EP0917567A1 - Thrombinaktivierbarer plasminogenaktivator - Google Patents
Thrombinaktivierbarer plasminogenaktivatorInfo
- Publication number
- EP0917567A1 EP0917567A1 EP97934509A EP97934509A EP0917567A1 EP 0917567 A1 EP0917567 A1 EP 0917567A1 EP 97934509 A EP97934509 A EP 97934509A EP 97934509 A EP97934509 A EP 97934509A EP 0917567 A1 EP0917567 A1 EP 0917567A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- plasminogen activator
- thrombin
- plasmin
- cleavage
- modified
- 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- Withdrawn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
- 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
-
- 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
- the invention relates to a thrombin-activatable plasminogen activator, medicaments for the treatment of thromboembolic disorders, pharmaceutical compositions which contain such a plasminogen activator, and their use
- Tissue plasminogen activator (t-PA) is a multi-domain serine protease that catalyzes the conversion of plasminogen to plasmin and is used for fibrinolytic therapy
- t-PA fibinolysis is partially regulated by the interaction between t-PA and plasminogen activator inhibitor 1 (PAI-1, a serine protease inhibitor from the Se family). 1 on t-PA occurs essentially via amino acids 296-302.
- PAI-1 plasminogen activator inhibitor 1
- a mutation of this region reduces the inhibitory influence of PAI-1 on t-PA (EL Madison et al (1990)) on the mechanism of the interaction between the amino acid region 296-302 of Comprehensive investigations were carried out in t-PA with PAI-1 (see also EL Madison, Nature 339 (1989) 721-723; RV Schohet, Thrombosis and Hae ostasis 71 (1994) 124-128, CJ Refino, Thrombosis and Haemostasis 70 (1993) 313-319, NF Paoni, Protein Engineering 6 (1993) 529-534 and Thrombosis and Haemostasis 70 (1993) 307-312, WF Bennett, J Biol Chem 266 (1991) 5191-3201, D Eastman, Biochemistry 31 (1992) 419-422).
- Unmodified human t-PA (hereinafter referred to as t-PA) consists of 527 amino acids in its form in the plasma and can be split into two chains by plasmin, which are then held together by a disulfide bridge.
- the A chain (also called heavy chain) consists of four structural domains.
- the finger domains (amino acids 1-49) show certain similarities with the finger structures in fibronectin.
- the growth factor domain (amino acids 50-86) is to a certain extent homologous to murine and human epidermal growth factors
- the Kringledomanes (amino acids 87 - 261) are largely homologous to the fourth and fifth Kringledomain of plasminogen.
- the finger and Kringle-2 domains of t-PA are in the fibrin formation and particularly involved in the stimulation of proteolytic activity by fibrin.
- the B chain of t-PA (amino acids 276 - 527, protease domain) is a serine protease and largely homologous to the B chains of urokinase and plasmin (TJ R Harris (1987) and J Krause (1988)
- t-PA catalytic activation of plasminogen to plasmin
- the mechanism of action of t-PA is in vivo
- t-PA activates plasminogen to plasmin.
- Plasmin cleaves fibers to soluble fibrin cleavage products.
- t-PA is formed between amino acid 275 (arginine) and 276 (isoleucm ) split and thereby activated. The two partial chains remain connected by a cysteine bridge
- the stimulability of the activity by fibrin, fibrin cleavage products is an essential feature of t-PA, which distinguishes t-PA from the other known plasminogen activators, such as urokinase or streptokinase.
- the stimulability can be further improved by modifying the amino acid sequence of t-PA
- a measure of the stimulability is the ratio of the catalytic efficiency (K cat / K m ) in the presence and in the absence of fibrin
- K ca t is the rate constant of the catalytic reaction and K m is the Michaelis constant
- the stimulability of t-PA can be modified by modifying the Amino acids 292 and / or 305 increase 19 to 81 times (EL Madison et al, Science 262 (1993) 419-421
- t-PA derivatives which are modified in the region of the amino acids 272-280, in particular in the region 274-277 and additionally in the region of the glycosylation sites (117-119 and 184-186).
- Such t PA derivatives have an improved proteolytic and plasminogenolytic activity, a reduced sensitivity to inhibition, an improved affinity for fibrin and / or an improved fibrin dependency of the plasminogenolytic activity
- thrombin-activatable plasminogen activators which are modified in such a way that they contain a thrombin cleavage site for activation. It is further stated that although the growth factor domain (EGF domain) can be deleted in such t-PA derivatives, the fibrin binding domain (finger domain) and the Kringles structures must be retained.
- plasminogen can be activated by introducing a thrombin cleavage site from thrombin to plasmin. Since thrombin is contained in the blood clot, this activation should mainly take place on the blood clot.
- modified plasminogen would have to be administered to the patient in large quantities
- the object of the invention is to provide improved plasminogen activators which, with high specificity and effectiveness, are able to dissolve blood clots in vivo.
- the object is achieved by a plasminogen activator, which starts from human tissue plasminogen activator
- plasminogen activator can be cleaved by thrombin and is converted into the two-chain form by such cleavage
- b) is modified so that the zymogenicity compared to human t-PA is at least 1.2 times, preferably 2 times greater, and c) whose fibrin binding is reduced to such an extent that the plasminogen activator can penetrate more than 50% into a blood clot.
- Such a plasminogen activator acts specifically on the blood clot and therefore shows significantly fewer side effects than the known plasminogen activators.
- the starting point is the sequence of the human tissue plasminogen activator.
- “starting from human tissue plasminogen activator” means that the sequence of the plasminogen activator according to the invention is derived from the sequence of the human plasminogen activator.
- the plasminogen activator according to the invention is modified compared to human tissue plasminogen activator so that the cleavage of the plasminogen activator according to the invention by plasmin between the amino acids Pl (275) and PT (276) is reduced.
- the cleavage by plasmin is preferably reduced by 10% or more, particularly preferably by 20% or more and very particularly preferably by 50% or more. It is not necessary for the plasmin cleavage to be completely eliminated. In particular, thrombin activation can be improved by plasmin cleavage of the plasminogen activator according to the invention.
- cleavage by plasmin can be determined in an in vitro test.
- the plasminogen activator is incubated with increasing amounts of plasmin for five minutes at 25 ° C and then SDS electrophoresis is carried out in an acrylamide gel with 12.5 - 15% acrylamide, depending on the size of the plasminogen activator (U. Kohnert et al., Protein Engineering 5 (1992) 93-100).
- Plasmin can also be reduced by modifying positions P4 - P3 * (amino acids 272 - 278).
- P2 is preferably converted into a small, preferably hydrophobic and / or non-aromatic amino acid, such as P.
- a thrombin cleavage can surprisingly be achieved.
- (F) P2 hydrophobic amino acid (F, H, G, V, L, I, T, A or P, particularly preferably P)
- (R) Pl R or K, preferably R.
- (K) P2 ' V, L, I or K, preferably V
- P4 is particularly preferably converted into V, P2 into P, P2 'into V. This results in the particularly preferred cleavage point (272-278) VQPRIVG (SEQ ID NO: 1).
- a thrombin cleavage site can also be introduced according to the prior art, but a corresponding mutation is preferably introduced in the region of amino acids 264-288.
- a modification of t-PA to introduce a - finity to thrombin can also be done by modifying loops 459-471, autolysis loop 417-425 and / or amino acids Q 475, K 505 and / or E 506
- the specificity of an enzyme for its substrate essentially depends on the sequence of the cleavage site (primary sequence). For serine proteases, such as thrombm and plasmin, the PI residue is the essential specific determinant.
- the specificity of folded protein substrates also depends on the characteristic contact between the enzyme (Thrombin or plasmin) and substrate (plasminogen activator) as well as on the conformation and flexibility of the cleavage site Since the primary specificities of plasmin and thrombin are very similar (both cleave after arginine at the Pl position), it is not readily possible to use plasminogen activators to obtain, in which the cleavage by plasmin (plasma inactivability) is reduced and the cleavage by thrombin (thrombin inactivability) is present or is substantially greater than the plasma inactivability (at least factor 2-10). Surprisingly, however, it was found that by modification at the secondary binding sites between E nzyme and substrate and modifications of the structural and dynamic properties of
- the introduction of a thrombin-specific cleavage site is preferably carried out with a simultaneous reduction in the plasmin cleavage by changing the primary specificity by mutations in the activation loop between amino acids 264 and 288.
- the mutations mentioned above in the range 272-277 (P4-P2 ') are particularly preferred.
- Thrombin cleavage can be improved by modifying the flexibility and / or accessibility of the binding loop.
- G 265 (mutation leads to less flexibility) or R 267 (mutation leads to a change or cleavage of the salt bridge between G 265 and E 410) is particularly preferred for this purpose Insertions in the range between 264 and 267 are also preferred.
- R 267 is particularly preferably modified in S (D. Lamba et al, J Mol. Biol 258 (1996) 117-135)
- a mutation with which flexibility can be increased is the change in R 267 in S, preferably in connection with mutations in P4 in F, P3 in G, P2 in P and P2 'in V.
- Thrombin cleavage and specificity can be further improved by changing the secondary specific binding sites.
- the loop 459 - 471 can be deleted completely or partially. This loop consists of the amino acids
- region PQANLH (SEQ CD NO: 3) is preferably completely or at least partially deleted (preferably H being retained) or its amino acid sequence is changed.
- GLSQASQGIPRIV SEQ ID NO: 7).
- the sequence GLRQYSQAQGIPRIV (SEQ ID NO: 8) is also preferred for this area.
- amino acids G and I are inserted between Q and P (original sequence: Q and F) for extension.
- Such an insertion is preferably carried out at any point in the range between 264 and 276.
- cleavage site SEQ ID NO: 1 with one or more, preferably all of the mutations P4 in F, P3 in G, P2 in P and P2 'in V and the mutation R 267 in S is combined.
- the plasminogen activator additionally contains a mutation which indicates the activity of the single-chain form but not the activity the two-chain form is drastically reduced and the zymogenicity is improved by a factor of 1.2, preferably by a factor of 2 or more.
- Zymogenicity is the quotient of the activity of the two-chain form and the activity of the single-chain form. The activity is determined amidolytically.
- a plasminogen activator achieves high selectivity and effectiveness of thrombus dissolution in vivo with drastically reduced side effects.
- Suitable and preferred mutations of the single-chain form are described, for example, in E.L. Madison et al., Science 262 (1993) 419-421.
- zymogenicity ratio of the amidolytic activity of the two-chain form to the activity of the single-chain form of the plasminogen activator
- it is particularly preferred also for all plasminogen activators derived from human tissue plasminogen activator to K 429 in Q and / or H 417 in T. modify and / or prevent or interfere with the interaction between K 429 and H 417.
- Particularly preferred compounds according to the invention contain the cleavage sites (272-278) VQPRIVG (SEQ ED NO: 1) with the additional mutation K 429 in Q and / or H 417 in T.
- the reduction in fibrin binding can be done by deleting or mutating the domain of t-PA which is specific for fibrin binding (fibrin binding domain, finger domain) in such a way that fibrin binding via the finger domain cannot or only to a small extent (none functional finger domain).
- the plasminogen activator can penetrate the clot (preferably more than 50%) and is distributed evenly. There it is cleaved by thrombin and unfolds its activity in the active two-chain form.
- This specific mode of action increases the potency of the plasminogen activator and, in particular, drastically reduces side effects.
- the penetration of the plasminogen activator according to the invention into a clot can be determined in an in vitro model. The extent of clot penetration and distribution in the clot can be determined visually.
- the plasminogen activator described in US Pat. No. 5,223,256 is used as the standard for assessment, which penetrates into the clot, distributes itself homogeneously and thus by definition represents the 100% value (determined at a concentration of 3 ⁇ g / ml).
- recombinant human tissue plasminogen activator according to EP-B 0 093 619 is used, which by definition is not included in the clot penetrates and essentially binds to the surface.
- the investigation of the clot penetration is carried out as described in Example 3c.
- a comparison of these standards shows that "non-penetration into the clot" means that the vast majority (80% or more) of the plasminogen activator is in the first quarter of the clot, whereas with a "homogeneous distribution" at least 50% of the plasminogen activator penetrate further into the clot and are thus located in the remaining three quarters.
- a plasminogen activator according to the invention which additionally shows no or only very low and non-specific fibrin binding.
- Such molecules penetrate the inside of the clot and thus ensure an efficient activation of plasminogen to plasmin in the clot.
- plasminogen activators are based, for example, on the protease domain of t-PA (WO 96/17928) or on a substance which essentially contains the Kringle 2 domain and the protease domain, but not the finger domain, as t-PA domains (WO 90/09437, U.S. Patent 5,223,256, EP-B 0 297 066, EP-B 0 196 920).
- the plasminogen activator according to the invention is additionally modified so that it cannot be inhibited by PA1-1.
- a modification is preferably carried out by mutating amino acids 296-302 (Madison, EL et. Al., Proc. Natl. Acad. Sci. USA 87 (1990) 3530-3533) and particularly preferably by replacing amino acids 296-299 (KHRR ) by AAAA (WO 96/01312)
- the compounds according to the invention are thrombolytically active proteins which, in contrast to t-PA (Alteplase), are preferably administered as iv. Bolus injection are suitable. They are effective in a lower dose and show practically the same thrombolytic effect as a clinically customary infusion of Alteplase
- the compounds according to the invention an extraordinarily valuable thrombolytic agent for the treatment of all thromboembolic disorders.
- the use of such variants opens up the possibility of using thrombolysis by the compounds according to the invention even in the case of less acute life-threatening diseases, such as deep leg vein thrombosis.
- the use of thrombolytics on the basis of the compounds according to the invention can now take place much more widely than hitherto, since this is a main obstacle to - lü ⁇
- the compounds according to the invention can advantageously also be used in acute diseases, such as heart attack or pulmonary embolism.
- the plasminogen activators used according to the invention can be produced in eukaryotic or prokaryotic cells according to the methods familiar to the person skilled in the art.
- the compounds according to the invention are preferably produced by genetic engineering. Such a process is described, for example, in WO 90/09437, EP-A 0 297 066, EP-A 0 302 456, EP-A 0 245 100 and EP-A 0 400 545, which are the subject of the disclosure for such production processes .
- Mutations can be introduced into the cDNA of t-PA or a derivative thereof by "oligonucleotide-directed site-specific mutagenesis".
- the "site-specific mutagenesis” is, for example, by Zoller and Smith (1984), modified from T.A. Kunkel (1985) and Morinaga et al. (1984).
- the method of PCR mutagenesis which is described, for example, in Ausubel et al (1991), is also suitable
- the nucleic acid obtained in this way serves to express the plasminogen activator used according to the invention if it is present on an expression vector suitable for the host cell used.
- nucleic acid sequence of the protein according to the invention can additionally be modified. Such modifications are, for example
- nucleic acid sequence to introduce different restriction enzyme recognition sequences to facilitate the steps of ligation, cloning and mutagenesis
- the glycosylated plasminogen activators used according to the invention are produced in eukaryotic host cells.
- the non-glycosylated plasminogen activators used according to the invention are either produced in eukaryotic host cells, the glycosylated product initially obtained thereby having to be deglycosylated by methods familiar to the person skilled in the art, or preferably by expression in non-glycosylating host cells, particularly preferably in prokaryotic host cells.
- E. coli, Streptomyces spec. are prokaryotic host organisms. or Bacillus subtilis.
- the prokaryotic cells are fermented in a conventional manner and, after the bacteria have been digested, the protein is isolated in a conventional manner. If the protein is obtained in an inactive form (inclusion bodies), it is solubilized and naturalized according to the methods familiar to the person skilled in the art. It is also possible according to the methods familiar to the person skilled in the art to secrete the protein from the microorganisms as the active protein.
- An expression vector suitable for this preferably contains a signal sequence which is suitable for the secretion of proteins in the host cells used, and the nucleic acid sequence which codes for the protein.
- the protein expressed with this vector is secreted either into the medium (for gram-positive bacteria) or into the periplasmatic space (for gram-negative bacteria).
- the signal sequence and the sequence coding for the t-PA derivative according to the invention there is expediently a sequence which codes for a cleavage site which allows the protein to be split off either during processing or by treatment with a protease.
- the selection of the base vector into which the DNA sequence coding for the plasminogen activator according to the invention is introduced depends on the host cells used later for expression. Suitable plasmids and the minimum requirements placed on such a plasmid (e.g. origin of replication, restriction sites) are known to the person skilled in the art. In the context of the invention, a cosmid, the replicative double-stranded form of phage ( ⁇ , Ml 3) or other vectors known to the person skilled in the art can also be used instead of a plasmid.
- the inclusion bodies that form from the soluble cell particles separate, solubilize the inclusion bodies containing plasminogen activator by treatment with denaturing agents under reducing conditions, then derivatize with GSSG and renaturate the plasminogen activator by adding GSH and denaturing agents in non-denaturing concentration or L-arginine.
- Such methods for activating t-PA and derivatives from inclusion bodies are described, for example, in EP-A 0 219 874 and EP-A 0 241 022. However, other methods of obtaining the active protein from the inclusion bodies can also be used.
- the plasminogen activators according to the invention are preferably purified in the presence of L-arginine, in particular at an arginine concentration of 10-1000 mmol / l.
- Foreign proteins are preferably separated off by affinity chromatography and particularly preferably via an adsorber column on which ETI (Erythrina Trypsin Inhibitor) is immobilized.
- Sepharose® for example, is used as the carrier material.
- Cleaning via an ETI adsorber column has the advantage that the ETI adsorber column material can be loaded directly from the concentrated renaturation batch even in the presence of such high arginine concentrations as 0.8 mol / 1.
- the plasminogen activators according to the invention are preferably purified via an ETI adsorber column in the presence of 0.6-0.8 mol / 1 arginine.
- the solution used here preferably has a pH of more than 7, particularly preferably between 7.5 and 8.6.
- the plasminogen activators according to the invention are eluted from the ETI column by lowering the pH both in the presence and in the absence of arginine.
- the pH is preferably in the acidic range, particularly preferably between pH 4.0 and 5.5.
- Another object of the invention is a pharmaceutical composition containing a thrombolytically active protein according to the invention, the protein preferably containing the protease domain and optionally the Kringel 2 domain of the human tissue plasminogen activator as the only structure causing the thrombolytic activity.
- the plasminogen activators used according to the invention can be formulated for the production of therapeutic agents in a manner familiar to the person skilled in the art, the compounds according to the invention usually being combined with a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier typically contain an effective one Amount of 0.1-7 mgkg, preferably 0.7-5 mg / kg and particularly preferably 1-3 mg / kg body weight as a dose.
- the therapeutic compositions are usually in the form of sterile, aqueous solutions or sterile, soluble dry formulations such as lyophilisates.
- the compositions usually contain a suitable amount of a pharmaceutically acceptable salt with which an isotonic solution is prepared.
- Buffers such as arginine buffer, phosphate buffer for stabilizing a suitable pH Value (preferably 5.5-7.5) can be used by any person skilled in the art to determine the dosage of the compounds according to the invention. It depends, for example, on the type of application (infusion or bolus) and the duration of the therapy. Because of their prolonged half-life (with respect to degradation in vivo), the compounds according to the invention are particularly suitable for a bolus application (single bolus, multiple bolus) .
- a suitable form for a bolus application is, for example, an ampoule which contains 25-1000 mg of the compound according to the invention, a substance which is soluble of the plasminogen activator improved (such as arginine) and buffer contained.
- the application is preferably intravenous, but also subcutaneously, intramuscularly or intraarterially. Plasminogen activators according to the invention can also be infused or applied locally
- the compounds according to the invention can be used as a multiple bolus (preferably as a double bolus). Suitable time intervals are between 20 and 180 minutes, an interval between 30 and 90 minutes is particularly preferred and an interval between 30 and 60 minutes is particularly preferred administration as an infusion over a period of 1 h - 2 days is possible
- the compounds according to the invention are particularly suitable for the treatment of all thromboembolic diseases, such as, for example, acute heart attack, cerebral infarction, pulmonary embolism, deep leg vein thrombosis, acute arterial occlusion, etc.
- the compounds according to the invention are particularly preferred for the treatment of subchronic thromboembolic diseases in which prolonged thrombolysis is carried out must be applied
- anticoagulation such as, for example, hepan and / or an inhibitor of platelet aggregation, which increases the vascular effect with few side effects.
- anticoagulant such as, for example, hepan and / or an inhibitor of platelet aggregation
- the administration of anticoagulants can take place at the same time or after the administration of the compound according to the invention.
- substances which promote blood circulation or substances which improve the microcirculation are to be understood as examples which describe the subject matter of the invention even after modifications.
- R-PA is further understood to mean a recombinant plasminogen activator which consists of the domains K2 and P of human t-PA.
- the production of such plasminogen activators is described, for example, in US Pat. No. 5,223,256
- FIG. 1 is a schematic representation of the plasma clot penetration and lysis model.
- the pressure was generated through a buffer chamber (hatched area).
- the mixing of the buffer with the plasma over the clot was carried out avoided by installing a bubble trap
- 1 buffer tank, 2nd peristaltic pump, 3 bubble trap, 4 injection syringe for the fibrinolytic, 5: pipette tip with clot (cross-hatched area), 6 hose clamp, 7: pressure element
- FIG. 2 shows the cleavage of r-PA (F274P, K277V) (A) or r-PA (B) by thromboma (for more see Example 8)
- FIG. 3 shows the cleavage of r-PA (F274P, K277V) (A) or r-PA (B) by plasmin (for more details see Example 9)
- FIG. 4 shows the cleavage of r-PA (P272V, F274P, K277V) by thrombin (for more see Example 10) example 1
- the starting plasmid pA27fd contains the following components: tac promoter, lac operator region with an ATG start codon, the coding region for the t-PA mutein, consisting of the Kringle 2 domain and the Protease domain and the fd transcription terminator.
- the starting vector represents the plasmid pkk 223-3.
- fragment A the large BamHI fragment
- fragment B the vector linearized with Pvu I
- Table 1 lists the oligonucleotides used and the resulting mutations.
- the heteroduplex batch was transformed together with the plasmid pUBS520 into E. coli (Brinkmann et al., Gene 85 (1989) 109).
- the transformants were selected by adding ampicillin and kanamycin (50 ⁇ g / ml each) to the nutrient medium.
- buffer 50 mmol / 1 Tris-HC1 pH8, 50 mmol / 1 EDTA
- the insoluble protein fractions were collected by centrifugation again and resuspended in the above-mentioned buffer by sonication.
- the suspension was coated with V * volume of application buffer (250 mmol / 1 Tris-HCl pH 6.8, 10 mmol / 1 EDTA, 5% SDS, 5% Mercaptoethanol, 50% glycerin and 0.005% bromophenol blue) were added and analyzed using a 12.5% SDS polyacrylamide gel.
- application buffer 250 mmol / 1 Tris-HCl pH 6.8, 10 mmol / 1 EDTA, 5% SDS, 5% Mercaptoethanol, 50% glycerin and 0.005% bromophenol blue
- the rabbit model of neck vein thrombolysis established by D. Collen (J. Clin Invest 71 (1983) 368-376) was used.
- a radiolabelled thrombus was generated in the animals in the neck vein.
- the animals were subcutaneously anticoagulated with 100 IU / kg heparin.
- Alteplase recombinant wild-type tissue plasminogen activator, "t-PA", commercially available as Actilyse® from Thomae, Biberach, Germany
- t-PA tissue plasminogen activator
- the protein described in Example 1 streptokinase (commercially available as Streptase® from Behring, Marburg, Germany ) or solvents (0.2 M arginine phosphate buffer) were administered intravenously to the rabbits
- the placebo group received an intravenous single bolus injection of 1 mg / kg solvent.
- the Alteplase group received a total dose of 1.45 mg / kg intravenously, of which 0.2 mg / kg as an initial bolus injection and 0.75 mg / kg as 30 minute infusion, followed directly by 0.5 mg / kg as a 60 minute continuous infusion (total infusion 90 minutes).
- the streptokinase group received a 60-minute intravenous infusion of 64,000 IU / kg.
- the group with the protein according to the invention received an intravenous single bolus injection.
- thrombolysis thrombolysis
- Blood samples for obtaining plasma were taken before therapy and two hours after the start of therapy.
- the activated thromboplastin time was measured using standard methods.
- Blood loss due to thrombolytic therapy was also quantified.
- a defined skin incision of 4 cm long and 0.3 cm deep was made to the animals on the thigh with the aid of a template and a scalpel. The resulting bleeding came to a standstill as a result of natural coagulation A sponge was placed on the wound, which soaked up the blood from the bleeding newly started by thrombolysis.
- the sample is adjusted to the respectively required protein concentration by adding buffer (0.06 M Na2HPÜ4, pH 7.4, 5 mg / ml BSA (bovine serum albumin), 0.01% Tween® 80).
- buffer 0.06 M Na2HPÜ4, pH 7.4, 5 mg / ml BSA (bovine serum albumin), 0.01% Tween® 80.
- 0.1 ml of the sample was mixed with 1 ml of human fibrinogen solution (IMCO) (2 mg / ml 0.006 M Na2HP04, pH 7.4, 0.5 mg / ml BSA, 0.01% Tween® 80) and 5 min. incubated at 37 ° C.
- IMCO human fibrinogen solution
- the substances according to the invention are investigated under conditions which are quite similar to the in vivo conditions.
- the substances are placed in the plasma over the clot under the influence of a peristaltic pressure similar to the pressure caused by the heartbeat.
- Branch A contains the 1 ml pipette tip filled with the plasma clot, which closes this branch.
- Branch B is a blind line running parallel to branch A. The pressure was set to 10 mbar using a hose clamp in branch B. Plasma (1 ml) was added to the clot. The pump was turned on and the stability of each clot checked for 15 minutes.
- the fibrinolytic (final plasma concentration between 0.5 and 10 and 20 ⁇ g / ml for the proteins of Example 1 or CHO-t-PA) was injected using a 1 ml tuberculin syringe with a hypodermic needle for intramuscular injection (Braun, Melsungen, FRG) carefully injected into the plasma.
- the clot lysis time was calculated as the time difference between the addition of the fibrinolytic enzyme and the drop in pressure to 50% of the value before the addition of the fibrinolytic.
- the pressure was determined using a water-calibrated piezoelectric pressure detection system and documented using a computer-aided documentation program.
- 800 ⁇ l human citrate plasma (healthy donor) are mixed with 75 ⁇ l Ca buffer (50 mmol / l Tris / HCl, pH 7.2, 0.25 mol / 1 CaCl 2 ), 20 ⁇ l gelatin solution (10% w / v) in 0.9% NaCl) and 100 ml thrombin solution (8 U / ml, 0.05 mol / 1 sodium citrate / HCl, pH 6.5, 0.15 mol / 1 NaCl). 800 ⁇ l of this mixture are carefully transferred into a 2 ml column (Pierce, Rockfort, EL, USA). A plasma clot is formed by incubation for three hours at 37 ° C.
- Ca buffer 50 mmol / l Tris / HCl, pH 7.2, 0.25 mol / 1 CaCl 2
- 20 ⁇ l gelatin solution (10% w / v) in 0.9% NaCl
- 100 ml thrombin solution 8 U /
- 2 ml buffer (0.008 mol / 1 Na 2 HPO 4 , 0.001 mol / 1 KH 2 PO 4 , 0.003 mol / 1 KC1, 0, 137 mol / 1 NaCl, 0.1% bovine serum albumin, 0.01% Tween® 80) are adjusted to the desired concentrations (0, 0.5, 1, 2 and 3 ⁇ g / ml) with the plasminogen activator, which was previously inhibited with Glu-Gly-Arg-chloromethyl ketone, and 1 ml of this solution is applied to the surface of the clot . The remaining buffer is discarded.
- the surface of the clot is washed with 2 ml PBS buffer (0.008 mol / 1 Na HPO, 0.001 mol 1 KH 2 PO 4 , 0.003 mol / 1 KC1 and 0.137 mol / 1 NaCl) and the protein is fixed by adding 2 ml glutardialdehyde Solution in PBS.
- the clot surface is then washed with 2 ml of 50 mmol / 1 Tris / HCl, pH 8.0 and incubated with 1 ml of peroxidase-labeled polyclonal antibodies against t-PA (250 mU / ml). After washing the clot with 1 ml of PBS, the antibody-bound protein is determined by incubation with 3-amino-9-ethylcarbazole, which is converted into an insoluble red color by peroxidase.
- Plasminogen activators according to the invention are not concentrated on the surface of the clot, but penetrate into the clot and are distributed evenly.
- the intensity of the immunologically colored part of the clot increases with increasing concentration of the plasminogen activators according to the invention in the plasma
- the thrombolytically active proteins from Example 1 are tested for their ability to bind to fibrin and compared with this property also with Alteplase
- Samples of Alteplase and a protein according to the invention were prepared as solutions of 1.5 ⁇ g protein ml.
- Samples (100 ⁇ l) of the thrombolytically active protein were then each prepared with 770 ⁇ l buffer (0.05 M Tris / HCl, pH 7.4, 0, 15 NaCl, 0.01% Tween® 80), 10 ⁇ l bovine serum albumin solution (100 mg / ml), 10 ⁇ l aprotinin (3.75 mg / ml), 10 ⁇ l bovine thrombin (concentration 100 U / ml) and increasing amounts of Fibrinogen (10 ⁇ g / ml to 300 ⁇ g / ml) mixed. All solutions were water. It is known that thrombin converts fibrinogen into an insoluble fibrin clot
- the components were mixed and incubated at 37 ° C for one hour.
- the supernatant was then separated from the fibrin clot by centrifugation (15 minutes, 13,000 rpm, at 4 ° C) and the amount of plasminogen activator protein present in the supernatant was determined by a standard ELISA certainly
- the fibrinogen fragments acting as a stimulator were produced by treating human fibrinogen with cyanogen bromide (lg human fibrinogen, 1.3 g CNBr in 100 ml water) in 70% v / v formic acid over a period of 17 hours at room temperature, followed by dialysis against distilled water
- Eprobe ( E Probe t ⁇ E BV t) - ( E Probe 0 - E BV o)
- test buffer 0.1 mol / 1 Tris, pH 7.5, 15% Tween® 80
- the activity in the presence of the t-PA stimulator is divided by the activity in the absence of the t-PA stimulator.
- the dilution should be such that an almost identical absorbance is achieved in both preparations
- the activity is measured in the same way both in the absence and in the presence of the stimulator.
- the stimulation factor F is calculated as follows E sample with stimulant x V he d ünnung Pro be -with stimulator
- the specific activity is the quotient of plasminogenolytic activity (KU / ml) and protein concentration (mg / ml).
- the thrombolysis by the proteins of Example 1 produced in E. coli can be evaluated in a dog model of thrombosis of the left coronary artery induced by electrical stimulation.
- the samples were then 1: 1 (v / v) with SDS sample buffer (0.125 mol 1 Tris / HCl, pH 8.8, 4.6% (w / v) SDS, 4 mol / 1 urea, 0.1% bromophenol blue, 0.3 mol / 1 dithioerythritol) mixed, incubated for 3 min at 95 ° C and analyzed by SDS-polyacrylamide gel electrophoresis.
- SDS sample buffer 0.125 mol 1 Tris / HCl, pH 8.8, 4.6% (w / v) SDS, 4 mol / 1 urea, 0.1% bromophenol blue, 0.3 mol / 1 dithioerythritol
- FIG. 2 The cleavage of r-PA (F274P, K277V) by thrombin is shown in FIG. 2.
- the data show that r-PA (F274P, K277V) is completely converted into the two-chain form by increasing amounts of thrombin.
- the protease and kringle 2 domains of thrombin-cleaved r-PA (F274P, K277V) run at the same level as the corresponding domains of the two-chain form of r-PA (r-PA (tc)) produced by plasmin digestion.
- r-PA F274P, K277V
- r-PA (tc) two-chain form of r-PA, which was obtained by incubating r-PA with plasmin.
- r-PA F274P, K277V
- A is cleaved much more poorly by plasmin. No significant cleavage of r-PA (F274P, K277V) by plasmin was observed in the incubation with 0.025 U and 0.1 U plasmin.
- Molecular weight standard lysozyme (14,307 Da), soybean trypsin inhibitor (20,100 Da), triose phosphate isomerase (26,626 Da), aldolase (39,212 Da), glutamate dehydrogenase (55,562 Da), fructose-6-phosphate kinase (85,204 Da), ß- Galactosidase (116,353 Da), ⁇ - 2nd Macroglobulin (170,000 Da).
- r-PA (tc) two-chain form of r-PA, which was obtained by incubating r-PA with plasmin-Sepharose.
- r-PA 40 ⁇ g of r-PA (P272V, F274P, K277V) were preincubated for 15 min at 37 ° C and mixed with the units below of bovine thrombin (Sigma), which was also preincubated for 15 min at 37 ° C, and mixed for 30 min Incubated at 37 ° C.
- the samples were then mixed 1: 1 (v / v) with SDS sample buffer (0.125 mol / 1 Tris / HCl, pH 8.8, 4.6% (w / v) SDS, 4 mol 1 urea, 0.1% bromophenol blue, 0.3 mol / 1 dithioerythritol) mixed, incubated for 3 min at 95 ° C and analyzed by SDS-polyacrylamide gel electrophoresis.
- SDS sample buffer 0.125 mol / 1 Tris / HCl, pH 8.8, 4.6% (w / v) SDS, 4 mol 1 urea, 0.1% bromophenol blue, 0.3 mol / 1 dithioerythritol
- r-PA The cleavage of r-PA (P272V, F274P, K277V) by thrombin is shown in FIG. 4.
- the data show that r-PA (P272V, F274P, K277V) is completely converted into the two-chain form by increasing amounts of thrombin.
- the protease and kringle 2 domains of thrombin-cleaved r-PA (P272V, F274P, K277V) run at the same level as the corresponding domains of the two-chain form of r-PA (r-PA (tc)) produced by plasmin digestion. ).
- Lane 5 r-PA (P272 V, F274P, K277 V) + thrombin buffer
- Lane 7 r-PA (P272V, F274P, K277V) + 0.55 NIH units of thrombin
- Lane 8 r-PA (P272V, F274P, K277V) + 2.74 NTH units of thrombin
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Enzymes And Modification Thereof (AREA)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97934509A EP0917567A1 (de) | 1996-08-02 | 1997-07-21 | Thrombinaktivierbarer plasminogenaktivator |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96112487 | 1996-08-02 | ||
EP96112487 | 1996-08-02 | ||
EP97934509A EP0917567A1 (de) | 1996-08-02 | 1997-07-21 | Thrombinaktivierbarer plasminogenaktivator |
PCT/EP1997/003925 WO1998005762A1 (de) | 1996-08-02 | 1997-07-21 | Thrombinaktivierbarer plasminogenaktivator |
Publications (1)
Publication Number | Publication Date |
---|---|
EP0917567A1 true EP0917567A1 (de) | 1999-05-26 |
Family
ID=8223069
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97934509A Withdrawn EP0917567A1 (de) | 1996-08-02 | 1997-07-21 | Thrombinaktivierbarer plasminogenaktivator |
Country Status (11)
Country | Link |
---|---|
EP (1) | EP0917567A1 (tr) |
JP (1) | JP2000504941A (tr) |
KR (1) | KR20000029755A (tr) |
CN (1) | CN1226926A (tr) |
AU (1) | AU3769797A (tr) |
BR (1) | BR9711015A (tr) |
CA (1) | CA2262751A1 (tr) |
TR (1) | TR199900193T2 (tr) |
TW (1) | TW450996B (tr) |
WO (1) | WO1998005762A1 (tr) |
ZA (1) | ZA976876B (tr) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR19990081421A (ko) * | 1998-04-29 | 1999-11-15 | 성재갑 | 동물세포를 이용한 인간 혈소판 생성 촉진인자(tpo)의 제조방법 |
CN101886066A (zh) * | 2009-05-13 | 2010-11-17 | 南京大学 | 组织型纤溶酶原激活物环饼结构-2对脑卒中的治疗作用及制备方法 |
AU2018271424A1 (en) | 2017-12-13 | 2019-06-27 | Playable Pty Ltd | System and Method for Algorithmic Editing of Video Content |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5200340A (en) * | 1987-05-22 | 1993-04-06 | Zymogenetics, Inc. | Thrombin-activated tissue plasminogen activators |
JPH02438A (ja) * | 1987-10-09 | 1990-01-05 | Collaborative Res Inc | 修飾した低分子量プラスミノーゲン活性化因子およびその製造方法 |
US5504001A (en) * | 1987-11-25 | 1996-04-02 | Zymogenetics, Inc. | Hybrid plasminogen activator |
US4999194A (en) * | 1988-01-14 | 1991-03-12 | Collaborative Research, Inc. | Two-chain urokinase plasminogen activators for treatment of thrombotic disease |
GB8927722D0 (en) * | 1989-12-07 | 1990-02-07 | British Bio Technology | Proteins and nucleic acids |
GB9222758D0 (en) * | 1992-10-29 | 1992-12-09 | British Bio Technology | Proteins and nucleic acids |
-
1997
- 1997-07-21 EP EP97934509A patent/EP0917567A1/de not_active Withdrawn
- 1997-07-21 AU AU37697/97A patent/AU3769797A/en not_active Abandoned
- 1997-07-21 CN CN97196933A patent/CN1226926A/zh active Pending
- 1997-07-21 JP JP10507530A patent/JP2000504941A/ja active Pending
- 1997-07-21 CA CA002262751A patent/CA2262751A1/en not_active Abandoned
- 1997-07-21 KR KR1019997000868A patent/KR20000029755A/ko not_active Application Discontinuation
- 1997-07-21 WO PCT/EP1997/003925 patent/WO1998005762A1/de not_active Application Discontinuation
- 1997-07-21 BR BR9711015A patent/BR9711015A/pt not_active IP Right Cessation
- 1997-07-21 TR TR1999/00193T patent/TR199900193T2/tr unknown
- 1997-07-28 TW TW086110829A patent/TW450996B/zh not_active IP Right Cessation
- 1997-08-01 ZA ZA976876A patent/ZA976876B/xx unknown
Non-Patent Citations (1)
Title |
---|
See references of WO9805762A1 * |
Also Published As
Publication number | Publication date |
---|---|
KR20000029755A (ko) | 2000-05-25 |
WO1998005762A1 (de) | 1998-02-12 |
CA2262751A1 (en) | 1998-02-12 |
BR9711015A (pt) | 1999-08-17 |
JP2000504941A (ja) | 2000-04-25 |
CN1226926A (zh) | 1999-08-25 |
TW450996B (en) | 2001-08-21 |
TR199900193T2 (tr) | 1999-03-22 |
AU3769797A (en) | 1998-02-25 |
ZA976876B (en) | 1999-02-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
DE68923298T2 (de) | Gewebe-plasminogenaktivator mit zymogenen oder fibrinspezifischen eigenschaften. | |
DE69031127T2 (de) | Aktivierbare fibrinolytische und antithrombotische proteine | |
EP0966536B1 (de) | Faktor x-analoge mit modifizierter proteasespaltstelle | |
DE3382760T2 (de) | Herstellung von funktionellen menschlichen Urokinasepolypeptiden. | |
DE69434332T2 (de) | Thrombin mutanten | |
DE3689943T2 (de) | Degradierte Spezies eines Gewebeplasminogenaktivators, pharmazeutische Zubereitung und Verfahren zur Herstellung derselben. | |
DE68923741T2 (de) | Fledermausspeichel-Plasminogenaktivatoren. | |
DE3613390A1 (de) | Human-gewebeplasminogen-aktivator-mutanten | |
EP1012303B1 (de) | Faktor x-deletionsmutanten und analoge davon | |
WO1990009437A1 (de) | Gewebs-plasminogenaktivator-derivat | |
DE3853100T2 (de) | Gewebs-Plasminogen-Aktivator-Analoge mit modifizierten Wachstumsfaktordomänen. | |
EP0669394B1 (de) | Bifunktionelle Urokinasevarianten mit verbesserten fibrinolytischen Eigenschaften und thrombinhemmender Wirkung | |
DE68918279T2 (de) | Plasminogenaktivator-varianten und verfahren zu ihrer herstellung. | |
DE3852625T2 (de) | Vektoren und Verbindungen zur Expression von Zymogen-Formen von menschlichem Protein C. | |
WO1996001312A1 (de) | Nicht-glykosylierte plasminogenaktivator-derivate und ihre verwendung bei erhöhtem blutungsrisiko | |
WO1998005762A1 (de) | Thrombinaktivierbarer plasminogenaktivator | |
DE69321134T2 (de) | Plasminogen-derivate die durch thrombin aktivierbar sind | |
DE3886755T2 (de) | Verfahren zur behandlung von gefässkrankheiten. | |
DE4442665A1 (de) | Chimäre Proteine mit fibrinolytischen und thrombinhemmenden Eigenschaften | |
DE69105121T2 (de) | Gewebeplasminogenaktivator mit fibrin-spezifischen eigenschaften. | |
DE4440892A1 (de) | Proteine mit fibrinolytischen und gerinnungshemmenden Eigenschaften | |
WO1999009184A1 (de) | Plasminogenaktivator mit verbesserter zymogenität und verminderter fibrinbindung | |
WO1999009185A1 (de) | Plasminogenaktivator mit verbesserter zymogenität | |
DE69215537T2 (de) | t-PA SUBSTITUTIONSVARIANTEN MIT VERBESSERTER FIBRINSPEZIFITÄT | |
EP0400545B1 (de) | Gewebs-Plasminogen-Aktivator-Derivat |
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: 19990210 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU NL PT SE |
|
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: 20030201 |