# US 8,569,237 B2 · Chimeric hirudin proteins

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- Number: US 8,569,237 B2
- Document ID: US8569237B2
- Status: Granted
- Kind: B2
- Application number: 12/619,919
- Filed: 2009-11-17
- Published: 2010-06-03
- Granted: 2013-10-29
- Earliest claimed: 2008-11-17
- Assignees: Canadian Blood Services
- Applicants: William P. Sheffield
- Inventors: William P. Sheffield
- Web page: https://patentgrep.com/patent/US8569237B2

## CPC

- C07K 14/815: from leeches, e.g. hirudin, eglin
- A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C07K 2319/50: containing protease site
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention relates to a chimeric hirudin protein comprising a carrier attached to the N-terminus of hirudin, with an intervening plasmin cleavage site. The chimeric hirudin protein contains a relatively inactive form of hirudin. However, when such chimeric hirudin protein being cleaved by plasmin in the vicinity of a clot and, ultimately causing the release of active hirudin and the reduction of the size of the clot. The chimeric hirudin protein exhibited much slower clearance in mice than unfused wild-type hirudin.

## Claims

### Claim 1

A chimeric hirudin protein comprising in this specific order:
a carrier protein having a molecular weight equal to or greater than 40 kDa and being physiologically acceptable;
a plasmin cleavage site covalently attached through its amino terminus to the carrier protein and consisting of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO: 13;
a scissile peptide bond covalently attached to the carboxyl terminus of said plasmin cleavage site; and
a latent hirudin covalently and directly attached by its amino terminus to said scissile peptide bond on the carboxyl terminus of said plasmin cleavage site, wherein when cleaved at said scissile peptide bond in the presence of plasmin the latent hirudin is released from said chimeric hirudin protein as activated hirudin.

### Claim 2 (depends on 1)

The chimeric hirudin protein of claim 1, wherein said carrier is a protein.

### Claim 3 (depends on 2)

The chimeric hirudin protein of claim 2, wherein the protein is a plasma protein.

### Claim 4 (depends on 3)

The chimeric hirudin protein of claim 3, wherein said plasma protein is albumin or alpha-1-acid glycoprotein.

### Claim 5

The chimeric hirudin protein of claim, wherein said hirudin is a hirudin variant 3.

### Claim 6 (depends on 1)

A method for limiting the growth of a clot in a subject in need thereof, said method comprising the step of administering to the subject the chimeric hirudin protein of claim 1 wherein said administration results in limiting the growth of said clot in the subject due to ability of said chimeric hirudin protein to reduce the size of the blood clot.

### Claim 7 (depends on 6)

The method of claim 6, wherein the chimeric hirudin protein is administered with a thrombolytic agent.

### Claim 8 (depends on 6)

The method of claim 6, wherein the chimeric hirudin protein is administered after the thrombolytic agent.

### Claim 9 (depends on 1)

A method for reducing the size of a clot in a subject in need thereof, said method comprising the step of administering to the subject the chimeric hirudin protein of claim 1 wherein said administration results in reducing the size of said clot in the subject due to ability of said chimeric hirudin protein to reduce the size of the blood clot.

### Claim 10 (depends on 9)

The method of claim 9, wherein the chimeric hirudin protein is administered with a thrombolytic agent.

### Claim 11 (depends on 9)

The method of claim 9, wherein the chimeric hirudin protein is administered after a thrombolytic agent.