Tissue factor based prothrombin time reagent

• US 5,314,695 A
• Filed: 10/29/1991
• Issued: 05/24/1994
• Est. Priority Date: 11/13/1990
• Status: Expired due to Term

• Current Assignee: Corvas Internation Incorporated
• Original Assignee: Corvas International, Inc. (Bausch Health Cos., Inc.)
• Inventors: Brown, Scott M.
• Primary Examiner(s): Page, Thurman K.
• Assistant Examiner(s): KISHORE, GOLLAMUDI S
• Application Number:
  US07/784,326
• Time in Patent Office: 938 Days
• Field of Search: 424/450, 428/402.2, 264/4.1, 264/4.3, 264/4.6, 514/2, 514/21, 530/381, 436/18, 436/69
• US Class Current: 424/450
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61K 9/1277 Processes for preparing; Proliposomes
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: C07K 14/745 Blood coagulation or fibrinolysis factors
• CPC Class Codes: G01N 2333/745 Assays involving non-enzymic blood coagulation factors
• CPC Class Codes: G01N 2405/04 Phospholipids, i.e. phosphoglycerides
• CPC Class Codes: G01N 33/5432 Liposomes or microcapsules
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]

Abstract

Prothrombin time reagents are provided which comprise novel liposome compositions in which tissue factor is associated with an inserted into the phospholipid bilayer of the liposomes. Methods for their preparation are also provided. The liposome compositions may be adjusted to allow maximum coagulant activity and sensitivity to extrinsic coagulation factors.

Claims

1. A prothrombin time reagent containing purified natural or recombinant tissue factor which comprises a liposome composition comprising:

(a) a phospholipid mixture comprising;

(i) from about 20 to about 95 mole percent phosphatidylcholine,

(ii) from about 2.5 to about 50 mole percent phosphatidylethanolamine,

(iii) from about 2.5 to about 50 mole percent phosphatidylserine, and

(iv) from about 0 to 40 mole percent phosphatidylglycerol; and

(b) from about 0.1 μ

g to about 3 μ

g of tissue factor per mg phospholipid mixture.

2. A prothrombin time reagent according to claim 1 further comprising from about 0.5 percent to about 1.5 percent glycine.

3. A prothrombin time reagent according to claim 1 or 2 further comprising a carbohydrate cryopreservative selected from a group consisting of trehalose, maltose, lactose, glucose, and mannitol.

4. A prothrombin time reagent according to claim 3 wherein the carbohydrate cryopreservative comprises about 50 mM to 250 mM trehalose.

5. A prothrombin time reagent according to claim 4 wherein said phospholipid mixture comprises from about 5 to about 15 mole percent phosphatidylethanolamine;

from about 5 to about 20 mole percent phosphatidylserine, from about 10 to 25 mole percent phosphatidylglycerol and the remainder phosphatidylcholine.

6. A prothrombin time reagent according to claim 4 wherein said phospholipid mixture comprises about 8 to about 12 mole percent phosphatidylethanolamine, from about 3 to about 10 mole percent phosphatidyl serine, from about 14 to about 20 mole percent phosphatidylglycerol, and from about 58 to about 75 mole percent phosphatidylcholine.

7. A prothrombin time reagent according to claim 1 or 2 wherein said phospholipid mixture comprises about 8 to about 12 mole percent phosphatidylethanolamine, from about 3 to about 10 mole percent phosphatidyl serine, from about 14 to about 20 mole percent phosphatidylglycerol, and from about 58 to about 75 mole percent phosphatidylcholine.

8. A prothrombin time reagent according to claim 7 wherein said tissue factor is recombinant tissue factor.

9. A prothrombin time reagent according to claim 4, comprising from about 0.6 to about 1.2 percent (w/v) glycine.