EP0125254A1 - Enzyme derivatives and their use in the treatment of thrombosis - Google Patents
Enzyme derivatives and their use in the treatment of thrombosisInfo
- Publication number
- EP0125254A1 EP0125254A1 EP19830903395 EP83903395A EP0125254A1 EP 0125254 A1 EP0125254 A1 EP 0125254A1 EP 19830903395 EP19830903395 EP 19830903395 EP 83903395 A EP83903395 A EP 83903395A EP 0125254 A1 EP0125254 A1 EP 0125254A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- activator
- modified
- plasminogen activator
- unmodified
- human
- 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- Withdrawn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
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)
- C12N9/6456—Plasminogen activators
- C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
-
- 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 a plasminogen activator, processes for preparing the activator and pharmaceutical compositions containing it.
- US Patent No. 4 326 033 discloses a method of increasing the biological alf life of the fibrinolytic agent urokinase which consists of chemically treating urokinase to remove or degrade carbohydrate units in the urokinase molecule. The patent states that this treatment results in the retention of 20% of the fibrinolytic activity of the original starting material.
- Urokinase is one of many glycoproteins, and there is no indication in the above US Patent that the treatment described therein would have any applicability to other glycoproteins.
- Tissue-type plasminogen activator (hereinafter referred to as t-PA) is, unlike urokinase, similar in its immunolo ical and biological nature to human extrinsic plasminogen activator normally present in human blood. T-PA can be readily isolated from the culture fluid of human melanoma cells, and has been shown to have a potent thrombolytic effect in animals and man.
- tissue-type plasminogen activator t-PA
- t-PA tissue-type plasminogen activator
- ii) Enzymatic treatment of t-PA with a mixture of exo lycosidases, selected for example from (3-galactosidase, ⁇ -mannosidase, -fucosidase, B-N-acetylglucosaminidase or neuraminidase, or one or more endoglycosidases, such as endoglycosidase D (from Diplococcus pneumoniae) or endoglycosidase H (from Streptomyces plicatus).
- endoglycosidase D from Diplococcus pneumoniae
- endoglycosidase H from Streptomyces plicatus
- T-PA can itself be obtained according to the method described in Published European Patent Application No. 0 041 766, which describes the production of a preferred form of t-PA, namely melanoma plasminogen activator, from human melanoma cells.
- the proportion of retained fibrinolytic activity of the structurally modified t-PA depends to some extent on the method of modification and the efficiency of recovery and purification. We have found that by employing the method of paragraph (i) above, and carefully monitoring the physical conditions of the degradation process, we can obtain 70% or more of retained fibrinolytic activity.
- modified t-PA of this invention can be formulated into pharmaceutical compositions in accordance with standard procedures.
- the invention also provides a pharmaceutical composition which comprises structurally modified t-PA as defined herein together with a pharmaceutically acceptable carrier.
- the composition is preferably adapted for intravenous administration to human beings.
- compositions for intravenous administration are solutions of the modified t-PA in sterile isotonic aqueous buffer.
- the composition may also include a solubilizing agent to keep the modified t-PA in solution and a local anaesthetic such as lignocaine to ease pain at the site of injection.
- the modified t-PA will be supplied in unit dosage form for example as a dry powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of t-PA in activity units.
- modified t-PA is to be administered by infusion
- it will be dispensed with an infusion bottle containing sterile pharmaceutical grade 'Water for Injection'.
- modified t-PA is to be administered by injection, it is dispensed with an ampoule of sterile water for injection.
- the injectable or infusable* composition will be made up by mixing the ingredients prior to administration.
- the quantity of material administered will depend upon the amount of fibrinolysis required and the speed with which it is required, the seriousness of the thromboembolic condition and the position and size of the clot.
- the precise dose to be employed and mode of administration may be decided according to the circumstances by the physician supervising treatment.
- a patient being treated for a medium size thrombus will generally receive a daily dose of from 0.10 to 1.0 mg kg" 1 of body weight either by injection in up to eight doses or by infusion.
- the invention provides a therapeutic or prophylactic method of treating thrombosis in human or non-human animals, which comprises treating an animal with an effective, non-toxic amount of the structurally modified plasminogen activator of the invention.
- Melanoma plasminogen activator was purified to 70-90% purity from cultured Bowes melanoma cells by chromatography using zinc-chelate agarose, and lysine - Sepharose.
- the purified MPA was then subjected to carbohydrate degradation, based on published procedures (Biochem. Biophys. Res. Commun. , j57_, 55, 1974) which were modified in order to obtain a high degree of recovery of- enzyme activity.
- Purified MPA was dissolved in a buffer containing 0.1 M sodium phosphate, 0.15M sodium chloride, 0.01% Tween 80, pH 6.0 at a concentration of about 0.5-1.0 mg/mi. Any contaminating ions present in the MPA preparation can be removed by prior gel filtration or dialysis. To this solutiorr ⁇ was added a solution of 0.1 M sodium periodate in the above buffer to a final concentration of 10 mM. The solution was kept at 4 ⁇ C in the dark for one hour. During this period the enzymic activity, as measured by chromogenic substrate assay, ' dropped to 70% of the original.
- the modified activator was found to be equally active as the native activator.
- Chromogenic substrate assay - MPA was assayed by spectrophotometric measurement of the rate of release of p-nitroaniline from the substrate S-2288 (Kabi Diagnostica) when incubated with the enzyme in 0.1 M triethanolamine buffer, pH 8.0 at 25 ⁇ C.
- the substrate concentration was 1 mM.
- mice Male Sprague-Dawley rats (300-400 g) were anaesthetized with pentobarbitone sodium (60 mg/kg i.p.). One carotid artery was cann ⁇ lated for collection of blood samples. One femoral vein was cannulated for injection of heparin (50 U/kg) and compound under test. Approximately 5 min after heparinization, a pre-dose blood sample (0.8 ml) was taken and mixed with 0.1 volumes 129 mM trisodium citrate. The compound under test was then injected (1 ml/kg) over 10s. Further blood samples were taken exactly 1,2,4,8,16,30 and 60 min later.
- Heparin treatment 50 U/kg was repeated after the 30 min sample to maintain cannula patency. All citrated blood samples were kept on ice until the end of each experiment, then centrifuged at 1700 g for 15 min at 4 ⁇ to obtain plasma. The euglobulin fraction was precipitated by adding 0.1 ml each plasma to 1.82 ml ice-cold 0.011% (v/v) acetic acid in water. After 30 min standing in ice, all tubes were centrifuged at 1700 g for 15 min at 4°.
- Fibrin plates were prepared from 0.4% (w/v) human fibrinogen (Kabi, Grade L, Flow Laboratories, Scotland) dissolved in 0.029 M barbitone in 125 mM NaCl, pH 7.4, pipetted (9 ml) into 10 x 10 cm square plastic dishes (Sterilin) and clotted by rapid mixing with 0.3 ml bovine thrombin (50 NIH units/ml, Parke-Davis, UK). Plates were incubated at 370 for 18-24h usually, but longer if required, and stained with aqueous bro ophenol blue. For each lysis zone, two diameters perpendicular to each other were measured using Vernier calipers.
- Fig. 1 shows the clearance of native and modified MPA from the bloodstream of the rat.
- the modified MPA is cleared less rapidly than the native material.
- Thrombolysis in vivo is generally considered to be a prolonged event which requires significant concentrations of activator to be present over a long period; the initial rapid clearance phase (which is essentially identical for both modified and native MPA) is of less importance than the second phase.
- Fig. 1 shows that after the first ca. 10 minutes, the plasma, concentration of modified MPA is on average six times that of native MPA and that the rate of clearance is significantly slower for the modified form.
Abstract
On modifie structuralement un activant plasminogène de type tissulaire de façon à enlever ou à dégrader au moins une partie de la portion hydrate de carbone de la molécule activante. L'activant modifié possède une activité fibrinolytique conservée au moins à 50 % en comparaison avec l'activant non modifié, couplée avec une demi vie biologique accrue.A tissue-type plasminogen activator is structurally modified so as to remove or degrade at least part of the carbohydrate portion of the activating molecule. The modified activator has at least 50% conserved fibrinolytic activity in comparison with the unmodified activator, coupled with an increased biological half-life.
Description
ENZYME DERIVATIVES AND THEIR USE IN THE TREATMENT OF THROMBOSIS
This invention relates to a plasminogen activator, processes for preparing the activator and pharmaceutical compositions containing it.
US Patent No. 4 326 033 (Abbott Laboratories) discloses a method of increasing the biological alf life of the fibrinolytic agent urokinase which consists of chemically treating urokinase to remove or degrade carbohydrate units in the urokinase molecule. The patent states that this treatment results in the retention of 20% of the fibrinolytic activity of the original starting material.
Urokinase is one of many glycoproteins, and there is no indication in the above US Patent that the treatment described therein would have any applicability to other glycoproteins.
We have now discovered that removal or degradation of carbohydrate units in an unrelated glycoprotein to urokinase, namely tissue-type plasminogen activator, produces unexpectedly high retention of fibrinolytic activity as compared to the unmodified activator, coupled with increased biological half life.
Tissue-type plasminogen activator (hereinafter referred to as t-PA) is, unlike urokinase, similar in its immunolo ical and biological nature to human extrinsic plasminogen activator normally present in human blood. T-PA can be readily isolated from the culture fluid of human melanoma cells, and has been shown to have a potent thrombolytic effect in animals and man.
According to the present invention, there is provided structurally modified tissue-type plasminogen activator (t-PA) wherein at least part of the carbohydrate portion of the t-PA molecule is removed or degraded, the modified t-PA having at least 50% of retained fibrinolytic activity compared to unmodified t-PA.
The required modification of t-PA can be carried out in various ways, but preferred methods are as follows:-
i) Chemical degradation of carbohydrate units utilising, for example, sodium periodate. This method of degradation is based on published procedures (Biochem. Biophys. Res. Commun., 57, 55, 1974).
ii) Enzymatic treatment of t-PA with a mixture of exo lycosidases, selected for example from (3-galactosidase, α-mannosidase, -fucosidase, B-N-acetylglucosaminidase or neuraminidase, or one or more endoglycosidases, such as endoglycosidase D (from Diplococcus pneumoniae) or endoglycosidase H (from Streptomyces plicatus).
iii) Preparation from cultured cells, for example, melanoma cells, grown in the presence of an inhibitor of protein glycosylation, such as tunicamycin.
iv) Preparation by recombinant DNA techniques in bacterial systems. This can be carried out by cloning the genetic information for t-PA into prokaryotes.
T-PA can itself be obtained according to the method described in Published European Patent Application No. 0 041 766, which describes the production of a preferred form of t-PA, namely melanoma plasminogen activator, from human melanoma cells.
The proportion of retained fibrinolytic activity of the structurally modified t-PA depends to some extent on the method of modification and the efficiency of recovery and purification. We have found that by employing the method of paragraph (i) above, and carefully monitoring the physical conditions of the degradation process, we can obtain 70% or more of retained fibrinolytic activity.
The modified t-PA of this invention can be formulated into pharmaceutical compositions in accordance with standard procedures.
Accordingly, the invention also provides a pharmaceutical composition which comprises structurally modified t-PA as defined herein together with a pharmaceutically acceptable carrier. The composition is preferably adapted for intravenous administration to human beings.
Typically, compositions for intravenous administration are solutions of the modified t-PA in sterile isotonic aqueous buffer. Where necessary the composition may also include a solubilizing agent to keep the modified t-PA in solution and a local anaesthetic such as lignocaine to ease pain at the site of injection. Generally, the modified t-PA will be supplied in unit dosage form for example as a dry powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of t-PA in activity units. Where the modified t-PA is to be administered by infusion, it will be dispensed with an infusion bottle containing sterile pharmaceutical grade 'Water for Injection'. Where the modified t-PA is to be administered by injection, it is dispensed with an ampoule of sterile water for injection. The injectable or infusable* composition will be made up by mixing the ingredients prior to administration.
The quantity of material administered will depend upon the amount of fibrinolysis required and the speed with which it is required, the seriousness of the thromboembolic condition and the position and size of the clot. The precise dose to be employed and mode of administration may be decided according to the circumstances by the physician supervising treatment. However, in general, a patient being treated for a medium size thrombus will generally receive a daily dose of from 0.10 to 1.0 mg kg"1 of body weight either by injection in up to eight doses or by infusion.
In a further aspect the invention provides a therapeutic or prophylactic method of treating thrombosis in human or non-human animals, which comprises treating an animal with an effective, non-toxic amount of the structurally modified plasminogen activator of the invention.
The following example illustrates the invention:-
OM?I
Example
Melanoma plasminogen activator (MPA) was purified to 70-90% purity from cultured Bowes melanoma cells by chromatography using zinc-chelate agarose, and lysine - Sepharose.
The purified MPA was then subjected to carbohydrate degradation, based on published procedures (Biochem. Biophys. Res. Commun. , j57_, 55, 1974) which were modified in order to obtain a high degree of recovery of- enzyme activity.
Purified MPA was dissolved in a buffer containing 0.1 M sodium phosphate, 0.15M sodium chloride, 0.01% Tween 80, pH 6.0 at a concentration of about 0.5-1.0 mg/mi. Any contaminating ions present in the MPA preparation can be removed by prior gel filtration or dialysis. To this solutiorr~was added a solution of 0.1 M sodium periodate in the above buffer to a final concentration of 10 mM. The solution was kept at 4©C in the dark for one hour. During this period the enzymic activity, as measured by chromogenic substrate assay,' dropped to 70% of the original. One-tenth of the volume of glycerol was added followed by ethanola ine to a final concentration of 20-50 mM. The activity returned to approximately 100%. The solution was then gel filtered on a column of Sephadex G-25 equilibrated with the described phosphate buffer in order to remove excess reagents. About 70-90% of the original activity was recovered.
On human fibrin plates containing human plasminogen, the modified activator was found to be equally active as the native activator.
OMH
Methods
Chromogenic substrate assay - MPA was assayed by spectrophotometric measurement of the rate of release of p-nitroaniline from the substrate S-2288 (Kabi Diagnostica) when incubated with the enzyme in 0.1 M triethanolamine buffer, pH 8.0 at 25θC. The substrate concentration was 1 mM.
Assay of fibrinolytic activity in the bloodstream of rats
Male Sprague-Dawley rats (300-400 g) were anaesthetized with pentobarbitone sodium (60 mg/kg i.p.). One carotid artery was cannύlated for collection of blood samples. One femoral vein was cannulated for injection of heparin (50 U/kg) and compound under test. Approximately 5 min after heparinization, a pre-dose blood sample (0.8 ml) was taken and mixed with 0.1 volumes 129 mM trisodium citrate. The compound under test was then injected (1 ml/kg) over 10s. Further blood samples were taken exactly 1,2,4,8,16,30 and 60 min later. Heparin treatment (50 U/kg) was repeated after the 30 min sample to maintain cannula patency. All citrated blood samples were kept on ice until the end of each experiment, then centrifuged at 1700 g for 15 min at 4θ to obtain plasma. The euglobulin fraction was precipitated by adding 0.1 ml each plasma to 1.82 ml ice-cold 0.011% (v/v) acetic acid in water. After 30 min standing in ice, all tubes were centrifuged at 1700 g for 15 min at 4°. The supernatants were poured away, the inner walls of each tube carefully wiped dry and each precipitate redissolved in 0.4 ml 0.1 M triethanolamine HC1 buffer, pH 8.0, containing 0.05%
(w/v) sodium azide. Aliquots (20μl) were then applied to fibrin plates in quadruplicate. Fibrin plates were prepared from 0.4% (w/v) human fibrinogen (Kabi, Grade L, Flow Laboratories, Scotland) dissolved in 0.029 M barbitone in 125 mM NaCl, pH 7.4, pipetted (9 ml) into 10 x 10 cm square plastic dishes (Sterilin) and clotted by rapid mixing with 0.3 ml bovine thrombin (50 NIH units/ml, Parke-Davis, UK). Plates were incubated at 370 for 18-24h usually, but longer if required, and stained with aqueous bro ophenol blue. For each lysis zone, two diameters perpendicular to each other were measured using Vernier calipers. All diameters for each sample were averaged, and this mean converted to- fibrinolytic activity by reference to a calibration curve. The latter was obtained by adding known amounts of the compound under test to a stock of plasma pooled from at least ten rats. These standards were processed using the same methods and. at" the same time as the experimental samples. To construct the calibration curve, diameters (mm) were plotted against log^g concentration of compound. The plasma concentration of compound in each experimental sample was expressed as a percentage of that expected on the basis of the dose given and the assumption of 35 ml plasma/kg body weight for each rat.
"gtJRE. OMH
Results
Fig. 1 shows the clearance of native and modified MPA from the bloodstream of the rat. The modified MPA is cleared less rapidly than the native material.
Thrombolysis in vivo is generally considered to be a prolonged event which requires significant concentrations of activator to be present over a long period; the initial rapid clearance phase (which is essentially identical for both modified and native MPA) is of less importance than the second phase. Fig. 1 shows that after the first ca. 10 minutes, the plasma, concentration of modified MPA is on average six times that of native MPA and that the rate of clearance is significantly slower for the modified form.
Claims
1. Structurally modified tissue-type plasminogen activator, wherein at least part of the carbohydrate portion of the activator molecule is removed or degraded, the modified activator having at least 50% retained fibrinolytic activity compared to unmodified activator.
2. Plasminogen activator according to claim 1, having at least 70% retained fibrinolytic activity compared to unmodified activator.
3. Plasminogen activator according to claim 1 or claim 2, in the form of modified* melanoma plasminogen activator.
4. A process for preparing a modified plasminogen activator"according to any one of claims 1 to 3, which comprises, a) chemically degrading carbohydrate units on the unmodified activator using alkali metal periodate, b) enzymatically treating the unmodified activator with a mixture of exoglycosidases, and/or endoglycosidase^ , c) growing cells in the presence of an inhibitor of protein glycosylation, or d)* preparing the activator by recombinant DNA techniques in bacterial systems.
5. A pharmaceutical composition comprising modified activator according to any one of claims 1 to 3, together with a pharmaceutically acceptable carrier.
6. A modified plasminogen activator according to any one of claims 1 to 3, for use in treating thrombosis in human or non-human animals.
7. 'A method of treating thrombosis in human or non-human animals, which comprises treating an animal with an effective non-toxic amount of an activator according to any one of claims 1 to 3.
OMH
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB8230801 | 1982-10-28 | ||
| GB8230801 | 1982-10-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0125254A1 true EP0125254A1 (en) | 1984-11-21 |
Family
ID=10533890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP19830903395 Withdrawn EP0125254A1 (en) | 1982-10-28 | 1983-10-25 | Enzyme derivatives and their use in the treatment of thrombosis |
Country Status (2)
| Country | Link |
|---|---|
| EP (1) | EP0125254A1 (en) |
| WO (1) | WO1984001786A1 (en) |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4753879A (en) * | 1984-08-27 | 1988-06-28 | Biogen N.V. | Modified tissue plasminogen activators |
| DK175327B1 (en) * | 1984-10-01 | 2004-08-23 | Integrated Genetics Inc | Replicable expression vector and recombinant DNA molecule encoding active human uterine tissue plasminogen activator, mammalian cell transformed with the vector, and method of producing active human tissue plasminogen activator (TPA) |
| US5244806A (en) * | 1985-08-26 | 1993-09-14 | Eli Lilly And Company | DNA encoding novel tissue plasminogen activator derivatives having kringles 1 and 2 deleted, vectors and host cells |
| US4960702A (en) * | 1985-09-06 | 1990-10-02 | Codon | Methods for recovery of tissue plasminogen activator |
| GB8528321D0 (en) * | 1985-11-18 | 1985-12-24 | Ciba Geigy Ag | Modified fibrinolytic agents |
| ZA869626B (en) * | 1985-12-23 | 1987-10-28 | Chiron Corp | Peptide plasminogen activators |
| WO1987004722A1 (en) * | 1986-01-31 | 1987-08-13 | Genetics Institute, Inc. | Novel thrombolytic proteins |
| US5258298A (en) * | 1986-01-31 | 1993-11-02 | Genetics Institute, Inc. | Deletion and glycosylation mutant of human tissue plasminogen activator |
| US5002887A (en) * | 1986-01-31 | 1991-03-26 | Genetics Institute, Inc. | Truncated thrombolytic proteins |
| US5837518A (en) * | 1986-01-31 | 1998-11-17 | Genetics Institute, Inc. | Thrombolytic proteins |
| US5071972A (en) * | 1986-01-31 | 1991-12-10 | Genetics Institute, Inc. | DNA sequences encoding novel thrombolytic proteins |
| US5589361A (en) * | 1986-03-18 | 1996-12-31 | Genentech, Inc. | Human tissue-type plasminogen activator variant |
| IE81073B1 (en) * | 1986-03-18 | 2000-01-12 | Genentech Inc | Modified human tissue-type plasminogen activator and its preparation |
| EP0275282A1 (en) * | 1986-07-16 | 1988-07-27 | Celltech Limited | Process for purifying a plasminogen activator |
| US7084251B1 (en) | 1987-02-12 | 2006-08-01 | Genentech, Inc. | Methods and Deoxyribonucleic acid for the preparation of tissue factor protein |
| US6994988B1 (en) | 1987-02-12 | 2006-02-07 | Genetech, Inc. | Methods and deoxyribonucleic acid for the preparation of tissue factor protein |
| NO882226L (en) * | 1987-05-22 | 1988-11-23 | Zymogenetics Inc | FIBRINOLYTIC PROTEINS. |
| ES2058180T3 (en) * | 1987-06-04 | 1994-11-01 | Zymogenetics Inc | MUTANT T-PA WITH KRINGLE REPLACEMENT. |
| IE67036B1 (en) * | 1987-06-04 | 1996-05-15 | Zymogenetics Inc | Tissue plasminogen activator analogs having modified growth factor domains |
| US4935237A (en) * | 1988-03-21 | 1990-06-19 | Genentech, Inc. | Processes for the preparation of t-PA mutants |
| IL87276A (en) * | 1987-08-03 | 1995-07-31 | Fujisawa Pharmaceutical Co | Analog of tissue plasminogen activator comprising only the kringle 2 and protease domain dna encoding the same processes for the preparation thereof and pharmaceutical compositions containing the same |
| JP2708749B2 (en) * | 1987-08-10 | 1998-02-04 | エーザイ株式会社 | Injectable composition containing modified tPA |
| IL88247A0 (en) * | 1987-11-06 | 1989-06-30 | Genentech Inc | Novel human tissue-type plasminogen activator variant |
| US5648254A (en) * | 1988-01-15 | 1997-07-15 | Zymogenetics, Inc. | Co-expression in eukaryotic cells |
| WO1989007641A1 (en) * | 1988-02-10 | 1989-08-24 | Genzyme Corporation | Enhancement of the therapeutic properties of glycoprotein |
| US5037646A (en) * | 1988-04-29 | 1991-08-06 | Genentech, Inc. | Processes for the treatment of vascular disease |
| US5242688A (en) * | 1990-12-24 | 1993-09-07 | Eli Lilly And Company | Method of treating thromboembolic disorders by administration of diglycosylated t-pa variants |
| AU1131397A (en) * | 1995-11-30 | 1997-06-19 | Eisai Co. Ltd. | Sugar chain-deficient tissue plasminogen activator derivatives |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3015699C2 (en) * | 1979-04-26 | 1982-07-15 | Asahi Kasei Kogyo K.K., Osaka | Manufacture of a plasminogen activator |
| US4370417A (en) * | 1980-04-03 | 1983-01-25 | Abbott Laboratories | Recombinant deoxyribonucleic acid which codes for plasminogen activator |
| US4326033A (en) * | 1980-05-05 | 1982-04-20 | Abbott Laboratories | Modified urokinase having extended activity and method of making |
| NL8003402A (en) * | 1980-06-11 | 1982-01-04 | Leuven Res & Dev Vzw | NEW PLASMINOGEN ACTIVATOR AND PHARMACEUTICAL PREPARATION WITH THROMBOLYTIC ACTION. |
-
1983
- 1983-10-25 WO PCT/GB1983/000273 patent/WO1984001786A1/en unknown
- 1983-10-25 EP EP19830903395 patent/EP0125254A1/en not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| See references of WO8401786A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1984001786A1 (en) | 1984-05-10 |
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