EP0815211A1 - Neue, definierte enzymmischungen zur zellgewinnung und für die wundbehandlung - Google Patents
Neue, definierte enzymmischungen zur zellgewinnung und für die wundbehandlungInfo
- Publication number
- EP0815211A1 EP0815211A1 EP96907436A EP96907436A EP0815211A1 EP 0815211 A1 EP0815211 A1 EP 0815211A1 EP 96907436 A EP96907436 A EP 96907436A EP 96907436 A EP96907436 A EP 96907436A EP 0815211 A1 EP0815211 A1 EP 0815211A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- collagenase
- cells
- gly
- pro
- enzymes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 96
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 90
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 90
- 206010052428 Wound Diseases 0.000 title claims abstract description 13
- 208000027418 Wounds and injury Diseases 0.000 title claims abstract description 13
- 241001465754 Metazoa Species 0.000 claims abstract description 14
- 239000012634 fragment Substances 0.000 claims abstract description 12
- 241000193159 Hathewaya histolytica Species 0.000 claims abstract description 8
- 102000029816 Collagenase Human genes 0.000 claims description 106
- 108060005980 Collagenase Proteins 0.000 claims description 106
- 229960002424 collagenase Drugs 0.000 claims description 97
- 229940088598 enzyme Drugs 0.000 claims description 86
- 238000002955 isolation Methods 0.000 claims description 53
- 102000016387 Pancreatic elastase Human genes 0.000 claims description 34
- 108010067372 Pancreatic elastase Proteins 0.000 claims description 34
- 230000000694 effects Effects 0.000 claims description 33
- 238000012360 testing method Methods 0.000 claims description 20
- 239000000758 substrate Substances 0.000 claims description 16
- 108090001092 clostripain Proteins 0.000 claims description 12
- 108010035532 Collagen Proteins 0.000 claims description 11
- 102000008186 Collagen Human genes 0.000 claims description 11
- 102000016942 Elastin Human genes 0.000 claims description 11
- 108010014258 Elastin Proteins 0.000 claims description 11
- 229920001436 collagen Polymers 0.000 claims description 11
- 229920002549 elastin Polymers 0.000 claims description 11
- 238000001962 electrophoresis Methods 0.000 claims description 9
- 241000283690 Bos taurus Species 0.000 claims description 8
- 238000001502 gel electrophoresis Methods 0.000 claims description 7
- YMMPZCZOLKBHAP-IHPCNDPISA-N (2s)-2-[[(2s)-1-[2-[[2-[[(2s)-1-[2-(phenylmethoxycarbonylamino)acetyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]acetyl]pyrrolidine-2-carbonyl]amino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)CNC(=O)CNC(=O)[C@H]1N(C(=O)CNC(=O)OCC=2C=CC=CC=2)CCC1 YMMPZCZOLKBHAP-IHPCNDPISA-N 0.000 claims description 6
- 108010010803 Gelatin Proteins 0.000 claims description 4
- 239000008273 gelatin Substances 0.000 claims description 4
- 229920000159 gelatin Polymers 0.000 claims description 4
- 235000019322 gelatine Nutrition 0.000 claims description 4
- 235000011852 gelatine desserts Nutrition 0.000 claims description 4
- 210000002435 tendon Anatomy 0.000 claims description 4
- 108010027805 Azocoll Proteins 0.000 claims description 3
- ZLFQNOJSYZSINX-PVJKAEHXSA-N (2s)-2-[[(2s)-1-[2-[[(2s)-2-[[(e)-3-(furan-2-yl)prop-2-enoyl]amino]-4-methylpentanoyl]amino]acetyl]pyrrolidine-2-carbonyl]amino]propanoic acid Chemical compound N([C@@H](CC(C)C)C(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(O)=O)C(=O)\C=C\C1=CC=CO1 ZLFQNOJSYZSINX-PVJKAEHXSA-N 0.000 claims description 2
- 108010089930 2-furanacryloyl-leucyl-glycyl-prolyl-alanine Proteins 0.000 claims description 2
- 101710097834 Thiol protease Proteins 0.000 claims description 2
- 230000007691 collagen metabolic process Effects 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 description 77
- 210000003494 hepatocyte Anatomy 0.000 description 38
- 210000001519 tissue Anatomy 0.000 description 34
- 239000000872 buffer Substances 0.000 description 27
- 238000002360 preparation method Methods 0.000 description 25
- 210000004185 liver Anatomy 0.000 description 22
- 238000000034 method Methods 0.000 description 17
- 210000000496 pancreas Anatomy 0.000 description 13
- 241000700159 Rattus Species 0.000 description 12
- 210000004153 islets of langerhan Anatomy 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 210000002919 epithelial cell Anatomy 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 210000002889 endothelial cell Anatomy 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 102000035195 Peptidases Human genes 0.000 description 7
- 108091005804 Peptidases Proteins 0.000 description 7
- 239000006285 cell suspension Substances 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 210000004881 tumor cell Anatomy 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 6
- 239000001639 calcium acetate Substances 0.000 description 6
- 235000011092 calcium acetate Nutrition 0.000 description 6
- 229960005147 calcium acetate Drugs 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000004365 Protease Substances 0.000 description 5
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 5
- 229940079919 digestives enzyme preparation Drugs 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 4
- 235000011130 ammonium sulphate Nutrition 0.000 description 4
- 235000021028 berry Nutrition 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- 230000007717 exclusion Effects 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000001155 isoelectric focusing Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 230000010412 perfusion Effects 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 210000003954 umbilical cord Anatomy 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108090000145 Bacillolysin Proteins 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 102000035092 Neutral proteases Human genes 0.000 description 3
- 108091005507 Neutral proteases Proteins 0.000 description 3
- 206010039509 Scab Diseases 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 210000001789 adipocyte Anatomy 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 230000008033 biological extinction Effects 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 230000003915 cell function Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 239000013522 chelant Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 3
- 229960002591 hydroxyproline Drugs 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 210000005228 liver tissue Anatomy 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 230000001338 necrotic effect Effects 0.000 description 3
- 210000004923 pancreatic tissue Anatomy 0.000 description 3
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 230000006920 protein precipitation Effects 0.000 description 3
- 238000011403 purification operation Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- UBAZGMLMVVQSCD-UHFFFAOYSA-N carbon dioxide;molecular oxygen Chemical compound O=O.O=C=O UBAZGMLMVVQSCD-UHFFFAOYSA-N 0.000 description 2
- 238000013375 chromatographic separation Methods 0.000 description 2
- 230000003366 colagenolytic effect Effects 0.000 description 2
- 239000002131 composite material Substances 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 210000003890 endocrine cell Anatomy 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- WRMRXPASUROZGT-UHFFFAOYSA-N monoethylglycinexylidide Chemical compound CCNCC(=O)NC1=C(C)C=CC=C1C WRMRXPASUROZGT-UHFFFAOYSA-N 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 2
- 210000003606 umbilical vein Anatomy 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 108010047303 von Willebrand Factor Proteins 0.000 description 2
- 102100036537 von Willebrand factor Human genes 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000050051 Chelone glabra Species 0.000 description 1
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000002045 Endothelin Human genes 0.000 description 1
- 108050009340 Endothelin Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 208000002260 Keloid Diseases 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 1
- 229930064664 L-arginine Natural products 0.000 description 1
- 235000014852 L-arginine Nutrition 0.000 description 1
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 239000008156 Ringer's lactate solution Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000016571 aggressive behavior Effects 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000000013 bile duct Anatomy 0.000 description 1
- 210000003445 biliary tract Anatomy 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- ZEWYCNBZMPELPF-UHFFFAOYSA-J calcium;potassium;sodium;2-hydroxypropanoic acid;sodium;tetrachloride Chemical compound [Na].[Na+].[Cl-].[Cl-].[Cl-].[Cl-].[K+].[Ca+2].CC(O)C(O)=O ZEWYCNBZMPELPF-UHFFFAOYSA-J 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000006041 cell recruitment Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 1
- 230000003246 elastolytic effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- ZUBDGKVDJUIMQQ-UBFCDGJISA-N endothelin-1 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)NC(=O)[C@H]1NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@@H](CC=2C=CC(O)=CC=2)NC(=O)[C@H](C(C)C)NC(=O)[C@H]2CSSC[C@@H](C(N[C@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N2)=O)NC(=O)[C@@H](CO)NC(=O)[C@H](N)CSSC1)C1=CNC=N1 ZUBDGKVDJUIMQQ-UBFCDGJISA-N 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- YQDHCCVUYCIGSW-LBPRGKRZSA-N ethyl (2s)-2-benzamido-5-(diaminomethylideneamino)pentanoate Chemical compound NC(=N)NCCC[C@@H](C(=O)OCC)NC(=O)C1=CC=CC=C1 YQDHCCVUYCIGSW-LBPRGKRZSA-N 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 210000002907 exocrine cell Anatomy 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 210000003547 hepatic macrophage Anatomy 0.000 description 1
- 230000002706 hydrostatic effect Effects 0.000 description 1
- 210000003405 ileum Anatomy 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 210000001117 keloid Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 229960004194 lidocaine Drugs 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229940105631 nembutal Drugs 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 231100000065 noncytotoxic Toxicity 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000277 pancreatic duct Anatomy 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 102000014452 scavenger receptors Human genes 0.000 description 1
- 108010078070 scavenger receptors Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000001631 vena cava inferior Anatomy 0.000 description 1
- 229960001134 von willebrand factor Drugs 0.000 description 1
- 230000029663 wound healing Effects 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)
- C12N9/6448—Elastases, e.g. pancreatic elastase (3.4.21.36); leukocyte elastase (3.4.31.37)
-
- 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
-
- 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
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the use of defined mixtures of purified enzymes from Clostridium histolyticum for the reproducible, standardized extraction of cells or tissue fragments from human or animal tissue and the use of these purified enzymes for wound treatment.
- the normally used and recommended collagenase-containing preparations (2) are obtained from culture filtrates of the bacterial strain Clostridium histolyticum and, in addition to various collagenases and proteases (3a), (3b), also contain cleavage products of these enzymes formed by proteolysis, as well as others, some of which are harmful acting and unknown components.
- Collagenase fraction and purified trypsin from bovine pancreas isolated.
- the collagenase fraction used was enriched However, 2 was not defined in terms of its content of various enzymes and also contained, for example, small amounts of clostripain.
- the success of the isolation was not significantly different from that which was obtained when using undefined compositions containing collagenase. Satisfactory tissue disintegration could not be achieved with individual components of the mixture.
- trypsin for cell isolation is not without problems, since this proteolytic enzyme attacks membrane proteins. For example, insulin binding to liver membranes and adipocytes is negatively influenced (5).
- Hefley (6), (7) used mixtures of purified collagenase fractions to isolate bone cells from the skull cap of mice (6). Earlier (7) the author described that a mixture of a purified collagenase fraction can be used together with a neutral protease to successfully isolate these cells. In both studies, however, eluates from column fractionations were used, whose content of various collagenases, which differ in their substrate specificity, and of other components was not known.
- the invention relates to the individual enzymes collagenase HP, collagenase AZ and elastase, in high purity and mixtures thereof.
- Collagenase HP has a specific activity of at least 20 U / mg, preferably at least 50 U / mg in the Grclumann and Nordwig (11) test with the synthetic hexapeptide Z-Gly-Pro-Gly-Gly-Pro-Ala as substrate. For pharmaceutical purposes, it preferably has a specific activity of 100 U / mg or more.
- Collagenase AZ has a specific activity of at least 10 U / mg, preferably at least 30 U / mg in the test according to Mandl et al. (12) using azocoll as substrate. For phar a- for therapeutic purposes, it preferably has a specific activity of 50 U / mg or more.
- Elastase has a specific activity of at least 2 U / mg, preferably at least 5 U / mg in the test with elastin from the neck band of the cattle as a substrate. For pharmaceutical purposes, it preferably has a specific activity of 12 U / mg or more.
- the invention further relates to the use of a mixture of collagenase HP and elastase, optionally with the addition of collagenase AZ and / or clostripain, for the isolation of cells or tissue fragments from human or animal tissue.
- the invention further relates to the direct or indirect use of these enzymes, alone or as a component of mixtures, for medical applications, e.g. in wound treatment.
- the mixtures can, for example, be packaged in vials in lyophilized form in such a way that they are sufficient for disintegration from an individual rat liver (approx. 9-11 g wet weight of the organ).
- Suitable mixtures are those which consist of at least two of the purified enzymes collagenase HP (50-300 U; preferably 70-170 U) and elastase (5-70 U, preferably 10-25 U) and optionally an addition of collagenase AZ (1-20, preferably 2-8 U) and / or clostripain (10-280 U, preferably 20-50 U) contain.
- the numbers given indicate the amounts in one unit of the mixture - for example in a vial - available.
- the purity criterion for the enzymes used is their respective specific activity and their uniformity detection in the electrophoretic methods commonly used for this purpose (SDS gel electrophoresis, isoelectric focusing and electrophoresis on agarose gel).
- the specific activity of the purified enzymes is up to 100 times higher than that of the starting material.
- a major advantage is that a time-consuming and costly inspection of batches is no longer necessary, since the production of purified enzymes is based on known properties. This eliminates the workload that is necessary for the functional characterization of the cells, or at least it is reduced. For these reasons, the number of animal experiments can be reduced in many areas.
- tissue fragments and / or cells are suitable for the use of the mixtures of purified enzymes according to the invention:
- the biliary tract the blood system, glands, the vascular system, the brain, the skin, the heart, the intes inu, Langerhans' islets, the liver, the lungs, the stomach, the spleen, muscles, the Umbilical cord, nerves, the kidney, the pancreas, the spinal cord, the thyroid, the terminal ileum, tumor tissue, the uterus, the digestive tract and the tongue.
- the cells or tissue fragments isolated with the aid of the mixtures described are particularly suitable for use in cell and tissue transplantation, as well as in gene therapy (e.g. islets of Langerhans, islet cells, hepatocytes, tumor cells, adipocytes), in immunotherapy or in Wound treatment.
- gene therapy e.g. islets of Langerhans, islet cells, hepatocytes, tumor cells, adipocytes
- immunotherapy e.g. islets of Langerhans, islet cells, hepatocytes, tumor cells, adipocytes
- Wound treatment e.g. islets of Langerhans, islet cells, hepatocytes, tumor cells, adipocytes
- hybrids according to the invention are particularly advantageous.
- a well-defined mixture of 2 collagenases with different substrate specificity and one elastase has proven to be ideally suited for the isolation of hepatocytes from rat liver (application examples A, D, E) and human liver (application example F) from bile duct epithelial cells
- Rat livers application example G
- endothelial cells from human umbilical cords
- application examples I and islet cells from porcine pancreas
- Another main area of application is wound treatment.
- the enzymes are used in the highest possible purity.
- collagenase HP and collagenase AZ were collected in separate fractions.
- the two separate enzyme fractions of collagenase HP and collagenase AZ were concentrated to 50-100 ml with the aid of an ultrafiltration membrane (cutoff limit 10,000 Da), then dialyzed against water and again through an ultrafiltration membrane (cutoff limit 10,000 Da) to approx. 10 liters concentrated.
- the collagenase HP was eluted from the column with buffer C (20 mM TRIS / HCl + 200 mM NaCl, pH 7.5) in purified form.
- Collagenase HP got its name * HP "because of its property to break down the hexapeptide Z-Gly-Pro-Gly-Gly-Pro-Ala particularly well. It is therefore suitable for a specific detection of the activity.
- Collagenase HP is characterized in that it can only convert denatured collagens such as gelatin or azocoll to a small extent, but it attacks bovine tendon collagen and cleaves the synthetic peptides mentioned in Example 2b between glycine and glycine or between any amino acid and glycine with high conversion.
- collagenase HP together with collagenase AZ (vice-versa, i.e. also any mixture of at least collagenase HP and AZ) has an over-additive effect on the degradation of native collagen. This synergistic effect in vitro is also important when used for tissue integration (e.g. application example G).
- the specific activity of collagenase HP is a maximum of 146 U / mg in the test with the synthetic substrate Z-Gly-Pro-Gly-Gly-Pro-Ala (11). This corresponds to an approximately 100-fold increase in their specific activity compared to the starting material.
- the collagenase HP purified in this way forms only a single band both in SDS gel electrophoresis and in isoelectric focusing and electrophoresis on agarose gel.
- the fine purification of collagenase AZ was carried out on the anion exchanger Mono Q (HR 10/10, Pharmacia) and with the aid of the FPLC device from Pharmacia.
- a mixture of 1 ml of buffer D and 1 ml of the fraction containing collagenase AZ (from the metal chelate described above) was added to the column, which had been equilibrated with buffer D (20 mM calcium acetate, pH 7.2). Affinity chromatography).
- collagenase AZ was eluted from the column with buffer E (20 mM calcium acetate, pH 5.0).
- the collagenase AZ received its designation * AZ "due to its property of degrading the substrate azocolla particularly well. It is characterized in that it converts denatured collagens such as gelatin or azocolla, but also bovine tendon collagen well. However, it is capable of small synthetic peptides such as 2 -Furanacryloyl-Leu-Gly-Pro-Ala, 4-phenyl-azobenzyloxy-carbonyl-Pro Leu-Gly-Pro-Arg and Z-Gly-Pro-Gly-Gly-Pro-Ala.
- collagenase AZ The specific activity of collagenase AZ is a maximum of 82 U / mg in that of Mandl et al. (12) developed test using Azokoll as substrate. This corresponds to an approximately 80-fold increase in their specific activity compared to the starting material.
- the collagenase AZ purified in this way forms only a single band both in SDS gel electrophoresis and in isoelectric focusing and electrophoresis on agarose gel. Its molecular weight determined by SDS gel electrophoresis is 111,000 Da. Their isoelectric point is at pH 5.9-6.1.
- the first cleaning step of elastase was carried out by protein precipitation with ammonium sulfate, as described under l.a. described.
- the protein precipitate AI is dissolved in 1 L of water, concentrated to about 170 mL via an ultrafiltration membrane (cutoff limit 10,000 Da) and dialyzed against a 0.1 mM calcium acetate solution. The enzyme solution was then again concentrated over an ultrafiltration membrane (cut-off limit 10,000 Da) to approximately 50 mL and then lyophilized.
- the fine purification was carried out by gel chromatography on SEPHADEX G100 or G200 (Pharmacia).
- the elastase is characterized in that it can break down elastin with high conversion. Their specific activity (see 3.c.) was 18 U / mg. This corresponds to an approximately 80-fold increase in their specific activity compared to the starting material.
- the elastase purified in this way forms only a single band in SDS gel electrophoresis as well as in isoelectric focusing and electrophoresis on agarose gel.
- the molecular weight of the elastase determined by SDS electrophoresis is 35,000 Da. c. Activity determination
- the factor F is determined by completely dissolving 10 mg elastin of a certain batch with the aid of elastase and measuring the corresponding extinction difference ⁇ E.
- the multiplication of this factor F by the extinction difference ⁇ E gives the dissolved milligram amount of elastin per mL test batch.
- Clostripain was isolated by the method described by Ullmann and Jakubke (14).
- the thiol protease clostripain is characterized in that it cleaves specifically behind the amino acid L-arginine in polypeptide chains and in synthetically produced substrates.
- Hepatocytes were isolated using the standard Berry and Friend method (9) and the modification by Seglen (16).
- Wistar rats 200-280 g were anesthetized by ip injection of nembutal (35 mg pentobarbital / kg body weight).
- the vena portae was cannulated, and the following carbogen-gassed solutions with a pH value of 7.4 ⁇ were subjected to constant hydrostatic pressure (12 cm water column, variable flow rate) 0.05 infused at a temperature of 36-36.8 ° C after opening of the inferior vena cava:
- Carbogen gas mixture of 95% 0 2 and 5% C0 2 (v / v)
- the enzyme mixture was a lyophilizate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 87.5 mL cell buffer.
- the proportion of vital hepatocytes, hepatocyte-cell pairs or multicellular aggregates from hepatocytes was determined microscopically after resuspension of the cell precipitate obtained in a Burker count (staining with 0.08% trypan blue, for 2 minutes). Result
- the enzyme mixture used was a lyophilisate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was in 87.5 ml cell buffer (analogous to example A).
- the success of the isolation of hepatocytes was measured using the following five parameters: 1. microscopic determination of the proportion of vital hepatocytes (in% of the total number of hepatocytes in the cell suspension obtained by means of a trypan blue exclusion test), 2. determination of the total number of isolated hepatocytes, 3. determination measurement of the total number of hepatocytes isolated per gram of animal weight, 4. determination of the proportion of single cells (in% based on all forms of aggression in the cell suspension obtained, ie compared to two and multiple aggregates of hepatocytes), and 5. recording of the success rate Tissue disintegration necessary perfusion time with the collagenase solution.
- Hepatocytes were isolated using the standard Berry and Friend method (9) and the modification by Seglen (16).
- the enzyme mixture used was a lyophilizate of 130 U.
- the quality of the cell suspension obtained was further characterized with regard to the function of the hepatocytes (21, 22) by the following parameters: ATP content, energy charge (EC), lidocaine metabolism to monoethylglycine xylidide (MEGX), and absorption of cholyItaurin ((3 ⁇ , 7 ⁇ , 12 ⁇ -trihydroxy-5ß-cholan-24-oyD-2-aminoethanesulfonic acid) at a substrate concentration of 21 ⁇ -M.
- the hepatocytes isolated with an enzyme mixture according to the invention not only after assessment by the simple isolation success, but also after evaluation of various cell functions, e.g. certain functions of differentiated mass transport, or certain metabolic performances of cytochrome P450-dependent enzymes of the hepatocytes, are completely intact.
- Hepatocytes were isolated by the Berry and Friend method (9) and the modifications by Seglen (16) using a Biopsy perfusion technique (21).
- the enzyme mixture used was a lyophilizate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 50 mL cell buffer.
- the success of the isolation of human hepatocytes was determined according to the following parameters: proportion of vital cells after the trypan blue exclusion test and cell yield per g of liver.
- biliary epithelial cells from the rat liver were isolated according to the method described in reference (18).
- hepatocytes were first removed enzymatically from the tissue, and in a second step biliary epithelial cells were isolated from the remaining tissue with trypsin.
- the enzyme mixture for removing the hepatocytes was a lyophilizate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 87.5 mL cell buffer.
- the use of the enzyme mixture described made it extremely easy to isolate biliary epithelial cells from the remaining vascular system using trypsin, since the residual tissue obtained after the first step was practically free of hepatocytes.
- Human umbilical vein endothelial cells were isolated using the method of Jaffe (17). For this purpose, the umbilical cords (20 - 30 cm long) were halved, filled in pairs with the respective enzyme solution and incubated for 15 min at 37 ° C and 5% CO 2 in the incubator. The detached cells were removed and kept in primary culture for evaluation. The yield of living and Divisible cells were determined after washing the non-adherent cells after four days in culture.
- the enzyme mixture was a lyophilizate of 130 U collagenase HP, 5 U collagenase AZ and 21 U elastase, which was dissolved in 87.5 mL cell isolation buffer.
- the confluent cell lawn consists of over 95% endothelial cells, such as FACS studies with antibodies against factor VIII-related antigen (Von Willebrand factor) or two endothelium-specific surface antigens (EN-4, PAL-E) or with a fluorescent ligand of the scavenger receptor (dil-Ac-LDL).
- the yield (n 2) achieved with the above-mentioned mixture of purified enzymes exceeded that which had been obtained with enzyme preparations containing collagenase and having an undefined composition.
- the cell density after 4 days was 1.8 x IO 4 per cm 2 . If you use previously known methods, you get a cell density that is significantly lower. The confluence of the monolayer was reached earlier (already after 5-6 days) when using the above mixture.
- the prepared cells were functionally checked in the first passage (endothelin production, release of LDH). All values were within the normal range. The functions of the cells were therefore indistinguishable from results from control experiments in which cells with preparations containing collagenase containing an undefined composition were isolated.
- Tumor cells were isolated from human tumors according to protocol (23).
- the enzyme mixture used was a lyophilizate of 130 U
- Collagenase HP 5 U collagenase AZ and 21 U elastase, which was dissolved in 28.4 L cell isolation buffer (Ringer's lactate / PBS).
- the success of the isolation was determined on the basis of the proportion of vital cells after the trypan blue exclusion test and counting of the number of lymphocytes. Directly comparative cell isolations were carried out using the enzyme mixture according to the invention and a preparation of an undefined composition containing collagenase, which is very suitable for this purpose, in 4 different tumor tissues.
- the enzyme mixture according to the invention can be used very well to isolate tumor cells from human tumors.
- the collagenase solution acts on the pancreatic tissue for a long time after infusion through the pancreatic duct under particularly well-standardized conditions.
- the islands which have already been detached are continuously discharged into a reservoir at a lower temperature, and after the disintegration has ended, the islands which have already been partially freed from their matrix in the pancreatic tissue are completely detached and isolated using light mechanical treatment.
- the success of the isolation can be determined on the basis of the size distribution of the intact cell aggregates obtained and other customary parameters.
- mixtures could also be used successfully, for example those composed of collagenase HP (3-48 U / cm 2 ) and elastase (1-12 U / cm 2 ).
- Example K The results obtained on the degrading effect of the necrotic tissue (Example K) were characterized further in an in vitro model.
- the release of 4-hydroxyproline (20) was determined after 2, 4, 6 and 24 hours by the action of collagenase HP, collagenase AZ and / or elastase on the burn scab mentioned in Example K.
- the purified enzymes and enzyme mixtures according to the invention can also be used in therapeutic areas, such as in wound treatment or for the treatment of keloids or fibrils. They can be applied externally or injected.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Materials For Medical Uses (AREA)
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19509584 | 1995-03-16 | ||
DE19509584 | 1995-03-16 | ||
DE19532906A DE19532906A1 (de) | 1995-03-16 | 1995-09-07 | Neue, definierte Enzymmischungen zur Zellgewinnung und für die Wundbehandlung |
DE19532906 | 1995-09-07 | ||
PCT/EP1996/001044 WO1996028543A1 (de) | 1995-03-16 | 1996-03-12 | Neue, definierte enzymmischungen zur zellgewinnung und für die wundbehandlung |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0815211A1 true EP0815211A1 (de) | 1998-01-07 |
Family
ID=26013440
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP96907436A Withdrawn EP0815211A1 (de) | 1995-03-16 | 1996-03-12 | Neue, definierte enzymmischungen zur zellgewinnung und für die wundbehandlung |
Country Status (13)
Country | Link |
---|---|
US (1) | US6146626A (no) |
EP (1) | EP0815211A1 (no) |
JP (1) | JPH11501517A (no) |
AU (1) | AU702514B2 (no) |
BR (1) | BR9607143A (no) |
CA (1) | CA2213723A1 (no) |
CZ (1) | CZ280397A3 (no) |
EA (1) | EA000583B1 (no) |
HU (1) | HUP9901188A2 (no) |
IL (1) | IL117479A (no) |
NO (1) | NO974260L (no) |
PL (1) | PL322271A1 (no) |
WO (1) | WO1996028543A1 (no) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5718897A (en) * | 1995-06-07 | 1998-02-17 | Trustees Of Tufts College | Enhancing keratinocyte migration and proliferation |
US5830741A (en) * | 1996-12-06 | 1998-11-03 | Boehringer Mannheim Corporation | Composition for tissue dissociation containing collagenase I and II from clostridium histolyticum and a neutral protease |
US7364565B2 (en) * | 2001-07-27 | 2008-04-29 | Ramot At Tel Aviv University Ltd. | Controlled enzymatic removal and retrieval of cells |
US7226605B2 (en) * | 2001-07-27 | 2007-06-05 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Botulinum toxin in the treatment or prevention of acne |
US7811560B2 (en) * | 2006-01-30 | 2010-10-12 | Auxilium Us Holdings, Llc | Compositions and methods for treating collagen-mediated diseases |
US8323642B2 (en) * | 2006-12-13 | 2012-12-04 | Depuy Mitek, Inc. | Tissue fusion method using collagenase for repair of soft tissue |
US20100159564A1 (en) * | 2007-11-30 | 2010-06-24 | Dwulet Francis E | Protease resistant recombinant bacterial collagenases |
AU2010328269B2 (en) | 2009-12-08 | 2014-07-10 | Smith & Nephew Orthopaedics Ag | Enzymatic wound debriding compositions with enhanced enzymatic activity |
NZ733709A (en) | 2012-01-12 | 2022-10-28 | Auxilium Int Holdings Inc | Clostridium histolyticum enzymes and methods for the use thereof |
CN110545722B (zh) | 2017-03-01 | 2023-02-03 | 恩多风投有限公司 | 用于评估和治疗橘皮组织的装置和方法 |
JP7227918B2 (ja) | 2017-03-28 | 2023-02-22 | エンド ベンチャーズ リミテッド | コラゲナーゼ産生の改良された方法 |
CN109694845A (zh) * | 2018-12-25 | 2019-04-30 | 中国医学科学院北京协和医院 | 用于分离单细胞的试剂盒及其应用 |
WO2023228873A1 (ja) * | 2022-05-24 | 2023-11-30 | 国立研究開発法人国立成育医療研究センター | 肝細胞の製造方法、肝細胞の接着性改善剤、および肝細胞 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5162205A (en) * | 1986-04-26 | 1992-11-10 | Sankyo Company, Limited | Human pancreatic elastase I |
JPH0673456B2 (ja) * | 1986-04-26 | 1994-09-21 | 三共株式会社 | ヒト・膵臓エラスタ−ゼ▲i▼ |
CA2138948A1 (en) * | 1992-06-22 | 1994-01-06 | Hun-Chi Lin | Molecular cloning of the genes responsible for collagenase production from clostridium histolyticum |
JP3028852B2 (ja) * | 1994-06-24 | 2000-04-04 | ロシュ ダイアグノスティックス コーポレーション | ヒストリチクス菌から得られるコラゲナーゼおよび2つの他のプロテアーゼの精製混合物 |
-
1996
- 1996-03-12 AU AU51066/96A patent/AU702514B2/en not_active Ceased
- 1996-03-12 EP EP96907436A patent/EP0815211A1/de not_active Withdrawn
- 1996-03-12 CZ CZ972803A patent/CZ280397A3/cs unknown
- 1996-03-12 HU HU9901188A patent/HUP9901188A2/hu unknown
- 1996-03-12 US US08/913,396 patent/US6146626A/en not_active Expired - Fee Related
- 1996-03-12 CA CA002213723A patent/CA2213723A1/en not_active Abandoned
- 1996-03-12 EA EA199700241A patent/EA000583B1/ru not_active IP Right Cessation
- 1996-03-12 JP JP8527270A patent/JPH11501517A/ja active Pending
- 1996-03-12 WO PCT/EP1996/001044 patent/WO1996028543A1/de not_active Application Discontinuation
- 1996-03-12 PL PL96322271A patent/PL322271A1/xx unknown
- 1996-03-12 BR BR9607143A patent/BR9607143A/pt not_active Application Discontinuation
- 1996-03-14 IL IL11747996A patent/IL117479A/xx active IP Right Grant
-
1997
- 1997-09-15 NO NO974260A patent/NO974260L/no not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO9628543A1 * |
Also Published As
Publication number | Publication date |
---|---|
NO974260D0 (no) | 1997-09-15 |
JPH11501517A (ja) | 1999-02-09 |
WO1996028543A1 (de) | 1996-09-19 |
AU5106696A (en) | 1996-10-02 |
CZ280397A3 (cs) | 1998-04-15 |
US6146626A (en) | 2000-11-14 |
MX9706725A (es) | 1997-11-29 |
EA000583B1 (ru) | 1999-12-29 |
AU702514B2 (en) | 1999-02-25 |
BR9607143A (pt) | 1997-11-25 |
IL117479A (en) | 1999-12-22 |
NO974260L (no) | 1997-11-14 |
CA2213723A1 (en) | 1996-09-19 |
PL322271A1 (en) | 1998-01-19 |
EA199700241A1 (ru) | 1998-02-26 |
HUP9901188A2 (hu) | 1999-08-30 |
IL117479A0 (en) | 1996-07-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
DE69431714T2 (de) | Verfahren und zusammensetzung zur verdauung von gewebe | |
DE69004329T2 (de) | Verfahren zur Herstellung von antimikrobiellen Partikeln, antimikrobielle Mittel und Anwendungen. | |
DE69735308T2 (de) | Enzymzusammensetzung für die freisetzung von zellen aus geweben. | |
DE69823361T2 (de) | Herstellung von kollagen | |
EP0815211A1 (de) | Neue, definierte enzymmischungen zur zellgewinnung und für die wundbehandlung | |
US20030203008A1 (en) | Preparation of collagen | |
DE3886175T2 (de) | Trigramin, ein Polypeptid, das die Aggregation der Blutplättchen hemmt. | |
DE68918922T3 (de) | Biologische Unterlage für Zellkulturen, bestehend aus durch Thrombin koagulierte Plasmaproteine, ihre Verwendung für die Keratinozytenkultur, deren Rückgewinnung und Transport für therapeutische Verwendungszwecke. | |
DE69333239T2 (de) | Reinigung von plasmafraktion | |
CH635747A5 (de) | Verfahren zur herstellung einer blutgerinnungsfoerdernden praeparation aus menschlichem blutplasma. | |
WO1996034093A1 (en) | Composition containing collagenase and chymopapain for isolating hepatocytes and pancreatic islet cells | |
DE2619667A1 (de) | Pharmazeutisches mittel | |
DE2201993A1 (de) | Enzympraeparat und Verfahren zu dessen Herstellung | |
DE60038312T2 (de) | Faktor x-analog mit einer verbesserten fähigkeit, aktiviert zu werden | |
EP0101063A2 (de) | Neues Polypeptid mit Wirkung auf das Immunsystem, Verfahren zu seiner Isolierung und Reinigung, seine Verwendung und dieses enthaltende Mittel | |
WO2000039290A2 (de) | Pharmazeutische formulierung enthaltend ein hyaluronsäure-spaltendes enzym mikrobieller herkunft | |
DE19532906A1 (de) | Neue, definierte Enzymmischungen zur Zellgewinnung und für die Wundbehandlung | |
DE3306944A1 (de) | Fibrinolytisch aktives mittel und verfahren zu seiner herstellung | |
JPWO2002102845A1 (ja) | 絹フィブロイン由来機能性ポリペプチドの製造法及びその利用 | |
DE69230887T2 (de) | Escharase enthaltene proteolytische mischung und methode seiner isolierung | |
DE69127346T2 (de) | Behandlung thrombotischer ereignisse | |
WO1989006538A1 (en) | Process for producing a therapeutically active ingredient, in particular for cicatrization or for treatment in geriatry, and a therapeutic preparation containing said active ingredient | |
WO1998001108A1 (de) | Definierte enzymmischungen für kosmetische zwecke | |
DE19716098A1 (de) | Fibroblasten mit einem Fremdgen enthaltende Zusammensetzung zur Behandlung von Wunden | |
DE4002166A1 (de) | Verfahren zur herstellung einer suspension zur rekonstruktion von zellmembranen |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19970822 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB IT LI NL PT SE |
|
17Q | First examination report despatched |
Effective date: 20000623 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20020522 |