# US 7,745,192 B2 · Prothrombin activating protein

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- Number: US 7,745,192 B2
- Document ID: US7745192B2
- Status: Granted
- Kind: B2
- Application number: 10/406,031
- Filed: 2003-04-02
- Published: 2004-03-04
- Granted: 2010-06-29
- Earliest claimed: 2003-04-02
- Assignees: Venomics PTY Limited
- Inventors: Paul Pantaleone Masci; John De Jersey; Martin Lavin
- Web page: https://patentgrep.com/patent/US7745192B2

## CPC

- A61L 15/32: Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin {(A61L15/225 takes precedence)}
- A61L 17/10: containing macromolecular materials
- A61L 26/0047: {Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042}
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/6408: {Serine endopeptidases (3.4.21)}
- 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)
- A61L 2400/04: Materials for stopping bleeding
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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 invention relates to snake venom protease polypeptides and nucleic acid sequences encoding same. This invention also relates to methods of making and using the snake venom proteases, e.g., to promote haemostasis and prevent blood loss such as during surgery or for treatment of wounds resulting from accidents and other types of injury or trauma.

## Claims

### Claim 1

A composition comprising an isolated snake venom protease (SVP), wherein the SVP comprises one light chain and one heavy chain,
wherein the light chain shares at least 95% sequence identity with the light chain sequence set forth by residues 41-179 of SEQ ID NO: 2 and the heavy chain shares at least 86% sequence identity with the heavy chain sequence set forth by residues 210-467 of SEQ ID NO:2, wherein the SVP comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO: 19-21,
wherein the SVP does not require calcium, factor Va, or phospholipid for activity, and the SVP is an active SVP capable of processing prothrombin to thrombin, and
wherein the composition has less than 30% contaminating protein.

### Claim 2 (depends on 1)

The composition of the SVP of claim 1, wherein the SVP includes a propeptide domain.

### Claim 3 (depends on 1)

The composition of the SVP of claim 1, wherein the light and heavy chains are on the same polypeptide chain.

### Claim 4 (depends on 1)

The composition of the SVP of claim 1, wherein the light and heavy chains are on different polypeptide chains.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the SVP comprises one or more of the following domains:
a first or propeptide domain which has at least 95% sequence identity with residues 1-40 of SEQ ID NO: 2;
a light chain cleavage site between residues 40 and 41 of SEQ ID NO: 2;
a domain which shares at least 95% sequence identity with residues 41-85 of SEQ ID NO: 2;
a domain which shares at least 95% sequence identity with residues 86-122 of SEQ ID NO: 2;
a domain which has at least 95% sequence identity with residues 123-165 of SEQ ID NO: 2;
a domain which has at least 95% sequence identity with residues 166-179 of SEQ ID NO: 2;
a domain which consists of residues 180-182 of SEQ ID NO: 2; and
a domain which has at least 90% sequence identity with residues 183-209 of SEQ ID NO: 2.

### Claim 6 (depends on 1)

The composition of claim 1 wherein the SVP comprises residues H251, D309, and S406 of SEQ ID NO: 2.

### Claim 7 (depends on 1)

The composition of claim 1 wherein the SVP comprises a sequence which is the same as or differs at no more than 5 residues from the sequence of amino acids 292-305 of SEQ ID NO: 2.

### Claim 8 (depends on 1)

The composition of claim 1 wherein the SVP comprises a dimeric molecule of the light chain and the heavy chain wherein the light and heavy chains have been cleaved from a propolypeptide, wherein the cleavage is between residues corresponding to 40 and 41 of SEQ ID NO: 2.

### Claim 9 (depends on 1)

The composition of claim 1 wherein the SVP comprises a dimeric molecule of the light and the heavy chain having intra-chain Cys-Cys linkages between residues corresponding to 57 and 62, 90 and 101, 95 and 110, 112 and 121, 129 and 140, and 151 and 164 of the light chain and intra-chain Cys-Cys linkages between residues corresponding to 216 and 221, 236 and 252, 377 and 391, and 402 and 430 of the heavy chain, and inter-chain Cys-Cys linkages between the residue corresponding to 172 of the light chain and the residue corresponding to 329 of the heavy chain (all references are to the amino acid numbering of SEQ ID NO: 2).

### Claim 10 (depends on 1)

The composition of the SVP of claim 1, wherein the light chain is the light chain of SEQ ID NO:2.

### Claim 11 (depends on 1)

The composition of the SVP of claim 1, wherein the SVP heavy chain has at least 95% sequence identity with the heavy chain sequence from SEQ ID NO:2.

### Claim 12 (depends on 1)

The composition of the SVP of claim 1, wherein the SVP heavy chain differs at 10 or fewer residues from the heavy chain sequence from SEQ ID NO:2.

### Claim 13 (depends on 1)

The composition of the SVP of claim 1, wherein the SVP heavy chain is the heavy chain of SEQ ID NO:2.

### Claim 14 (depends on 1)

The composition of the SVP of claim 1, wherein the pH of the composition is between about 5 and 9.

### Claim 15 (depends on 14)

The composition of claim 14, wherein the pH of the composition is about 6.5 to 7.

### Claim 16 (depends on 1)

The composition of the SVP of claim 1, further comprising a polyol.

### Claim 17 (depends on 16)

The composition of claim 16, wherein the polyol is glycerol.

### Claim 18 (depends on 1)

The composition of the SVP of claim 1 further comprising a pharmaceutically acceptable carrier.

### Claim 19 (depends on 1)

A kit comprising the composition of claim 1 and one or more of: instructions for use; a diluent; devices or other materials for preparing the composition for administration;
pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

### Claim 20 (depends on 19)

The kit of claim 19, wherein the kit comprises one or more reagents selected from the group consisting of: one or more cofactors, an anti-microbial, an antibiotic, an antiviral, an antifungal, an antiparasitic agent, an anti-inflammatory agent, an antihistamine, an anti-fibrolytic agent, an analgesic, and a growth factor.

### Claim 21 (depends on 20)

The kit of claim 20, wherein the one or more cofactors is selected from the group consisting of: calcium, a phospholipid, and factor Va.

### Claim 22 (depends on 1)

A device upon which is disposed an amount of the composition of claim 1 wherein the amount is sufficient to inhibit bleeding when the device is brought in contact with a subject.

### Claim 23 (depends on 22)

The device of claim 22, wherein said device is any of a bandage, compress, wound dressing, suture, or an article of clothing.

### Claim 24 (depends on 1)

The composition of the SVP of claim 1, wherein the SVP comprises the light chain of SEQ ID NO:2 and the heavy chain of SEQ ID NO:2.

### Claim 25 (depends on 1)

The composition of claim 1 wherein the SVP further comprises the propeptide domain set forth by residues 1-40 of SEQ ID NO: 2.

### Claim 26 (depends on 1)

The composition of the SVP of claim 1, wherein the SVP comprises the amino acid sequence of SEQ ID NO:2.

### Claim 27 (depends on 1)

The composition of the SVP of claim 1, wherein the SVP has at least 90% sequence identity with SEQ ID NO:2.