U.S. patent application number 11/127544 was filed with the patent office on 2005-10-13 for methods and compositions for treating platelet-related disorders.
Invention is credited to Hanson, Stephen R..
Application Number | 20050228001 11/127544 |
Document ID | / |
Family ID | 22553421 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050228001 |
Kind Code |
A1 |
Hanson, Stephen R. |
October 13, 2005 |
Methods and compositions for treating platelet-related
disorders
Abstract
Pharmaceutical preparations comprising one or more agents that
reduce the number of circulating platelets to low normal or below
normal levels formulated in a delivery system are provided. The
pharmaceutical preparations are useful in the prophylactic and
therapeutic treatment of subjects for the purpose of inhibiting
vaso-occlusive events, including embolism, by reducing the number
of circulating platelets to low normal to below normal levels.
Inventors: |
Hanson, Stephen R.;
(Beaverton, OR) |
Correspondence
Address: |
FISH & RICHARDSON, PC
12390 EL CAMINO REAL
SAN DIEGO
CA
92130-2081
US
|
Family ID: |
22553421 |
Appl. No.: |
11/127544 |
Filed: |
May 11, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11127544 |
May 11, 2005 |
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10603401 |
Jun 25, 2003 |
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10603401 |
Jun 25, 2003 |
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09666223 |
Sep 21, 2000 |
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6585995 |
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60154929 |
Sep 21, 1999 |
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Current U.S.
Class: |
514/257 |
Current CPC
Class: |
A61K 31/519 20130101;
A61K 38/49 20130101; A61P 43/00 20180101; A61P 3/06 20180101; A61K
31/519 20130101; A61P 9/12 20180101; A61P 9/14 20180101; A61P 11/00
20180101; A61K 9/1647 20130101; A61K 38/49 20130101; A61K 31/00
20130101; A61P 7/02 20180101; A61K 31/60 20130101; A61P 13/12
20180101; A61K 31/522 20130101; A61K 31/557 20130101; A61K 31/4365
20130101; A61K 9/204 20130101; A61K 31/616 20130101; A61K 31/4365
20130101; A61K 45/06 20130101; A61K 31/4965 20130101; A61K 31/60
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61P 27/14 20180101; A61P
9/10 20180101; A61K 47/34 20130101 |
Class at
Publication: |
514/257 |
International
Class: |
A61K 031/519 |
Claims
What is claimed:
1. A pharmaceutical composition, comprising a platelet reducing
agent that reduces platelet number, wherein the composition is
formulated in a controlled release delivery system.
2. The pharmaceutical composition of claim 1, wherein the delivery
system is a time-release system, a delayed release system or a
sustained release system.
3. The pharmaceutical composition of claim 2, wherein the delivery
system comprises an erosional system or a diffusional system.
4. The pharmaceutical composition of claim 3, wherein the platelet
reducing agent is a cAMP phosphodiesterase inhibitor.
5. The pharmaceutical composition of claim 1, wherein the platelet
reducing agent is anagrelide
(6,7-dichloro-1,5-dihydroimidazo-[2,1-b]quin- azolin-2(3H)-one) or
a derivative of anagrelide.
6. The pharmaceutical composition of claim 5, wherein the platelet
reducing agent is anagrelide.
7. The pharmaceutical composition of claim 5, wherein the delivery
system is a sustained release system.
8. The pharmaceutical composition of claim 5, wherein the
derivative of anagrelide has an increased specificity towards a
megakaryocyte lineage-restricted cell than anagrelide.
9. The pharmaceutical composition of claim 5, wherein the
derivative of anagrelide is more effective at reducing levels of
platelets than anagrelide.
10. The pharmaceutical composition of claim 5, wherein the
derivative of anagrelide is
N-cyclohexyl-N-methyl-4-(7-oxy-1,2,3,5-tetrahydroimadazo-[2-
,1-b]quinazolin-2-one) or
6,7-dichloro-1,2,3,5-tetrahydroimidazo-[2,1-b]-q-
uinazolin-2-one.
11. The pharmaceutical composition of claim 5, wherein the
derivative of anagrelide is selected from among
1,2,3,5-tetrahydroimidazo[2,1-b]-quinaz- olin-2-one,
7-bromo-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-nitro-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-amino-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
6-hydroxy-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-hydroxy-1,2,3,5-tetrahydroimidazo-[2,1-b]-quinazolin-2-one,
8-bromo-6-[H]-1,2,3,4-tetrahydroimidazo-[2,1-b]-quinazolin-2-one,
6-methyl-7-nitro-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-bromo-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-chloro-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
6-chloro-7-bromo-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
6,7-dichloro-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-amino-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-amino-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
3-(carboxymethyl)-3,4-dihydro-5-methyl-4-methylene-1H-quinazolin-2-one,
3-(carboxymethyl)-4,5-dimethyl-1,2,3,4-tetrahydro-quinazoline-2-one,
2-chloro-3-carethoxymethyl-4,5-dimethyl-3,4-dihydroquinazoline,
5,6-dimethyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
3-(carbethoxymethyl)-3,4-dihydro-6-methylene-1H-quinazolin-2-one,
3-(carbethoxymethyl)-4,6-dimethyl-1,2,3,4-tetrahydroqionazolin-2-one,
2-chloro-3-carbethoxymethyl-4,6-dimethyl-3,4-dihydro-quinazoline,
5,7-dimethyl-1,2,3,5-tetrahydroimidazo-[2,1-b]quinazolin-2-one,
5-methyl-3-(carbethoxymethyl)-1,2,3,4-tetrahydroquinazolin-2-one,
2-chloro-3-carbethoxymethyl-5-methyl-3,4-dihyrdoquinazoline
hydrochloride and
6-methyl-1,2,3,5-tetrahydroimidazo-[2,1-b]quinazolin-2-one.
12. The pharmaceutical composition of claim 5, wherein the dose of
the agent is from about 0.01 mg/kg per day to 1000 mg/kg per
day.
13. The pharmaceutical composition of claim 5, wherein the
effective amount is in the range of 30 .mu.g/kg/day to 150
.mu.g/kg/day.
14. The pharmaceutical composition of claim 5, wherein the
effective amount is in the range of 1 .mu.g/kg/day to 10
.mu.g/kg/day.
15. The pharmaceutical composition of claim 5, wherein the
effective amount is in the range of 1 .mu.g/kg/day to 0.150
.mu.g/kg/day.
16. The pharmaceutical composition of claim 5, wherein the delivery
system administers an amount of the agent effective to reduce
platelet numbers to low normal levels in a subject.
17. The pharmaceutical composition of claim 5, wherein the delivery
system administers an amount of the agent effective to reduce
platelet numbers to below normal levels in a subject.
18. The pharmaceutical composition of claim 1, wherein the agent is
provided in an amount to reduce platelet levels to below
2.times.10.sup.5 platelets per .mu.l in a human subject.
19. The pharmaceutical composition of claim 1, wherein the agent is
provided in an amount to reduce platelet levels to below
1.times.10.sup.5 platelets per .mu.l in a human subject.
20. The pharmaceutical composition of claim 5, wherein the platelet
reducing agent is dispersed in a matrix.
21. The pharmaceutical composition of claim 20, wherein the matrix
is formulated to achieve a reduction in peak level of agent in the
subject.
22. The pharmaceutical composition of claim 20, wherein the matrix
is formulated to administer a dose of agent that maintains a
desired platelet count in a subject.
23. The pharmaceutical composition of claim 20, wherein the matrix
comprises a polymer or cholesterol.
24. The pharmaceutical composition of claim 20, wherein the matrix
comprises a binding agent.
25. The pharmaceutical composition of claim 24, wherein the binding
agent is selected from among polyethylene glycol,
polyvinylpyrollidone, hydroxymethylcellulose and
hydroxypropylmethylcellulose.
26. The pharmaceutical composition of claim 20, wherein the matrix
comprises a lubricating agent.
27. The pharmaceutical composition of claim 26, wherein the
lubricating agent is selected from among stearic acid, magnesium
stearate, calcium stearate.
28. The pharmaceutical composition of claim 20, wherein the matrix
comprises: (a) 20-80% cholesterol powder; (b) 20-80% cholesterol
prills, 100-1200 microns in diameter; (c) 0.1-5.0% biocompatible
binding agent; (d) 0.1-5.0% biocompatible lubricating agent.
29. The pharmaceutical composition of claim 5, wherein the delivery
system comprises one or more than one polymer selected from among a
poly(lactide-glycolide), a copolyoxalate, a polycaprolactone, a
polyesteramide, a polyorthoester, a polyhydroxybutyric acid or a
polyanhydride.
30. The pharmaceutical composition of claim 5, wherein the delivery
system comprises a lipid, a sterol, a fatty acid, a neutral fat, a
wax or a combination thereof.
31. The pharmaceutical composition of claim 5, wherein the delivery
system comprises a hydrogel delivery system.
32. The pharmaceutical composition of claim 5, wherein the delivery
system comprises microcapsules.
33. The pharmaceutical composition of claim 32, wherein the
microcapsules comprise one or more than one polymer selected from
among a poly(lactide-glycolide), a copolyoxalate, a
polycaprolactone, a polyesteramide, a polyorthoester, a
polyhydroxybutyric acid or a polyanhydride.
34. The pharmaceutical composition of claim 5, wherein the
composition is formulated for administration by a route selected
from among oral, rectal, topical, nasal, intradermal, intramuscular
and parenteral routes.
35. The pharmaceutical composition of claim 5, wherein the
composition is formulated for daily administration.
36. The pharmaceutical composition of claim 5, wherein the
composition is formulated to be administered in multiple doses per
day.
37. The pharmaceutical composition of claim 5, further comprising
aspirin.
38. The pharmaceutical composition of claim 5, further comprising
heparin or coumarin.
39. The pharmaceutical composition of claim 5, further comprising
another therapeutic compound selected from among an inhibitor of
platelet function, an anti-coagulant agent and a fibrinolytic
agent.
40. The pharmaceutical composition of claim 39, wherein the
inhibitor of platelet function is selected from among acadesine,
anipamil, argatroban, aspirin, clopidogrel, a cyclooxygenase
inhibitor, a nonsteroidal anti-inflammatory drug, the synthetic
compound FR-122047, danaparoid sodium, dazoxiben hydrochloride, a
diadenosine 5',5'"-P1,P4-tetraphosphat- e (Ap4A) analog,
difibrotide, dilazep dihydrochloride, 1,2-glyceryl dinitrate,
1,3-glyceryl dinitrate, dipyridamole, dopamine, 3-methoxytyramine,
efegatran sulfate, enoxaparin sodium, glucagon, a glycoprotein
IIb/IIIa antagonist, Ro-43-8857, L-700,462, ifetroban, ifetroban
sodium, iloprost, isocarbacyclin methyl ester,
isosorbide-5-mononitrate, itazigrel, ketanserin, BM-13.177,
lamifiban, lifarizine, molsidomine, nifedipine, oxagrelate,
prostaglandin E (PGE), a platelet activating factor antagonist,
lexipafant, prostacyclin (PGI2), a pyrazine, pyridinol carbamate,
abciximab, sulfinpyrazone, BN-50727, BN-52021, CV-4151, E-5510,
FK-409, GU-7, KB-2796, KBT-3022, KC-404, KF-4939, OP-41483,
TRK-100, TA-3090, TFC-612, ZK-36374, 2,4,5,7-tetrathiaoctane,
2,4,5,7-tetrathiaoctane 2,2-dioxide, 2,4,5-trithiahexane,
theophyllin, pentoxifyllin, a thromboxane inhibitor, a thromboxane
synthetase inhibitor, picotamide, sulotroban, ticlopidine,
tirofiban, trapidil, triclopidine, trifenagrel, trilinolein, a
3-substituted 5,6-bis(4-methoxyphenyl)-1,2,4-triazine, an antibody
to glycoprotein IIb/IIIa, an anti-serotonin drug, dipyridamole,
clofibrate, caffeine and ticlopidine.
41. The pharmaceutical composition of claim 40, wherein the
inhibitor of platelet function is selected from the group
consisting of aspirin, abciximab, clopidogrel and dipyridamole.
42. The pharmaceutical composition of claim 40, wherein the
inhibitor of platelet function is clopidogrel.
43. The pharmaceutical composition of claim 39, wherein the
anti-coagulant agent is selected from among ardeparin sodium,
bivalirudin, bromindione, coumarin, dalteparin sodium, desirudin,
dicumarol, heparin, lyapolate sodium, nafamostat mesylate,
phenprocoumon, sulfatide, tinzaparin sodium and warfarin
sodium.
44. The pharmaceutical composition of claim 39, wherein the
fibrinolytic agent is selected from among ancrod, anistreplase,
bisobrin lactate, brinolase, a Hageman factor fragment,
prostacyclin, molsidomine, streptokinase, a tissue plasminogen
activator (TPA) and urokinase.
45. The pharmaceutical composition of claim 39, wherein the
therapeutic compound is an angiotensin converting enzyme (ACE)
inhibitor.
46. The pharmaceutical composition of claim 45, wherein the ACE
inhibitor is selected from among an acylmercapto proline, a
mercaptoalkanoyl proline, a carboxyalkyl dipeptide, a carboxyalkyl
dipeptide mimic and a phosphinylalkanoyl proline.
47. The pharmaceutical composition of claim 45, wherein the ACE
inhibitor is selected from among captopril, zofenopril, enalapril,
lisinopril, quinapril, ramipril, perindopril, cilazapril,
benazapril, fosinopril and trandolopril.
48. The pharmaceutical composition of claim 39, wherein the
therapeutic compound is a diadenosine 5',5'"-P1,P4-tetraphosphate
(Ap4A) analog or a cyclooxygenase inhibitor.
49. The pharmaceutical composition of claim 39, wherein the
anti-coagulant is a glycosoaminoglycan.
50. The pharmaceutical composition of claim 5, wherein the delivery
system is formulated to administer an amount of the agent effective
to reduce platelet numbers in a subject by at least 10%.
51. The pharmaceutical composition of claim 5, wherein the delivery
system is formulated to administer an amount of the agent effective
to reduce platelet numbers in a subject by at least 20%.
52. The pharmaceutical composition of claim 5, wherein the delivery
system is formulated to administer an amount of the agent effective
to reduce platelet numbers in a subject by at least 50%.
53. A method of treating a subject to inhibit a vaso-occlusive
event, comprising administering to a subject in need of such
treatment a controlled release delivery system comprising
anagrelide or an anagrelide derivative, wherein the delivery system
is formulated to administer an amount of the anagrelide or the
anagrelide derivative effective to reduce platelet count in the
subject by at least 10% of pre-treatment levels.
54. The method of claim 53, wherein the platelet count is reduced
to at least a low normal level.
55. The method of claim 53, wherein the subject has a normal
platelet count prior to treatment.
56. The method of claim 53, wherein the subject has an above normal
platelet count prior to treatment.
57. The method of claim 53, wherein the subject is a human.
58. The method of claim 53, wherein the subject has vascular
disease.
59. The method of claim 58, wherein the vascular disease is
selected from the group consisting of arteriosclerosis,
cardiovascular disease, cerebrovascular disease, renovascular
disease, mesenteric vascular disease, pulmonary vascular disease,
ocular vascular disease and peripheral vascular disease.
60. The method of claim 53, wherein the subject has had a primary
vaso-occlusive event.
61. The method of claim 53, wherein the subject has a condition
selected from the group consisting of hypercholesterolemia,
hypertension and atherosclerosis.
62. The method of claim 53, wherein the subject will undergo an
elective surgical procedure.
63. The method of claim 62, wherein the surgical procedure is
selected from the group consisting of coronary angiography,
coronary stent placement, coronary by-pass surgery, carotid artery
procedure, peripheral stent placement, vascular grafting,
thrombectomy, peripheral vascular surgery, vascular surgery, organ
transplant, artificial heart transplant, vascular angioplasty,
vascular laser therapy, vascular replacement and vascular
stenting.
64. The method of claim 53, wherein the subject has undergone a
surgical procedure.
65. The method of claim 64, wherein the surgical procedure is
selected from the group consisting of coronary angiography,
coronary stent placement, coronary by-pass surgery, carotid artery
procedure, peripheral stent placement, vascular grafting,
thrombectomy, peripheral vascular surgery, vascular surgery, organ
transplant, artificial heart transplant, vascular angioplasty,
vascular laser therapy, vascular replacement and vascular
stenting.
66. The method of claim 53, wherein the effective amount is in the
range of 1 .mu.g/kg/day to 10 .mu.g/kg/day.
67. The method of claim 53, wherein the effective amount is in the
range of 1 .mu.g/kg/day to 0.150 .mu.g/kg/day.
68. The method of claim 53, wherein the controlled release delivery
system further comprises another therapeutic compound selected from
among an inhibitor of platelet function, an anti-coagulant agent
and a fibrinolytic agent.
69. The method of claim 68, wherein the inhibitor of platelet
function is selected from among acadesine, anipamil, argatroban,
aspirin, clopidogrel, a cyclooxygenase inhibitor, a nonsteroidal
anti-inflammatory drug, the synthetic compound FR-122047,
danaparoid sodium, dazoxiben hydrochloride, a diadenosine
5',5'"-P1,P4-tetraphosphate (Ap4A) analog, difibrotide, dilazep
dihydrochloride, 1,2-glyceryl dinitrate, 1,3-glyceryl dinitrate,
dipyridamole, dopamine, 3-methoxytyramine, efegatran sulfate,
enoxaparin sodium, glucagon, a glycoprotein IIb/IIIa antagonist,
Ro-43-8857, L-700,462, ifetroban, ifetroban sodium, iloprost,
isocarbacyclin methyl ester, isosorbide-5-mononitrate, itazigrel,
ketanserin, BM-13.177, lamifiban, lifarizine, molsidomine,
nifedipine, oxagrelate, prostaglandin E (PGE), a platelet
activating factor antagonist, lexipafant, prostacyclin (PGI2), a
pyrazine, pyridinol carbamate, abciximab, sulfinpyrazone, BN-50727,
BN-52021, CV-4151, E-5510, FK-409, GU-7, KB-2796, KBT-3022, KC-404,
KF-4939, OP-41483, TRK-100, TA-3090, TFC-612, ZK-36374,
2,4,5,7-tetrathiaoctane, 2,4,5,7-tetrathiaoctane 2,2-dioxide,
2,4,5-trithiahexane, theophyllin, pentoxifyllin, a thromboxane
inhibitor, a thromboxane synthetase inhibitor, picotamide,
sulotroban, ticlopidine, tirofiban, trapidil, triclopidine,
trifenagrel, trilinolein, a 3-substituted
5,6-bis(4-methoxyphenyl)-1,2,4-triazine, an antibody to
glycoprotein IIb/IIIa, an anti-serotonin drug, dipyridamole,
clofibrate, caffeine and ticlopidine.
70. The method of claim 69, wherein the inhibitor of platelet
function is selected from the group consisting of aspirin,
abciximab, clopidogrel and dipyridamole.
71. The method of claim 53, wherein the controlled release delivery
system comprises a matrix in which the anagrelide or the anagrelide
derivative is dispersed.
72. The method of claim 71, wherein the matrix is formulated to
achieve a reduction in peak level of agent in the subject.
73. The method of claim 53, wherein the delivery system comprises
one or more than one polymer selected from among a
poly(lactide-glycolide), a copolyoxalate, a polycaprolactone, a
polyesteramide, a polyorthoester, a polyhydroxybutyric acid or a
polyanhydride.
74. The method of claim 53, wherein the delivery system comprises a
lipid, a sterol, a fatty acid, a neutral fat, a wax or a
combination thereof.
75. The method of claim 53, wherein the delivery system comprises a
hydrogel delivery system.
76. The method of claim 53, wherein the delivery system comprises
microcapsules.
77. The method of claim 76, wherein the microcapsules comprise one
or more than one polymer selected from among a
poly(lactide-glycolide), a copolyoxalate, a polycaprolactone, a
polyesteramide, a polyorthoester, a polyhydroxybutyric acid or a
polyanhydride.
78. The method of claim 53, wherein the delivery system is
formulated for administration by a route selected from among oral,
rectal, topical, nasal, intradermal, intramuscular and parenteral
routes.
79. The method of claim 53, wherein platelet count is reduced by at
least 20%.
80. The method of claim 53, wherein platelet count is reduced by at
least 50%.
81. The method of claim 53, wherein platelet count is reduced to
below 200.times.10.sup.3 platelets per .mu.l.
82. The method of claim 53, wherein platelet count is reduced to
below 150.times.10.sup.3 platelets per .mu.l.
83. The method of claim 53, wherein platelet count is reduced to
below 100.times.10.sup.3 platelets per .mu.l.
84. The method of claim 53, wherein platelet count is reduced by at
least 10% and to an amount above 200.times.10.sup.3 platelets per
.mu.l.
85. The method of claim 53, wherein platelet count is reduced by at
least 10% and to an amount below 200.times.10.sup.3 platelets per
.mu.l.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of copending U.S. patent
application Ser. No. 10/603,401, filed Jun. 25, 2003, to Stephen R.
Hanson, entitled "METHODS AND COMPOSITIONS FOR TREATING
PLATELET-RELATED DISORDERS," which is a continuation of U.S. patent
application Ser. No. 09/666,223, filed Sep. 21, 2000, to Stephen R.
Hanson, entitled "METHODS AND COMPOSITIONS FOR TREATING
PLATELET-RELATED DISORDERS," now U.S. Pat. No. 6,585,995, which
issued Jul. 1, 2003, and which claims benefit of priority under 35
U.S.C. .sctn. 19(e) to U.S. provisional application Ser. No.
60/154,929 to Stephen R. Hanson, filed Sep. 21, 1999, entitled
"METHODS AND COMPOSITIONS FOR TREATING PLATELET-RELATED DISORDERS."
The benefit of priority is claimed to each of these applications.
The disclosure of each of these applications is incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to methods and products for treatment
and/or prevention of platelet related thrombotic and other
vaso-occlusive disorders.
BACKGROUND OF THE INVENTION
[0003] Conditions resulting from thrombotic or thromboembolic
events are the leading causes of illness and death in adults in
western civilization. A great deal of effort and monetary resources
have been directed towards understanding the mechanisms involved in
vascular occlusive diseases involving thrombotic and thromboembolic
events. These efforts have yielded a number of promising
therapeutic agents. Notwithstanding the effort and financial
resources that have been invested, these conditions still account
for the vast majority of illness and death in the adult populations
of developed nations.
[0004] Platelets are an important cellular component of blood
involved in hemostasis as well as thrombotic or thromboembolic
events. Abnormally high platelet counts such as those that result
from hematological proliferative disorders such as for example
essential thrombocythemia have been recognized as an important risk
factor in thrombus formation. Furthermore, it has long been
accepted that aspirin, which is known to inhibit cyclooxygenase and
thereby prevents production of thromboxane A.sub.2 in platelets,
lowers the incidence of thrombotic and thromboembolic events. For
platelets, therapeutic regimens thus far reported have as their aim
an inhibition of platelet function (e.g., inhibition of platelet
adhesion, aggregation or factor release). An alternative modality
is the reduction in platelet count in patients with abnormally high
levels in certain hematological malignancies to levels
approximating normal levels. Therapeutic intervention for reducing
platelet count to low normal or below normal levels in subjects
without myeloproliferative disorders has not been proposed
primarily since normal platelet count has been thought to be
critical to normal hemostasis.
SUMMARY OF THE INVENTION
[0005] The invention in a broad aspect involves the surprising
discovery that subjects, including those with normal levels of
circulating platelets, can unexpectedly derive medical benefit from
a reduction in platelet count to below normal levels, without
serious adverse consequences as a result of the platelet count
reduction. The benefit may be proportional or correlative to the
reduction in platelet count in a broad safety range. Thus in
situations where it is desirable to inhibit a pathological
condition or process mediated in part by normal levels of
circulating platelets, subjects can be treated to lower platelet
count preferably to a below normal level, thereby inhibiting the
development, progression or propagation of the condition or
accelerating or enhancing its regression. The methods of the
invention are also useful for reducing the incidence of abnormal
vessel growth induced by the presence of platelets.
[0006] A method is provided for treating a subject to reduce the
risk of developing an adverse condition or to inhibit the
progression and consequences of an adverse condition mediated at
least in part by platelets. In some aspects, the subject is treated
to reduce platelet count to low normal levels, while in other
aspects the subject is treated to reduce platelet count to below
normal levels. In one embodiment, the subject is treated with a
pharmaceutical agent.
[0007] In one aspect, the invention provides a method for treating
a subject to inhibit a vaso-occlusive event. Inhibiting a
vaso-occlusive event means to prevent the formation of a
vaso-occlusive event, to reduce progression and consequences of an
already established vaso-occlusive event or to induce regression of
a vaso-occlusive event. The invention also provides other methods
aimed at reducing morbidity or mortality of subjects from
vaso-occlusive events such as but not limited to thrombotic events
which may lead to total or partial vessel blockage by thrombus, or
arterial stenosis due to excessive cell proliferation.
[0008] The methods of the invention comprise administering to a
subject in need of such treatment an agent that reduces platelet
count in the subject. The agent is administered in an amount
effective to reduce platelet count in the subject to at least a low
normal level. Such reductions in platelet count will reduce
morbidity and/or mortality and thereby provide patient outcome
benefit.
[0009] As used herein, a vaso-occlusive event includes a
pathological partial occlusion (including a narrowing) or complete
occlusion of a blood vessel, a stent or a vascular graft. A
vaso-occlusive event intends to embrace thrombotic and
thromboembolic events, and the vascular occlusion disorders or
conditions to which they give rise. Thus, a vaso-occlusive event is
intended to embrace all vascular occlusive disorders resulting in
partial or total vessel occlusion from thrombotic or thromboembolic
events, except those that are related to high platelet count due to
a hematological proliferative disorder. A thrombotic event as used
herein is meant to embrace both a local thrombotic event and a
distal thrombotic event (e.g., a thromboembolic event such as for
example an embolic stroke). A vaso-occlusive event also includes
abnormal blood vessel growth induced by the presence of platelets
and the factors they secrete. An example of this latter form of
vaso-occlusive event is intimal hyperplasia which results in a
narrowing of the blood vessels (i.e., reduction in the diameter of
blood vessels either locally or throughout an extended segment of
the vessel) due to a hyperproliferation of cells of the intimal
layer of the blood vessel wall.
[0010] Preferably, the subject is otherwise free of symptoms
calling for treatment with the agent. In some embodiments, the
subject is preferably free of symptoms associated with a
hematological proliferative disorder such as for example
myeloproliferative disease. Preferably, the subject is a human
subject, but is not so limited. In another embodiment, the subject
is apparently healthy. In preferred embodiments, the subjects do
not have abnormally elevated platelet levels (i.e., a platelet
count that is higher than the normal range) that are caused by a
hematological proliferative disorder. Thus, preferably, the
subjects do not have a hematological proliferative disorder. In an
important embodiment, the subject has a normal platelet count prior
to treatment. In some embodiments, the subject has a higher
platelet count than the mean normal level but is still considered
within the normal range. As an example, a subject with a platelet
count of 450.times.10.sup.3 platelets per .mu.l is considered to be
at the high end of the normal range and is intended to be treated
by the methods of the invention. In some embodiments, the subject
may have a platelet count above the usual range, but without any
underlying hematological proliferative disorder. In another
embodiment, the subject is not a post-menopausal female.
[0011] In some aspects, the invention intends to treat subjects who
are at risk of a vaso-occlusive event. These subjects may or may
not have had a previous vaso-occlusive event. The invention
embraces the treatment of subjects prior to a vaso-occlusive event,
at a time of a vaso-occlusive event and following a vaso-occlusive
event. Thus, as used herein, the "treatment" of a subject is
intended to embrace both prophylactic and therapeutic treatment,
and can be used both to limit or to eliminate altogether the
symptoms or the occurrence of a vaso-occlusive event. In one
embodiment, the subject may exhibit symptoms of a vaso-occlusive
event.
[0012] The invention also intends to embrace the treatment of a
subject that has an abnormally elevated risk of a vaso-occlusive
event such as a thrombotic event. The subject may have vascular
disease. The vascular disease may be selected from the group
consisting of arteriosclerosis, cardiovascular disease,
cerebrovascular disease, renovascular disease, mesenteric vascular
disease, pulmonary vascular disease, ocular vascular disease or
peripheral vascular disease.
[0013] In another embodiment, the subject has had a primary
vaso-occlusive event such as a primary thrombotic event. The agent
may be administered to a subject following a primary vaso-occlusive
event. The method of the invention embraces treatment of a subject
to reduce the risk of a secondary thrombotic event or to inhibit
the propagation of an existing thrombotic event. The thrombotic
event may be selected from the group consisting of arterial
thrombosis, coronary thrombosis, heart valve thrombosis, coronary
stenosis, stent thrombosis and graft thrombosis. The vaso-occlusive
event also includes disorders or conditions that may arise from a
thrombotic event or a thromboembolic event and in this regard a
vaso-occlusive event includes but is not limited to myocardial
infarction, stroke and transient ischemic attack. In an important
embodiment the vaso-occlusive event is myocardial infarction. In
one embodiment, the subject has had a myocardial infarction. A
subject who has hypercholesterolemia, hypertension or
atherosclerosis also can be treated by the methods of the
invention.
[0014] In yet another embodiment, the subject is one who will
undergo an elective surgical procedure. The agent may be
administered to such a subject prior to the elective surgical
procedure. The method of the invention can also be directed towards
a subject who has undergone a surgical procedure. As used herein, a
surgical procedure is meant to embrace those procedures that have
been classically regarded as surgical procedures as well as
interventional cardiology procedures such as arteriography,
angiography, angioplasty and stenting. Thus, the surgical
procedure, whether elective or not, can be selected from the group
consisting of coronary angiography, coronary stent placement,
coronary by-pass surgery, carotid artery procedure, peripheral
stent placement, vascular grafting, thrombectomy, peripheral
vascular surgery, vascular surgery, organ transplant, artificial
heart transplant, vascular angioplasty, vascular laser therapy,
vascular replacement, prosthetic valve replacement and vascular
stenting.
[0015] In a preferred embodiment, the agent is anagrelide. In one
embodiment, the agent is a derivative of anagrelide. In important
embodiments, the agent is not a 2-aryl benzo[b]thiophene. In other
important embodiments, the agent is not raloxifene hydrochloride.
However, the agent is not an MPL pathway inhibitory agent (i.e.,
the agent does not impact upon the signal transduction pathway
involving thrombopoietin and the MPL receptor).
[0016] The agent is administered in an amount effective to reduce
platelet count, in the subject, preferably to at least low normal
levels. In one embodiment, the agent is administered in an amount
ranging from 30 .mu.g/kg/day to 150 .mu.g/kg/day. In another
embodiment, the agent is administered in an amount ranging from 1
.mu.g/kg/day to 150 .mu.g/kg/day. In some embodiments, these latter
ranges are preferred when the agent is anagrelide or an anagrelide
derivative.
[0017] In one embodiment, the agent is administered in an amount
effective to reduce the platelet count to at least low normal
levels if the subject has a normal platelet count prior to
treatment.
[0018] In some embodiments, the agent is administered in an amount
effective to reduce the platelet count to below normal levels if
the subject has an above normal platelet count prior to treatment.
In these latter embodiments, the subject may not have a
hematological proliferative disorder, but is not so limited.
[0019] Depending upon the particular embodiment, the platelet count
is reduced anywhere from at least 10% to 95% of pre-treatment
levels. In particular embodiments, the platelet count is reduced by
at least 90%, at least 80%, at least 70%, at least 60%, at least
50%, at least 40%, at least 30%, at least 20%, or at least 10%. In
some important embodiments, the platelet count is reduced by more
than 10%. In another embodiment, platelet count is reduced by more
than 30% or by more than 40%.
[0020] In some embodiments that embrace the treatment of a human
subject, platelet count is preferably reduced to below
200.times.10.sup.3 platelets per .mu.l, and in still others to
below 150.times.10.sup.3 platelets per .mu.l. In still another
embodiment, platelet count is reduced to below 100.times.10.sup.3
platelets per .mu.l of blood in a human subject. In embodiments in
which the platelet count is reduced to a low normal level this is
defined as 10% less than the mean normal platelet count. In other
embodiments, the platelet count is reduced to below normal
levels.
[0021] In yet another embodiment, the agent is administered in an
amount effective to reduce the platelet count by at least 10% and
to an amount above 200.times.10.sup.3 platelets per .mu.l. In other
embodiments, the agent is administered in an amount effective to
reduce the platelet count by at least 10% and below
200.times.10.sup.3 platelets per .mu.l. In other embodiments, the
agent is administered in an amount effective to reduce the platelet
count by at least 20% and to below 200.times.10.sup.3 platelets per
.mu.l.
[0022] The agent of the invention can be administered
simultaneously or consecutively with another therapeutic compound
such as an agent which would normally be indicated for the subject.
Such agents include agents for treating vascular disease or
vascular complications (i.e., complications resulting from such
disease). In some important embodiments, the agent for treating
vascular disease or vascular complications is an anti-thrombotic
agent. The anti-thrombotic agent may be selected from the group
consisting of an anti-coagulant agent, a fibrinolytic agent and an
inhibitor of platelet function, but is not so limited. Thus, in one
embodiment, the agent is administered with an inhibitor of platelet
function. The inhibitor of platelet function may be selected from
the group consisting of aspirin, abciximab, clopidogrel and
dipyridamole. In another embodiment, the agent may be administered
with an anti-coagulant agent. The anti-coagulant may be selected
from the group consisting of glycosaminoglycans (e.g., heparins)
and vitamin K antagonists. In a further embodiment, the agent is
administered with a fibrinolytic agent, such as but not limited to
one selected from the group consisting of plasminogen activators
such as tissue plasminogen activator (TPA), streptokinase and
urokinase, plasmin and plasminogen. Depending upon the embodiment,
the agent of the invention may be administered before,
simultaneously with or following administration of the agent for
treating vascular disease or vascular complications.
[0023] Other useful categories of such agents include but are not
limited to anti-inflammatory agents, anti-thrombotic agents,
anti-platelet agents, fibrinolytic agents, lipid reducing agents,
direct thrombin inhibitors, glycoprotein IIb/IIIa receptor
inhibitors, agents that bind to cellular adhesion molecules and
inhibit the ability of white blood cells to attach to such
molecules, calcium channel blockers, beta-adrenergic receptor
blockers, cyclooxygenase-2 inhibitors, and angiotensin system
inhibitors.
[0024] In one embodiment, the agent is administered following a
primary vaso-occlusive event such as a thrombotic event. The agent
can be administered in a number of ways, including enteral and
parenteral routes. In some preferred embodiments, the agent is
administered in a sustained release device.
[0025] The invention also provides a number of pharmaceutical
preparations comprising agents that reduce platelet count. The
pharmaceutical preparations of the invention comprise one or more
agents that reduce platelet count and a pharmaceutically acceptable
carrier. The agent is present in the pharmaceutical preparation in
an amount effective to reduce platelet count. In important
embodiments of the invention, the pharmaceutical preparation
comprises the agent in an amount effective to reduce platelet count
to low normal levels or to below normal levels.
[0026] In yet a further aspect, the invention provides a sustained
release device that comprises an agent that reduces platelet count
in a subject, wherein the agent is released for at least 7 days. In
one embodiment, the sustained release device further comprises an
agent for treating vascular disease or vascular complications. The
agent for treating vascular disease or vascular complications may
be an anti-thrombotic agent but is not so limited. In one
embodiment, the anti-thrombotic is selected from the group
consisting of an anti-coagulant agent, a fibrinolytic agent and an
inhibitor of platelet function.
[0027] Preferably, the agent is released from the sustained release
device in an amount effective to reduce platelet count in a subject
to low normal or below normal levels. In one embodiment the agent
is anagrelide or a derivative of anagrelide. Depending upon the
embodiment and the nature of the agent, the sustained release
device may release the agent at a rate ranging from 30 .mu.g/kg/day
to 150 .mu.g/kg/day. In other embodiments, the agent may be
released at a rate ranging from 1 .mu.g/kg/day to 150 .mu.g/kg/day.
In one embodiment, the sustained release device releases the agent
for at least 30 days. In other embodiments, the agent is released
for at least 6 months, for at least 1 year, for at least 2 years or
for at least 5 years or more. Preferably, the agent is released in
an effective amount that does not affect platelet function.
[0028] In another aspect, a subject's blood is treated
extracorporeally to reduce platelet count to low normal or below
normal levels, using procedures such as pheresis or adsorption of
platelets and removal. Subjects, target platelet count and
concurrent therapies are as described above. This aspect of the
invention is particularly suited to acute therapy, although it is
not so limited.
DETAILED DESCRIPTION OF THE INVENTION
[0029] Unexpectedly, the invention involves the discovery that
treating human subjects, especially those who do not have a
hematological proliferative disorder, to induce a lower platelet
count (such as to low normal levels or in some preferred instances
to below normal levels) can have desirable medical benefit without
significant adverse side effects.
[0030] The invention is premised in part on the discovery that a
reduction in platelet count in a subject, such as for example to
low normal and more preferably below normal levels, reduces the
risk of a vaso-occlusive event such as a thrombotic event in the
subject without significant adverse side effects. As used herein, a
vaso-occlusive event is an event that is characterized by or
results in a decrease in the internal diameter of blood vessels
either locally or systemically to an extent which impedes blood
flow in a subject and which for the purposes of the invention is of
a pathological nature. Thus, a vaso-occlusive event embraces
pathological narrowing or occlusion of a stent, a vascular graft or
a blood vessel. As used herein, "pathological narrowing or
occlusion" refers to narrowing or occlusion which is abnormal
and/or disease-related. A vaso-occlusive event includes events
which cause blood vessel narrowing or occlusion (such as thrombotic
events, thromboembolic events and intimal hyperplasia) as well as
conditions which result from such blood vessel narrowing (such as
myocardial infarction and ischemic stroke).
[0031] A thrombotic event is an event associated with the formation
or presence of a thrombus in a subject, particularly when present
in the vasculature. A thrombus is an aggregation of blood factors,
primarily platelets and fibrin with entrapment of cellular
elements, frequently causing vascular obstruction at the point of
its formation. Thrombotic events embrace thrombosis at a primary
site as well as at a distal site (i.e., thromboembolism).
Thrombosis collectively refers to diseases caused by the formation,
development, or presence of a thrombus. Thromboembolism refers to
diseases characterized by the blocking of a vessel, other than at
the initial site of thrombus formation, by a thrombus which has
been carried to the distal site by the blood current. As used
herein, the term thrombosis is intended to embrace
thromboembolism.
[0032] Thrombotic events, including thromboembolic events, can be
serious medical conditions particularly since they can cause a
reduction in blood flow to critical organs including the brain and
myocardium. Examples of thrombotic events include but are not
limited to arterial thrombosis, including stent and graft
thrombosis, cardiac thrombosis, coronary thrombosis, heart valve
thrombosis and venous thrombosis. Cardiac thrombosis is thrombosis
in the heart. Arterial thrombosis is thrombosis in an artery.
Coronary thrombosis is the development of an obstructive thrombus
in a coronary artery, often causing sudden death or a myocardial
infarction. Venous thrombosis is thrombosis in a vein. Heart valve
thrombosis is thrombosis on a heart valve. Stent thrombosis is
thrombosis resulting from and/or located in the vicinity of a
vascular stent. Graft thrombosis is thrombosis resulting from
and/or located in the vicinity of an implanted graft, particularly
a vascular graft.
[0033] Examples of conditions or disorders that result from
thrombotic events include but are not limited to myocardial
infarction, stroke, transient ischemic attacks, amaurosis fugax,
aortic stenosis, cardiac stenosis, coronary stenosis and pulmonary
stenosis. Stenosis is the narrowing or stricture of a duct or
canal. Coronary stenosis is the narrowing or stricture of a
coronary artery. Cardiac stenosis is narrowing or diminution of any
heart passage or cavity. Pulmonary stenosis is the narrowing of the
opening between the pulmonary artery and the right ventricle.
Aortic stenosis is narrowing of the aortic orifice of the heart or
of the aorta itself.
[0034] Vaso-occlusive events also include disorders in which the
blood vessel narrowing results not necessarily from a thrombus but
rather a thickening of the vessel wall such as with intimal
hyperplasia. Intimal hyperplasia refers to a condition
characterized by abnormal proliferation of the cells of the intimal
layer of the blood vessel wall.
[0035] Thus, one aspect of the invention relates to a method for
reducing the risk of a thrombotic event. In a particular
embodiment, the method reduces the risk of stroke. Stroke is a
condition resulting from the lack of oxygen to the brain, resulting
from one or more occlusive thrombi. Depending on the area of the
brain affected, stroke can result in a wide range of symptoms from
transient ischemic attacks to death (e.g., coma, reversible or
irreversible paralysis, speech problems or dementia). In preferred
embodiments, the stroke is non-hemorrhagic in nature.
[0036] The method of the invention in another embodiment relates to
reducing the risk of myocardial infarction. Myocardial infarction
refers to an irreversible injury to the heart muscle. Myocardial
infarction generally results from an abrupt decrease in coronary
blood flow following a thrombotic occlusion (e.g., a
thromboembolism) of a coronary artery. The thrombus, in many
instances, forms after the rupture of atherosclerotic plaques in
diseased coronary arteries. Such injury is highly correlated with
factors such as cigarette smoking, hypertension and lipid
accumulation.
[0037] Transient ischemic attack is a transient acute neurological
dysfunction resulting from a thromboembolism in the cerebral
circulation. Amaurosis fugax is the temporary monocular blindness
resulting from a thromboembolism in the retinal vasculature.
[0038] The methods of the invention can be used to reduce the risk
of a primary or a secondary vaso-occlusive event such as a
thrombotic event or to inhibit the progression of such an event. A
primary vaso-occlusive event refers to the first known
vaso-occlusive event experienced by the subject. A secondary
vaso-occlusive event refers to a vaso-occlusive event which occurs
in a subject known or diagnosed as having previously experienced a
vaso-occlusive event (i.e., a primary vaso-occlusive event).
[0039] According to the invention, the risk of a vaso-occlusive
event such as a thrombotic event is reduced by administering to a
subject an agent that reduces platelet count to a "low normal
level" and in some embodiments to a "below normal level." A
"normal" platelet count as used herein may be a level in a control
population, which preferably includes subjects having similar
characteristics as the treated individual, such as age and sex. The
"normal" level can also be a range, for example, where a population
is used to obtain a baseline range for a particular group into
which the subject falls. Thus, the "normal" value can depend upon a
particular population selected. Preferably, the normal levels are
those of apparently healthy subjects who have no prior history of
platelet-mediated disorders. Such "normal" levels then can be
established as preselected values, taking into account the category
in which an individual falls. Appropriate ranges and categories can
be selected with no more than routine experimentation by those of
ordinary skill in the art. Either the mean or another preselected
number within the range can be established as the normal
preselected value.
[0040] As used herein, the terms "platelet level," "platelet
number" and "platelet count" are used interchangeably to refer to
the number of platelets per a given volume of blood in a subject.
The platelet count may be referred to in a number of ways (e.g.,
per .mu.l of blood, per ml of blood, etc.). Generally, platelet
counts are referred to herein as the number of platelets per .mu.l
of blood (i.e., platelets per .mu.l); however, other units may be
used.
[0041] As is known in the art, the typical range for platelets in a
"healthy" human subject is about 150.times.10.sup.3 to
450.times.10.sup.3 platelets per .mu.l of blood (mean
300.times.10.sup.3 platelets per .mu.l). Thus, "below normal
levels" of platelets, as used herein, in this population is
typically less than 150.times.10.sup.3 platelets/.mu.l. "Low normal
levels" as used herein refer to a platelet count which is 10% less
than the mean normal platelet count. Thus, for the population just
mentioned, low normal levels would be 270.times.10.sup.3
platelets/.mu.l. Human subjects who have a platelet count of less
than 100.times.10.sup.3 platelets/.mu.l are considered
thrombocytopenic. Platelet counts of less than 25.times.10.sup.3
platelets/.mu.l indicate severe thrombocytopenia. The invention
intends to embrace reductions in platelet counts resulting in
platelet counts of equal to or less than 270.times.10.sup.3,
260.times.10.sup.3, 250.times.10.sup.3, 240.times.10.sup.3,
230.times.10.sup.3, 220.times.10.sup.3, 210.times.10.sup.3
platelets/.mu.l of blood. In preferred embodiments the platelet
counts are equal to or less than 200.times.10.sup.3,
190.times.10.sup.3, 180.times.10.sup.3, 170.times.10.sup.3,
160.times.10.sup.3, 150.times.10.sup.3, 140.times.10.sup.3,
130.times.10.sup.3, 120.times.10.sup.3, 110.times.10.sup.3,
100.times.10.sup.3 platelets/.mu.l. Nonetheless, it will be
understood that it may be desirable depending on factors such as
the particular disease, and the age, and the physical condition of
the subject that lower levels are desirable, such as platelet
counts equal to or less than 90.times.10.sup.3, 80.times.10.sup.3,
70.times.10.sup.3, 60.times.10.sup.3, 50.times.10.sup.3, and
25.times.10.sup.3 platelets/.mu.l of blood. In one embodiment,
platelet count is reduced to below 200.times.10.sup.3
platelets/.mu.l in a human subject. In more preferred embodiments,
the platelet count is reduced to below 150.times.10.sup.3
platelets/.mu.l while in some other even more preferred embodiments
the platelet count is reduced to below 100.times.10.sup.3
platelets/.mu.l in a human subject.
[0042] In some instances, it may be desirable to treat subjects
having a platelet count in the normal range in order to reduce
their platelet count and thereby reduce the risk of a
vaso-occlusive event even if the post-treatment platelet count is
still in the normal range. As an example, the methods of the
invention may be used to treat a subject who has a platelet count
of 450.times.10.sup.3 platelets/.mu.l which while high, is still in
the normal range. The subject may be treated in order to reduce the
platelet count to either a lower level within the normal range
(e.g., a low normal level, as described herein) or to a below
normal level.
[0043] Platelet reductions may also be measured as a percentage of
the pre-treatment platelet count in a subject. Thus the agents of
the invention may be administered in an amount effective to reduce
platelet count from at least 10% to at least 95% of pre-treatment
levels. In some embodiments, the agents are administered in an
amount effective to reduce platelet count by at least 20%, at least
30%, at least 40%, at least 50%, at least 60%, at least 70%, at
least 80%, or at least 90% of pre-treatment levels. In some
embodiments, the subjects are normal subjects who do not have an
abnormally high level of circulating platelets such as a platelet
count greater than 500.times.10.sup.3 platelets per .mu.l, or
greater than 600.times.10.sup.3 platelets per .mu.l which may be
due to a hematological proliferative disorder. However, in other
embodiments the subjects are normal subjects who have a high level
of circulating platelets which is still however within the normal
range. The invention intends to treat this latter group of subjects
provided the subject does not have a hematological proliferative
disorder such as myeloproliferative disease. In still other
embodiments, the subject may have a platelet count above the normal
range, yet not have a hematological proliferative disorder. In
preferred embodiments, platelets are reduced by at least 20% of
pre-treatment levels. In more preferred embodiments, platelets are
reduced by at least 20% to at least 90% of pre-treatment levels. In
still other embodiments, platelets are reduced by over 50% of
pre-treatment levels.
[0044] The subjects may be treated so as to achieve both a drop in
platelet count below an absolute level (such as for example below
200.times.10.sup.3 platelets per .mu.l) and a particular percentage
drop in platelet count relative to pre-treatment levels (such as
for example at least 10%). As an example, a subject may be treated
so as to reduce platelet count by at least 20% and to achieve a
platelet count of less than 200.times.10.sup.3 platelets per
.mu.l.
[0045] The invention intends to treat subjects who would benefit
from inhibiting the growth of an existing thrombus or lowering of
the risk of a vaso-occlusive event such as a thrombotic event. A
subject is a mammal including humans, nonhuman primates, dogs,
cats, sheep, goats, horses, cows, pigs or rodents. The preferred
subject is a human. The subject may be apparently healthy. An
apparently healthy subject is one who, at the time of treatment,
does not exhibit disease signs or symptoms. In other words, such
individuals, if examined by a medical professional, would be
characterized as healthy and free of symptoms of disease. The
apparently healthy subjects however may still demonstrate
particular risk factors which may place them at an elevated risk of
a thrombotic event. For example, such subjects may be apparently
healthy and still have a family history of thrombosis-related
disorders. Alternatively, the subject may have symptoms of
vaso-occlusive disease (such as chest pain, heart palpitations,
shortness or breath, as well as a wide range of other symptoms well
known to a medical practitioner of ordinary skill) or may have been
diagnosed with such disease.
[0046] In still other embodiments the subject is one who is
otherwise free of symptoms calling for treatment with an agent that
reduces platelet count in the subject. These subjects may not
necessarily be apparently healthy but at a minimum they do not
exhibit symptoms which ordinarily call for treatment specifically
with an agent which reduces platelet count. As an example, if the
agent is anagrelide, the subject can be otherwise free of signs,
symptoms or evidence of disorders for which anagrelide would
normally be prescribed (e.g., myeloproliferative disease).
Anagrelide has been prescribed previously for patients diagnosed
with essential thrombocythemia (ET). The hallmark of ET is an
abnormally high level of platelets in the circulation, generally
greater than about 600.times.10.sup.3 platelets per .mu.l of blood.
Involvement and expansion of other hemopoietic cell types is not
necessarily a common feature of ET. Splenomegaly may also be
observed in such patients. Secondary thrombocytosis is another
disease state associated with an abnormally elevated number of
platelets. This latter condition is distinguished from ET in that
it results from a variety of primary conditions such as recovery
from acute infection, malignant diseases including carcinoma and
lymphoma, hemolytic anemia, acute hemorrhage, iron deficiency,
response to certain drugs and chronic inflammatory disorders.
Subjects for whom the methods of the invention are not intended are
those diagnosed with conditions which already call for treatment
with an agent such as anagrelide, i.e., secondary thrombocytosis,
essential thrombocytosis, polycythemia vera, chronic myelogenous
leukemia and myelofibrosis. In other words, in some preferred
embodiments, the subject is not one who has, or who has been
diagnosed with, a hematological proliferative disorder (such as
myeloproliferative disease) which indicates the need for platelet
lowering therapy.
[0047] The subject can also be one who is at abnormally elevated
risk of a thrombotic event. The subject to be treated may be one
who is prone to a thrombotic event. Included in this category of
subjects are (1) those who have undergone a surgical procedure and
are immobilized following such a procedure, (2) those who have
chronic congestive heart failure, (3) those who have
atherosclerotic vascular disease, (4) those who have malignancy
preferably other than a hematological malignancy which results in
abnormally high platelet counts, and (5) those who are pregnant. A
large majority of human subjects prone to thrombotic events do not
manifest any observable perturbation in hemostasis.
[0048] One category of subjects with an abnormally elevated risk of
a thrombotic event is those subjects who have previously
experienced a primary thrombotic event. Subjects having an
abnormally elevated risk of a thrombotic event also include (i)
those who have inherited a disposition towards thrombosis, for
example those with a family history of thrombosis related
disorders, (ii) those who have acquired a risk of a thrombotic
event such as surgical patients, and (iii) those who engage in
lifestyle habits which are considered high risk indicators for
thrombosis.
[0049] One category of subjects with an abnormally elevated risk of
thrombosis is those subjects having vascular disease. Vascular
disease is a term which broadly encompasses all disorders of blood
vessels (collectively known as the vasculature) including small and
large arteries and veins, and blood flow. The most prevalent form
of vascular disease is arteriosclerosis, a condition associated
with the thickening and hardening of the arterial wall.
Arteriosclerosis or an arteriosclerotic condition as used herein
means classical atherosclerosis, accelerated atherosclerosis,
atherosclerosis lesions and any other arteriosclerotic conditions
characterized by undesirable endothelial and/or vascular smooth
muscle cell proliferation, including vascular complications of
diabetes. It is responsible for the majority of deaths in the
United States and in most westernized societies.
[0050] Arteriosclerosis of the large vessels is referred to as
atherosclerosis. Atherosclerosis is the predominant underlying
factor in disorders such as coronary artery disease, aortic
aneurysm, arterial disease of the lower extremities and
cerebrovascular disease. Other types of arteriosclerosis include
focal calcific arteriosclerosis (Monckeberg's sclerosis) and
arteriolosclerosis. Arterial diseases other than arteriosclerosis
include congenital structural defects, inflammatory or
granulomatous diseases (e.g., syphilitic aortitis), and small
vessel disorders such as hypertension and autoimmune diseases.
Disorders which are associated with early arteriosclerosis include
diabetes mellitus, hypertension, familial hypercholesterolemia,
familial combined hyperlipidemia, familial dysbetalipoproteinemia,
familial hypoalphalipoproteinemia, hypothyroidism, cholesterol
ester storage disease, systemic lupus erythrematosus,
homocysteinemia, chronic renal insufficiency, chronic vitamin D
intoxication, pseudoxanthoma elasticum, idiopathic arterial
calcification in infancy, aortic valvular calcification in the
elderly and Werner's syndrome.
[0051] Subjects with cardiovascular disease, cerebrovascular
disease and/or peripheral vascular disease (e.g., diabetic feet,
failed grafts) are also considered at abnormally high risk of a
thrombotic event. Cardiovascular disease refers to a number of
disorders of the heart and vascular system. Cerebrovascular disease
refers to a number of disorders of the blood vessels in the
cerebrum of the brain. Peripheral vascular disease encompasses
disorders of the peripheral vasculature including that of the lower
extremities.
[0052] The method of the invention can be used to treat subjects at
abnormally elevated risk of experiencing particular vaso-occlusive
events. For example, a subject with an abnormally elevated risk of
myocardial infarction can be treated according to the method of the
invention. Subjects may be treated prophylactically to reduce the
risk of a primary or secondary myocardial infarction. As used
herein, subjects having an abnormally elevated risk of myocardial
infarction include those with unstable angina, multiple coronary
risk factors, and Prinzmetal's variant angina. Less common
etiologic factors include hypercoagulability, coronary emboli,
collagen vascular disease, and cocaine abuse.
[0053] A subject with an abnormally elevated risk of stroke, for
example non-hemorrhagic stroke, can also be treated according to
the invention. Subjects having an abnormally elevated risk of an
ischemic stroke are a category determined according to conventional
medical practice; such subjects may also be identified in
conventional medical practice as having known risk factors for
stroke or having increased risk of cerebrovascular events. The
primary risk factors include hypertension, hypercholesterolemia,
and smoking. Subjects having an abnormally elevated risk of an
ischemic stroke also include individuals having any cardiac
condition that may lead to decreased blood flow to the brain, such
as atrial fibrillation, ventrical tachycardia, dilated
cardiomyopathy and other cardiac conditions requiring
anticoagulation. Subjects having an abnormally elevated risk of an
ischemic stroke also include individuals having conditions
including arteriopathy or brain vasculitis, such as that caused by
lupus, congenital diseases of blood vessels, such as cadasil
syndrome, or migraine, especially prolonged episodes.
[0054] Another category of subjects with an abnormally elevated
risk of a thrombotic event are those subjects who will undergo or
those who have already undergone a surgical or mechanical
interventional procedure for the purposes of vessel repair and/or
revascularization. Such procedures may be therapeutic or diagnostic
in nature, and thus can also be elective or emergency treatments,
and most likely involve the risk of formation of thrombi or the
release of emboli. Procedures which fall into this category include
but are not limited to vascular surgery including peripheral
vascular surgery, vascular grafting, vascular laser therapy,
vascular replacement, including prosthetic valve replacement, and
vascular stenting, ventricular assist procedures, artificial heart
transplant, heart and other organ transplants which require an
interfacing of the transplanted organ with the vasculature of the
transplant recipient, thrombectomy, coronary angiography, coronary
and peripheral stent placements, carotid artery procedures
including carotid endarterectomy, brain angiography, neurosurgical
procedures in which blood vessels are compressed or occluded,
cardiac catheterization, vascular angioplasty, including balloon
angioplasty, and coronary by-pass surgery. In addition to the risk
of thrombus formation during or immediately following the surgical
procedure, there also exists a risk to subjects who have undergone
a surgical procedure and are currently immobilized following the
procedure. Thus the invention seeks to embrace treatment of the
subject prior to, during and following surgical procedures.
[0055] Other factors which predispose subjects to abnormally
elevated risk of a thrombotic event are genetic risk factors and
lifestyle habits. Inherited conditions can generally be regarded as
hypercoaguable states or pre-thrombotic states. The pre-thrombotic
subject can sometimes be identified if they present with a personal
history of early (i.e., adolescent or as a young adult) and/or
repeated thromboembolic events in the absence of an overt
pre-disposing condition, and/or a family history of thrombosis
related conditions. Subjects who have experienced pain in walking,
ischemia (i.e., a deficiency of blood flow to an area of the body
due to functional constriction or obstruction of a blood vessel),
gangrene (i.e., a death of tissue, usually considerable in mass and
generally associated with loss of blood flow) and chest pain, may
be regarded as having a personal history of arterial thrombosis or
stroke, and are thus also at risk of a thrombotic event. Risk
factors for a thrombotic event also include inheritable
hematological abnormalities such as deficiency and/or dysfunction
in any number of factors including anti-thrombin III, protein C,
protein S and clotting factor V. Cardiovascular abnormalities,
i.e., congenital structural abnormalities of the cardiovascular
system, are also considered risk factors for thrombotic events.
Vascular abnormalities such as atherosclerotic plaque ruptures are
also considered a risk factor.
[0056] Lifestyle risk factors include smoking, failure to exercise
and diet to the extent that it affects other risk factors such as
obesity, high cholesterol, hyperlipidemia and high blood pressure
(i.e., hypertension). High cholesterol (i.e.,
hypercholesterolemia), high blood pressure (i.e., hypertension),
hyperlipidemia, and obesity are most certainly also induced by a
variety of non-dietary causative elements including genetic and
environmental factors.
[0057] A hyperlipidemic subject is defined as one whose cholesterol
and triglyceride levels equal or exceed the limits set as described
herein for both the hypercholesterolemic and hypertriglyceridemic
subjects. A hypercholesterolemic subject (i.e., one with high
cholesterol) has either an LDL (i.e., low-density lipoprotein)
level of >160 mg/dL, or an LDL level of >130 mg/dL and at
least two risk factors selected from the group consisting of male
gender, family history of premature coronary heart disease,
cigarette smoking (more than 10 cigarettes per day), hypertension,
low HDL (<35 mg/dL), diabetes mellitus, hyperinsulinemia,
abdominal obesity, high lipoprotein (a), and a personal history of
cerebrovascular disease or occlusive peripheral vascular disease. A
hypertriglyceridemic subject has a triglyceride (TG) level of
>250 mg/dL.
[0058] Subjects who are hypertensive (i.e., those that have high
blood pressure) are also at risk of a thrombotic event. A
hypertensive subject is one who experiences persistently high
arterial blood pressure. Hypertension may have no known cause, in
which case it is referred to as essential or idiopathic
hypertension. Alternatively, hypertension may be associated with
other primary diseases, in which case it is referred to as
secondary hypertension. It is generally considered a risk factor
for the development of heart disease, peripheral vascular disease,
stroke and kidney disease. In adults, a diastolic pressure below 85
mmHg is considered normal, between 85 and 89 mmHg is considered
high normal, 90 to 104 mmHg is considered mild hypertension, 105 to
114 mmHg is considered moderate hypertension and 115 mmHg or
greater is considered severe hypertension. When the diastolic
pressure is below 90 mmHg, a systolic pressure below 140 mmHg
indicates normal blood pressure, between 140 and 159 mmHg is
borderline isolated systolic hypertension and 160 mmHg or higher is
isolated systolic hypertension. Thus, generally, normal subjects
are those with a blood pressure of 140/90 or less.
[0059] Other risk factors which contribute to an elevated risk of
thrombotic events, and the disorders which underlie such thrombotic
events (e.g., arteriosclerosis), include hyperlipidemia,
hyperglycemia and diabetes mellitus, stress and personality, low
index of high density lipoproteins (HDL), male gender, age,
hyperinsulinemia, high lipoprotein (a) and a personal history of
cerebrovascular disease or occlusive peripheral vascular disease.
Hyperglycemia is a condition associated with too high a level of
glucose in the blood, sometimes indicative of uncontrolled
diabetes. It occurs when the body does not have enough insulin or
cannot effectively use insulin to metabolize glucose. This
condition may be associated with diabetes mellitus, Cushing's
disease, and Cushing's syndrome. Signs of hyperglycemia are
significant thirst, dry mouth, and frequent urination. Normal
asymptomatic human subjects who are at least 50 years of age, and
more preferably 60 years of age, are also at increased risk for
thrombosis.
[0060] Subjects at risk of having intimal hyperplasia as well as
those having intimal hyperplasia are also intended to be treated
according to the methods of the invention. Thus, the method of the
invention can be used to treat subjects who have or are at risk of
having intimal hyperplasia, as well as to reduce the risk of
intimal hyperplasia. One common form of intimal hyperplasia is
atherosclerosis.
[0061] The invention also intends to treat, in other aspects,
subjects who have had a primary vaso-occlusive event or who are
currently experiencing a vaso-occlusive event, including subjects
who have been diagnosed with thrombosis or as having a thrombotic
event. The invention can also be used to treat subjects that
manifest an abnormal healing of blood vessels.
[0062] The treatment method of the invention involves the
administration to a subject of an agent that reduces circulating
platelet count in the subject. Agents which reduce platelet count
are herein sometimes referred to as platelet reducing agents.
Preferably such agents have the specific effect of reducing only
platelet count without affecting levels of other cell types,
although it should be understood that an agent may also reduce
levels of other cell types provided these latter reductions do not
induce unacceptable levels of adverse side effects associated with
such reduction in other cell types. For example, the agent may
reduce levels of megakaryocytes, the precursors of platelets, and
such reduction should not have any undesirable side effect. As
another example, an agent may be cytotoxic for a megakaryocyte
lineage restricted cell, such as a platelet, and another blood
cell, or a common precursor of these two cell types, in which case
the agent is acceptable only if platelet count can be reduced to
below normal levels without unacceptable levels of side effects
associated with such reduction in the other cell type. In still
another example, the agent may inhibit megakaryocyte function. It
will be apparent to persons of ordinary skill in the art how to
select and distinguish between such agents.
[0063] Agents already known to reduce platelet count include but
are not limited to (1) cAMP phosphodiesterase inhibitors (e.g.,
anagrelide),
6,7-dichloro-1,5-dihydroimidazo-[2,1-b]quinazolin-2(3H)-one or
6,7-dichloro-1,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2-one (U.S.
Pat. Nos. 3,932,407; 4,146,718; RE31,617, Haematologica (1992)
77:40-3), (2) antibodies to cell surface receptors specifically
expressed by platelets or megakaryocytes such as glycoprotein
IIb/IIIa receptor antibodies, (3) most chemotherapeutic anti-cancer
drugs such as busulphan (Br. J. Haematol. 1986 62:229-37),
hydroxyurea (N Engl J Med 1995 332:1132-6), hepsulfan,
phosphorus-32 (Br J Radiol 1997 70:1169-73), pipobroman (Scand J.
Haematol 1986 37:306-9), cyclophosphamide (J Cell Physiol 1982
112:222-8), certain alkylating agents and certain antimetabolites,
(4) cytokines, growth factors and interleukins such as
alpha-interferon (Cancer Immunol Immunother 1987 25:266-73),
gamma-interferon, transforming growth factor-beta, neutrophil
activating peptide-2 and its analogs (U.S. Pat. No. 5,472,944),
macrophage inflammatory protein and its analogs (U.S. Pat. No.
5,306,709), (5) compounds secreted by either platelets or
megakaryocytes such as platelet-factor 4 (U.S. Pat. No. 5,185,323),
transforming growth factor-beta, the 12-17 kD glycoprotein produced
by megakaryocytes, thrombin and thrombospondin and its amino (1-174
amino acid) terminal fragment (J Lab Clin Med 1997 129:231-8), and
(6) other agents including anti-cheloid agents such as Tranilast
(Rizaben) (J Dermatol 1998 25:706-9); forskolin and spleen
anti-maturation factor (U.S. Pat. No. 4,088,753).
[0064] All the aforementioned agents may be suitable for use in the
method of the invention to reduce normal platelet count in a
subject preferably to a below normal level with the purpose of
preventing or treating a vaso-occlusive event such as a thrombosis.
In some instances these benefits are achieved by reducing the
platelet count to low normal while in other more preferred
instances the platelet count is reduced to below normal levels. It
should be understood that the agents useful in the invention may be
capable of affecting platelet function as well as reducing platelet
count. However, preferably, such agents are used in a dose,
formulation and administration schedule which favor the platelet
count reducing activity of the agent and do not impact
significantly, if at all, on platelet function.
[0065] Another category of agents which reduce platelet counts is
MPL pathway inhibitory agents. However, the invention does not
intend to embrace this category of agent in the methods provided
herein.
[0066] A preferred agent is anagrelide. Although anagrelide is
capable of affecting platelet function, it is used in the
compositions and methods of the invention in a dose, formulation
and administration schedule which reduces platelet count
(preferably to below normal levels) without significantly impacting
upon platelet function. Analogs (e.g., derivatives) of anagrelide
which are as effective or more effective than the parent compound
are also intended for use in the method of the invention.
Preferably, such analogs would also be screened for an increased
potency and specificity towards the megakaryocyte lineage with
limited side effects. Synthesis of anagrelide analogs can be
accomplished through routine chemical modification methods such as
those routinely practiced in the art. Analogs of anagrelide have
been reported by a number of groups. Jones et al. reported the
synthesis of an analog, RS-82856
(N-cyclohexyl-N-methyl-4-(7-oxy-1,2,3,5-tetrahydroimidazo-[2,1-b-
]quinazolin-2-one (J. Med. Chem. 1987 30:295-303). Other inhibitors
of platelet CAMP20 phosphodiesterases synthesized by directed
replacement of side chains on anagrelide have been reported by
Meanwell et al. (J. Med. Chem. 1992 35:2672-87). Other anagrelide
analogs have been documented in U.S. Pat. Nos. 3,932,407; 4,146,718
and RE31,617. For example, Beverung, Jr. et al. (U.S. Pat. No.
RE31,617 (1984)) discloses optionally substituted
1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-ones and
6(H)-1,2,3,4-tetrahydropyrimido[2,1-b]quinazolin-2-ones. These
include 1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-bromo-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-nitro-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-amino-1,2,3,5-tetrahydroimidazo-[2,1-b]-quinazolin-2-one,
6-hydroxy-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-hydroxy-1,2,3,5-tetrahydroimidazo-[2,1-b]-quinazolin-2-one,
8-bromo-6-[H]-1,2,3,4-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
6-methyl-7-nitro-1,2,3,5-tetrahydroimidazo-[2,1-b]-quinazolin-2-one,
7-bromo-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-chloro-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
6-chloro-7-bromo-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
6,7-dichloro-1,2,3,5-tetrahydro-imidazo[2,1-b]-quinazolin-2-one,
7-amino-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
7-amino-6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
3-(carboxymethyl)-3,4-dihydro-5-methyl-4-methylene-1H-quinazolin-2-one,
3-(carboxymethyl)-4,5-dimethyl-1,2,3,4-tetrahydroquinazoline-2-one,
2-chloro-3-carbethoxymethyl-4,5-dimethyl-3,4-dihydroquinazoline,
5,6-dimethyl-1,2,3,5-tetrahydroimidazo[2,1-b]-quinazolin-2-one,
3-(carbethoxymethyl)-3,4-dihydro-6-methylene-1H-quinazolin-2-one,
3-(carbethoxymethyl)-4,6-dimethyl-1,2,3,4-tetrahydroqionazolin-2-one,
2-chloro-3-carbethoxymethyl-4,6-dimethyl-3,4-dihydroquinazoline,
5,7-dimethyl-1,2,3,5-tetrahydroimidazo-[2,1-b]quinazolin-2-one,
5-methyl-3-(carbethyoxymethyl)-1,2,3,4-tetrahydroquinazolin-2-one,
2-chloro-3-carbethoxymethyl-5-methyl-3,4-dihyrdoquinazoline
hydrochloride and
6-methyl-1,2,3,5-tetrahydroimidazo-[2,1-b]quinazolin-2-one.
[0067] Many of the above-listed agents while capable of reducing
platelet count can also impact upon other cell lineages,
particularly other hemopoietic cell lineages. It is preferred that
the agents used in the methods of the invention are provided or
administered in a manner which limits effects on other cell types.
One way this can be accomplished is to identify agents which while
perhaps not exclusive for the megakaryocyte lineage, have limited
specificity for other cell lineages. Most of the agents listed
above fall into this category. Another way of limiting unnecessary
side effects is to administer the agent in the maximal dose which
reduces platelet count and which does not impact upon other cell
types. Such determination can be made using in vitro clonogenic
assays such as those described herein, which are standard in the
art. Yet another way of providing specificity is to conjugate an
otherwise non-specific agent with a molecule which will target the
non-specific agent to megakaryocytes and platelets. Potential
targeting molecules are those which normally bind receptors
uniquely expressed on cells of the megakaryocyte lineage.
[0068] The methods of the invention utilize this library technology
to identify small molecules including small peptides which bind to
receptor ligand binding sites. One advantage of using libraries for
antagonist identification is the facile manipulation of millions of
different putative candidates of small size in small reaction
volumes (i.e., in synthesis and screening reactions). Another
advantage of libraries is the ability to synthesize antagonists
which might not otherwise be attainable using naturally occurring
sources, particularly in the case of non-peptide moieties.
[0069] Methods for preparing libraries of molecules are well known
in the art and many libraries are commercially available. Libraries
of interest in the invention include synthetic organic
combinatorial libraries. Libraries are also meant to include, but
are not limited to, synthetic small molecule libraries and chemical
libraries. The libraries can also comprise cyclic carbon or
heterocyclic structure and/or aromatic or polyaromatic structures
substituted with one or more of the above-identified functional
groups.
[0070] Small molecule combinatorial libraries may also be
generated. A combinatorial library of small organic compounds is a
collection of closely related analogs that differ from each other
in one or more points of diversity and are synthesized by organic
techniques using multi-step processes. Combinatorial libraries
include a vast number of small organic compounds. One type of
combinatorial library is prepared by means of parallel synthesis
methods to produce a compound array. A "compound array" as used
herein is a collection of compounds identifiable by their spatial
addresses in Cartesian coordinates and arranged such that each
compound has a common molecular core and one or more variable
structural diversity elements. The compounds in such a compound
array are produced in parallel in separate reaction vessels, with
each compound identified and tracked by its spatial address.
Examples of parallel synthesis mixtures and parallel synthesis
methods are provided in U.S. Ser. No. 08/177,497, filed Jan. 5,
1994 and its corresponding PCT published patent application
WO95/18972, published Jul. 13, 1995 and U.S. Pat. No. 5,712,171
granted Jan. 27, 1998 and its corresponding PCT published patent
application WO96/22529, which are hereby incorporated by
reference.
[0071] One way of testing putatively useful agents is to perform in
vitro assays in which platelets or platelet precursors (e.g.,
megakaryocytes, or megakaryocyte precursors) are exposed to a
compound after which their morphology (for example using an
appropriate cell staining technique such as Wright's stain), number
(for example using a Coulter counter) and/or colony forming ability
are tested. These latter assays can be performed using either cell
lines known to differentiate into the megakaryocyte lineage, or to
the megakaryocyte lineage, several of which have been established
in the prior art and examples of which include the Ba/F3 and
UT-7/GM cell lines, or primary hemopoietic tissue, such as bone
marrow. The number and quality of megakaryocyte colonies can be
determined as a function of the presence and absence of the library
member. Preferably, the assays are carried out by culturing the
cells in a semi-solid culture in an amount of thrombopoietin
sufficient to stimulate maximal megakaryocyte colony growth from
the cell population. The library member is then titrated into the
cultures in order to determine the amount necessary to reduce
megakaryocyte colony formation. In this manner, in addition to the
amount of antagonist necessary to inhibit megakaryocyte growth
altogether, one can also determine that amount which inhibits the
growth by a particular percentage. For example, if it desirable to
reduce megakaryocyte growth and proliferation by 50% in order to
achieve a reduction in platelet count in vivo, then the assay can
be used to determine that amount of antagonist necessary to inhibit
megakaryocyte colony growth by 50%. An important benefit of a
clonogenic assay, such as that described herein, is the ability to
analyze the effect of the library member on a wide variety of
hemopoietic cell types. Since it is possible, with a correct
cocktail of growth factors, to stimulate the growth of a variety of
hemopoietic lineages in culture, the effect of the library member
on each lineage can be studied. Thus, library members can be
further screened for their selective action on the megakaryoctye
lineage. Clonogenic assays such as those described herein are
routinely employed by artisans of ordinary skill. Each of the
aforementioned in vitro screening assays is amenable to
high-throughput screening.
[0072] Another way of measuring the biological activity of the
synthetic compound is to perform in vivo assays in which animals,
preferably mice, are injected, for example intravenously, with the
compound and then analyzed for megakaryocyte growth and
proliferation or platelet production. Hemopoietic populations, such
as bone marrow and spleen, can be harvested from treated animals
and plated into in vitro semi-solid clonogenic cultures in order to
determine the effect of the library member on megakaryocytes.
Preferably the number and quality of megakaryocyte colonies derived
from test animals should be compared to that of animals injected
with control carrier (e.g., saline). Alternatively, animals can be
assayed directly for platelet counts. This can be done in a number
of ways including by bleeding the animals (usually from the tail or
retro-orbital vein) and counting the number of platelets either
manually using a hemocytometer or through the use of an automated
cell counter, such as a Coulter counter. Adverse side effects can
also be tested in animals injected with putative antagonists in
this manner. One possible adverse side effect may be an inability
to clot due to a severe reduction in platelets. To assess clotting
function, standard bleeding assays can be employed which measure
the time required for bleeding from an experimentally induced wound
to clot and thus stop. Platelet count and bleeding assays are
routinely performed in human subjects as a measure of platelet
count and platelet activity. Human subjects with a platelet count
of more than 100.times.10.sup.3 platelets per .mu.l of blood are
generally asymptomatic and their bleeding times are within the
normal range. Bleeding times of less than 10 minutes are considered
normal. When platelet count falls below 100.times.10.sup.3
platelets per .mu.l, the bleeding time is extended and appears to
be linearly related to the platelet count. Human subjects with a
platelet count of less than 50.times.10.sup.3 platelets per .mu.l
experience easy bruising, while those with a platelet count of less
than 20.times.10.sup.3 platelets per .mu.l are prone to spontaneous
internal bleeding. Platelet count and bleeding assays are routinely
practiced by those of ordinary skill in the art and are taught in
Harrison's Principles of Internal Medicine, Isselbacher, McGraw
Hill, New York (1994).
[0073] Physical methods also exist for reducing platelet count.
These methods include platelet-pheresis which is the centrifugal
separation of platelets from other blood cellular components.
Platelet-pheresis provides the benefit of effecting platelet
reduction in a short period of time. This may be desirable for a
subject unexpectedly scheduled for an elective surgery. Other
physical methods for reducing platelet count involve the use of
adsorption of platelets onto solid state matrices coated with
binding partners specific for platelets. As an example, platelets
may be removed from blood using positive selection affinity
filtration. Such an approach may involve applying peripheral blood
to a column containing a solid matrix to which is coupled a growth
factor such as thrombopoietin. Another example of affinity
chromatography may involve elution of blood over an affinity
matrix, having as the solid state Sephadex G-10, coated with
fibrinogen, fibronectin, or preferably, an Arg-Gly-Asp tripeptide,
all of which are known to bind platelets. This latter approach has
been reported by Besselink et al. for binding of human platelets.
(J. Biomater. Sci. Polym. Ed. (1995) 7:551-562). Other binding
partners for platelets which could be used in an adsorption
technique to separate platelets from blood are glycoprotein
IIb/IIIa antagonists such as Ro-43-8857 and L-700,462 reported by
Cook et al. (Thromb. Haemost. (1993) 70:838-47). Yet another form
of affinity separation useful in the removal of platelets from
blood is immune affinity which uses a solid matrix coupled to an
antibody specific for platelets and/or megakaryocytes such as an
anti-glycoprotein IIb/IIIa receptor, or a fragment thereof.
[0074] Thus, in this aspect of the invention, a subject's blood is
removed, depleted of platelets, and then returned until overall
platelet count is below normal.
[0075] The invention provides pharmaceutical preparations of the
agents of the invention. These pharmaceutical preparations comprise
the agent of the invention and also a pharmaceutically acceptable
carrier. The pharmaceutical preparations may be administered in
effective amounts. The effective amount will depend upon the mode
of administration, the particular condition being treated and the
desired outcome. It will also depend upon, as discussed above, the
stage of the condition, the age and physical condition of the
subject, the nature of concurrent therapy, if any, and like factors
well known to the medical practitioner. For prophylactic
applications, it is that amount sufficient to delay the onset of;
inhibit the progression of, or halt altogether the particular
condition being treated, thereby producing patient benefit. For
therapeutic applications, it is that amount sufficient to achieve a
medically desirable result, thereby producing patient benefit. In
some instances, patient benefit may be measured by a reduction in
morbidity and/or mortality. In some cases this is a decrease in
cell maturation and/or proliferation. In the case of
megakaryocytes, the medically desirable result may be to inhibit
thrombosis via blocking of megakaryoctye maturation,
endoreduplication and/or proliferation. In other cases, it is an
increase in platelet consumption, elimination or death. Ultimately,
the amount which is administered is one effective for reducing
platelet count to low normal and more preferably below normal
levels in a subject, without a significant level of adverse side
effects.
[0076] Generally, doses of active compounds of the present
invention would be from about 0.01 mg/kg per day to 1000 mg/kg per
day. It is expected that doses ranging from 1-500 mg/kg, and
preferably doses ranging from 1-100 mg/kg, and even more preferably
doses ranging from 1-50 mg/kg, will be suitable. In most preferred
embodiments, the agents will be administered in doses ranging from
1 .mu.g/kg/day to 10 mg/kg/day, with even more preferred doses
ranging from 1 .mu.g/kg/day to 0.150 .mu.g/kg/day and from 30
.mu.g/kg/day to 150 .mu.g/kg/day depending upon the purpose of the
intervention and the subject to be treated. The latter dose range
is preferred if the agent is anagrelide. A variety of
administration routes are available. The methods of the invention,
generally speaking, may be practiced using any mode of
administration that is medically acceptable, meaning any enteral or
parenteral mode that produces effective levels of the active
compounds without causing clinically unacceptable adverse effects.
Such modes of administration include oral, rectal, topical, nasal,
intrapulmonary, intracavitary, transdermal, intradermal,
transmucosal, subcutaneous, intravenous, intraarterial,
intramuscular, or local routes. The term "parenteral" includes
subcutaneous, intravenous, intramuscular, or infusion. Injectable
routes such as intravenous or intramuscular routes are not
particularly suitable for long-term therapy and prophylaxis. They
could, however, be preferred in situations where oral
administration is contra-indicated. Oral administration will be
preferred for prophylactic or therapeutic treatment because of the
convenience to the patient as well as the dosing schedule.
[0077] Compositions suitable for oral administration may be
presented as discrete units, in both immediate release or
controlled release formulations such as capsules, tablets,
lozenges, each containing a predetermined amount of the active
agent. Other compositions include suspensions in aqueous liquids or
non-aqueous liquids such as a syrup, elixir or an emulsion.
[0078] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions as
well as injectable drug delivery devices such as controlled release
preparations. Examples of non-aqueous solvents are propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers
include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, Ringer's dextrose,
dextrose and sodium chloride, lactated Ringer's or fixed oils.
Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers (such as those based on Ringer's
dextrose), and the like. Preservatives and other additives may also
be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, and inert gases and the like. Lower doses will
result from other forms of administration, such as intravenous
administration. In the event that a response in a subject is
insufficient at the initial doses applied, higher doses (or
effectively higher doses by a different, more localized delivery
route) may be employed to the extent that patient tolerance
permits. Multiple doses per day are contemplated to achieve
appropriate systemic levels of compounds.
[0079] The agents that reduce platelet count may be combined,
optionally, with a pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" as used herein means one or
more compatible solid or liquid filler, diluents or encapsulating
substances which are suitable for administration into a human. The
term "carrier" denotes an organic or inorganic ingredient, natural
or synthetic, with which the active ingredient is combined to
facilitate the application. The components of the pharmaceutical
compositions also are capable of being co-mingled with the agents
of the present invention, and with each other, in a manner such
that there is no interaction which would substantially impair the
desired pharmaceutical efficacy.
[0080] When administered, the pharmaceutical preparations of the
invention are applied in pharmaceutically-acceptable amounts and in
pharmaceutically-acceptably compositions. Such preparations may
routinely contain salt, buffering agents, preservatives, compatible
carriers, and optionally other therapeutic agents. When used in
medicine, the salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically-acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically-acceptable salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts.
[0081] Other delivery systems can include immediate release or
controlled release formulations. Examples of controlled release
formulations include time-release, delayed release or sustained
release delivery systems. Such systems can reduce toxicity,
increase efficacy and avoid repeated administrations of the
platelet reducing agent, reducing peak-related side effects and
increasing convenience to the subject and the physician. Many types
of release delivery systems are available and known to those of
ordinary skill in the art. They include but are not limited to
polymer base systems such as poly(lactide-glycolide),
copolyoxalates, polycaprolactones, lipids, polyesteramides,
polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.
Microcapsules of the foregoing polymers containing drugs are
described in, for example, U.S. Pat. No. 5,075,109, and non-polymer
systems such as melted and recrystallized sterols including
cholesterol. Delivery systems also include non-polymer systems that
are: lipids including sterols such a cholesterol, cholesterol
esters and fatty acids or neutral fats such as mono- di- and
tri-glycerides; hydrogel release systems; silastic systems; peptide
based systems; wax coatings; compressed tablets using conventional
binders and excipients; partially fused implants; and the like.
Specific examples include, but are not limited to: (a) erosional
systems in which the platelet reducing agent is contained in a form
within a matrix such as those described in U.S. Pat. Nos.
4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in
which an active component permeates at a controlled rate from a
polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974
and 5,407,686. In addition, pump-based hardware delivery systems
can be used, some of which are adapted for implantation.
[0082] For example, U.S. Pat. No. 4,452,775 discloses that the
relative proportions of active agent and matrix components can be
varied within defined ranges depending on the macromolecule to be
administered and the desired rate and duration of release. The
macromolecular active agent may comprise 0.1-10.0%, by weight, of
the delivery system. The actual amount of active agent incorporated
in the delivery system will depend on the particular active agent,
the desired effect, and, to a limited extent, the desired duration
of release; a preferred amount of active agent is 2 to 5%, by
weight, of the system. The relative proportions of the matrix
components may be varied within the following ranges:
[0083] (a) 20-80% cholesterol powder;
[0084] (b) 20-80% cholesterol prills, 100-1200 microns in
diameter;
[0085] (c) 0.1-5.0% biocompatible binding agent;
[0086] (d) 0.1-5.0% biocompatible lubricating agent.
[0087] A preferred matrix composition comprises, by weight:
[0088] (a) 40-60% cholesterol powder;
[0089] (b) 40-60% cholesterol prills having diameters of 200-800
microns;
[0090] (c) 0.1-1.0% biocompatible binding agent; and
[0091] (d) 0.5-1.5% biocompatible lubricating agent.
[0092] An especially preferred matrix composition comprises, by
weight:
[0093] (a) 48-52% cholesterol powder;
[0094] (b) 48-52% cholesterol prills having diameters of 420-710
microns;
[0095] (c) 0.4-0.6% biocompatible binding agent; and
[0096] (d) 0.8-1.0% biocompatible lubricating agent.
[0097] The size of the cholesterol prill may vary from about
100-1200 microns in diameter. A preferred range is about 400-700
microns. The choices of matrix component proportions and
cholesterol prill size are governed in large part by the
macromolecule to be delivered, the desired release rate and
duration of release, and the body site at which the system is to be
placed. Those skilled in the art can readily determine the rate of
diffusion of active agents through the cholesterol matrix when
wetted by the biological fluids of the targeted body site, and
adjust the ratios and sizes of matrix components to achieve a
delivery system which is best suited to the particular active agent
and site of prills, binder, lubricant and active agent for a
particular application.
[0098] U.S. Pat. No. 4,452,775 discloses that a number of binders
and lubricating agents are known. The binding agent used may be
chosen from among, for example, the U.S.P. grade polyethylene
glycols having molecular weights between 1,250 (PEG 1250) and 8,000
(PEG 8000), polyvinylpyrollidone, hydroxymethylcellulose,
hydroxypropylmethylcellulos- e, and the materials commercially
known under the trademark Pluronic.TM. with molecular weights
between 3,800 (Pluronic.TM. L101) and 14,000 (Pluronic.TM. F127). A
preferred binding agent is PEG 6000. Depending on the method of
manufacture, incorporation in the matrix of an effective amount of
a lubricating agent may facilitate production. Suitable lubricants
include for example, stearic acid, magnesium stearate, calcium
stearate, or Sterotex.TM.. Preferred among these are stearic acid
and magnesium stearate. The foregoing lists of binders and
lubricants are not intended to be exhaustive of the materials which
are compatible with the scope and intention of this invention, but
merely set out examples to illustrate the types of lubricants and
binders which may be used.
[0099] Use of a long-term sustained release implant (or device) may
be particularly suitable for treatment of subjects at elevated risk
of a vaso-occlusive event such as one resulting from a thrombotic
event. These subjects would include subjects scheduled for elective
vascular surgery. Long-term release, as used herein, means that the
implant is constructed and arranged to deliver levels of the active
ingredient for at least 1 week, in some instances for at least 30
days, and in others for at least 60 days. In some aspects of the
invention that involve longer-term treatment and prevention, it is
desirable that the sustained release device release effective
amounts of agent for at least 6 months, 1 year, 2 years or in some
cases, 5 years or more. Long-term sustained release implants are
well-known to those of ordinary skill in the art and include some
of the release systems described above.
[0100] Regardless of the particular agent used in the methods of
the invention, administration via a sustained release device is
preferable in some instances because it can reduce the peak levels
of agent which are often observed with single bolus administrations
(such as non-continuous injection or oral delivery). A reduction in
the peak level of agent in the subject also reduces the likelihood
of side effects. As an example, a side effect resulting from the
ingestion of anagrelide is diarrhea. Sustained release of
anagrelide would minimize this side effect.
[0101] The agent of the invention should be administered for a
length of time sufficient to provide either or both therapeutic and
prophylactic benefit to the subject. Generally, the agent is
administered for at least one day. In some instances, particularly
where a subject has had a vaso-occlusive event or where the subject
is at risk of such an event, the agent may be administered for the
remainder of the subject's life. The rate at which the agent is
administered may vary depending upon the needs of the subject and
the mode of administration. For example, it may be necessary in
some instances to administer higher and more frequent doses of the
agent to a subject for example during or immediately following a
vaso-occlusive event (such as a myocardial infarction), provided
still that such doses reduce platelet count but do not
significantly affect platelet function. On the other hand, it may
be desirable to administer lower doses in order to maintain a
desired platelet count once it is achieved. In still other
embodiments, the same dose of agent may be administered throughout
the treatment period which as described herein may extend
throughout the lifetime of the subject. The frequency of
administration may vary depending upon the characteristics of the
subject. The agent may be administered daily, every 2 days, every 3
days, every 4 days, every 5 days, every week, every 10 days, every
2 weeks, every month, or more, or any time therebetween as if such
time was explicitly recited herein.
[0102] In other aspects, the agents of the invention are
administered with another agent, preferably an agent that would
normally be indicated for the subject. In some embodiments, the
agents may be administered substantially simultaneously with the
other therapeutic agents. By substantially simultaneously, it is
meant that a platelet reducing agent of the invention is
administered to a subject close enough in time with the
administration of the other therapeutic agent, whereby the two
compounds may exert an additive or even synergistic effect, e.g.,
reducing platelet function by decreasing platelet count and
inhibiting their ability to aggregate. In other embodiments, the
platelet reducing agents of the invention can be administered
before or after the administration of the other therapeutic
agent.
[0103] The agents of the invention may be administered with several
categories of therapeutic agents, although preferably these agents
are those which would normally be indicated for the subject.
Generally, these agents are those which are useful and which are
currently indicated for treating vascular disorders and vascular
complications. These agents can be classified in terms of their
function or in terms of the disorders for which they are indicated.
Several useful categories of such agents include but are not
limited to anti- inflammatory agents, anti-thrombotic agents,
anti-platelet agents, fibrinolytic agents, lipid reducing agents,
direct thrombin inhibitors, glycoprotein IIb/IIIa receptor
inhibitors, agents that bind to cellular adhesion molecules and
inhibit the ability of white blood cells to attach to such
molecules, calcium channel blockers, beta-adrenergic receptor
blockers, cyclooxygenase-2 inhibitors, and angiotensin system
inhibitors.
[0104] One broad category of agents which may be administered with
the platelet reducing agents of the invention is anti-thrombotic
agents. Anti-thrombotic agents are defined as agents which prevent
the formation of a blood thrombus via a number of potential
mechanisms and they include fibrinolytic agents, anti-coagulant
agents and inhibitors of platelet function.
[0105] Fibrinolytic agents are defined as agent that lyse a
thrombus (e.g., a blood clot), usually through the dissolution of
fibrin by enzymatic action. Examples of thrombolytic agents include
but are not limited to ancrod, anistreplase, bisobrin lactate,
brinolase, Hageman factor (i.e. factor XII) fragments, molsidomine,
plasminogen activators such as streptokinase, tissue plasminogen
activators (TPA) and urokinase, and plasmin and plasminogen.
Anti-coagulant agents also include inhibitors of factor Xa, factor
TFPI, factor VIIa, factor IXc, factor Va, factor VIIa as well as
inhibitors of other coagulation factors.
[0106] Anti-coagulant agents are agents which inhibit the
coagulation pathway by impacting negatively upon the production,
deposition, cleavage and/or activation of factors essential in the
formation of a blood clot. Anti-coagulant agents include but are
not limited to vitamin K antagonists such as coumarin and coumarin
derivatives (e.g., warfarin sodium); glycosoamino-glycans such as
heparins both in unfractionated form and in low molecular weight
form; ardeparin sodium, bivalirudin, bromindione, coumarin
dalteparin sodium, desirudin, dicumarol, lyapolate sodium,
nafamostat mesylate, phenprocoumon, sulfatide, and tinzaparin
sodium.
[0107] Other "anti-coagulant" and/or "fibrinolytic" agents include
plasminogen (to plasmin via interactions of prekallikrein,
kininogens, Factors XII, XIIIa, plasminogen proactivator, and
tissue plasminogen activator[TPA]); streptokinase; urokinase:
anisoylated plasminogen-streptokinase activator complex;
pro-urokinase; (Pro-UK); rTPA (alteplase or activase; r denotes
recombinant); rPro-UK; abbokinase; eminase; streptase; anagrelide
hydrochloride; bivalirudin; dalteparin sodium; danaparoid sodium;
dazoxiben hydrochloride; efegatran sulfate; enoxaparin sodium;
ifetroban; ifetroban sodium; tinzaparin sodium; retaplase;
trifenagrel; warfarin; and dextrans.
[0108] Still other anti-coagulant agents include, but are not
limited to, ancrod; anti-coagulant citrate dextrose solution;
anticoagulant citrate phosphate dextrose adenine solution;
anticoagulant citrate phosphate dextrose solution; anticoagulant
heparin solution; anticoagulant sodium citrate solution; ardeparin
sodium; bivalirudin; bromindione; dalteparin sodium; desirudin;
dicumarol; heparin calcium; heparin sodium; lyapolate sodium;
nafamostat mesylate; phenprocoumon; tinzaparin sodium; warfarin
sodium.
[0109] Heparin may stabilize symptoms in evolving stroke, but
anticoagulants are useless (and possibly dangerous) in acute
completed stroke, and are contraindicated in hypertensives because
of the increased possibility of hemorrhage into the brain or other
organs. Although the timing is controversial, anticoagulants may be
started to prevent recurrent cardiogenic emboli. Clot lysing
agents, including tissue plasminogen activator and streptokinase,
are being evaluated for the very early treatment of acute stroke.
Nimodipine has recently been shown to improve survival and clinical
outcome after ischemic stroke.
[0110] Inhibitors of platelet function are agents that impair the
ability of mature platelets to perform their normal physiological
roles (i.e., their normal function). Platelets are normally
involved in a number of physiological processes such as adhesion,
for example, to cellular and non-cellular entities, aggregation,
for example, for the purpose of forming a blood clot, and release
of factors such as growth factors (e.g., platelet-derived growth
factor (PDGF)) and platelet granular components. One subcategory of
platelet function inhibitors are inhibitors of platelet aggregation
which are compounds which reduce or halt the ability of platelets
to associate physically with themselves or with other cellular and
non-cellular components, thereby precluding the ability of a
platelet to initiate the formation of a thrombus.
[0111] Examples of useful inhibitors of platelet function include
but are not limited to acadesine, anagrelide (if given at doses
exceeding 10 mg/day), anipamil, argatroban, aspirin, clopidogrel,
cyclooxygenase inhibitors such as nonsteroidal anti-inflammatory
drugs and the synthetic compound FR-122047, danaparoid sodium,
dazoxiben hydrochloride, diadenosine 5',5'"-P1,P4-tetraphosphate
(Ap4A) analogs, difibrotide, dilazep dihydrochloride, 1,2- and
1,3-glyceryl dinitrate, dipyridamole, dopamine and
3-methoxytyramine, efegatran sulfate, enoxaparin sodium, glucagon,
glycoprotein IIb/IIIa antagonists such as Ro-43-8857 and L-700,462,
ifetroban, ifetroban sodium, iloprost, isocarbacyclin methyl ester,
isosorbide-5-mononitrate, itazigrel, ketanserin and BM-13.177,
lamifiban, lifarizine, molsidomine, nifedipine, oxagrelate,
prostaglandin E (PGE), platelet activating factor antagonists such
as lexipafant, prostacyclin (PGI2), pyrazines, pyridinol carbamate,
ReoPro (i.e., abciximab), sulfinpyrazone, synthetic compounds
BN-50727, BN-52021, CV-4151, E-5510, FK-409, GU-7, KB-2796,
KBT-3022, KC-404, KF-4939, OP-41483-, TRK-100, TA-3090, TFC-612 and
ZK-36374, 2,4,5,7-tetrathiaoctane, 2,4,5,7-tetrathiaoctane
2,2-dioxide, 2,4,5-trithiahexane, theophyllin, pentoxifyllin,
thromboxane and thromboxane synthetase inhibitors such as
picotamide and sulotroban, ticlopidine, tirofiban, trapidil and
triclopidine, trifenagrel, trilinolein, 3-substituted
5,6-bis(4-methoxyphenyl)-1,2,4-triazines, antibodies to
glycoprotein IIb/IIIa as well as those disclosed in U.S. Pat. No.
5,440,020, anti-serotonin drugs, dipyridamole, clofibrate, caffeine
and ticlopidine.
[0112] "Anti-inflammatory" agents include alclofenac; alclometasone
dipropionate; algestone acetonide; alpha amylase; amcinafal;
amcinafide; amfenac sodium; amiprilose hydrochloride; anakinra;
anirolac; anitrazafen; apazone; balsalazide disodium; bendazac;
benoxaprofen; benzydamine hydrochloride; bromelains; broperamole;
budesonide; carprofen; cicloprofen; cintazone; cliprofen;
clobetasol propionate; clobetasone butyrate; clopirac; cloticasone
propionate; cormethasone acetate; cortodoxone; deflazacort;
desonide; desoximetasone; dexamethasone dipropionate; diclofenac
potassium; diclofenac sodium; diflorasone diacetate; diflumidone
sodium; diflunisal; difluprednate; diftalone; dimethyl sulfoxide;
drocinonide; endrysone; enlimomab; enolicam sodium; epirizole;
etodolac; etofenamate; felbinac; fenamole; fenbufen; fenclofenac;
fenclorac; fendosal; fenpipalone; fentiazac; flazalone; fluazacort;
flufenamic acid; flumizole; flunisolide acetate; flunixin; flunixin
meglumine; fluocortin butyl; fluorometholone acetate; fluquazone;
flurbiprofen; fluretofen; fluticasone propionate; furaprofen;
furobufen; halcinonide; halobetasol propionate; halopredone
acetate; ibufenac; ibuprofen; ibuprofen aluminum; ibuprofen
piconol; ilonidap; indomethacin; indomethacin sodium; indoprofen;
indoxole; intrazole; isoflupredone acetate; isoxepac; isoxicam;
ketoprofen; lofemizole hydrochloride; lomoxicam; loteprednol
etabonate; meclofenamate-sodium; meclofenamic acid; meclorisone
dibutyrate; mefenamic acid; mesalamine; meseclazone;
methylprednisolone suleptanate; momiflumate; nabumetone; naproxen;
naproxen sodium; naproxol; nimazone; olsalazine sodium; orgotein;
orpanoxin; oxaprozin; oxyphenbutazone; paranyline hydrochloride;
pentosan polysulfate sodium; phenbutazone sodium glycerate;
pirfenidone; piroxicam; piroxicam cinnamate; piroxicam olamine;
pirprofen; prednazate; prifelone; prodolic acid; proquazone;
proxazole; proxazole citrate; rimexolone; romazarit; salcolex;
salnacedin; salsalate; salycilates; sanguinarium chloride;
seclazone; sermetacin; sudoxicam; sulindac; suprofen; talmetacin;
talniflumate; talosalate; tebufelone; tenidap; tenidap sodium;
tenoxicam; tesicam; tesimide; tetrydamine; tiopinac; tixocortol
pivalate; totmetin; tolmetin sodium; triclonide; triflumidate;
zidometacin; glucocorticoids; and zomepirac sodium. One preferred
anti-inflammatory agent is aspirin.
[0113] "Lipid reducing" agents include gemfibrozil, cholystyramine,
colestipol, nicotinic acid, probucol, lovastatin, fluvastatin,
simvastatin, atorvastatin, pravastatin, cirivastatin.
[0114] "Direct thrombin inhibitors" include hirudin, hirugen,
hirulog, agatroban, PPACK, thrombin aptamers.
[0115] "Glycoprotein IIb/IIIa receptor inhibitors" are both
antibodies and non-antibodies, and include but are not limited to
ReoPro (abcixamab), lamifiban, tirofiban.
[0116] "Calcium channel blockers" are a chemically diverse class of
compounds having important therapeutic value in the control of a
variety of diseases including several cardiovascular disorders,
such as hypertension, angina, and cardiac arrhythmias
(Fleckenstein, Cir. Res. v. 52, (suppl. 1), p. 13-16 (1983);
Fleckenstein, Experimental Facts and Therapeutic Prospects, John
Wiley, New York (1983); McCall, D., Curr Pract Cardiol, v. 10, p.
1-11 (1985)). Calcium channel blockers are a heterogeneous group of
drugs that prevent or slow the entry of calcium into cells by
regulating cellular calcium channels (Remington, The Science and
Practice of Pharmacy, Nineteenth Edition, Mack Publishing Company,
Eaton, Pa., p. 963 (1995)). Most of the currently available calcium
channel blockers, and useful according to the present invention,
belong to one of three major chemical groups of drugs, the
dihydropyridines, such as nifedipine, the phenyl alkyl amines, such
as verapamil, and the benzothiazepines, such as diltiazem. Other
calcium channel blockers useful according to the invention,
include, but are not limited to, amrinone, amlodipine, bencyclane,
felodipine, fendiline, flunarizine, isradipine, nicardipine,
nimodipine, perhexilene, gallopamil, tiapamil and tiapamil
analogues (such as 1993RO-11-2933), phenyloin, barbiturates, and
the peptides dynorphin, omega-conotoxin, and omega-agatoxin, and
the like and/or pharmaceutically acceptable salts thereof.
[0117] "Beta-adrenergic receptor blocking agents" are a class of
drugs that antagonize the cardiovascular effects of catecholamines
in angina pectoris, hypertension, and cardiac arrhythmias.
Beta-adrenergic receptor blockers include, but are not limited to,
atenolol, acebutolol, alprenolol, befunolol, betaxolol, bunitrolol,
carteolol, celiprolol, medroxalol, indenolol, labetalol,
levobunolol, mepindolol, methypranol, metindol, metoprolol,
metrizoranolol, oxprenolol, pindolol, propranolol, practolol,
sotalol, nadolol, tiprenolol, tomalol, timolol, bupranolol,
penbutolol, trimepranol,
2-(3-(1,1-dimethyl-ethyl)-amino-2-hydroxypropoxy-
)-3-pyridenecarbonitrile HCl,
1-butylamino-3-(2,5-dichlorophenoxy)-2-propa- nol,
1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)-phenoxy)-2-propanol-
, 3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,
2-(3-t-butylamino-2-hydroxypropyl-thio)-4-(5-carbamoyl-2-thienyl)thiazol,
and 7-(2-hydroxy-3-t-butylaminpropoxy)phthalide. The
above-identified compounds can be used as isomeric mixtures, or in
their respective levorotating or dextrorotating form.
[0118] Cyclooxygenase-2 (COX-2) is a recently identified form of a
cyclooxygenase. "Cyclooxygenase" is an enzyme complex present in
most tissues that produces various prostaglandins and thromboxanes
from arachidonic acid. Non-steroidal, anti-inflammatory drugs exert
most of their anti-inflammatory, analgesic and antipyretic activity
and inhibit hormone-induced uterine contractions and certain types
of cancer growth through inhibition of the cyclooxygenase (also
known as prostaglandin G/H synthase and/or prostaglandin-
endoperoxide synthase). Initially, only one form of cyclooxygenase
was known, the "constitutive enzyme" or cyclooxygenase-1 (COX-1).
It was originally identified in bovine seminal vesicles.
[0119] Cyclooxygenase-2 (COX-2) has been cloned, sequenced and
characterized initially from chicken, murine and human sources
(See, e.g., U.S. Pat. No. 5,543,297, issued Aug. 6, 1996 to
Cromlish et al., and assigned to Merck Frosst Canada, Inc.,
Kirkland, Calif., entitled: "Human cyclooxygenase-2 cDNA and assays
for evaluating cyclooxygenase-2 activity"). This enzyme is distinct
from COX-1. COX-2 is rapidly and readily inducible by a number of
agents including mitogens, endotoxin, hormones, cytokines and
growth factors. As prostaglandins have both physiological and
pathological roles, the constitutive enzyme, COX-1, is responsible,
in large part, for endogenous basal release of prostaglandins and
hence is important in their physiological functions such as the
maintenance of gastrointestinal integrity and renal blood flow. By
contrast, it is believed that the inducible form, COX-2, is mainly
responsible for the pathological effects of prostaglandins where
rapid induction of the enzyme would occur in response to such
agents as inflammatory agents, hormones, growth factors, and
cytokines. Therefore, it is believed that a selective inhibitor of
COX-2 has similar anti-inflammatory, antipyretic and analgesic
properties to a conventional non-steroidal anti-inflammatory drug,
and in addition inhibits hormone-induced uterine contractions and
also has potential anti-cancer effects, but with reduced side
effects. In particular, such COX-2 inhibitors are believed to have
a reduced potential for gastrointestinal toxicity, a reduced
potential for renal side effects, a reduced effect on bleeding
times and possibly a decreased potential to induce asthma attacks
in aspirin-sensitive asthmatic subjects, and are therefore useful
according to the present invention.
[0120] A number of selective "COX-2 inhibitors" are known in the
art. These include, but are not limited to, COX-2 inhibitors
described in U.S. Pat. No. 5,474,995 "Phenyl heterocycles as COX-2
inhibitors"; U.S. Pat. No. 5,521,213 "Diaryl bicyclic heterocycles
as inhibitors of cyclooxygenase-2"; U.S. Pat. No. 5,536,752 "Phenyl
heterocycles as COX-2 inhibitors"; U.S. Pat. No. 5,550,142 "Phenyl
heterocycles as COX-2 inhibitors"; U.S. Pat. No. 5,552,422 "Aryl
substituted 5,5 fused aromatic nitrogen compounds as
anti-inflammatory agents"; U.S. Pat. No. 5,604,253
"N-benzylindol-3-yl propanoic acid derivatives as cyclooxygenase
inhibitors"; U.S. Pat. No. 5,604,260
"5-methanesulfonamido-1-indanones as an inhibitor of
cyclooxygenase-2"; U.S. Pat. No. 5,639,780 "N-benzyl indol-3-yl
butanoic acid derivatives as cyclooxygenase inhibitors"; U.S. Pat.
No. 5,677,318 "Diphenyl-1,2-3-thiadiazoles as anti-inflammatory
agents"; U.S. Pat. No. 5,691,374
"Diaryl-5-oxygenated-2-(5H)-furanones as COX-2 inhibitors"; U.S.
Pat. No. 5,698,584 "3,4-diaryl-2-hydroxy-2,5-dihy- drofurans as
prodrugs to COX-2 inhibitors"; U.S. Pat. No. 5,710,140 "Phenyl
heterocycles as COX-2 inhibitors"; U.S. Pat. No. 5,733,909
"Diphenyl stilbenes as prodrugs to COX-2 inhibitors"; U.S. Pat. No.
5,789,413 "Alkylated styrenes as prodrugs to COX-2 inhibitors";
U.S. Pat. No. 5,817,700 "Bisaryl cyclobutenes derivatives as
cyclooxygenase inhibitors"; U.S. Pat. No. 5,849,943 "Stilbene
derivatives useful as cyclooxygenase-2 inhibitors"; U.S. Pat. No.
5,861,419 "Substituted pyridines as selective cyclooxygenase-2
inhibitors"; U.S. Pat. No. 5,922,742
"Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2
inhibitors"; U.S. Pat. No. 5,925,631 "Alkylated styrenes as
prodrugs to COX-2 inhibitors"; all of which are commonly assigned
to Merck Frosst Canada, Inc. (Kirkland, Calif.). Additional COX-2
inhibitors are also described in U.S. Pat. No. 5,643,933, assigned
to G. D. Searle & Co. (Skokie, Ill.), entitled: "Substituted
sulfonylphenyl-heterocycles as cyclooxygenase-2 and 5-lipoxygenase
inhibitors."
[0121] A number of the above-identified COX-2 inhibitors are
prodrugs of selective COX-2 inhibitors, and exert their action by
conversion in vivo to the active and selective COX-2 inhibitors.
The active and selective COX-2 inhibitors formed from the
above-identified COX-2 inhibitor prodrugs are described in detail
in WO 95/00501, published Jan. 5, 1995, WO 95/18799, published Jul.
13, 1995 and U.S. Pat. No. 5,474,995, issued Dec. 12, 1995. Given
the teachings of U.S. Pat. No. 5,543,297, entitled: "Human
cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2
activity," a person of ordinary skill in the art would be able to
determine whether an agent is a selective COX-2 inhibitor or a
precursor of a COX-2 inhibitor, and therefore part of the present
invention.
[0122] An "angiotensin system inhibitor" is an agent that
interferes with the function, synthesis or catabolism of
angiotensin II. These agents include, but are not limited to,
angiotensin-converting enzyme (ACE) inhibitors, angiotensin II
antagonists, angiotensin II receptor antagonists, agents that
activate the catabolism of angiotensin II, and agents that prevent
the synthesis of angiotensin I from which angiotensin II is
ultimately derived. The renin-angiotensin system is involved in the
regulation of hemodynamics and water and electrolyte balance.
Factors that lower blood volume, renal perfusion pressure, or the
concentration of Na.sup.+ in plasma tend to activate the system,
while factors that increase these parameters tend to suppress its
function.
[0123] Angiotensin I and angiotensin II are synthesized by the
enzymatic renin-angiotensin pathway. The synthetic process is
initiated when the enzyme renin acts on angiotensinogen, a
pseudoglobulin in blood plasma, to produce the decapeptide
angiotensin I. Angiotensin I is converted by angiotensin converting
enzyme (ACE) to angiotensin II (angiotensin-[1-8] octapeptide). The
latter is an active pressor substance which has been implicated as
a causative agent in several forms of hypertension in various
mammalian species, e.g., humans.
[0124] Angiotensin (renin-angiotensin) system inhibitors are
compounds that act to interfere with the production of angiotensin
II from angiotensinogen or angiotensin I or interfere with the
activity of angiotensin II. Such inhibitors are well known to those
of ordinary skill in the art and include compounds that act to
inhibit the enzymes involved in the ultimate production of
angiotensin II, including renin and ACE. They also include
compounds that interfere with the activity of angiotensin II, once
produced. Examples of classes of such compounds include antibodies
(e.g., to renin), amino acids and analogs thereof (including those
conjugated to larger molecules), peptides (including peptide
analogs of angiotensin and angiotensin I), pro-renin related
analogs. Among the most potent and useful renin-angiotensin system
inhibitors are renin inhibitors, ACE inhibitors, and angiotensin II
antagonists. In a preferred embodiment of the invention, the
renin-angiotensin system inhibitors are renin inhibitors, ACE
inhibitors, and angiotensin II antagonists.
[0125] "Angiotensin II antagonists" are compounds which interfere
with the activity of angiotensin II by binding to angiotensin II
receptors and interfering with its activity. Angiotensin II
antagonists are well known and include peptide compounds and
non-peptide compounds. Most angiotensin II antagonists are slightly
modified congeners in which agonist activity is attenuated by
replacement of phenylalanine in position 8 with some other amino
acid; stability can be enhanced by other replacements that slow
degeneration in vivo. Examples of angiotensin II antagonists
include: peptidic compounds (e.g., saralasin,
[(San.sup.1)(Val.sup.5)(Ala- .sup.8)] angiotensin-(1-8) octapeptide
and related analogs); N-substituted imidazole-2-one (U.S. Pat. No.
5,087,634); imidazole acetate derivatives including
2-N-butyl-4-chloro-1-(2-chlorobenzile) imidazole-5-acetic acid (see
Long et al., J. Pharmacol. Exp. Ther. 247(1), 1-7 (1988));
4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid and
analog derivatives (U.S. Pat. No. 4,816,463); N2-tetrazole
beta-glucuronide analogs (U.S. Pat. No. 5,085,992); substituted
pyrroles, pyrazoles, and tryazoles (U.S. Pat. No. 5,081,127);
phenol and heterocyclic derivatives such as 1,3-imidazoles (U.S.
Pat. No. 5,073,566); imidazo-fused 7-member ring heterocycles (U.S.
Pat. No. 5,064,825); peptides (e.g., U.S. Pat. No. 4,772,684);
antibodies to angiotensin II (e.g., U.S. Pat. No. 4,302,386); and
aralkyl imidazole compounds such as biphenyl-methyl substituted
imidazoles (e.g., EP Number 253,310, Jan. 20, 1988); ES8891
(N-morpholinoacetyl-(-1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl
(35, 45)-4-amino-3-hydroxy-5-cyclohexa-pentanoyl-N-hexylamide,
Sankyo Company, Ltd., Tokyo, Japan); SKF108566
(E-alpha-2-[2-butyl-1-(carboxy phenyl) methyl]
1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid, Smith Kline
Beecham Pharmaceuticals, PA); Losartan (DUP753/MK954, DuPont Merck
Pharmaceutical Company); Remikirin (RO42-5892, F. Hoffman LaRoche
AG); A.sub.2 agonists (Marion Merrill Dow) and certain non-peptide
heterocycles (G. D. Searle and Company).
[0126] "Angiotensin converting enzyme" (ACE), is an enzyme which
catalyzes the conversion of angiotensin I to angiotensin II. ACE
inhibitors include amino acids and derivatives thereof, peptides,
including di- and tri-peptides and antibodies to ACE which
intervene in the renin-angiotensin system by inhibiting the
activity of ACE thereby reducing or eliminating the formation of
pressor substance angiotensin II. ACE inhibitors have been used
medically to treat hypertension, congestive heart failure,
myocardial infarction and renal disease. Classes of compounds known
to be useful as ACE inhibitors include acylmercapto and
mercaptoalkanoyl prolines such as captopril (U.S. Pat. No.
4,105,776) and zofenopril (U.S. Pat. No. 4,316,906), carboxyalkyl
dipeptides such as enalapril (U.S. Pat. No. 4,374,829), lisinopril
(U.S. Pat. No. 4,374,829), quinapril (U.S. Pat. No. 4,344,949),
ramipril (U.S. Pat. No. 4,587,258), and perindopril (U.S. Pat. No.
4,508,729), carboxyalkyl dipeptide mimics such as cilazapril (U.S.
Pat. No. 4,512,924) and benazapril (U.S. Pat. No. 4,410,520),
phosphinylalkanoyl prolines such as fosinopril (U.S. Pat. No.
4,337,201) and trandolopril.
[0127] "Renin inhibitors" are compounds which interfere with the
activity of renin. Renin inhibitors include amino acids and
derivatives thereof, peptides and derivatives thereof, and
antibodies to renin. Examples of renin inhibitors that are the
subject of United States patents are as follows: urea derivatives
of peptides (U.S. Pat. No. 5,116,835); amino acids connected by
nonpeptide bonds (U.S. Pat. No. 5,114,937); di- and tri-peptide
derivatives (U.S. Pat. No. 5,106,835); amino acids and derivatives
thereof (U.S. Pat. Nos. 5,104,869 and 5,095,119); diol sulfonamides
and sulfinyls (U.S. Pat. No. 5,098,924); modified peptides (U.S.
Pat. No. 5,095,006); peptidyl beta-aminoacyl aminodiol carbamates
(U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S. Pat. No.
5,075,451); fluorine and chlorine statine or statone containing
peptides (U.S. Pat. No. 5,066,643); peptidyl amino diols (U.S. Pat.
Nos. 5,063,208 and 4,845,079); N-morpholino derivatives (U.S. Pat.
No. 5,055,466); pepstatin derivatives (U.S. Pat. No. 4,980,283);
N-heterocyclic alcohols (U.S. Pat. No. 4,885,292); monoclonal
antibodies to renin (U.S. Pat. No. 4,780,401); and a variety of
other peptides and analogs thereof (U.S. Pat. Nos. 5,071,837,
5,064,965, 5,063,207, 5,036,054, 5,036,053, 5,034,512, and
4,894,437).
[0128] Agents that bind to cellular adhesion molecules and inhibit
the ability of white blood cells to attach to such molecules
include polypeptide agents. Such polypeptides include polyclonal
and monoclonal antibodies, prepared according to conventional
methodology. Such antibodies already are known in the art and
include anti-ICAM 1 antibodies as well as other such antibodies
(see earlier discussion on antibodies).
[0129] Other than aspirin, ticlopidine is another antiplatelet
agent that has been shown to be beneficial for stroke treatment.
Endarterectomy may be indicated in patients with 70 to 99 percent
narrowing of a symptomatic internal carotid artery. However, most
authorities agree that carotid endarterectomy is not indicated in
patients with TIAs that are referable to the basilar-vertebral
system, in patients with significant deficits from prior strokes,
or in patients in whom a stroke is evolving.
[0130] HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is
the microsomal enzyme that catalyzes the rate limiting reaction in
cholesterol biosynthesis (HMG-COA to mevalonate). An HMG-CoA
reductase inhibitor inhibits HMG-CoA reductase, and as a result
inhibits the synthesis of cholesterol. A number of HMG-COA
reductase inhibitors has been used to treat individuals with
hypercholesterolemia. More recently, HMG-CoA reductase inhibitors
have been shown to be beneficial in the treatment of stroke (Endres
M, et al., Proc Natl Acad Sci USA, 1998, 95:8880-5).
[0131] HMG-CoA reductase inhibitors useful for co-administration
with the agents of the invention include, but are not limited to,
simvastatin (U.S. Pat. No. 4,444,784), lovastatin (U.S. Pat. No.
4,231,938), pravastatin sodium (U.S. Pat. No. 4,346,227),
fluvastatin (U.S. Pat. No. 4,739,073), atorvastatin (U.S. Pat. No.
5,273,995), cerivastatin, and numerous others described in U.S.
Pat. No. 5,622,985, U.S. Pat. No. 5,135,935, U.S. Pat. No.
5,356,896, U.S. Pat. No. 4,920,109, U.S. Pat. No. 5,286,895, U.S.
Pat. No. 5,262,435, U.S. Pat. No. 5,260,332, U.S. Pat. No.
5,317,031, U.S. Pat. No. 5,283,256, U.S. Pat. No. 5,256,689, U.S.
Pat. No. 5,182,298, U.S. Pat. No. 5,369,125, U.S. Pat. No.
5,302,604, U.S. Pat. No. 5,166,171, U.S. Pat. No. 5,202,327, U.S.
Pat. No. 5,276,021, U.S. Pat. No. 5,196,440, U.S. Pat. No.
5,091,386, U.S. Pat. No. 5,091,378, U.S. Pat. No. 4,904,646, U.S.
Pat. No. 5,385,932, U.S. Pat. No. 5,250,435, U.S. Pat. No.
5,132,312, U.S. Pat. No. 5,130,306, U.S. Pat. No. 5,116,870, U.S.
Pat. No. 5,112,857, U.S. Pat. No. 5,102,911, U.S. Pat. No.
5,098,931, U.S. Pat. No. 5,081,136, U.S. Pat. No. 5,025,000, U.S.
Pat. No. 5,021,453, U.S. Pat. No. 5,017,716, U.S. Pat. No.
5,001,144, U.S. Pat. No. 5,001,128, U.S. Pat. No. 4,997,837, U.S.
Pat. No. 4,996,234, U.S. Pat. No. 4,994,494, U.S. Pat. No.
4,992,429, U.S. Pat. No. 4,970,231, U.S. Pat. No. 4,968,693, U.S.
Pat. No. 4,963,538, U.S. Pat. No. 4,957,940, U.S. Pat. No.
4,950,675, U.S. Pat. No. 4,946,864, U.S. Pat. No. 4,946,860, U.S.
Pat. No. 4,940,800, U.S. Pat. No. 4,940,727, U.S. Pat. No.
4,939,143, U.S. Pat. No. 4,929,620, U.S. Pat. No. 4,923,861, U.S.
Pat. No. 4,906,657, U.S. Pat. No. 4,906,624 and U.S. Pat. No.
4,897,402, the disclosures of which patents are incorporated herein
by reference.
[0132] Nitric oxide (NO) has been recognized as a messenger
molecule with many physiologic roles, in the cardiovascular,
neurologic and immune systems (Griffith, T M et al., J Am Coll
Cardiol, 1988, 12:797-806). It mediates blood vessel relaxation,
neurotransmission and pathogen suppression. NO is produced from the
guanidino nitrogen of L-arginine by NO synthase (Moncada, S and
Higgs, E A, Eur J Clin Invest, 1991, 21:361-374). Agents that
upregulate endothelial cell Nitric Oxide Synthase include, but are
not limited to, L-arginine, rho GTPase function inhibitors (see
International Application WO 99/47153, the disclosure of which is
incorporated herein by reference), and agents that disrupt actin
cytoskeletal organization (see International Application WO
00/03746, the disclosure of which is incorporated herein by
reference).
[0133] "Co-administering," as used herein, refers to administering
simultaneously two or more compounds of the invention (e.g.,
anagrelide, and an agent known to be beneficial in the treatment
of, for example, a cardiovascular condition e.g., an
anticoagulant), as an admixture in a single composition, or
sequentially, close enough in time so that the compounds may exert
an additive or even synergistic effect, i.e., on reducing
cardiomyocyte cell-death in a cardiovascular condition.
[0134] It should be understood that the preceding is merely a
detailed description of certain preferred embodiments. It therefore
should be apparent to those of ordinary skill in the art that
various modifications and equivalents can be made without departing
from the spirit and scope of the invention. It is intended that the
invention encompass all such modifications within the scope of the
appended claims.
[0135] All references, patents and patent applications and
publications that are cited or referred to in this application are
incorporated in their entirety herein by reference.
* * * * *