Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-12-10
2001-06-19
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C424S900000, C530S350000, C530S381000, C436S829000
Reexamination Certificate
active
06248353
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of incorporating membrane proteins into preformed liposomes. It further relates to methods of making a prothrombin time (PT) reagent using purified, reconstituted natural or recombinant human tissue factor (rTF). More particularly, the invention relates to the reconstitution of tissue factor (TF) into phospholipid liposomes to produce a tissue factor-based PT reagent.
BACKGROUND OF THE INVENTION
Membrane proteins are critical for cellular function and include receptors, ion pumps, electron transport proteins, signal transducers and regulators of the intracellular environment. The isolation and reconstitution of these proteins into membranes has been well studied and is well documented.
Liposomes are a general category of vesicles which comprise one or more lipid bilayers surrounding an aqueous space. Liposomes include unilamellar vesicles composed of a single membrane or lipid bilayer, and multilamellar vesicles (MLVs) composed of many concentric membranes (or lipid bilayers). Liposomes are commonly prepared from phospholipids. Due to unique characteristics of these vesicles, liposomes have been widely used as a model membrane for investigating the properties of biomembranes and for studying the functions of membrane proteins.
There are essentially four presently known mechanisms for incorporating, i.e., reconstituting, proteins into liposomes. See Rigaud, J- L., et al., “Liposomes as Tools for the Reconstitution of Biological Systems,” p. 71-88, in Liposomes as Tools in Basic Research and Industry, ed. Philippot, J. R. and Schuber, F., CRC Press, Boca Raton, Fla. (1995). One method involves the use of an organic solvent. However, such procedures often result in the denaturation of the proteins. A second method uses mechanical means to produce large and small unilamellar vesicles from MLVs by swelling of the dry phospholipid films in excess buffer. Such mechanical means include sonication of MLVs, forcing multilamellar lipid vesicles through a French press, or cycles of freeze-thawing or dehydration-rehydration. Drawbacks with sonication include variability and inactivation of certain proteins by sonication as well as production of small liposomes. A third process involves the direct incorporation of proteins into preformed small unilamellar liposomes, also termed spontaneous incorporation. Such methods are usually catalyzed by low cholate or lysolecithin concentrations. Problems with these methods include the wide size distribution of the proteoliposomes, heterogeneous distribution of the protein among the liposomes and presence of the non-phospholipid impurities, required for an effective protein incorporation, that would affect performance of those liposomes. The fourth and most often used method of incorporating proteins into liposomes involves the use of detergents. In such a method, the proteins and phospholipids are cosolubilized in a detergent to form micelles. The detergent is then removed, resulting in the spontaneous formation of bilayer vesicles with the protein incorporated therein. The detergent is incorporated into liposome as well as the protein and thus, these methods require removal of the detergent by methods such as dialysis, gel exclusion chromatography or adsorption on hydrophobic resins. The methods that use detergent are very slow because the detergent removal must be as complete as possible and also because a phase change that takes place during this process slows detergent removal even further. The detergent is also difficult to remove completely. Another disadvantage is that one cannot control the orientation of protein incorporated into the liposomes by using the detergent methods.
Liposomes have several properties which make them useful in various applications. The most important of these characteristics are the uniform controllable size and the surface characteristics which can control the biological fate of the liposomes. These properties make liposomes preferred carriers for drug delivery systems and the basis for reagents for assays. For example, liposomes containing tissue factor have been used as reagents for prothrombin time (PT) assays for testing coagulation of blood. In these cases, the phospholipid constituent of the liposomes is used as a substitute for platelet phospholipids, which are essential for normal hemostasis in vivo. For example, Dade Behring Inc. presently produces INNOVIN® for use in PT determinations and prothrombin time-based assays. This product is prepared from purified human tissue factor produced in
E. coli
combined with synthetic phospholipids (thromboplastin), calcium, buffers and stabilizers.
Coagulation of blood occurs by two pathways, the intrinsic pathway and the extrinsic pathway. In the intrinsic (endogenous or foreign contact dependent) pathway the chain of events leading to coagulation is set in motion merely by exposure of plasma to nonendothelial surfaces, such as glass in vitro or collagen fibers in basement membranes in vivo. In contrast, the extrinsic (exogenous or tissue-dependent) pathway is initiated when, as a result of outside injury to the vessel wall, tissue juice becomes mixed with components of the blood plasma.
It has been observed that the tissues of vertebrates, when added to citrated plasma and recalcified, will profoundly accelerate clotting time. This tissue constituent which has been observed to activate the coagulation protease cascades by the extrinsic pathway is commonly referred to as thromboplastin or tissue factor (TF).
Tissue factor is a membrane-associated glycoprotein which functions by forming a complex with blood coagulation factors VII and VIIa. The complexing of these factors greatly enhances the proteolytic activity of factors VII and VIIa. Functional activity of tissue factor has an absolute dependence on the presence of phospholipids. Bach, Ronald R.,
Initiation of Coagulation by Tissue Factor, CRC Critical Reviews in Biochemistry
1988; 23 (4): pp. 339-368. The factor VII/VIIa/tissue factor complex activates a series of specific enzymes that comprise the extrinsic and common pathways of the coagulation cascades ultimately leading to the formation of thrombin, fibrin, platelet activation, and finally clot formation. Nemerson, Yale,
Tissue Factor and Hemostasis, Blood
1988; 71:pp. 1-8.
Screening tests for coagulation disorders are designed to detect a significant abnormality in one or more of the clotting factors and to localize this abnormality to various steps in the coagulation pathway. Commonly used screening tests for this purpose include the activated partial thromboplastin time (APTT) and the prothrombin time (PT). Diagnostic tests such as the PT test, utilize this series of enzymatic events in vitro under controlled conditions to diagnose disfunctions in the blood coagulation system of patients. In the PT test, the time it takes for clot formation to occur, is the Prothrombin time or “PT value”.
The PT test is performed by adding tissue thromboplastin with calcium to plasma. This initiates clotting by activating Factor VII which in turn activates Factor X which in the presence of Factor V, lead to the conversion of prothrombin to thrombin. The thrombin which is so produced converts fibrinogen to fibrin. PT therefore bypasses the intrinsic clotting pathway and is normal in patients with deficiencies of Factors XII, XI, IX and VIII. PT is abnormal in patients with deficiencies of Factors VII, X, V, prothrombin or fibrinogen. Tissue thromboplastin is a phospholipid extract (from rabbit brain or lung and human brain or placenta) to which calcium has been added. It is usually provided in a lyophilized form and must be reconstituted with distilled water.
The prothrombin time (PT) test is the most commonly performed assay in the coagulation laboratory.
PT assay reagents are particularly useful in rapid screening tests to detect single or combined deficiencies of the extrinsic coagulation system indicative of hereditary and acquired coagulation disorders, liver disease or vitamin K deficiency. PT assay re
Buckley Linda
Carlson Karen Cochrane
Dade Behring Inc.
Lowen Cara
Schnizer Holly
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