# US 5,705,355 A · Hirudin, pharmaceutical compositions comprising it and their use

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- Number: US 5,705,355 A
- Document ID: US5705355A
- Status: Granted
- Kind: A
- Application number: 08/481,867
- Filed: 1995-06-07
- Granted: 1998-01-06
- Earliest claimed: 1984-03-27
- Assignees: Transgene S.A.
- Inventors: Paul Tolstoshev; Richard Harvey; Michael Courtney; Jean-Pierre Lecocq
- Web page: https://patentgrep.com/patent/US5705355A

## CPC

- C07K 14/815: from leeches, e.g. hirudin, eglin
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention relates to vectors for cloning, in a host cell, hirudin or an analog of hirudin, characterized in that it comprises the gene for encoding hirudin or an analog of hirudin, and the elements for the expression of this gene in said host cell, said coding gene beginning, after the starting sequence, by an Ile codon and a Thr codon. This invention also relates to the process for the preparation of hirudin or analogs of hirudin as well as the hirudin or anologs of hirudin obtained.

## Claims

### Claim 1

A substantially pure hirudin having an amino acid sequence as follows:
______________________________________ 1 5 10 ATT ACT TAC ACT GAT TGT ACA GAA TCG GGT Ile THr Tyr Thr Asp Cys Thr Glu Ser Gly 15 20 CCA AAT TTG TGC CTC TGC GAG GGA AGC AAT Gln Asn Leu Cyc Leu Cyc Glu Gly Ser Asn 25 30 GTT TGC CCT AAA GGC AAT AAG TGC ATA TTG Val Cys Gly Lys Gly Asn Lys Cys Ile Leu 35 40 GGT TCT AAT GGA AAG GGC AAC CAA TGT GTC Gly Ser Asn Gly Lys Gly Asn Gln Cys Val 45 50 ACT GGC GAA GGT ACA CCG AAC CCT GAA AGC Thr Gly Glu Gly Thr Pro Asn Pro Glu Ser 55 60 CAT AAT AAC GGC GAT TTC GAA GAA ATT CCA His Asn Asn Gly Asp Phe Glu Glu Ile Pro 65 GAA GAA TAT TTA CAA Glu Glu Tyr Leu Gln. ______________________________________

### Claim 2

A substantially pure variant hirudin having an amino acid sequence as follows:
__________________________________________________________________________ 1 5 10 ATT ACT TAC ACT GAT TGT ACA GAA TCG GGT CCA AAT TTG TGC Ile Thr Tyr Thr Asp Cys Thr Glu Ser Gly Gln Asn Leu Cys 15 20 25 CTC TGC GAG GGA AGC AAT GTT TGC CCT AAA GGC AAT AAG TGC Leu Cys Glu Gly Ser Asn Val Cys Gly Lys Gly Asn Lys Cys 30 35 40 ATA TTG GGT TCT AAT GGA AAG GGC AAC CAA TGT GTC ACT GGC Ile Leu Gly Ser Asn Gly Lys Gly Asn Gln Cys Val Thr Gly 45 50 55 GAA GGT ACA CCG AAC CCT GAA AGC CAT AAT AAC GGC GAT TTC Glu Gly Thr Pro Asn Pro Glu Ser His Asn Asn Gly Asp Phe 60 65 GAA GAA ATT CCA GAA GAA TAT TTA CAA Glu Glu Ile Pro Glu Glu Tyr Leu Gln __________________________________________________________________________
wherein the amino acid Ile in position 1 is replaced by the amino acid Val and the amino acid Thr in position 2 is replaced by the amino acid Val.

### Claim 3 (depends on 1)

The hirudin of claim 1, wherein said hirudin does not contain any glycosylation or sulfation.

### Claim 4 (depends on 1)

A pharmaceutical composition comprising the hirudin of claim 1.

### Claim 5 (depends on 1)

A method for diagnosing clot formation in man or animal, comprising:
providing labeled hirudin, wherein said hirudin is the hirudin of claim 1;
administering a diagnostically effective amount of said labeled hirudin to said man or animal under conditions such that said labeled hirudin selectively binds to thrombin; and
detecting said labeled hirudin, which is selectively bound to thrombin, thereby visualizing the formation of a clot.

### Claim 6 (depends on 1)

An extracorporeal blood circuit, wherein at least part of the circuit in contact with the blood is coated with the hirudin of claim 1.

### Claim 7 (depends on 1)

A process for the separation of a clotting factor from blood or from a blood fraction, wherein the separation is performed in the presence of the hirudin of claim 1.