | Eli Lilly And Company | Protein C derivatives |
US6630138B2 (en) | 2000-02-11 | 2003-10-07 | Eli Lilly And Company | Protein C derivatives |
WO2002028416A1 (fr) * | 2000-09-30 | 2002-04-11 | Mochida Pharmaceutical Co., Ltd. | Traitements preventifs/remedes associes a l'anemie hemolytique |
WO2002032461A3 (fr) * | 2000-10-18 | 2002-09-26 | Maxygen Aps | Molecules de proteine c ou de type proteine c activee |
US6933367B2 (en) | 2000-10-18 | 2005-08-23 | Maxygen Aps | Protein C or activated protein C-like molecules |
US7226999B2 (en) | 2000-10-18 | 2007-06-05 | Maxygen Aps | Protein C or activated protein C-like molecules |
WO2006044294A3 (fr) * | 2004-10-14 | 2006-09-28 | Lilly Co Eli | Analogues de la proteine humaine c |
WO2023171719A1 (fr) * | 2022-03-08 | 2023-09-14 | 学校法人自治医科大学 | Séquence de protéine c activée |
Also Published As
Publication number | Publication date |
---|---|
EP1090130A1 (fr) | 2001-04-11 |
AR029627A1 (es) | 2003-07-10 |
BR0006088A (pt) | 2001-03-20 |
CA2338799A1 (fr) | 2000-11-09 |
KR20010053345A (ko) | 2001-06-25 |
IL140326A0 (en) | 2002-02-10 |
HUP0102444A2 (hu) | 2001-10-28 |
PE20010066A1 (es) | 2001-02-02 |
JP2002542832A (ja) | 2002-12-17 |
HUP0102444A3 (en) | 2003-09-29 |
AU4188500A (en) | 2000-11-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6630138B2 (en) | Protein C derivatives | |
US6693075B1 (en) | Modified vitamin K-dependent polypeptides | |
US9249404B2 (en) | Coagulation factor X polypeptides with modified activation properties | |
EP1651252B1 (fr) | Variants de proteine c activee avec activite cytoprotectrice normale mais avec activite anticoagulante reduite | |
EP2086568B1 (fr) | Variantes de protéine c activée avec une activité cytoprotectrice normale mais une activité anticoagulante réduite | |
US20030100506A1 (en) | Modified Vitamin K-dependent polypeptides | |
US20060204489A1 (en) | Protein C derivatives | |
CA2071630C (fr) | Proteine c hybride | |
EP1238065B1 (fr) | Analogue de facteur x a activation amelioree | |
US6841371B2 (en) | Protein C derivatives | |
WO2000066754A1 (fr) | Dérivés de protéine c | |
US6998122B1 (en) | Protein C derivatives | |
JP4680329B2 (ja) | 血管障害の治療方法 | |
MXPA00012789A (en) | Protein c derivatives | |
WO2006044294A2 (fr) | Analogues de la proteine humaine c | |
WO2004044190A2 (fr) | Polypeptides de proteine c de type zymogene |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
WWE | Wipo information: entry into national phase |
Ref document number: 140326 Country of ref document: IL Ref document number: 09719911 Country of ref document: US |
|
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/2000/012789 Country of ref document: MX |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000921591 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1020007015115 Country of ref document: KR |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2338799 Country of ref document: CA Kind code of ref document: A Ref document number: 2338799 |
|
WWP | Wipo information: published in national office |
Ref document number: 2000921591 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1020007015115 Country of ref document: KR |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1020007015115 Country of ref document: KR |