KR840006438A - Thrombolytic - Google Patents

Abstract

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Description

Thrombolytic

As this is a public information case, the full text was not included.

Figures 1 to 6 show the relative fibrinolytic activity of six proteases, such as HM-27, HM-89, HM-45, HM-54, HM-15, and HM-64, at various pH values. graph.

Claims (13)

White amorphous powder protease F-III-1-HM-27, characterized by the following composition and properties: (A) Substrate-specific and enzymatic activity plasminogen activating casein with fibrinolytic activity against coagulated fibrin, tosyl-L-argininemethyl esmer hydrochloride, N-α-tosyl-L-lysine mester hydrochloride, Strong activity against L- pyroglutamylglyciyl-L-arginine-P-nitroanilide hydrochloride and HD-valyl-L-leucine-L-lysine P-nitroanilide dihydrochloride; No activity on N-benzoyl-L-alaninemethylester and N-benzoyl-L-tyrosine ester. (B) Concern about dissolving action of fiber for coagulated fiber as substrate, PH is about 8, PH and stability range is 5 ~ 12. (C) The optimum operating temperature for the dissolution of cellulose on the coagulated cellulose in PH7.8 is about 50 ° C. The DLRH operating temperature range is 30 ~ 60 ° C. (D) Fully deactivated after heating at 70 ℃ for 60 minutes. (E) Molecular weight: 32,400 ± 2,000 (E) Ultraviolet absorption spectrum, maximum absorption at wavelength of about 280 NM and minimum absorption at wavelength of about 250 nm. (G) Isoelectric point at PH3.6 ± 0.1. (H) Sensitivity to inhibitors, including complete inhibitory action, on cellulose-induced side effects on coagulated cellulose: lima-companpsin inhibitor, difluorophosphate, soybean trypsin inhibitor, anti-pine, leupeptin and trasiluldaso Strong inhibitory substances: Tanbatintrypsin inhibitors and trans-4- (aminoethyl) cyclohexanecarboxylic acid Weak inhibitory substances: ε-amino caproic acid, chymostatin and pepstatin hardly inhibit Substances: disodium ethyldiamine tetraacetate and N-ethylmaleimide. Amino acid composition (mole%): aspartic acid 15.25%, threonine 6.09%, serine 10.08%, golutamic acid 7.04%, proline 0.55%, glycine 12.66%, alanine 6.90%, cysteine 1.36%, valine 10.33%, methionine 0.36 %, Isoleucine 7.89%, leucine 3.73%, tyrosine 4.63%, phenylalanine 3.09%, tryptophan 0.83%, lysine 0.65%, histidine 2.73%, arginine 4.19%, provided that this data includes unavoidable analytical errors. Elementary composition (wt%): carbon 48.61%, hydrogen 6.58%, nitrogen 14.75%, sulfur 2.03%, but this data includes unavoidable analytical errors. White amorphous powder protease F-0-HM-45 characterized by the following composition and properties: (A) Substrate-specific and enzymatic activity plasminogen activating activity with fibrinolytic activity against coagulated fibrin Casein, tosyl-L-arginine meryl ester hydrochloride, strong activity against N-benzoyl-L tyrosineethylester; Extremely weak activity against pyroglutamylglycyl-L-arginine-P-nitroanilide hydrochloride and HD-valyl-L-leucine-L-lysine-P-nitroanilide dihydrochloride; N-α-tosyl-L- Little activity on lysine methyl ester hydrochloride and N-benzyl-L-alanine methyl ester. (B) The appropriate pH range of the dissolution action for cellulose on coagulated cellulose as a substrate is about 8 to about 10 and the pH stability range is 4 to 12. (C) In the dissolution action for cellulose on the coagulated cellulose at PH 7.8, the appropriate temperature range is about 50 ~ 60 ℃ and the action temperature is 30 ~ 60 ℃. (D) Fully deactivated after heating at 70 ℃ for 60 minutes. (E) Molecular weight: 23,500 ± 2,000 (F) Ultraviolet absorption spectrum ‥ Maximum absorption at wavelength of about 280 nm, minimum absorption at wavelength of about 250 nm. (G) Isoelectric point at PH4.1 ± 0.1. (H) Susceptibility to inhibitors, including complete inhibitory action, in fibrinolytic action for coagulated fibrin: limacomtrypsin inhibitor, difluorophosphate, soybean trypsin inhibitor, anti-pine, leupeptin and trasilul And chymosatin a substance which gives a rather strong inhibitory effect: trans-4- (aminoethyl) cyclohexanecarboxymic acid and ε-amino caproic acid; Weak inhibitory substances: lanbaektrimcin inhibitors, femstatin and -N-ethylmaleimide; Almost Inhibitory Substance: Somediumdium ethyldiaminetetraacetate. Amino acid composition (mole%): aspartic acid 12.26%, threonine 12.22%, serine 9.38%, glutamic acid 3.97%, proline 0.40%, glycine 13.48%, alanine 12.95%, cislane 1.44%, valine 7.03%, methionine 0.55 %, Isoleucine 7.89%, leucine 5.83%, tyrosine 3.91%, phenylalanine 2.00%, tryptophan 0.64%, lysine 0.44%, histidine 2.45%, arginine 3.98%, provided that this data includes unavoidable analytical errors. Elementary composition (wt%): carbon 48.30%, hydrogen 6.84%, nitrogen 15.88%, sulfur 2.07%, but this data includes unavoidable analytical errors. White amorphous powder protease F-I-1-HM-54, characterized by the following composition and properties: (A) substrate specificity and enzymatic activity with fibrinolytic activity against coagulated fibrin; Plasminogen activation; Strong activity against casein, tosyl-L-arginine methylester hydrochloride, and N-benzoyl-L tyrosineethylester; Extremely weak activity against N-α-tosyl-L-lysinemethylester hydrochloride and L- pyroglutamylglyciyl-L-arginine-P-nitroanilide hydrochloride; Little activity against H-D-valyl-L-leucine-L-lysine-P-nitroanilide dihydrochloride and N-benzyl-L-alaninemethylester. (B) The appropriate pH range of the dissolution action for cellulose on coagulated cellulose as a substrate is about 8 to about 10 and the pH stability range is 4 to 12. (C) In the dissolution action for cellulose on the coagulated cellulose at PH 7.8, the appropriate temperature range is about 50 ~ 60 ℃ and the action temperature is 30 ~ 60 ℃. (D) Fully deactivated after heating at 70 ℃ for 60 minutes. (E) Molecular weight: 27,500 ± 2,000 (F) Ultraviolet absorption spectrum ‥ Maximum absorption at wavelength of about 280 nm, minimum absorption at wavelength of about 250 nm. (G) Isoelectric point at PH4.1 ± 0.1. (H) Sensitivity to inhibitors, including complete inhibitory action, in fibrin side effects on coagulated fibrin: limacomtrypsin inhibitor, difluorophosphate and N-ethylmaleimide; Somewhat stronger inhibitory substances; Soybean Trypsin Inhibitor and Leupeptin Inhibitor Substance: Lanva Trypsin Inhibitor Trans-4- (Aminoethyl) Cyclohexanecarboxylic Acid and Chemostatin Inhibitor: Antipine, Trasylol ε-Amino Capro Acid pepstatin and somewhatdadium ethyldiaminetetraacetate. Amino acid composition (mole%): aspartic acid 15.65%, threonine 8.25%, serine 11.30%, golutamic acid 6.05%, proline 0.37%, golysine 15.39%, alanine 9.49%, cysteine 1.09%, valine 5.46%, methionine 0.97%, isoleucine 6.54%, leucine 7.95%, tyrosine 3.69%, phenylalanine 1.11%, tryptophan 0.98%, lysine 0.50%, histidine 2.75%, arginine 2.46%, but this data includes unavoidable analytical errors. Elementary composition (wt%): carbon 48.93%, hydrogen 6.65%, nitrogen 15.95%, sulfur 1.34%, but this data includes unavoidable analytical errors. White amorphous powder protease F-I-2-HM-15, characterized by the following composition and properties: (A) substrate specificity and enzymatic activity with fibrinolytic activity against coagulated fibrin; Plasminogen activation; Strong activity against casein, tosyl-L-arginine methylester hydrochloride, and N-benzoyl-L tyrosineethylester; Extremely weak activity against N-α-tosyl-L-lysinemethylester hydrochloride and L- pyroglutamylglyciyl-L-arginine-P-nitroanilide hydrochloride; Little activity against H-D-valyl-L-leucine-L-lysine-P-nitroanilide dihydrochloride and N-benzyl-L-alaninemethylester. (B) The appropriate pH range of the dissolution action for cellulose on coagulated cellulose as a substrate is about 8 to about 10 and the pH stability range is 4 to 12. (C) In the dissolution action for cellulose on the coagulated cellulose at PH 7.8, the appropriate temperature range is about 50 ~ 60 ℃ and the action temperature is 30 ~ 60 ℃. (D) Fully deactivated after heating at 70 ℃ for 60 minutes. (E) Molecular weight: 27,000 ± 2,000 (F) Ultraviolet absorption spectrum ‥ Maximum absorption at wavelength of about 280 nm, minimum absorption at wavelength of about 250 nm. (G) Isoelectric point at PH3.9 ± 0.1. (H) Sensitivity to inhibitors, including complete inhibitory action, in fibrin side effects on coagulated fibrin: limacomtrypsin inhibitor, difluorophosphate and N-ethylmaleimide; Somewhat stronger inhibitory substances; Leuupeptin and lanback trypsin inhibitor Substances with mild inhibitory activity: soybean trypsin inhibitor, anti-pine, trans-4- (aminoethyl) cyclohexanecarboxylic acid, ε-aminocaproic acid, chymosstatin, pepstatin and disodium Ethyldiaminetetraacetate; Inhibitory substance: Trasylol Amino acid composition (mole%): aspartic acid 16.06%, threonine 8.46%, serine 10.85%, golutamic acid 6.13%, proline 0.41%, golysine 15.41%, alanine 9.45%, cysteine 1.67%, valine 5.51%, methionine 0.82%, isoleucine 6.64%, leucine 8.01%, tyrosine 3.79%, phenylalanine 1.11%, tryptophan undetectable amount, lysine 0.53%, histidine 2.73%, arginine 2.42%, but this data includes unavoidable analytical errors. Elementary composition (wt%): carbon 46.15%, hydrogen 6.64%, nitrogen 16.02%, sulfur 2.05%, provided that this data includes unavoidable analytical errors. White amorphous powder protease F-II-HM-64 characterized by the following composition and properties: (A) substrate specificity and enzymatic activity with fibrinolytic activity against coagulated fibrin; Plasminogen activation; Good activity against casein, tosyl-L-arginine methylester hydrochloride; Weak activity against N-α-tosyl-L-lysinemethylester hydrochloride and L- pyroglutamylglycosyl-L-arginine-P-nitroanilide hydrochloride; Extremely weak activity against H-D-valyl-L-leucine-L-lysine-P-nitroanilide dihydrochloride; Little activity against N-benzoyl-L-alaninemethylester and N-benzoyl-L-tyrosineethylester. (B) The appropriate pH range of the dissolution action for cellulose on coagulated cellulose as a substrate is about 7 to about 8 and the pH stability range is 5 to 12. (C) In the dissolution action for cellulose on the coagulated cellulose at PH 7.8, the appropriate temperature range is about 50 ~ 60 ℃ and the action temperature is 30 ~ 60 ℃. (D) Fully deactivated after heating at 70 ℃ for 60 minutes. (E) Molecular weight: 27,800 ± 2,000 (F) Ultraviolet absorption spectrum ‥ Maximum absorption at wavelength of about 280 nm, minimum absorption at wavelength of about 250 nm. (G) Isoelectric point at PH3.8 ± 0.1. (H) Sensitivity to inhibitors, including complete inhibitory action, in fibrin side effects on coagulated fibrin: limacomtrypsin inhibitor, difluorophosphate and N-ethylmaleimide; Somewhat stronger inhibitory substances; Inhibitors of trasylol, lanback trypsin, trans-4- (aminoethyl) cyclohexanecarboxylic acid, chymosatin and pepsterin Weak inhibitors: anti-pine and ε-aminocaproic acids, non-inhibiting substances: Ruupeptin and Disodium Ethyl Diamine Tetraacetate Amino acid composition (mole%): aspartic acid 14.81%, threonine 8.40%, serine 12.05%, golutamic acid 6.04%, proline 0.44%, golysine 14.48%, alanine 10.27%, cysteine 0.80%, valine 7.77%, methionine 0.98%, isoleucine 5.96% leucine 7.46%, tyrosine 3.24%, phenylalanine 0.58%, tryptophan 1.63%, lysine 0.45%, histidine 2.30%, arginine 2.34%, but this data includes unavoidable analytical errors. Elementary composition (wt%): Carbon 48.23%, Hydrogen 6.53%, Nitrogen 15.93%, Sulfur 1.43%, but this data includes unavoidable analytical errors. White amorphous powder protease F-III-HM-89 characterized by the following composition and properties: (A) substrate specificity and enzymatic activity with fibrinolytic activity against coagulated fibrin; Plasminogen activation; Extremely weak activity against casein, tosyl-L-arginine methylester hydrochloride, and N-N-α-tosyl-L-lysinemethylester hydrochloride and L- pyroglutamyl golysyl -L-arginine-P-nitroanilide hydrochloride ; Extremely weak activity against H-D-valyl-L-leucine-L-lysine-P-nitroanilide dihydrochloride; Little activity against N-benzoyl-L-alaninemethylester and N-benzoyl-L-tyrosineethylester. (B) Appropriate pH range of coagulation solution for coagulation as substrate is about 8 and PH stability range is 4 ~ 12. (C) In the dissolution action for cellulose on the coagulated cellulose at PH 7.8, the appropriate temperature range is about 50 ~ 60 ℃ and the action temperature is 30 ~ 60 ℃. (D) Fully deactivated after heating at 70 ℃ for 60 minutes. (E) Molecular weight: 32,800 ± 2,000 (F) Ultraviolet absorption spectrum ‥ Maximum absorption at wavelength of about 280 nm, minimum absorption at wavelength of about 250 nm. (G) Isoelectric point at PH3.5 ± 0.1. (H) Sensitivity to inhibitors, including complete inhibitory action, on the side effects for fibrin to coagulated fibrin: limacomitpsin inhibitor, difluorophosphate, soybean trypsin inhibitor, antipine, leupeptin and trasylol; Somewhat stronger inhibitory substances; Trans-4- (aminoethyl) cyclohexanecarboxylic acid; Substances with mild inhibitory activity: Lanback trypsin inhibitor, ε-aminocaproic acid, chymostatin and pepstatin: Substances without inhibitory activity: disodium ethyl diaminetetraacetate laacetate and N-ethylmaleimide Amino acid composition (mole%): aspartic acid 14.99%, threonine 6.24%, serine 10.39%, golutamic acid 7.63%, proline 0.57%, golysine 12.89%, alanine 6.57%, cysteine 1.40%, valine 10.67%, methionine 1.34%, Isoleucine 7.62% Leucine 3.41%, Tyrosine 4.83%, Phenylalanine 2.67%, Tryptophan 1.09%, Lysine 1.67%, Histidine 2.12%, Arginine 3.90%, provided that this data includes unavoidable analytical errors. Elementary Composition (wt%): Carbon 47.53%, Hydrogen 6.55%, Nitrogen 14.59%, Sulfur 2.06%, but this data includes unavoidable analytical errors Proteases FO-HM-45, F-I-1-HM-54, F-I-2-HM-15, F-II-HM-64, F-III-1-HM-27 and F-III-2 -Enzymatic substance with thrombolytic activity, consisting of at least one cold protease as an active ingredient (A) Extract the earthworm tissue with the extractant solution to obtain an extract solution containing various proteases, (B) A polar organic solvent is added to the extract solution to produce precipitates containing various proteases, (C) The precipitates were fractionated by anion exchange chromatography to obtain five fibrinolytic active isolates, three of which were protease F-0-HM-45, FI-1-HM-54 and FI. Contains one of -2-HM-15, (D) Each of the isolates containing one of the proteases F-0-HM-45, FI-1-HM-54 and FI-2-HM-15 is passed through an anion exchange packed column to obtain the active ingredient. Adsorbed to the anion exchanger (E) The active ingredient adsorbed to each anion exchange in step (d) was eluted by concentration gradient technique using sodium chloride, and the protease F-0-HM-45, FI-1-HM-54 and FI-2 After obtaining a isolate containing one of the following: (F) Continuous dissolution of fibrinolysis, in which separation containing the protease F-0-HM-45, FI-1-HM-54 or FI-2-HM-15 is purified using a gel ion exchanger, respectively. Method for preparing active prorease F-0-HM-45, FI-1-HM-54 and FI-2-HM-15. The method according to claim 8, wherein the separation is passed through the column packed with the gel ion exchanger to adsorb the active ingredient onto the gel ion exchanger, and the eluting solution is eluted by eluting the active ingredient using a concentration gradient technique using ammonium sulfate. Process for producing step (d) as desalination treatment. (A) Extract the earthworm tissue with the extractant solution to obtain the extract solution containing various proteases, (B) add a polar organic solvent to this extract solution to produce precipitates containing various proteases; (C) fractionate the precipitate by anion exchange chromatography to obtain five fibrinolytic activity isolates, one of which contains protease F-II-HM-64. (D) adsorb the active ingredient to the gel ion exchanger by passing the isolate containing the protease F-II-HM-64 through a column packed with the gel ion exchanger, (E) eluting the active ingredient by concentration gradient technique using ammonium sulfate to obtain a isolate containing protease F-II-HM-64. (F) dethermal treatment of the isolate containing this protease; (G) Adsorb the active ingredient to the adsorbent by passing the desalted isolate through a column packed with an adsorbent for affinity chromatography. (H) eluting the active ingredient by concentration gradient method using sodium chloride to obtain a isolate containing protease F-II-HM-64. (I) A method for producing protease F-II-HM-64 having fibrinolytic activity in a continuous step of purifying the isolate containing protease F-II-HM-64 using a gel ion exchanger. (A) Extract the earthworm tissue with the extractant solution to obtain the extract solution containing various proteases (B) A polar organic solvent is added to this extract solution to produce precipitates containing various proteases. (D) The precipitates were fractionated by anion exchange and chromatography to obtain five fibrinolytic activity isolates, one of which was protease F-III-1-HM-27 and F-III-2-HM-. It contains 87 (D) Purification of the isolate containing the above protease by affinity chromatography (E) Pass the separation purified by affinity chromatography in step (d) through a column packed with gel ion exchanger to adsorb proteases to the gel ion exchanger. (E) eluting the protease by concentration gradient method using ammonium sulfate to obtain a isolate containing protease F-III-1-HM-27 or protease F-III-2-HM-89, respectively. (G) A method for the fractionation treatment of proteases F-III-1-HM-27 and F-III-2-HM-89 having fibrinolytic activity in a continuous step of desalting the isolate containing this protease. (A) Extract the earthworm tissue with the extractant solution to obtain the extract solution containing various proteases, (B) add a polar organic solvent to this extract solution to produce precipitates containing various proteases; (C) Fractionation of precipitates by anion exchange chromatography to obtain five isolates with fibrinolytic properties containing at least one of several proteases. (D) salting each of these secretions to produce precipitates containing various proteases; (E) dissolve this precipitate in a buffer solution to make a solution. (F) After gel filtration of this solution, (G) Protease F-0-HM-45, FI-1-HM-54, FI-2-HM-15M, F-II- A method for the fractionation treatment of an enzyme substance having fibrinolytic activity mainly prepared by mixing HM-64 or F-III-1-HM-27 and F-III-1-HM-89. Protease F-0-HM-45, FI-1-HM-54, FI-2-HM-15M, F-II-HM-64 or F-III-1-HM-27 and F-III-1-HM A thrombolytic drug that uses at least one protease as an active ingredient, and is at least one physiologically ingestible one of -89. ※ Note: It is to be disclosed based on the initial application.