IL88925A - Hirudin derivative and pharmaceutical composition containing same - Google Patents
Hirudin derivative and pharmaceutical composition containing sameInfo
- Publication number
- IL88925A IL88925A IL8892589A IL8892589A IL88925A IL 88925 A IL88925 A IL 88925A IL 8892589 A IL8892589 A IL 8892589A IL 8892589 A IL8892589 A IL 8892589A IL 88925 A IL88925 A IL 88925A
- Authority
- IL
- Israel
- Prior art keywords
- gly
- glu
- thr
- cys
- leu
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Hydrogenated Pyridines (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Plural Heterocyclic Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A hirudin derivative which has the N-terminal amino-acid sequence Leu-Thr-Tyr-Thr-Asp has high biological activity. This hirudin derivative can be obtained very efficiently by production in genetically engineered yeasts.
Description
Hoe 88/F 0 3 A HIRUDIN DERIVATIVE AND. PHARMACEUTICAL COMPOSITIOH CONTAINING SAME • IT η jan .^nsim T»qprn ρ-η-ρ,π m m HOECHST A TJENGESELLSCHAFT HOE 88/F 043 Dr. L/ml Description Derivatives of hirudin and the genetic engineering pre-"p a rat ion thereof are disclosed in the European Patent Application with the publication number (EP-A) 0 , 1 7 1 , 024, correspondi nq to IL 76056.
It has now been found that the hirudin derivative of the amino acid sequence 0 1 10 Leu- Thr-Tyr-Thr- Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys- 20 Leu- Cys- Glu- Gly- Ser- Asn- Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys- 30 40 lie- Leu- Gly- Ser- Asp- Gly- Glu- Lys- Asn-Gln-Cys-Val-Thr-Gly- 50 Glu-Gly-Thr-Pro- Lys-Pro-Gln- Ser- His- Asn- Asp- Gly- Asp- Phe- 60 Glu- Glu- I le- ro- Glu- Glu- Tyr- Leu- Gin has a number of advantages. The numbering used in EP-A 0 , 1 7 1 , 024 has been retained in this sequence. Hirudin and its derivatives differ in iological acti ity, which can be attributed to differences in the affinity for thrombin and/or differences in the stability. The hirudin derivative according to the invention i s , s u rp r i s i n g I y , distinguished by special activity.
It has also been found that the hirudin derivative according to the invention is particularly ad antageousl expressed in yeasts. Comparison experiments showed that analogous hirudin deri atives starting w th N-terminal Thr-Tyr or Ile-Tyr are expressed only in low yields.
Expression from yeast cells is advantageous not only because the hirudin derivative is secreted but also, and espec a y, ecause it is virtually quantitavely in the correctly folded form and has high activity.
Figure 1 shows cloning vectors useful for making a gene structure which codes for the yeast MFa precursor protein and the hirudin derivative according to this invention. Figure 2 shows a yeast expression vector containing this gene structure.
The hirudin derivative according to the invention can, of course, also be prepared by different methods, for example by expression in bacteria or in higher eukaryotic cells such as insect cells or animal cells. However, expression from yeast systems is preferred, for example using the yeast species listed in EP-A 0,248,227, for example, Pichia pastoris, Hansenula polymorphis, Schizosaccharo yces pombe or, preferably, Saccharomyces cerevisiae.
A large number of vectors are known for expression in yeasts, e. g. from EP-A 0,060,057, 0,008,632, 0,116,201, 0,121,884, 0,123,544 and 0,195,691. The preparation of the hirudin derivative according to the invention is described hereinafter using the yeast a factor system, but this is to be understood to be merely by way of example, since other expression systems can also be used in a manner known per se.
The structure of the yeast pheromone gene MFa is known from Kurjan and Herskovitz, Cell 30 (1982) 933-943, where the possibility of expression of other genes and the secretion of the gene products is also discussed. In this connection, reference may also be made to Brake et al., Proc. Natl. Acad. Sci 81 (1984), 4642-4646.
The yeast vectors which are advantageously used are so-called shuttle vectors which have an origin of replication of a bacterial plasmid and of a yeast plasmid, as well as genes for selection in both host systems. Furthermore, vectors of this type contain the promoter sequences necessary for the expression of foreign genes and, where appropriate, a terminator sequence for improving the yield, so that the heterologous gene - expediently fused to secretory signals - is located between the promoter and terminator.
The present invention is for selection Patent vis a vis Patent No.76059.
The invention is explained in detail by the Examples which follow. Percentage data relate to weight.
Example 1: Construction of the expression vector Firstly the DNA sequence I (Table 1) is synthesized by the phosp ite method. This DNA sequence codes for amino acids 49 to 80 of the MFa precursor protein and essentially corresponds to the natural ONA sequence.
DNA sequence I is initially used as a probe for isolating the gene for the a factor, and for this purpose is label-ed with ^P. This probe is used to i s o a t e* f r om a genomic Xgt11 yeast gene bank (as are now commercially available from, for example, Clontech Laboratories Inc., 4055 Fabian Way, Palo Alto, CA94303). For this purpose, λ g 111 phages which carry the a factor gene are identified in a plaque-h bridization experiment. Phages from plaques identified as positive are isolated and propagated, and the ONA is obtained. The latter is cleaved with EcoRI and analyzed on a 0.8% agarose gel. After a Southern transfer experiment, the membrane is hybridized with the ^P-labeled DNA sequence I. Phage DNA which has an appro mately 1.75 kb fragment which hybridizes with DNA sequence I is again cleaved with the enzyme, and the correspond ng fragment is isolated. The vector pUC 19 is opened with EcoRI and reacted with the 1.75 kb fragment using T4 ligase. The cloning vector 1 is obtained.
The cloning vectors which are listed in Table 2 were all constructed on the basis of a pUC plasmid. This table shows only the polyl inker region of these vectors in the usual 5'-3' direction, with the MFa sequences being indicated by dotted lines, and the hirudin sequences being indicated by broken lines. Full lines denote pUC and linker sequences. Fig. 1 shows these cloning vectors as diagrams (not drawn to scale) .
The strain E. coli 79/02 is transformed with the ligation mixture. White colonies are isolated, the pLasmid DNA is obtained from them, and plasmids which contain the 1.75 kb EcoRI fragment are identified. * the gene The natural DNA sequence of the precursor protein for MFa contains in the region of*amino acids 8 to 10 a Pstl cLeavage site and in the region of*amino acids 48/49 a Taql cleavage site. The fragment which codes for amino acids 9 to 48 of the MFa precursor sequence is now isolated from the isolated plasmid DNA by reaction with Pstl and Taql. The vector pUCl8 is opened with Pstl and Kpnl and is reacted with the Pstl-Taql fragment as well as with the synthet c DNA sequence I using T4 ligase. E. coli 79/02 is transformed with the ligation mixture. The transformation mixture is plated out on IPTG-Xgal-Ap plates.
White colonies are isolated, and the plasmid DNA of these clones is characterized by restriction analysis. In this way is obtained the cloning vector 2 which codes for amino acids 8 to 80 of the MFa precursor sequence.
The said coding sequence is cut out of the cloning vector 2 by reaction with Pstl and Kpnl, and is introduced into the ligation described below. For this purpose, the clo-ning vector 1 is reacted with EcoRI and partially with Pstl, and the fragment comprising the coding sequence for the first 8 amino acids of the MFa precursor sequence is isolated. In addition, the vector pUCl9 is opened with EcoRI and Kpnl and ligated with the two fragments described, resulting in the cloning vector 3. The latter codes for the complete MFa precursor sequence up to amino acid 80.
The starting material used for most of the hirudin sequence is the synthetic gene which is depicted in EP-A 0,171,024 as "DNA sequence I" and is shown in the present Table 1 as DNA sequence IV. The restriction enzyme cleavage sites in this sequence are emphasized by underlining: Accl cuts in the region of amino acids 1 to 3, BamHI cuts in the region of amino acids 30/31, and Sacl cuts starting with the last stop codon. The protruding sequence for Xbal is located at the 5' end of the gene, and the protruding sequence for Sail is located at the 3' end.
This synthetic gene was subcloned in two parts (Figures * the codons for 1 and 2 in EP-A 0,171,024). These subcLoning vectors are depicted in Table 2 under No. 4 (corresponding to Figure 2 of EP-A 0,171,024) and 6 (corresponding to Figure 1 of EP-A 0,171,024).
The cLoning vector 4 is opened with HincII and Hindlll, and the Linearized DNA is Ligated with DNA sequence II (Table 1). An Ncol cleavage site has been formed at the site which has undergone blunt-ended ligation in the cloning vector 5 obtained in this way.
The fragment coding for the hirudin part-sequence is cut out of the cloning vector 6 by total digestion with BamHI and Accl. This fragment is then ligated with the cloning vector 3 which has been opened with BamHI and Kpnl, and with DNA sequence III (Table 1). The last three codons in DNA sequence III are numbered in the same way as in DNA sequence IV (Table 1). This results in the cloning vector 7 which codes for the first 80 amino acids of the MFct precursor sequence and the first 30 amino acids of the hirudin derivative according to the invention, as has been confirmed by DNA sequence analysis.
The fragment which codes for amino acids 31 to 64 of hirudin is cut out of the cloning vector 5 with BamHI and Hindlll. This fragment is ligated into the cloning vector 7 which has been opened with the same enzymes, resulting in the cloning vector 8 which codes for the first 80 amino ac ds of the MFct precursor sequence and the complete sequence of the hirudin derivative according to the invention. The structure of this plasmid is confirmed by restriction analysis.
The plasmid Yep13 (Broach et al., Gene 8 (1979) 121) is opened with BamHI, and the protruding ends are filled in with lenow polymerase. The DNA is precipitated with ethanol and treated with bovine alkaline phosphatase.
The fragment coding for the hirudin derivative and the MFct precursor sequence is cut out of the cloning vector 8 (Table 2) with Ncol and EcoRI, and the protruding ends are filLed in as described.
The two blunt-ended DNA sequences are ligated together, resulting in plasmids potfHir17 and pctfHir18 (Figure 2 . These two plasmids differ only in the orientation of the inserted fragment.
It is possible to insert, as described in EP-A 0,171,024, a terminator downstream of the inserted sequence (Figures 4 to 6 of EP-A 0,171,024). Suitable for this purpose are the Ncol and/or BamHI cleavage sites.
After ampl fication of the plasmid DNA in E. coli MM294, the plasmid pctfHir17 is transformed into the I euc i ne-depen-dent yeast strains Y79 (a, trp1-1, leu2-1) (Cantrell et al., Proc. Acad. Natl. Sci. USA 82 (1985) 6250) and DM6-6 (ct/otl eu2-3, 112 : : u r a3 + / I eu2 : : I y s2 + , trp1~/trp1~, his3-11, 15/his3-11, 15, ura3~/ura3~, Iys2~/lys2~, a r g4- 17 / a r g4+ , ade1~/ade1+) (Maya Hanna, Dept. Mol. Biol. Massachusetts General Hospital, Boston, USA) by the lithium method of Ito, H. et al., J. Bacterid. 153 ( 1983) 163 Isol at i on of single colonies which are able to grow on se I e c t i v e medium without added leucine is carried out. Yea s t m i n i ma I medium is inoculated with the individual col on i es and i n-cubated at 28°C for 24 hours. The cells are spun dow n and the supernatant is examined in a thrombi n i n h i b i t i on assay for hirudin activity. The plasmid DNA from yea s t clones whose supernatant shows hirudin activ i t y i s re i s 0-lated and characterized by restriction analy sis. The t r ans formed yeast strains are used for the expres s i on test s which follow.
Example 2: Expression 10 ml of yeast complete medium is inoculated with eel Is taken from a fresh overnight culture of a st rain ob t a i ned as in Example 1, from selective medium, in such a way that an optical density OD^QQ = 0.1 is reached. The culture is shaken at 28°C for 8 hours and then 90 ml of fresh medium are added. The culture is then shaken for a further 20 hours. The cells are spun down, and the hirudin activity in the supernatant is determined.
Example 3: Working up Supernatant obtained as in Example 2 is acidified to pH 3 to 5 and applied to an adsorption column containing a porous adsorber resin composed of a copolymer of styrene ( R ) and d i v i ny I benzene ( DIAION HP 20) which has been equilibrated with 0.1 M acetic acid. Washing with Tris . HCl (pH 8.5) and 50 mM acetic acid is followed by elution with 30% strength isopropanol. The fractions containing the hirudin derivative are combined and purified on a Q-SEPH- ( R ) AROSE column which has been equilibrated with 20 mM piperazine . HCl (pH 6). Elution in this case is with a 0 - 0.25 M NaC gradient. The fractions containing the hirudin derivative are again combined and purified by HPLC on a C18 reversed phase chromatography column. The pure product obtained in this way is then subjected to automated protein sequence analysis.
Example 4: Comparison example If the procedure of Example 1 is used but with the following sequences in place of DNA sequence III (Table 1), then only minimal hirudin activity is detectable in the supernatant of the yeast culture. 1 2 (Pro) Leu Asp Lys Arg Thr (Tyr) 5' CT TTG GAT AAA AGA ACG T Ilia 3' CAT GGA AAC CTA TTT TCT TGC ATA ( pnl) (Accl) 1 2 (Pro) Leu Asp Lys Arg lie (Tyr) 5' CT TTG GAT AAA AGA ATA T Illb 3' CAT GGA AAC CTA TTT TCT TAT ATA (Kpnl) When DNA sequence 111 b is used, t he vectors corresponding to cloning vectors 7 and 8 (Table 2) do not conta in the Accl cleavage site.
DNA sequences 50 55 C GAT GTT GCT GTT TTG CCA TTC TCC TA CAA CGA CAA AAC GGT AAG AGG (Taql) 60 65 AAC AGT ACT AAT AAC GGT TTA TTG TTC TTG TCA TGA TTA TTG CCA AAT AAC AAG 70 ATT AAT ACT ACT ATT GCT AGC ATT GCT TAA TTA TGA TGA TAA CGA TCG TAA CGA 75 80 GCT AAA GAA GAA GGG GTA C 3' CGA TTT CTT CTT CCC 5' (Kpnl) II. 5 ' CATGGA 3 ' 3 ' GTACCTTCGA 5 ' (Hindlll) (Pro) Leu Asp Lys Arg Leu Thr (Tyr) III. 5' CT TTG GAT AAA AGA CTT ACG T 3' 3* CAT GGA AAC CTA TTT TCT GAA TGC ATA 5' (Kpnl) (Accl) DNA sequence IV Tr iplet No.
Amino ac d 0 1 2 3 4 5 Nucleotide No. Met Thr Tyr Thr Asp Cys 1 10 20 Cod. strand 5' 1 CT AGA ATG ACG TAT ACT GAC 1 Non- cod. strand 3 T TAC TGC ATA TGA CTG 6 7 8 9 10 11 12 13 14 15 Thr Glu Ser Gly Gin Asn Leu Cys Leu Cys 30 40 50 ACT GAA TCT GGT CAG AAC CTG TGC CTG TGC TGA ' CTT AGA CCA GTC TTG GAC ACG GAC ACG 16 17 18 19 20 21 22 23 24 25 Glu Gly Ser Asn Val Cys Gly Gin Gly Asn 60 70 80 GAA GGA TCT AAC GTT TGC GGC CAG GGT AAC CTT CCT AGA TTG CAA ACG CCG GTC CCA TTG 26 27 28 29 30 31 32 33 34 35 Lys Cys lie Leu Gly Ser Asp Gly Glu Lys 90 100 110 AAA TGC ATC CTT GGA TCC GAC GGT GAA AAG TTT ACG TAG GAA CCT AGG CTG CCA CTT TTC 36 37 38 39 40 41 42 43 44 45 Asn Gin Cys Val Thr Gly Glu Gly Thr Pro 120 130 140 AAC CAG TGC GTT ACT GGC GAA GGT ACC CCG TTG GTC ACG CAA TGA CCG CTT CCA TGG GGC 46 47 48 49 50 51 52 53 54 55 Lys Pro Gin Ser His Asn Asp Gly Asp Phe 150 160 170 AAA CCG CAG TCT CAT AAC GAC GGC GAC TTC TTT GGC GTC AGA GTA TTG CTG CCG CTG AAG 56 57 58 59 60 61 62 63 64 Glu Glu He Pro Glu Glu Tyr Leu Gin Stp 180 190 200 GAA GAG ATC CCT GAG GAA TAC CTT CAG TAA CTT CTC TAG GGA CTC CTT ATG GAA GTC ATT Stp 210 TAG AGC TCG 3' ATC TCG AGC AGC T 5* Table 2: Cloning vectors No. pUC 1 19 -Ε···(1.75 kb a- Fragment )·· -E- 2 18 -Κ· · · (α-80-49) · · ·Τ· · · (a-48-8) · · -P- 3 19 -B-K- ·· (a-80-49) ·· ·Τ· ·· (a-48-8) ·· ·Ρ· · ·Ε- 4 8 -B (Hir31-64)-S-Hc-Hd- 5 8 -B (Hir31-64)-S-N-Hd- 6 12 -B (Hir30-3) A X-A- 7 19 - Hd- B (Hir30-3) A K · · · (a- 80-8) · · -P 8 19 -Hd-N-S--- (Hir64-3)---A---K- · · (a-80-8) · · ... MFot sequences hirudin sequences Abbreviations for restriction enzymes A = Accl B = BamHI E EcoRI He = Hindi Hd = Hindlll K = Kpnl N = Ncol P = Pstl S = Sail T = Taql X = Xbal 88925/2 - 11 - 88/F 043 PATENT
Claims (2)
1. CLAIMS A hirudin derivative with the amino acid sequence 0 1 10 Leu- Thr- Tyr- Thr- Asp- Cys- Thr- Glu- Ser- Gly- Gin- Asn- Leu-.Cys- 20 Leu- Cys- Glu- Gly- Ser- Asn- Val- Cys- Gly- Gin- Gly- Asn- Lys- Cys- 30 40 Ile- Leu- Gly- Ser- Asp- Gly- Glu- Lys- Asn- Gin- Cys- Val- Thr- Gly- 50 Glu-Gly-Thr-Pro-Lys- Pro- Gin- Ser- His- Asn- Asp- Gly- Asp- Phe- 60 Glu- Glu- lie- Pro- Glu- Glu- Tyr- Leu- Gin DNA coding for the polypeptide having the amino acid sequence as claimed in claim 1. Vectors containing a DNA sequence as claimed in claim
2. A process for the preparation of a polypeptide as claimed in claim 1, which comprises expression of a DNA as claimed in claim 2 in a host cell. The process as claimed in claim 4 , wherein the host cell is a yeast cell. Attorneys for Appl icant
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HU851341A HU198085B (en) | 1984-04-18 | 1985-04-11 | Process for producing new hirudine derivatives with anticoagulant effect and pharmaceutical compositions comprising same as active ingredient |
| DE3805540 | 1988-02-23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| IL88925A true IL88925A (en) | 1995-11-27 |
Family
ID=25865106
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL8892589A IL88925A (en) | 1985-04-11 | 1989-01-11 | Hirudin derivative and pharmaceutical composition containing same |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0324712B1 (en) |
| AT (1) | ATE87938T1 (en) |
| DE (2) | DE19775033I2 (en) |
| DK (1) | DK172350B1 (en) |
| ES (1) | ES2055149T3 (en) |
| FI (1) | FI96956C (en) |
| IL (1) | IL88925A (en) |
| LU (1) | LU90127I2 (en) |
| NL (1) | NL970027I2 (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3835815A1 (en) * | 1988-10-21 | 1990-04-26 | Hoechst Ag | NEW ISOHIRUDINE |
| JP3187044B2 (en) * | 1989-12-01 | 2001-07-11 | ビーエーエスエフ アクチェンゲゼルシャフト | Hirudin mutein and hirudin polyalkylene glycol complex |
| DE4140381A1 (en) * | 1991-12-07 | 1993-06-09 | Hoechst Ag, 6230 Frankfurt, De | NEW SYNTHETIC ISOHIRUDINE WITH IMPROVED STABILITY |
| DE4404168A1 (en) * | 1994-02-10 | 1995-08-17 | Hoechst Ag | Hirudin derivatives and process for their preparation |
| DE19529997C1 (en) | 1995-08-16 | 1997-04-10 | Hoechst Ag | A method for inactivating carboxypeptidase Y in hirudin-containing culture broths |
| DE19543737A1 (en) * | 1995-11-24 | 1997-05-28 | Hoechst Ag | Process for the ultrafiltration of biological matrices containing peptides or proteins |
| DE19544233A1 (en) * | 1995-11-28 | 1997-06-05 | Hoechst Ag | Process for using the yeast ADH II promoter system for the biotechnological production of heterologous proteins in high yields |
| EP0787569B1 (en) | 1996-01-31 | 2002-10-02 | Sumitomo Bakelite Company Limited | Method of producing epoxy resin-encapsulated semiconductor device |
| DE19607239A1 (en) | 1996-02-27 | 1997-08-28 | Behringwerke Ag | Pharmaceutical composition containing hirudin and process for its preparation |
| DE19944870A1 (en) | 1999-09-18 | 2001-03-29 | Aventis Pharma Gmbh | Signal sequences for the production of Leu-hirudin via secretion by E. coli in the culture medium |
| DE10033195A1 (en) * | 2000-07-07 | 2002-03-21 | Aventis Pharma Gmbh | Bifunctional fusion proteins from hirudin and TAP |
| US7638618B2 (en) | 2001-02-20 | 2009-12-29 | Sanofi-Aventis Deutschland Gmbh | Nucleic acids encoding a hirudin and pro-insulin as superscretable peptides and for parallel improvement of the exported forms of one or more polypeptides of interest |
| US7202059B2 (en) | 2001-02-20 | 2007-04-10 | Sanofi-Aventis Deutschland Gmbh | Fusion proteins capable of being secreted into a fermentation medium |
| JP2006096668A (en) * | 2002-11-08 | 2006-04-13 | Ono Pharmaceut Co Ltd | Medicine comprising combination of elastase inhibitor with enzyme inhibitor of blood coagulation system and/or fibrinolysis system |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3438296A1 (en) * | 1984-04-18 | 1985-11-07 | Hoechst Ag, 6230 Frankfurt | NEW POLYPEPTIDES WITH A BLOOD-CLOTHING EFFECT, METHOD FOR THE PRODUCTION OR THEIR RECOVERY, THEIR USE AND THE CONTAINERS THEREOF |
| EP0209061B1 (en) * | 1985-07-17 | 1994-01-12 | Hoechst Aktiengesellschaft | Peptides having an anticoagulant activity, process for their preparation, obtention, their use and agents containing them |
-
1989
- 1989-01-11 FI FI890127A patent/FI96956C/en not_active IP Right Cessation
- 1989-01-11 DE DE19775033C patent/DE19775033I2/en active Active
- 1989-01-11 EP EP89710003A patent/EP0324712B1/en not_active Expired - Lifetime
- 1989-01-11 IL IL8892589A patent/IL88925A/en active IP Right Grant
- 1989-01-11 ES ES89710003T patent/ES2055149T3/en not_active Expired - Lifetime
- 1989-01-11 DE DE8989710003T patent/DE58903988D1/en not_active Expired - Lifetime
- 1989-01-11 AT AT89710003T patent/ATE87938T1/en active
- 1989-01-12 DK DK013189A patent/DK172350B1/en not_active IP Right Cessation
-
1997
- 1997-07-23 NL NL970027C patent/NL970027I2/en unknown
- 1997-08-20 LU LU90127C patent/LU90127I2/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NL970027I1 (en) | 1997-10-01 |
| FI96956C (en) | 1996-09-25 |
| FI890127A (en) | 1989-07-14 |
| ATE87938T1 (en) | 1993-04-15 |
| NL970027I2 (en) | 1998-04-01 |
| DE58903988D1 (en) | 1993-05-13 |
| DK13189A (en) | 1989-07-14 |
| DE19775033I2 (en) | 2011-01-20 |
| LU90127I2 (en) | 1997-10-06 |
| FI890127A0 (en) | 1989-01-11 |
| EP0324712A3 (en) | 1990-07-04 |
| DK172350B1 (en) | 1998-04-06 |
| ES2055149T3 (en) | 1994-08-16 |
| EP0324712B1 (en) | 1993-04-07 |
| DK13189D0 (en) | 1989-01-12 |
| FI96956B (en) | 1996-06-14 |
| EP0324712A2 (en) | 1989-07-19 |
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