Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
2002-02-28
2004-06-29
Fonda, Kathleen K. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
C536S017900, C600S481000
Reexamination Certificate
active
06756208
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to neutral glycolipids as antithrombotic factors for prevention or inhibition of thrombosis and as anti-inflammatory agents. More specifically the invention relates to neutral glycolipids such as glucosylceramide, globotriaosylceramide, galactosylceramide, lactosylceramide and the like as antithrombotic factors and the use of the neutral glycolipids for prevention or inhibition of thrombosis and as anti-inflammatory agents. The present invention also relates to methods for screening for individuals at risk of thrombosis and relates to methods of screening for antithrombolytic agents from candidate neutral glycolipids.
BACKGROUND OF THE INVENTION
Poor anticoagulant response to activated protein C (APC), termed APC resistance, is detected in 20 to 50% of venous thrombosis patients, (1) and it can be present in patients with normal factor V genotype (2-4) or with the factor V polymorphism, arginine to glutamine substitution at amino acid 506, (5-7) or with a variety of acquired conditions, eg., oral contraceptive use, (8) autoantibody against APC, (9) etc. APC resistance is also associated with increased risk of ischemic stroke in subjects with normal R506-factor V. (10, 11) Severe deficiency of protein C or protein S causes life-threatening thrombosis. (12-15) Thus, the protein C pathway provides a major physiologic anticoagulant mechanism for down-regulation of thrombin generation in which APC proteolytically inactivates factors Va and VIIIa. (16)
Blood coagulation reactions are stimulated by phospholipid membrane surfaces as is the anticoagulant protein C pathway. (17) However, procoagulant and anticoagulant complexes may be differentially affected by different membrane phospholipid components. (18-20) Because we found that high density lipoprotein (HDL) exhibits anticoagulant cofactor activity for APC:protein S (21) we decided to evaluate further the influence of plasma lipids and lipoproteins on the protein C pathway.
Plasma lipoproteins contain glycolipids as well as phospholipids. (22-24) Glycolipids can play critical roles as bioregulators of a variety of processes such as cell proliferation, cell mobility and apoptosis. (23) Glycolipid molecules present their highly varied saccharide residues on the surface of lipoprotein particles as well as on cell surfaces, exposing saccharides to interactions with other cells, antibodies, bacterial toxins, and viral envelope proteins. (23) Several hundred glycolipids are known. However, the relevance of glycolipids to the blood coagulation system is currently unknown. Studies of the relationship between glycolipids and risks of venous thrombosis or of the influence of glycolipids on the APC:protein S anticoagulant pathway have not been reported.
This present invention demonstrates the relationship between thrombosis and plasma levels of neutral glycolipids. The present invention demonstrates that a neutral glycolipid deficiency is a potential risk factor for thrombosis and gives rise to the novel broad concept that neutral glycolipids may contribute to regulation of thrombin generation, blood coagulation, thrombosis and inflammation.
SUMMARY OF THE INVENTION
One object of the invention is to provide a neutral glycolipid as an antithrombotic or anti-inflammatory factor.
Another object of the invention is to provide a neutral glycolipid having the formula: R—sugar-linked ceramide as an antithrombotic or anti-inflammatory factor.
The invention further provides pharmaceutical compositions comprising neutral glycolipids, alone or in combination with at least one anticoagulant, antithrombotic agent, thrombolytic agent, antiplatelet drug, anti-inflammatory drug, high density lipoprotein or portion thereof, or combinations thereof.
The present invention provides neutral glycolipids in vesicle form alone or in combination with one or more lipids to form an antithrombotic or anti-inflammatory vesicle. The vesicles may be provided in the form of a pharmaceutical composition alone or in combination with at least one anticoagulant, antithrombotic agent, thrombolytic agent, antiplatelet drug, anti-inflammatory drug, high density lipoprotein or portion thereof, or combinations thereof.
The invention further provides nutritional supplements or dietary supplements comprising one or more neutral glycolipids and/or one or more vesicles comprising one or more neutral glycolipids.
The present invention provides a method of determining an individual at risk for thrombosis by determination of a below-normal level of a neutral glycolipid in a biological specimen, a below-normal level indicative of a risk factor for thrombosis in the individual.
The invention further provides a method of determining a neutral glycolipid concentration in a biological specimen.
Another aspect of the invention is a method of enhancing antithrombotic or anti-inflammatory activity in a subject comprising administration of at least one neutral glycolipid.
The invention further relates to a method for screening for candidate neutral glycolipids having antithrombotic activity.
The invention further provides an animal model for thrombosis comprising an animal deficient in glucosylceramide.
REFERENCES:
patent: WO 98/56365 (1998-12-01), None
patent: WO 00/53264 (2000-09-01), None
Debuchi H, Fernandez JA, Pabinger I, Heit JA and Griffin JH. “Plasma Glucosylceramide Deficiency as Potential Risk Factor for Venous Thrombosis and Modulator of Anticoagulant Protein C Pathway.” Blood 97: 1907-14, 2001.
Deguchi H, Fernandez JA, and Griffin JH. “Neutral glycosphingolipid-dependent inactivation of coagulation factor Va by activated protein C and protein S.” J Biol Chem 277:8861-65, 2002.
Griffin JH, Kojima K, Banka CL, Curtiss LK, Fernandez JA. “High-Density lipoprotein enhancement of anticoagulant activities of plasma protein S and activated protein C.” J. Clin Invest. 103:219-227, 1999.
Fernandez JA, Kojima K, Petaja J, Hackeng TM, Griffin JH. “Cardiolipin enhances protein C pathway anticoagulant activity.” Blood Cells Mol Dis. 26:115-23, 2000.
Deguchi H, Fernandez JA, Hackeng TM, Banka CL, Griffin JH. “Cardiolipin is a normal component of human plasma lipoproteins.” Proc. Natl Acad Sci USA. 97:1743-1748, 2000.
Smirnov MD, Esmon CT. “Phosphatidylethanolamine incorporation into vesicles selectively enhances factor Va inactivation by activated protein C.” J Biol Chem. 269:816-819, 1994.
Svensson PJ, Dahlback B. “Resistance to activated protein C as a basis for venous thrombosis.” N Engl J. Med. 330:517-522, 1994.
Clarke JTR. “The glycosphingolipids of human plasma lipoproteins.” Can J Biochem. 59:412-417, 1981.
Dawson G, Kruski AW, Scanu AM. “Distribution of glycosphinogolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias.” J Lipid Res. 17:125-131, 1976.
Hakomori S, Igarashi Y. “Functional role of glycosphingolipids in cell recognition and signaling.” J Biochem. 118:1091-1103, 1995.
Chatterjee S. “Sphingolipids in atherosclerosis and vascular biology.” Arterioscler Thromb Vasc Biol. 18:1523-1533, 1998.
Heran, C. et al., Antithrombotic efficacy of RPR208566, a novel factor Xa inhibitor, in a rat model of carotid artery thrombosis. Eu. J. Pharm. 389:201-207 2000.
Phillips, D. J. et al., Protein S, an antithrombotic factor, is synthesized and released by neural tumor cells. J. Neurochem. 61:344-347 1993.
Deguchi Hiroshi
Fernandez Jose
Griffin John H.
Fonda Kathleen K.
Morgan & Finnegan L.L.P.
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