Plasma glucosylceramide deficiency as risk factor for thrombosis and modulator of anticoagulant protein C

• US 20020177563A1
• Filed: 02/28/2002
• Published: 11/28/2002
• Est. Priority Date: 02/28/2001
• Status: Active Grant

• Current Assignee: Hiroshi Deguchi, John H. Griffin, Jose Fernandez
• Original Assignee: Hiroshi Deguchi, John H. Griffin, Jose Fernandez
• Inventors: Fernandez, Jose, Griffin, John H., Deguchi, Hiroshi
• Granted Patent: US 6,756,208 B2
• Time in Patent Office: Days
• US Class Current: 514/42
• CPC Class Codes: A61K 31/739 Lipopolysaccharides
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: C07H 15/10 containing unsaturated carbon-to-carbon bonds

Abstract

The present invention has determined that exogenously added glycosylceramide (GlcCer) and other neutral glycolipids such as the homologous Glc-containing globotriaosylceramide (Gb3Cer), dose-dependently prolonged clotting times of normal plasma in the presence but not absence of APC:protein S, indicating GlcCer or Gb3Cer can enhance protein C pathway anticoagulant activity. In studies using purified proteins, inactivation of factor Va by APC:protein S was enhanced by GlcCer alone and by GlcCer, globotriaosylceramide, lactosylceramide, and galactosylceramide in multicomponent vesicles containing phosphatidylserine and phosphatidylcholine. Thus, the present invention provides neutral glycolipids such as GlcCer and Gb3Cer, as anticoagulant cofactors that contribute to the antithrombotic activity of the protein C pathway. The present invention has also determined that a deficiency of plasma GlcCer is a risk factor for thrombosis. Methods are provided to determine individuals at risk for thrombosis, methods of treatment as well as methods of screening for antithrombotic factors from neutral glycolipids.

Claims

1. An antithrombotic factor comprising at least one neutral glycolipid.

2. The antithrombotic factor according to claim 1, wherein the neutral glycolipid comprises a formula:

R—

sugar—

linkage—

ceramide, wherein R is hydrogen or at least one saccharide unit.

3. The antithrombotic factor according to claim 2, wherein the sugar is a hexose sugar.

4. The antithrombotic factor according to claim 3, wherein the hexose sugar is glucose, galactose, allose, altrose, mannose, gulose, idose or talose.

5. The antithrombotic factor according to claim 2, wherein the sugar moiety is selected from a D-isomer, or a D-glucose linked covalently to ceramide.

6. The antithrombotic factor according to claim 2, wherein R is selected from the group consisting of:

(a) 1 to 20 saccharide units;

1 to 6 saccharide units;

or 1 to 3 saccharide units;

(b) multiple saccharide units that are linear or complexed branched units; and

(c) at least one saccharide unit selected from the group consisting of glucose, galactose, lactose, allose, altrose, mannose, gulose, idose, talose, globotriose, erythrose, threose, ribose, arabinose, xylose, lyxose, sedoheptose, sedoheptulose and combinations thereof.

7. The antithrombotic factor according to claim 2, wherein the linkage is selected from the group consisting of (a) a covalent bond;

and (b) a beta-1 linkage directly between the sugar and ceramide.

8. The antithrombotic factor according to claim 2, wherein the saccharide unit is linked by an alpha-1,4 or beta-1,4 linkage to the sugar.

9. The antithrombotic factor according to claim 2, wherein a fatty acid side chain of the ceramide is saturated, monounsaturated or polyunsaturated.

10. The antithrombotic factor according to claim 9, wherein the fatty acid side chain has 16, 18, 22, or 24 carbon atoms.

11. The antithrombotic factor according to claim 1, wherein the neutral glycolipid is glucosylceramide, globotriaosylceramide, lactosylceramide or galactosylceramide.

12. The antithrombotic factor according to claim 1, wherein the neutral glycolipid or glycolipids are in vesicle form.

13. The antithrombotic factor according to claim 12, wherein the vesicle further comprises at least one lipid.

14. The antithrombotic factor according to claim 13, wherein the lipid is phosphatidylserine, phosphatidylcholine, cholesterol, cholesterol ester, triglyceride, diacylglyceride, sphingomyelin, phosphatidylethanolamine, lecithin, liposomes or combinations thereof.

15. The antithrombotic factor according to claim 13, wherein the vesicle glycolipids and lipids are selected from a group consisting of:

(a) at least one neutral glycolipid and cholesterol;

(b) at least on neutral glycolipid and sphingomyelin;

(c) at least one neutral glycolipid, cholesterol and sphingomyelin;

(d) at least one neutral glycolipid, phosphatidylethanolamine and cholesterol;

(e) glucosylceramide, phosphatidylserine and phosphatidylcholine;

(f) globotriaosylceramide and sphingomyelin;

(g) globotriaosylceramide and sphingomyelin and cholesterol;

(h) glucosylceramide, sphingomyelin and cholesterol;

(i) lactosylceramide, sphingomyelin and cholesterol;

(j) lactosylceramide, phosphatidylethanolamine and cholesterol; and

(k) galactosylceramide, phosphatidylcholine and sphingomyelin.

16. The antithrombotic factor according to claim 13, wherein the neutral glycolipid comprises about 1 to about 99% of the weight of the vesicle, about 1 to about 50% of the weight of the vesicle, about 1 to about 10% of the weight of the vesicle, or about 1 to about 10% of the weight of the vesicle.

17. The antithrombotic factor according to claim 13, wherein the vesicle comprises about 80% to about 99% phosphatidylcholine;

or about 1% to about 10% phosphatidylserine.

18. The antithrombotic factor according to claim 13, wherein the vesicle comprises about 1 to about 10% neutral glycolipid, about 80% to about 99% phosphatidylcholine and about 0 to about 10% phosphatidylserine.

19. The antithrombotic factor according to claim 18, wherein the neutral glycolipid is selected from the group consisting of glucosylceramide, globotriaosylceramide, galactosylceramide, lactosylceramide, and combinations thereof.

31. A pharmaceutical composition comprising the antithrombotic factor according to claim 12 and a pharmaceutically acceptable carrier.

32. The pharmaceutical composition according to claim 31 further comprising at least one anticoagulant, antithrombotic agent, thrombolytic agent, antiplatelet drug, anti-inflammatory drug, high density lipoprotein or portion thereof, or combination thereof.

20. A pharmaceutical composition comprising at least one antithrombotic factor, wherein said at least one factor further comprises:

(a) at least one neutral glycolipid;

or

(b) at least one neutral glycolipid, wherein the neutral glycolipid comprises a formula;

R—

sugar—

linkage—

ceramide, wherein R is hydrogen or at least one saccharide unit;

and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition according to claim 20, wherein the antithrombotic factor is in a concentration selected from the group consisting of:

(a) about 0.01 μ

g/ml to about 1 mg/ml;

(b) about 0.05 μ

g/ml to about 500 μ

g/ml; and

(c) about 0.05 μ

g/ml to about 25 μ

g/ml.

22. The pharmaceutical composition according to claim 20, wherein the neutral glycolipid is present in an amount sufficient to increase a circulating blood level of neutral glycolipid by an amount selected from the group consisting of:

(a) at least about 0.1 μ

g/ml to about 15 μ

g/ml;

(b) at least about 0.5 μ

g/ml to about 10 μ

g/ml; and

(c) at least about 1 μ

g/ml to about 7 μ

g/ml.

23. The pharmaceutical composition according to claim 20, further comprising at least one anticoagulant, antithrombotic agent, thrombolytic agent, antiplatelet drug, anti-inflammatory drug, high density lipoprotein or portion thereof, or combinations thereof.

24. The pharmaceutical composition according to claim 23, wherein the anticoagulant is selected from the group consisting of protein C or analog thereof, plasma-derived protein C, recombinantly produced protein C, activated protein C or analog thereof, plasma-derived activated protein C, recombinantly produced activated protein C, protein S or analog thereof, plasma-derived protein S, recombinantly produced protein S, protein S comprising one or more chemically synthesized domains, tissue factor pathway inhibitor or analog thereof, antithrombin III or analog thereof, warfarin or analog thereof, heparin or analog thereof, glycosaminoglycan or analog thereof, soluble thrombomodulin or analog thereof, soluble endothelial protein C receptor or analog thereof and combinations thereof.

25. The pharmaceutical composition according to claim 24, wherein the activated protein C is in a concentration sufficient to raise a blood level of activated protein C in a subject by about 1.0 ng/ml to about 500 ng/ml.

26. The pharmaceutical composition according to claim 24, wherein the protein S is in a concentration to raise a blood level of protein S in a subject by about 0.1 μ

g/ml to about 20 μ

g/ml.

27. The pharmaceutical composition according to claim 24, comprising at least one neutral glycolipid, activated protein C and protein S.

28. The composition according to claim 23, wherein the antiplatelet agent is selected from the group consisting of aspirin, dixyridamole, clopidogrel, an inhibitor of platelet glycoprotein IIb-IIIa, an inhibitor of ADP receptors, and combinations thereof.

29. The composition according to claim 23, wherein the thrombolytic agent is selected from the group consisting of tissue plasminogen activator, tissue plasminogen activator analog, urokinase, urokinase analog, prourokinase, prourokinase analog, streptokinase, streptokinase analog, an acylated form of plasminogen, an acylated form of plasmin, acylated streptokinase-plasminogen complex, and combinations thereof.

30. The composition according to claim 23, wherein the portion of high density lipoprotein is selected from the group consisting of apolipoprotein A-I, apolipoprotein A-II, apolipoprotein B48, apolipoprotein B100, apolipoprotein C-I, apolipoprotein C-II, apolipoprotein C-III, apolipoprotein D, apolipoprotein E, apolipoprotein H, apolipoprotein J, apolipoprotein M and combinations thereof.

33. A method of determining an individual at risk for thrombosis comprising:

a) measuring a level of neutral glycolipid in a test biological specimen obtained from an individual;

b) comparing the level of said neutral glycolipid in said test biological specimen to a normal range of neutral glycolipid in a normal biological specimen, wherein a lower level of neutral glycolipid in the test biological specimen is indicative of a risk factor for thrombosis for the individual.

34. The method according to claim 33, wherein the biological specimen is plasma, serum, urine, cerebrospinal fluid, semen, lung fluid, lymph, saliva, or urine.

35. The method according to claim 33, wherein the thrombosis is venous thrombosis or arterial thrombosis.

36. The method according to claim 33, wherein the neutral glycolipid is selected from the group consisting of glucosylceramide, globotriaosylceramide, lactosylceramide and galactosylceramide.

37. The method according to claim 33, wherein a lower level of neutral glycolipid is:

below the 15^th percentile of the normal range of neutral glycolipid from a normal biological specimen, or below the 10^th percentile of the normal range of neutral glycolipid from a normal biological specimen.

39. The method according to claim 38, wherein the biological specimen is plasma, serum, cerebrospinal fluid, semen, lung, saliva, fluid, lymph or urine.

40. The method according to claim 38, wherein the thrombosis is venous thromobosis or arterial thrombosis.

41. The method according to claim 38, wherein the mean normal range of glucosylceramide is about 6.0 to about 7.0 μ

g/ml, and wherein the biological specimen is plasma.

42. The method according to claim 38, wherein a lower than normal level of glucosylceramide is about 4.6 μ

g/ml or less in plasma or about 4.2 μ

g/ml or less in plasma.

43. The method according to claim 38, wherein a lower level of glucosylceramide is:

below the 15^th percentile of the normal range;

or below the 10^th percentile of the normal range.

44. The method according to claim 38, further comprising chromatographic separation of said biological specimen into component parts, wherein said chromatographic separation comprises high performance liquid chromatography, thin layer chromatography, paper chromatography, gas chromatography, affinity chromatography, or affinity chromatography utilizing an antibody or antigen binding fragment thereof.

46. The method according to claim 45, wherein the neutral glycolipid is selected from the group consisting of glucosylceramide, lactosylceramide, globotriaosylceramide, and galactosylceramide.

47. The method according to claim 45, wherein the chromatographic separation is selected from the group consisting of:

HPLC;

HPLC utilizing silica beads;

thin layer chromatography;

affinity chromatography; and

immunoaffinity chromatography.

48. The method according to claim 47, wherein the silica beads have a particle size of about 10 microns and a pore size of about 125 Angstroms.

49. The method according to claim 47, wherein an affinity reagent for the affinity chromatography is verotoxin or shigatoxin.

50. The method according to claim 45, wherein the neutral glycolipid is quantified by evaporative light scattering detector.

38. A method of determining an individual at risk for thrombosis comprising:

A) measuring a level of glucosylceramide in a test biological specimen obtained from an individual;

B) comparing the level of said glucosylceramide to a normal range of glucosylceramide from a biological specimen, wherein a lower than normal level of glucosylceramide in the test biological specimen compared to a mean normal range in a normal biological specimen is indicative of a risk factor for thrombosis for the individual.

45. A method of determining a neutral glycolipid concentration in plasma or serum comprising:

a) extraction of lipids from plasma or serum to form a plasma lipid extract;

b) isolation of the neutral glycolipid by chromatographic separation of the plasma lipid extract; and

c) determination of the concentration of isolated neutral glycolipid by comparison to reference neutral glycolipid standards.

51. A method of enhancing antithrombotic or anti-inflammatory activity or reducing risk for thrombosis in an individual in need thereof, comprising:

administering to the individual at least one antithrombotic factor selected from the group consisting of;

(a) at least one neutral glycolipid;

(b) at least one neutral glycolipid, wherein the neutral glycolipid comprises a formula;

R—

sugar—

linkage—

ceramide, wherein R is hydrogen or at least one saccharide unit;

(c) at least one neutral glycolipid, wherein the neutral glycolipid or glycolipids are in vesicle form; and

(d) at least one neutral glycolipid, wherein the neutral glycolipid or glycolipids are in vesicle form, wherein the vesicle further comprises at least one lipid;

wherein, said antithrombotic factor is in an amount effective to enhance the antithrombotic or anti-inflammatory activity or to reduce the individual'"'"'s risk for thrombosis.

52. The method of enhancing antithrombotic or anti-inflammatory activity according to claim 51, further comprising the administration of at least one anticoagulant, antithrombotic agent, antiplatelet drug, thrombolytic agent, anti-inflammatory drug, high density lipoprotein or portions thereof, or combinations thereof.

53. The method of enhancing antithrombotic or anti-inflammatory activity according to claim 52, wherein the anticoagulant is selected from the group consisting of protein C or analog thereof, activated protein C or analog thereof, protein S or analog thereof, tissue factor pathway inhibitor or analog thereof, antithrombin III, or analog thereof, warfarin or analog thereof, heparin or analog thereof, glycosylaminoglycan-protein C receptor or analog thereof, and combinations thereof.

54. The method of enhancing antithrombotic or antiinflammatory activity according to claim 52, wherein the thrombolytic agent is selected from the group consisting of tissue plasminogen activator or analog thereof, urokinase or analog thereof, prourokinase or analog thereof, streptokinase or analog thereof, acylated plasminogen or analog thereof, acylated plasmin or analog thereof, and acylated streptokinase plasminogen complex.

55. The method of enhancing antithrombotic or anti-inflammatory activity according to claim 51, wherein the individual has a glucosylceramide deficiency.

56. The method of enhancing antithrombotic or anti-inflammatory activity according to claim 51, wherein said individual is selected from the group consisting of:

(a) an individual who is pregnant, taking an oral contraceptive, or is on hormone replacement therapy;

(b) an individual who has had recent surgery or recent trauma;

(c) an individual who has cancer, inflammatory bowel disease, Bechet'"'"'s disease, sepsis, bone fracture, Alzeheimer'"'"'s disease, vascular disease, hyperlipidemia, stroke or autoimmune disease;

(d) an individual who is taking an anticancer drug; and

(e) an individual who is taking an anticancer drug, wherein the anticancer drug is tamoxifen, progesterone and derivatives thereof, a selective estrogen receptor modifier, or combinations thereof.

57. The method of enhancing antithrombotic or anti-inflammatory activity according to claim 51, wherein the antithrombotic factor is administered in a dose of about 300 μ

g to about 300 mg.

58. The method of enhancing antithrombotic or anti-inflammatory activity in an individual according to claim 51, wherein the dosage of antithrombotic factor is an amount sufficient to raise a circulating neutral glycolipid concentration by about 0.01 μ

g/ml to about 15 μ

g/ml.

59. The method of reducing an individual'"'"'s risk for thrombosis according to claim 51, wherein the amount is sufficient to maintain the plasma level of glucosylceramide to above the 15^th percentile of the normal range of glucosylceramide in plasma, or above the 10^th percentile of the normal range of glucosylceramide in plasma.

60. The method of reducing an individual'"'"'s risk for thrombosis according to claim 51, wherein the antithrombotic factor is administered as a bolus, a continuous infusion, or a bolus followed by a continuous infusion.

61. A nutritional supplement comprising at least one neutral glycolipid.

62. The nutritional supplement according to claim 61, further comprising components selected from the group consisting of:

(a) active components of a conventional vitamin supplement, active components of a conventional mineral supplement, or combinations thereof;

(b) active components of a conventional vitamin supplement, wherein the vitamin is selected from the group consisting of vitamin A, vitamin E, vitamin C, vitamin D, folic acid, thiamin, riboflavin, niacin, vitamin B-6, vitamin B-12, biotin and combinations thereof;

(c) components of a conventional mineral supplement, wherein the mineral is selected from the group consisting of zinc, manganese, magnesium, iron, calcium, copper, iodine, phosphorous, and combinations thereof.

63. The nutritional supplement according to claim 61, further comprising a component selected from (a) processed food;

and (b) processed food, wherein the processed food is selected from the group consisting of;

milk, milk-derived products, cereal, flour, grain and infant formula

64. A method of nutritional supplementation comprising administration of an effective amount of at least one neutral glycolipid to a subject.

65. The method according to claim 64, wherein the amount is about 1 mg/day to about 500 mg/day of neutral glycolipid.

66. The method according to claim 64, wherein the amount is sufficient to increase the circulating blood level of a neutral glycolipid in a subject by at least about 0.1 μ

g/ml to about 15 μ

g/ml.

68. The animal model according to claim 67, wherein the glucosylceramide deficiency is induced by:

administration of an inhibitor of glucosylceramide synthase;

or genetic alteration of genes that regulate glucosylceramide synthesis, transport, processing, metabolism, or hydrolysis.

67. An animal model for thrombosis comprising an animal deficient in glucosylceramide.

69. A method of screening for antithrombotic factors from among candidate neutral glycolipids comprising:

a) adding vesicles containing a candidate neutral glycolipid to plasma in the presence of (i) fibrinogen, (ii) a member of the group consisting of;

activated protein C, plasma-derived activated protein C, and recombinantly produced activated protein C, and (iii) a member of the group consisting of protein S, plasma-derived protein S, and recombinantly produced protein S;

b) measuring clotting time after addition of a tissue factor or recombinant human tissue factor and calcium ions, wherein prolongation of clotting time in comparison to a control is indicative of the neutral glycolipid as on antithrombotic factor.

70. A method of screening for antithrombotic factors from candidate neutral glycolipids comprising:

(a) incubating the candidate neutral glycolipid with a plasma sample with;

(1) exogenous activated protein C, exogenous protein C and an exogenous reagent that transforms exogenous protein C to activated protein C or an exogenous reagent that transforms endogenous protein C to activated protein C;

(2) an exogenous reagent (I) which at least partially activates a coagulation factor of the blood coagulation system of said plasma sample; and

optionally;

(3) an exogenous substrate for an enzyme wherein the activity of said enzyme is influenced by neutral glycolipid, to prepare a final assay medium;

(b) measuring a substrate conversion rate for a coagulation factor directly or indirectly activated in step (a), the activity of which is influenced by neutral glycolipid; and

(c) comparing said substrate conversion rate measure in step (b) with a standard value obtained from plasma in the absence of said neutral glycolipid wherein when said substrate conversion rate obtained for said neutral glycolipid in step (b) is lower than the standard value, said candidate neutral glycolipid is an antithrombotic factor.

71. The method of claim 70, wherein reagent (I) is an activated partial thromboplastin time reagent comprising a phospholipid or phospholipids and a contact activator.

72. The method of claim 70, wherein the neutral glycolipid and plasma sample are incubated with the components of (a)(1), and, optionally, (a)(3) after a preincubation of the sample with exogenous reagent (I) for a time sufficient to activate the blood coagulation system of the sample.

73. The method of claim 70, wherein the neutral glycolipid and the plasma sample is incubated in step (a) with exogenous protein C, optionally together with a protein S.

74. The method of claim 70, wherein in step (b), the conversion of fibrinogen to fibrin is measured.

75. The method of claim 70, wherein said exogenous reagent (I) is:

(i) components necessary to activate the blood coagulation system of the sample via the intrinsic pathway, or

(ii) components necessary to activate the blood coagulation system via the extrinsic pathway, or

(iii) components necessary to activate the blood coagulation system of the sample via the intrinsic pathway and components necessary to activate the blood coagulation system via the extrinsic pathway,

and components (a)(1), and (a)(3) are added simultaneously with, or after exogenous reagent (I) has been allowed to incubate with the sample for a sufficient time to activate the intrinsic pathway, or the extrinsic pathway, or the intrinsic and the extrinsic pathways.

76. The method according to claim 70, wherein said exogenous substrate is present and optionally a fibrin polymerization inhibitor is added, and substrate conversion is measured.

77. The method according to claim 76, wherein said exogenous substrate is specific for Factor X_a and is optionally present together with a fibrin polymerization inhibitor.

78. The method according to claim 76, wherein said exogenous substrate is specific for thrombin and is optionally present with a fibrin polymerization inhibitor.

79. The method according to claims 75, wherein said exogenous substrate is present and optionally a fibrin polymerization inhibitor is added, and substrate conversion is measured.

80. The method according to claim 79, wherein said exogenous substrate is specific for Factor X_a and is optionally present together with a fibrin polymerization inhibitor.

81. The method according to claim 79, wherein said exogenous substrate is specific for thrombin and is optionally present with a fibrin polymerization inhibitor.

82. The method according to claims 70, further adding a fibrin polymerization inhibitor, and said exogenous substrate being a synthetic peptide, said synthetic peptide comprising a group that is specifically released and becomes analytically detectable upon action of an enzyme, said group being bound in an amide linkage in the carboxy terminal of the peptide and being selected from a chromogenic, fluorogenic or chemiluminogenic group.

83. The method according to claims 75, further adding a fibrin polymerization inhibitor, and said exogenous substrate being a synthetic peptide, said synthetic peptide comprising a group that is specifically released and becomes analytically detectable upon action of an enzyme, said group being bound in an amide linkage in the carboxy terminal of the peptide and being selected from a chromogenic, fluorogenic or chemiluminogenic group.

84. The method according to claim 70, wherein said exogenous reagent (I) is Factor XI_a, Factor X_a or Factor IX_a, optionally, in combination with exogenous prothrombin.

85. The method according to claim 70, wherein said exogenous reagent (I) is exogenous Factor X in combination with exogenous Factor IX_a, or a combination of Factor X, Factor IX_a and thrombin.

86. The method according to claim 70, wherein said exogenous reagent (I) comprises Factor IX_a or Factor X, and said exogenous substrate is a chromogenic substrate for Factor X_a.

87. The method according to claim 70, wherein said exogenous reagent (I) comprises Factor XI_a, Factor IX_a, or Factor X_a, optionally in combination with exogenous prothrombin and said exogenous substrate is a chromogenic thrombin substrate.

88. A method of screening for a antithrombotic factor from a candidate neutral glycolipid, comprising:

A) incubating a candidate neutral glycolipid with a reaction mixture comprising activated protein C, protein S, Factor Va and a calcium source for a period sufficient for an enzymatic reaction with Factor Va;

B) stopping the enzymatic reaction;

C) determining the amount of Factor Va inactivation, wherein an increase in Factor Va inactivation by greater than 5% compared with control in the absence of the candidate neutral glycolipid indicates the neutral glycolipid is an antithrombotic factor.

89. The method according to claim 88, wherein the determination of Factor Va inactivation employs standard clotting assays for Factor Va.

90. The method according to claim 88, wherein the determination of Factor Va inactivation employs standard prothrombinase assays.