U.S. patent application number 15/605579 was filed with the patent office on 2017-09-21 for modulation of apolipoprotein ciii (apociii) expression.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Thomas A. Bell, III, Rosanne M. Crooke, Kenneth W. Dobie, Mark J. Graham, Richard Lee, Adam Mullick.
Application Number | 20170268004 15/605579 |
Document ID | / |
Family ID | 47072800 |
Filed Date | 2017-09-21 |
United States Patent
Application |
20170268004 |
Kind Code |
A1 |
Mullick; Adam ; et
al. |
September 21, 2017 |
MODULATION OF APOLIPOPROTEIN CIII (APOCIII) EXPRESSION
Abstract
Provided herein are methods, compounds, and compositions for
reducing expression of ApoCIII mRNA and protein in an animal. Also
provided herein are methods, compounds, and compositions for
increasing HDL levels and/or improving the ratio of TG to HDL and
reducing plasma lipids and plasma glucose in an animal. Such
methods, compounds, and compositions are useful to treat, prevent,
delay, or ameliorate any one or more of cardiovascular disease or
metabolic disorder, or a symptom thereof.
Inventors: |
Mullick; Adam; (Carlsbad,
CA) ; Crooke; Rosanne M.; (Carlsbad, CA) ;
Graham; Mark J.; (San Clemente, CA) ; Dobie; Kenneth
W.; (Solana Beach, CA) ; Bell, III; Thomas A.;
(Encinitas, CA) ; Lee; Richard; (Oceanside,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Family ID: |
47072800 |
Appl. No.: |
15/605579 |
Filed: |
May 25, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14821641 |
Aug 7, 2015 |
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15605579 |
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14114187 |
Jan 6, 2014 |
9157082 |
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PCT/US2012/035694 |
Apr 27, 2012 |
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14821641 |
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61479817 |
Apr 27, 2011 |
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61595009 |
Feb 3, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/351 20130101;
A61P 3/06 20180101; C12N 2310/321 20130101; C12N 2310/346 20130101;
A61P 9/00 20180101; A61P 9/06 20180101; A61P 1/00 20180101; A61K
31/713 20130101; A61P 43/00 20180101; A61P 9/10 20180101; C12N
2310/11 20130101; A61K 45/06 20130101; A61P 21/00 20180101; A61P
25/00 20180101; A61P 1/18 20180101; A61P 9/04 20180101; A61P 29/00
20180101; C12N 2310/341 20130101; A61P 7/00 20180101; A61P 9/12
20180101; C12N 2310/315 20130101; A61K 31/7088 20130101; C12N
15/113 20130101; A61K 31/7088 20130101; A61K 2300/00 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713; A61K 31/7088
20060101 A61K031/7088; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of increasing HDL levels or improving the ratio of TG
to HDL by (a) selecting an animal in need thereof, and (b)
administering a compound targeting ApoCIII to the animal, whereby
HDL levels are increased or the ratio of TG to HDL is improved.
2. A method of preventing, delaying or ameliorating a
cardiovascular disease, disorder, condition or symptom thereof or
onset of a cardiovascular disease, disorder, condition or symptom
thereof in an animal comprising (a) selecting an animal in need
thereof, and (b) administering a compound targeting ApoCIII to the
animal, thereby increasing HDL levels in the animal, wherein the
cardiovascular disease, disorder, condition or symptom or the onset
of the cardiovascular disease, disorder, condition, or symptom
thereof, is prevented, delayed or ameliorated.
3. A method of reducing the risk of a cardiovascular disease,
disorder, condition or symptom in an animal comprising (a)
selecting an animal in need thereof, and (b) administering a
compound targeting ApoCIII to the animal, thereby raising HDL
levels in the animal, wherein the risk of a cardiovascular disease,
disorder, condition, or symptom thereof, is reduced.
4. A method of decreasing CETP levels by administering a compound
targeting ApoCIII to an animal, wherein CETP levels are
decreased.
5. A method of increasing ApoA1, PON1, fat clearance, chylomicron
triglyceride clearance, postprandial triglyceride clearance and/or
HDL comprising (a) selecting an animal in need thereof, and (b)
administering a compound targeting ApoCIII to the animal, wherein
ApoA1, PON1, fat clearance, chylomicron triglyceride clearance,
postprandial triglyceride clearance and/or HDL is increased.
6. A method of preventing, delaying or ameliorating pancreatitis
comprising (a) selecting an animal with, or at risk of,
pancreatitis, and (b) administering a compound targeting ApoCIII to
the animal, wherein the pancreatitis is prevented, delayed or
ameliorated.
7. A method of preventing, delaying or ameliorating pancreatitis
comprising (a) selecting an animal with, or at risk of,
pancreatitis, and (b) administering a compound targeting ApoCIII to
the animal, thereby increasing chylomicron clearance, wherein the
pancreatitis is prevented, delayed or ameliorated.
8. A compound targeting ApoCIII, for use in increasing HDL levels
or improving the ratio of TG to HDL by (a) selecting an animal in
need thereof, and (b) administering a compound targeting ApoCIII to
an animal, whereby HDL levels are increased or the ratio of TG to
HDL is improved.
9. A compound targeting ApoCIII, for use in preventing, delaying or
ameliorating a cardiovascular disease, disorder, condition or
symptom thereof or onset of a cardiovascular disease, disorder,
condition or symptom thereof in an animal comprising (a) selecting
an animal in need thereof, and (b) administering a compound
targeting ApoCIII to the animal, thereby increasing HDL levels in
the animal, wherein the cardiovascular disease, disorder, condition
or symptom or the onset of the cardiovascular disease, disorder,
condition, or symptom thereof, is prevented, delayed or
ameliorated.
10. A compound targeting ApoCIII, for use in reducing the risk of a
cardiovascular disease, disorder, condition or symptom in an animal
comprising (a) selecting an animal in need thereof, and (b)
administering a compound targeting ApoCIII to the animal, thereby
raising HDL levels in the animal, wherein the risk of a
cardiovascular disease, disorder, condition, or symptom thereof, is
reduced.
11. A compound targeting ApoCIII, for use in decreasing CETP levels
by administering a compound targeting ApoCIII to an animal, wherein
CETP levels are decreased.
12. A compound targeting ApoCIII, for use in increasing ApoA1,
PON1, fat clearance, chylomicron triglyceride clearance,
postprandial triglyceride clearance and/or HDL comprising (a)
selecting an animal in need thereof, and (b) administering a
compound targeting ApoCIII to the animal, wherein ApoA1, PON1, fat
clearance, chylomicron triglyceride clearance, postprandial
triglyceride clearance and/or HDL is increased.
13. A compound targeting ApoCIII, for use in preventing, delaying
or ameliorating pancreatitis comprising (a) selecting an animal
with, or at risk of, pancreatitis, and (b) administering a compound
targeting ApoCIII to the animal, wherein the pancreatitis is
prevented, delayed or ameliorated.
14. A compound targeting ApoCIII, for use in preventing, delaying
or ameliorating pancreatitis comprising (a) selecting an animal
with, or at risk of, pancreatitis, and (b) administering a compound
targeting ApoCIII to the animal, thereby increasing chylomicron
clearance, wherein the pancreatitis is prevented, delayed or
ameliorated.
15. The method or use of any preceding claim, wherein the animal
has, or is at risk for, hypertriglyceridemia.
16. The method or use of claim 15, wherein the animal has a
triglyceride level between 100-200 mg/dL, 100-300 mg/dL, 100-400
mg/dL, 100-500 mg/dL, 200-500 mg/dL, 300-500 mg/dL, 400-500 mg/dL,
500-1000 mg/dL, 600-1000 mg/dL, 700-1000 mg/dL, 800-1000 mg/dL,
900-1000 mg/dL, 500-1500 mg/dL, 1000-1500 mg/dL, 100-2000 mg/dL,
150-2000 mg/dL, 200-2000 mg/dL, 300-2000 mg/dL, 400-2000 mg/dL,
500-2000 mg/dL, 600-2000 mg/dL, 700-2000 mg/dL, 800-2000 mg/dL,
900-2000 mg/dL, 1000-2000 mg/dL, 1100-2000 mg/dL, 1200-2000 mg/dL,
1300-2000 mg/dL, 1400-2000 mg/dL, or 1500-2000 mg/dL.
17. The method or use of claim 15, wherein the hypertriglyceridemia
is Fredrickson Type II, IV or V.
18. The method or use of any preceding claim, wherein the animal
has a genetic defect leading to hypertriglyceridemia.
19. The method or use of claim 18, wherein the genetic defect is a
heterozygous LPL deficiency or an ApoCIII polymorphism.
20. The method or use of any preceding claim, wherein the animal
has a triglyceride level .gtoreq.500 mg/dL and a heterozygous LPL
deficiency.
21. The method or use of claim 5, 7, 12 or 14, wherein the
increased chylomicron clearance enhances clearance of postprandial
triglycerides and/or decreases postprandial triglycerides.
22. The method or use of any preceding claim, wherein ApoCIII has a
nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
23. The method or use of any preceding claim, wherein the compound
targeting ApoCIII is a modified oligonucleotide.
24. The method or use of claim 23, wherein the modified
oligonucleotide has a nucleobase sequence comprising at least 8
contiguous nucleobases of a nucleobase sequence of SEQ ID NO:
3.
25. The method or use of claim 23, wherein the nucleobase sequence
of the modified oligonucleotide is 80% complementary to a
nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
26. The method or use of claim 23, wherein the nucleobase sequence
of the modified oligonucleotide is 90% complementary to a
nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
27. The method or use of claim 23, wherein the nucleobase sequence
of the modified oligonucleotide is 100% complementary to a
nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
28. The method or use of any of claims 23-27, wherein the modified
oligonucleotide consists of a single-stranded modified
oligonucleotide.
29. The method or use of any of claims 23-28, wherein the modified
oligonucleotide consists of 12 to 30 linked nucleosides.
30. The method or use of claim 29, wherein the modified
oligonucleotide consists of 20 linked nucleosides.
31. The method or use of any of claims 23-30, wherein at least one
internucleoside linkage of the modified oligonucleotide is a
modified internucleoside linkage.
32. The method or use of claim 31, wherein each modified
internucleoside linkage of the modified oligonucleotide is a
phosphorothioate internucleoside linkage.
33. The method or use of any of claims 23-32, wherein at least one
nucleoside of the modified oligonucleotide comprises a modified
sugar.
34. The method or use of claim 33, wherein at least one modified
sugar is a bicyclic sugar.
35. The method or use of claim 33, wherein at least one modified
sugar comprises a 2'-O-methoxyethyl.
36. The method or use of any of claims 23-33, wherein at least one
nucleoside of the modified oligonucleotide comprises a modified
nucleobase.
37. The method or use of claim 36, wherein the modified nucleobase
is a 5-methylcytosine.
38. The method or use of claim 23, wherein the modified
oligonucleotide comprises: (a) a gap segment consisting of linked
deoxynucleosides; (b) a 5' wing segment consisting of linked
nucleosides; (c) a 3' wing segment consisting linked nucleosides;
wherein the gap segment is positioned immediately adjacent to and
between the 5' wing segment and the 3' wing segment and wherein
each nucleoside of each wing segment comprises a modified
sugar.
39. The method or use of claim 23, wherein the modified
oligonucleotide comprises: (a) a gap segment consisting of 8-12
linked deoxynucleosides; (b) a 5' wing segment consisting of 1-5
linked nucleosides; (c) a 3' wing segment consisting 1-5 linked
nucleosides; wherein the gap segment is positioned immediately
adjacent to and between the 5' wing segment and the 3' wing
segment, wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage.
40. The method or use of claim 23, wherein the modified
oligonucleotide comprises: (a) a gap segment consisting of 10
linked deoxynucleosides; (b) a 5' wing segment consisting of 5
linked nucleosides; (c) a 3' wing segment consisting 5 linked
nucleosides; wherein the gap segment is positioned immediately
adjacent to and between the 5' wing segment and the 3' wing segment
and wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage.
41. The method or use of claims 38-40, wherein the modified
oligonucleotide has a nucleobase sequence comprising at least 8
contiguous nucleobases of a nucleobase sequence of SEQ ID NO:
3.
42. A method of reducing the risk for a cardiovascular disease in
an animal comprising administering to the animal a therapeutically
effective amount of a compound comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, wherein
the modified oligonucleotide is complementary to an ApoCIII nucleic
acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the
compound administered to the animal reduces the risk for a
cardiovascular disease, by increasing HDL levels.
43. A method of reducing the risk for a cardiovascular disease or
pancreatitis in an animal comprising administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides,
and having a nucleobase sequence comprising at least 8 contiguous
nucleobases of SEQ ID NO: 3, and wherein the compound administered
to the animal reduces the risk for a cardiovascular disease or
pancreatitis, by increasing HDL levels and/or improving the ratio
of TG to HDL.
44. A method of preventing, treating, ameliorating or reducing at
least one symptom of a cardiovascular disease in an animal,
comprising administering to the animal a therapeutically effective
amount of a compound comprising a modified oligonucleotide
consisting of 12 to 30 linked nucleosides, wherein the modified
oligonucleotide is complementary to an ApoCIII nucleic acid, as
shown in SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the compound
administered to the animal prevents, treats, ameliorates or reduces
at least one symptom of the cardiovascular disease in the animal,
by increasing HDL levels.
45. A method of preventing, treating, ameliorating or reducing at
least one symptom of a cardiovascular disease in an animal,
comprising administering to the animal a therapeutically effective
amount of a compound comprising a modified oligonucleotide
consisting of 12 to 30 linked nucleosides, and having a nucleobase
sequence comprising at least 8 contiguous nucleobases of SEQ ID NO:
3, and wherein the compound administered to the animal prevents,
treats, ameliorates or reduces at least one symptom of the
cardiovascular disease in the animal, by increasing HDL levels in
the animal.
46. A compound comprising a modified oligonucleotide consisting of
12 to 30 linked nucleosides, wherein the modified oligonucleotide
is complementary to an ApoCIII nucleic acid as shown in SEQ ID NO:
1 or SEQ ID NO: 2, for use in reducing the risk for a
cardiovascular disease in an animal, and wherein the compound
reduces the risk for a cardiovascular disease by increasing HDL
levels.
47. A compound comprising a modified oligonucleotide consisting of
12 to 30 linked nucleosides, and having a nucleobase sequence
comprising at least 8 contiguous nucleobases of SEQ ID NO: 3, for
use in reducing the risk for a cardiovascular disease or
pancreatitis in an animal, wherein the compound reduces the risk
for a cardiovascular disease or pancreatitis by increasing HDL
levels and/or improving the ratio of TG to HDL.
48. A compound comprising a modified oligonucleotide consisting of
12 to 30 linked nucleosides, wherein the modified oligonucleotide
is complementary to an ApoCIII nucleic acid, as shown in SEQ ID NO:
1 or SEQ ID NO: 2, for use in preventing, treating, ameliorating or
reducing at least one symptom of a cardiovascular disease in an
animal, and wherein the compound prevents, treats, ameliorates or
reduces at least one symptom of the cardiovascular disease in the
animal by increasing HDL levels.
49. A compound comprising a modified oligonucleotide consisting of
12 to 30 linked nucleosides, and having a nucleobase sequence
comprising at least 8 contiguous nucleobases of SEQ ID NO: 3, for
use in preventing, treating, ameliorating or reducing at least one
symptom of a cardiovascular disease in an animal, wherein the
compound prevents, treats, ameliorates or reduces at least one
symptom of the cardiovascular disease in the animal by increasing
HDL levels in the animal.
50. The method or use of any preceding claim, wherein the symptoms
may be any one of, but not limited to, angina; chest pain;
shortness of breath; palpitations; weakness; dizziness; nausea;
sweating; tachycardia; bradycardia; arrhythmia; atrial
fibrillation; swelling in the lower extremities; cyanosis; fatigue;
fainting; numbness of the face; numbness of the limbs; claudication
or cramping of muscles; bloating of the abdomen; or fever.
51. A method of raising HDL levels and/or improving the ratio of TG
to HDL in an animal by administering to the animal a compound
consisting of SEQ ID NO: 3 to raise the HDL levels and/or improving
the ratio of TG to HDL in the animal.
52. A method of preventing, treating, ameliorating or reducing at
least one symptom of a cardiovascular disease in an animal by
administering to the animal a compound consisting of SEQ ID NO: 3
to prevent, treat, ameliorate or reduce at least one symptom of the
cardiovascular disease in the animal, by increasing HDL levels
and/or improving the ratio of TG to HDL in the animal.
53. A method of raising HDL levels and/or improving the ratio of TG
to HDL in an animal by administering to the animal a modified
oligonucleotide, having the sequence of SEQ ID NO: 3 wherein the
modified oligonucleotide comprises: (a) a gap segment consisting of
10 linked deoxynucleosides; (b) a 5' wing segment consisting of 5
linked nucleosides; (c) a 3' wing segment consisting 5 linked
nucleosides; wherein the gap segment is positioned immediately
adjacent to and between the 5' wing segment and the 3' wing
segment, wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage, wherein the modified oligonucleotide
raises the HDL levels and/or improving the ratio of TG to HDL in
the animal.
54. A method of preventing, treating, ameliorating or reducing at
least one symptom of a cardiovascular disease in an animal by
administering to the animal a modified oligonucleotide, having the
sequence of SEQ ID NO: 3 wherein the modified oligonucleotide
comprises: (a) a gap segment consisting of 10 linked
deoxynucleosides; (b) a 5' wing segment consisting of 5 linked
nucleosides; (c) a 3' wing segment consisting 5 linked nucleosides;
wherein the gap segment is positioned immediately adjacent to and
between the 5' wing segment and the 3' wing segment, wherein each
nucleoside of each wing segment comprises a 2'-O-methoxyethyl
sugar, wherein each cytosine is a 5-methylcytosine, and wherein
each internucleoside linkage is a phosphorothioate linkage, wherein
the modified oligonucleotide prevents, treats, ameliorates or
reduces at least one symptom in the animal with the cardiovascular
disease by raising the HDL levels and/or improving the ratio of TG
to HDL in the animal.
55. A method of raising HDL levels and/or improving the ratio of TG
to HDL in an animal by administering to the animal a compound
comprising a modified oligonucleotide consisting of 12 to 30 linked
nucleosides, wherein the modified oligonucleotide is complementary
to an ApoCIII nucleic acid, as shown in SEQ ID NO: 1 or SEQ ID NO:
2, to raise the HDL levels and/or improving the ratio of TG to HDL
in the animal.
56. A method of preventing, treating, ameliorating or reducing at
least one symptom of a cardiovascular disease in an animal by
administering to the animal a compound comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, wherein
the modified oligonucleotide is complementary to an ApoCIII nucleic
acid, as shown in SEQ ID NO: 1 or SEQ ID NO: 2, to prevent, treat,
ameliorate or reduce at least one symptom of the cardiovascular
disease in the animal, by raising the HDL levels and/or improving
the ratio of TG to HDL of the animal.
57. A method of decreasing CETP levels by administering to an
animal a compound having a nucleobase sequence comprising at least
8 contiguous nucleobases of SEQ ID NO: 3, wherein CETP levels are
decreased.
58. A method of increasing ApoA1, PON1, fat clearance, chylomicron
triglyceride clearance or HDL by administering to an animal a
compound having a nucleobase sequence comprising at least 8
contiguous nucleobases of SEQ ID NO: 3, wherein ApoA1, PON1, fat
clearance, chylomicron triglyceride clearance or HDL is
increased.
59. A method of decreasing CETP levels by administering to an
animal a compound having a nucleobase sequence consisting of SEQ ID
NO: 3, wherein CETP levels are decreased.
60. A method of increasing ApoA1, PON1, fat clearance, chylomicron
triglyceride clearance or HDL by administering to an animal a
compound consisting of at least 8 contiguous nucleobases of SEQ ID
NO: 3, wherein ApoA1, PON1, fat clearance, chylomicron triglyceride
clearance or HDL is increased.
61. A compound consisting of SEQ ID NO: 3, for use in raising HDL
levels and/or improving the ratio of TG to HDL in an animal.
62. A compound consisting of SEQ ID NO: 3, for use in preventing,
treating, ameliorating or reducing at least one symptom of a
cardiovascular disease in an animal by increasing HDL levels and/or
improving the ratio of TG to HDL in the animal.
63. A modified oligonucleotide, having the sequence of SEQ ID NO: 3
wherein the modified oligonucleotide comprises: (a) a gap segment
consisting of 10 linked deoxynucleosides; (b) a 5' wing segment
consisting of 5 linked nucleosides; (c) a 3' wing segment
consisting 5 linked nucleosides; wherein the gap segment is
positioned immediately adjacent to and between the 5' wing segment
and the 3' wing segment, wherein each nucleoside of each wing
segment comprises a 2'-O-methoxyethyl sugar, wherein each cytosine
is a 5-methylcytosine, and wherein each internucleoside linkage is
a phosphorothioate linkage, for use in raising HDL levels and/or
improving the ratio of TG to HDL in an animal.
64. A modified oligonucleotide, having the sequence of SEQ ID NO: 3
wherein the modified oligonucleotide comprises: (a) a gap segment
consisting of 10 linked deoxynucleosides; (b) a 5' wing segment
consisting of 5 linked nucleosides; (c) a 3' wing segment
consisting 5 linked nucleosides; wherein the gap segment is
positioned immediately adjacent to and between the 5' wing segment
and the 3' wing segment, wherein each nucleoside of each wing
segment comprises a 2'-O-methoxyethyl sugar, wherein each cytosine
is a 5-methylcytosine, and wherein each internucleoside linkage is
a phosphorothioate linkage, for use in preventing, treating,
ameliorating or reducing at least one symptom of a cardiovascular
disease in an animal by raising the HDL levels and/or improving the
ratio of TG to HDL in the animal.
65. A compound comprising a modified oligonucleotide consisting of
12 to 30 linked nucleosides, wherein the modified oligonucleotide
is complementary to an ApoCIII nucleic acid, as shown in SEQ ID NO:
1 or SEQ ID NO: 2, for use in raising HDL levels and/or improving
the ratio of TG to HDL in an animal.
66. A compound comprising a modified oligonucleotide consisting of
12 to 30 linked nucleosides, wherein the modified oligonucleotide
is complementary to an ApoCIII nucleic acid, as shown in SEQ ID NO:
1 or SEQ ID NO: 2, for use in preventing, treating, ameliorating or
reducing at least one symptom of a cardiovascular disease in an
animal by raising the HDL levels and/or improving the ratio of TG
to HDL of the animal.
67. A compound having a nucleobase sequence comprising at least 8
contiguous nucleobases of SEQ ID NO: 3, for use in decreasing CETP
levels in an animal.
68. A compound having a nucleobase sequence comprising at least 8
contiguous nucleobases of SEQ ID NO: 3, for use in increasing
ApoA1, PON1, fat clearance, chylomicron triglyceride clearance or
HDL in an animal.
69. A compound having a nucleobase sequence consisting of SEQ ID
NO: 3, for use in decreasing CETP levels in an animal.
70. A compound consisting of at least 8 contiguous nucleobases of
SEQ ID NO: 3, for use in increasing ApoA1, PON1, fat clearance,
chylomicron triglyceride clearance or HDL in an animal.
71. The method or use of any preceding claim, wherein the animal is
human.
72. The method or use of any preceding claim, wherein the
cardiovascular disease is aneurysm, angina, arrhythmia,
atherosclerosis, cerebrovascular disease, coronary heart disease,
hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia or
hypercholesterolemia.
73. The method or use of claim 72, wherein the dyslipidemia is
chylomicronemia.
74. The method or use of claim 73, wherein the animal is at risk
for pancreatitis.
75. The method or use of any preceding claim, wherein reducing
ApoCIII levels prevents, treats or ameliorates pancreatitis.
76. The method or use of any preceding claim, wherein reducing
ApoCIII levels enhance clearance of postprandial triglycerides.
77. The method or use of any preceding claim, wherein reducing
ApoCIII levels lowers postprandial triglycerides.
78. The method or use of any preceding claim, wherein the compound
is parenterally administered.
79. The method or use of claim 78, wherein the parenteral
administration is subcutaneous administration.
80. The method or use of any preceding claim, further comprising a
second agent.
81. The method or use of claim 80, wherein the second agent is
selected from an ApoCIII lowering agent, cholesterol lowering
agent, non-HDL lipid lowering agent, LDL lowering agent, TG
lowering agent, cholesterol lowering agent, HDL raising agent, fish
oil, niacin, fibrate, statin, DCCR (salt of diazoxide),
glucose-lowering agent or anti-diabetic agents.
82. The method or use of claim 81, wherein the animal fails to
respond to a maximally tolerated dose of the second agent.
83. The method or use of claim 80, wherein the second agent is
administered concomitantly or sequentially with the compound.
84. The method or use of any preceding claim, wherein the compound
is a salt form.
85. The method or use of any preceding claim, further comprising a
pharmaceutically acceptable carrier or diluent.
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled BIOL0130USC2SEQ_ST25.TXT, created on May 25, 2017
which is 8 Kb in size. The information in the electronic format of
the sequence listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] Provided herein are methods, compounds, and compositions for
reducing expression of Apolipoprotein CIII (ApoCIII) mRNA and
protein, and increasing HDL or HDL activity in an animal. Also,
provided herein are methods, compounds, and compositions for an
ApoCIII inhibitor for reducing ApoCIII related diseases or
conditions in an animal.
BACKGROUND
[0003] Lipoproteins are globular, micelle-like particles that
consist of a non-polar core of acylglycerols and cholesteryl esters
surrounded by an amphiphilic coating of protein, phospholipid and
cholesterol. Lipoproteins have been classified into five broad
categories on the basis of their functional and physical
properties: chylomicrons, very low density lipoproteins (VLDL),
intermediate density lipoproteins (IDL), low density lipoproteins
(LDL), and high density lipoproteins (HDL). Chylomicrons transport
dietary lipids from intestine to tissues. VLDLs, IDLs and LDLs all
transport triacylglycerols and cholesterol from the liver to
tissues. HDLs transport endogenous cholesterol from tissues to the
liver
[0004] Lipoprotein particles undergo continuous metabolic
processing and have variable properties and compositions.
Lipoprotein densities increase without increasing particle diameter
because the density of their outer coatings is less than that of
the inner core. The protein components of lipoproteins are known as
apolipoproteins. At least nine apolipoproteins are distributed in
significant amounts among the various human lipoproteins.
[0005] Apolipoprotein C-III (ApoCIII) is a constituent of HDL and
of triglyceride (TG)-rich lipoproteins. Elevated ApoCIII is
associated with hypertriglyceridemia. Accordingly, ApoCIII has a
role in hypertriglyceridemia, a risk factor for coronary artery
disease (Davidsson et al., J. Lipid Res. 2005. 46: 1999-2006).
ApoCIII slows clearance of triglyceride-rich lipoproteins by
inhibiting lipolysis, both through inhibition of lipoprotein lipase
and by interfering with lipoprotein binding to cell-surface
glycosaminoglycan matrix (Shachter, Curr. Opin. Lipidol., 2001, 12,
297-304).
[0006] The gene encoding human apolipoprotein C-III (also called
APOC3, APOC-III, ApoCIII, and APO C-III) was cloned in 1984 by
three research groups (Levy-Wilson et al., DNA, 1984, 3, 359-364;
Protter et al., DNA, 1984, 3, 449-456; Sharpe et al., Nucleic Acids
Res., 1984, 12, 3917-3932). The coding sequence is interrupted by
three introns (Protter et al., DNA, 1984, 3, 449-456). The human
ApoCIII gene is located approximately 2.6 kb to the 3' direction of
the apolipoprotein A-1 gene and these two genes are convergently
transcribed (Karathanasis, Proc. Natl. Acad. Sci. U.S.A, 1985, 82,
6374-6378). Also cloned was a variant of human apolipoprotein C-III
with a Thr74 to Ala74 mutation from a patient with unusually high
level of serum apolipoprotein C-III. As the Thr74 is
O-glycosylated, the Ala74 mutant therefore resulted in increased
levels of serum ApoCIII lacking the carbohydrate moiety (Maeda et
al., J. Lipid Res., 1987, 28, 1405-1409). Other variants or
polymorphisms that modulated Apo CIII expression were later
identified. Some of the polymorphsims elevated ApoCIII. Elevated
ApoCIII levels were associated with elevated triglyceride (TG)
levels and diseases such as cardiovascular disease, metabolic
syndrome, obesity and diabetes (Chan et al., Int J Clin Pract,
2008, 62:799-809; Onat et al., Atherosclerosis, 2003, 168:81-89;
Mendivil et al., Circulation, 2011, 124:2065-2072).
[0007] Five polymorphisms have been identified in the promoter
region of the gene: C (at position -641 of the gene) to A, G (at
position -630 of the gene) to A, T (at position -625 of the gene)
to deletion, C (at position -482 of the gene) to T and T (at
position -455 of the gene) to C. All of these polymorphisms are in
linkage disequilibrium with the SstI polymorphism in the 3'
untranslated region. The SstI polymorphic site distinguishes the S1
and S2 alleles and the S2 allele has been associated with elevated
plasma triglyceride levels (Dammerman et al., Proc. Natl. Acad.
Sci. U.S.A, 1993, 90, 4562-4566). The ApoCIII promoter is
downregulated by insulin and this polymorphic site abolishes the
insulin regulation. Thus the potential overexpression of ApoCIII
resulting from the loss of insulin regulation may be a contributing
factor to the development of hypertriglyceridemia associated with
the S2 allele (Li et al., J. Clin. Invest., 1995, 96, 2601-2605).
The T (at position -455 of the gene) to C polymorphism has been
associated with an increased risk of coronary artery disease
(Olivieri et al., J. Lipid Res., 2002, 43, 1450-1457). Other
polymorphisms in the human ApoCIII gene that have been associated
with elevated ApoCIII and/or triglyceride expression include: C (at
position 1100) to T, C (at position 3175) to G, T (at position
3206) to G, C (at positions 3238) to G, etc. (Tilly et al., J.
Lipid Res., 2003, 44:430-436; Waterworth et al., Arterioscler
Thromb Vasc Biol, 2000, 20:2663-2669; Petersen et al., N Engl J
Med, 2010, 362:1082-1089).
[0008] In addition to insulin, other regulators of ApoCIII gene
expression have been identified. A response element for the nuclear
orphan receptor rev-erb alpha has been located at positions -23 to
-18 of the gene, in the ApoCIII promoter region. Rev-erb alpha
decreases ApoCIII promoter activity (Raspe et al., J. Lipid Res.,
2002, 43, 2172-2179). The ApoCIII promoter region from positions
-86 to -74 of the gene is recognized by two nuclear factors CIIIb1
and CIIIB2 (Ogami et al., J Biol. Chem., 1991, 266, 9640-9646).
ApoCIII expression is also upregulated by retinoids acting via the
retinoid X receptor, and alterations in retinoid X receptor
abundance affects ApoCIII transcription (Vu-Dac et al., J Clin.
Invest., 1998, 102, 625-632). Specificity protein 1 (Sp1) and
hepatocyte nuclear factor-4 (HNF-4) have been shown to work
synergistically to transactivate the apolipoprotein C-III promoter
via the HNF-4 binding site (Kardassis et al., Biochemistry, 2002,
41, 1217-1228). HNF-4 also works in conjunction with SMAD3-SMAD4 to
transactivate the ApoCIII promoter (Kardassis et al., J. Biol.
Chem., 2000, 275, 41405-41414).
[0009] Transgenic and knockout mice have further defined the role
of ApoCIII in lipolysis. Overexpression of ApoCIII in transgenic
mice leads to hypertriglyceridemia and impaired clearance of
VLDL-triglycerides (de Silva et al., J Biol. Chem., 1994, 269,
2324-2335; Ito et al., Science, 1990, 249, 790-793). Knockout mice
with a total absence of the ApoCIII protein exhibited significantly
reduced plasma cholesterol and triglyceride levels compared with
wild-type mice and were protected from postprandial
hypertriglyceridemia (Maeda et al., J. Biol. Chem., 1994, 269,
23610-23616).
[0010] Total plasma ApoCIII levels have been identified as a major
determinant of serum triglycerides, and epidemiological studies
have demonstrated that ApoCIII and ApoB lipoproteins that have
ApoCIII as a component independently predict coronary heart disease
(Sacks et al., Circulation. 2000. 102: 1886-1892; Lee et al.,
Arterioscler Thromb Vasc Biol. 2003. 23: 853-858). Studies also
demonstrate that ApoCIII is a key determinant in the clearance of
triglyceride-rich lipoproteins and its remnants in
hypertriglyceridaemic states, including visceral obesity, insulin
resistance and the metabolic syndrome (Mauger et al., J. Lipid Res.
2006. 47: 1212-1218; Chan et al., Clin. Chem. 2002. 278-283; Ooi et
al., Clin. Sci. 2008. 114: 611-624).
[0011] Hypertriglyceridemia is a common clinical trait associated
with an increased risk of cardiometabolic disease (Hegele et al.
2009, Hum Mol Genet, 18: 4189-4194; Hegele and Pollex 2009, Mol
Cell Biochem, 326: 35-43) as well as of occurrence of acute
pancreatitis in the most severe forms (Toskes 1990, Gastroenterol
Clin North Am, 19: 783-791; Gaudet et al. 2010, Atherosclerosis
Supplements, 11: 55-60; Catapano et al. 2011, Atherosclerosis,
217S: S1-S44; Tremblay et al. 2011, J Clin Lipidol, 5: 37-44).
Examples of cardiometabolic disease include, but are not limited
to, diabetes, metabolic syndrome/insulin resistance, and genetic
disorders such as familial chylomicronemia, familial combined
hyperlipidemia and familial hypertriglyceridemia.
[0012] Hypertriglyceridemia is the consequence of increased
production and/or reduced or delayed catabolism of triglyceride
(TG)-rich lipoproteins: VLDL and, to a lesser extent, chylomicrons
(CM). Borderline high TGs (150-199 mg/dL) are commonly found in the
general population and are a common component of the metabolic
syndrome/insulin resistance states. The same is true for high TGs
(200-499 mg/dL) except that as plasma TG levels increase,
underlying genetic factors play an increasingly important etiologic
role. Very high TGs (.gtoreq.500 mg/dL) are most often associated
with elevated CM levels as well, and are accompanied by increasing
risk for acute pancreatitis. The risk of pancreatitis is considered
clinically significant if TG exceeds 880 mg/dL (>10 mmol) and
the European Atherosclerosis Society/European Society of Cardiology
(EAS/ESC) 2011 guidelines state that actions to prevent acute
pancreatitis are mandatory (Catapano et al. 2011, Atherosclerosis,
217S: S1-S44). According to the EAS/ESC 2011 guidelines,
hypertriglyceridemia is the cause of approximately 10% of all cases
of pancreatitis, and development of pancreatitis can occur at TG
levels between 440-880 mg/dL. Based on evidence from clinical
studies demonstrating that elevated TG levels are an independent
risk factor for atherosclerotic CVD, the guidelines from both the
National Cholesterol Education Program Adult Treatment Panel III
(NCEP 2002, Circulation, 106: 3143-421) and the American Diabetes
Association (ADA 2008, Diabetes Care, 31: S12-S54.) recommend a
target TG level of less than 150 mg/dL to reduce cardiovascular
risk.
[0013] ApoCIII-knockout mice had normal intestinal lipid absorption
and hepatic VLDL triacylglycerol secretion, but a rapid clearance
of VLDL triacylglycerols and VLDL cholesteryl esters from plasma
that may explain the observed hypolipidaemia (Gerritsen et al., J.
Lipid Res. 2005. 46: 1466-1473; Jong et al., J. Lipid Res. 2001.
42: 1578-1585). VLDL particles with ApoCIII have been cited to play
a major role in identifying the high risk of coronary heart disease
in hypertriglyceridemia (Campos et al., J. Lipid Res. 2001. 42:
1239-1249). A genome-wide association study found a naturally
occurring ApoCIII null mutation in Lancaster Amish people
demonstrated a favorable lipid profile and apparent
cardioprotection, with no obvious detrimental effects (Pollin et
al., Science. 2008. 322: 1702-1705). The mutation carriers are
observed to have lower fasting and postprandial serum triglycerides
and LDL-cholesterol, and higher levels of HDL-cholesterol.
[0014] The HDL class of lipoproteins comprises a heterogeneous and
polydisperse population of particles that are the most dense and
smallest of size (Havel and Kane. In, The Metabolic & Molecular
Bases of Inherited Disease. 8.sup.th Edition. McGraw-Hill, New
York, 2001:2705-16). HDL is a macromolecular complex of lipids
(cholesterol, triglycerides and phospholipids) and proteins
(apolipoproteins (apo) and enzymes). The surface of HDL contains
chiefly apolipoproteins A, C and E. The function of some of these
apoproteins is to direct HDL from the peripheral tissues to the
liver. Serum HDL levels can be affected by underlying genetic
causes (Weissglas-Volkov and Pajukanta, J Lipid Res, 2010,
51:2032-2057)
[0015] Epidemiological studies have indicated that increased levels
of HDL protect against cardiovascular disease or coronary heart
disease (Gordon et al., Am. J. Med. 1977. 62: 707-714). These
effects of HDL-cholesterol are independent of triglyceride and
LDL-cholesterol concentrations. In clinical practice, a low plasma
HDL-cholesterol is more commonly associated with other disorders
that increase plasma triglycerides, for example, central obesity,
insulin resistance, type 2 diabetes mellitus and renal disease
(chronic renal failure or nephrotic proteinuria) (Kashyap. Am. J.
Cardiol. 1998. 82: 42U-48U).
[0016] Currently, there are no known direct therapeutic agents that
affect the function of ApoCIII. The hypolipidemic effect of the
fibrate class of drugs has been postulated to occur via a mechanism
where peroxisome proliferator activated receptor (PPAR) mediates
the displacement of HNF-4 from the apolipoprotein C-III promoter,
resulting in transcriptional suppression of apolipoprotein C-III
(Hertz et al., J Biol. Chem., 1995, 270, 13470-13475). The statin
class of hypolipidemic drugs also lower triglyceride levels via an
unknown mechanism, which results in increases in lipoprotein lipase
mRNA and a decrease in plasma levels of apolipoprotein C-III
(Schoonjans et al., FEBS Lett., 1999, 452, 160-164). Consequently,
there remains a long felt need for additional agents capable of
effectively inhibiting apolipoprotein C-III function.
[0017] Antisense technology is emerging as an effective means for
reducing the expression of certain gene products and may therefore
prove to be uniquely useful in a number of therapeutic, diagnostic,
and research applications for the modulation of ApoCIII.
[0018] We have previously disclosed compositions and method for
inhibiting ApoCIII by antisense compounds in US 20040208856 (U.S.
Pat. No. 7,598,227), US 20060264395 (U.S. Pat. No. 7,750,141), and
WO 2004/093783. In the present application, we disclose the
unexpected result that antisense inhibition of ApoCIII resulted in
the elevation of HDL levels and decrease in postprandial
triglyceride levels. This result will be useful, for example, to
treat, prevent, delay, decrease or ameliorate any one or more
diseases, such as cardiovascular disease (e.g., coronary heart
disease or atherogenic diseases). For example, elevated
postprandial (non-fasting) triglyceride levels have been identified
as a significant risk factor for cardiovascular diseases (Bansal et
al., JAMA, 2007, 298:309-16; Nordestgaard et al., JAMA, 2007,
298:299-308), Also, in the present application, inhibition of
ApoCIII expression unexpectedly results in increased chylomicron
clearance and is therefore important in the prevention of
chylomicronemia (Chait et al., 1992, Adv Intern Med. 1992,
37:249-73), a dyslipidemic state caused by improper clearance of
chylomicron triglyceride. Severe forms of chylomicronemia can lead
to pancreatitis, a life-threatening condition. By inhibiting
intestinal ApoCIII, inhibition of lipoprotein lipase would be
reduced, and chylomicron triglyceride clearance would be enhanced,
thereby preventing pancreatitis.
SUMMARY OF THE INVENTION
[0019] Provided herein are methods of increasing HDL levels by
administering to an animal a compound targeting ApoCIII.
[0020] Certain embodiments provide a method of preventing,
treating, ameliorating, delaying the onset of or reducing the risk
of a cardiovascular disease, disorder or condition in an animal
comprising administering a compound targeting ApoCIII to the
animal. The compound administered to the animal prevents, treats,
ameliorates, delays the onset of, or reduces the risk of, the
cardiovascular disease, disorder or condition by increasing HDL
levels in the animal.
[0021] Certain embodiments provide a method of reducing the risk
for a cardiovascular disease in an animal comprising administering
to the animal a therapeutically effective amount of a compound
comprising a modified oligonucleotide consisting of 12 to 30 linked
nucleosides, wherein the modified oligonucleotide is complementary
to an ApoCIII nucleic acid. In certain embodiments, the ApoCIII
nucleic acid is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. In
certain embodiments, the compound comprises a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, and
having a nucleobase sequence comprising at least 8 contiguous
nucleobases of ISIS 304801 (SEQ ID NO: 3). In further embodiments,
the compound administered to the animal reduces the risk for a
cardiovascular disease, by increasing HDL levels.
[0022] Certain embodiments provide a method of preventing,
treating, ameliorating or reducing at least one symptom of a
cardiovascular disease in an animal, comprising administering to
the animal a therapeutically effective amount of a compound
comprising a modified oligonucleotide consisting of 12 to 30 linked
nucleosides, wherein the modified oligonucleotide is complementary
to an ApoCIII nucleic acid. In certain embodiments, the ApoCIII
nucleic acid is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. In
certain embodiments, the compound comprises a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, and
having a nucleobase sequence comprising at least 8 contiguous
nucleobases of ISIS 304801 (SEQ ID NO: 3). In further embodiments,
the compound administered to the animal prevents, treats,
ameliorates or reduces at least one symptom of the cardiovascular
disease in the animal, by increasing HDL levels in the animal.
[0023] Certain embodiments provide a method of raising HDL levels
in an animal by administering to the animal a compound consisting
of ISIS 304801 (SEQ ID NO: 3) to raise the HDL levels in the
animal.
[0024] Certain embodiments provide a method of preventing,
treating, ameliorating or reducing at least one symptom of a
cardiovascular disease in an animal by administering to the animal
a compound consisting of ISIS 304801 (SEQ ID NO: 3) to prevent,
treat, ameliorate or reduce at least one symptom of the
cardiovascular disease in the animal, by increasing HDL levels in
the animal.
[0025] Certain embodiments provide a method of raising HDL levels
in an animal by administering to the animal a modified
oligonucleotide, having the sequence of SEQ ID NO: 3 (ISIS 304801)
wherein the modified oligonucleotide comprises: a gap segment
consisting of 10 linked deoxynucleosides; a 5' wing segment
consisting of 5 linked nucleosides; a 3' wing segment consisting 5
linked nucleosides; wherein the gap segment is positioned
immediately adjacent to and between the 5' wing segment and the 3'
wing segment and wherein each nucleoside of each wing segment
comprises a 2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage, wherein the modified oligonucleotide
raises the HDL levels in the animal.
[0026] Certain embodiments provide a method of preventing,
treating, ameliorating or reducing at least one symptom of a
cardiovascular disease in an animal by administering to the animal
a modified oligonucleotide, having the sequence of ISIS 304801 (SEQ
ID NO: 3) wherein the modified oligonucleotide comprises: a gap
segment consisting of 10 linked deoxynucleosides; a 5' wing segment
consisting of 5 linked nucleosides; a 3' wing segment consisting 5
linked nucleosides; wherein the gap segment is positioned
immediately adjacent to and between the 5' wing segment and the 3'
wing segment and wherein each nucleoside of each wing segment
comprises a 2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage, wherein the modified oligonucleotide
prevents, treats, ameliorates or reduces at least one symptom in
the animal with the cardiovascular disease by raising the HDL
levels in the animal.
[0027] Certain embodiments provide a method of raising HDL levels
in an animal by administering to the animal a compound comprising a
modified oligonucleotide consisting of 12 to 30 linked nucleosides,
wherein the modified oligonucleotide is complementary to an ApoCIII
nucleic acid, as shown in SEQ ID NO: 1 or SEQ ID NO: 2, to raise
the HDL levels in the animal.
[0028] Certain embodiments provide a method of preventing,
treating, ameliorating or reducing at least one symptom of a
cardiovascular disease in an animal by administering to the animal
a compound comprising a modified oligonucleotide consisting of 12
to 30 linked nucleosides, wherein the modified oligonucleotide is
complementary to an ApoCIII nucleic acid, as shown in SEQ ID NO: 1
or SEQ ID NO: 2, to preventing, treating, ameliorating or reducing
at least one symptom of the cardiovascular disease in the animal,
by raising the HDL levels of the animal.
[0029] Certain embodiments provide a method of decreasing CETP
levels by administering a compound targeting ApoCIII to an animal.
In certain embodiments, the compound comprises a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, and
having a nucleobase sequence complementary to an ApoCIII nucleic
acid. In certain embodiments, the nucleobase sequence comprises at
least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In
certain embodiments, the compound consists of the nucleobases of
ISIS 304801 (SEQ ID NO: 3).
[0030] Certain embodiments provide a method of increasing ApoA1,
PON1, fat clearance, chylomicron triglyceride clearance, post
prandial triglyceride clearance or HDL by administering a compound
targeting ApoCIII to an animal. In certain embodiments, the
compound comprises a modified oligonucleotide consisting of 12 to
30 linked nucleosides, and having a nucleobase sequence
complementary to an ApoCIII nucleic acid. In certain embodiments,
the nucleobase sequence comprises at least 8 contiguous nucleobases
of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, the compound
consists of the nucleobases of ISIS 304801 (SEQ ID NO: 3).
[0031] Certain embodiments provide a method of preventing, delaying
or ameliorating pancreatitis comprising: (a) selecting an animal
with, or at risk of, pancreatitis, and (b) administering a compound
targeting ApoCIII to the animal, wherein the pancreatitis is
prevented, delayed or ameliorated.
[0032] Certain embodiments provide a method of preventing, delaying
or ameliorating pancreatitis comprising: (a) selecting an animal
with, or at risk of, pancreatitis, and (b) administering a compound
targeting ApoCIII to the animal, thereby increasing chylomicron
clearance, wherein the pancreatitis is prevented, delayed or
ameliorated.
[0033] In certain embodiments, the animal has, or is at risk for,
hypertriglyceridemia. In certain embodiments, the
hypertriglyceridemia is Fredrickson Type II, IV or V. In certain
embodiments, the animal has a genetic defect leading to
hypertriglyceridemia. In certain embodiments, the genetic defect is
a heterozygous LPL deficiency or an ApoCIII polymorphism. In
certain embodiments, the animal has a triglyceride level
.gtoreq.500 mg/dL and a heterozygous LPL deficiency.
[0034] In certain embodiments, the animal has a triglyceride level
between 100-200 mg/dL, 100-300 mg/dL, 100-400 mg/dL, 100-500 mg/dL,
200-500 mg/dL, 300-500 mg/dL, 400-500 mg/dL, 500-1000 mg/dL,
600-1000 mg/dL, 700-1000 mg/dL, 800-1000 mg/dL, 900-1000 mg/dL,
500-1500 mg/dL, 1000-1500 mg/dL, 100-2000 mg/dL, 150-2000 mg/dL,
200-2000 mg/dL, 300-2000 mg/dL, 400-2000 mg/dL, 500-2000 mg/dL,
600-2000 mg/dL, 700-2000 mg/dL, 800-2000 mg/dL, 900-2000 mg/dL,
1000-2000 mg/dL, 1100-2000 mg/dL, 1200-2000 mg/dL, 1300-2000 mg/dL,
1400-2000 mg/dL, or 1500-2000 mg/dL.
[0035] In certain embodiments, increased chylomicron clearance
enhances clearance of postprandial triglycerides and/or decreases
postprandial triglycerides.
[0036] Certain embodiments provide a use of a compound targeted to
ApoCIII for preventing, treating, ameliorating or reducing at least
one symptom of a cardiovascular disease, by increasing HDL
levels.
[0037] Certain embodiments provide a use of a compound targeted to
ApoCIII for increasing HDL levels in an animal.
[0038] Certain embodiments provide a use of a compound targeted to
ApoCIII for the preparation of a medicament for increasing HDL
levels in an animal.
[0039] Certain embodiments provide a use of a compound targeted to
ApoCIII for the preparation of a medicament for improving the ratio
of TG to HDL.
DETAILED DESCRIPTION OF THE INVENTION
[0040] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0041] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference for the portions of the document
discussed herein, as well as in their entirety.
Definitions
[0042] Unless specific definitions are provided, the nomenclature
utilized in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Where permitted, all
patents, applications, published applications and other
publications, GENBANK Accession Numbers and associated sequence
information obtainable through databases such as National Center
for Biotechnology Information (NCBI) and other data referred to
throughout in the disclosure herein are incorporated by reference
for the portions of the document discussed herein, as well as in
their entirety.
[0043] Unless otherwise indicated, the following terms have the
following meanings:
[0044] "2'-O-methoxyethyl" (also 2'-MOE,
2'-O(CH.sub.2).sub.2--OCH.sub.3 and 2'-O-(2-methoxyethyl)) refers
to an O-methoxy-ethyl modification of the 2' position of a furosyl
ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.
[0045] "2'-O-methoxyethyl nucleotide" means a nucleotide comprising
a 2'-O-methoxyethyl modified sugar moiety.
[0046] "3' target site" refers to the nucleotide of a target
nucleic acid which is complementary to the 3'-most nucleotide of a
particular antisense compound.
[0047] "5' target site" refers to the nucleotide of a target
nucleic acid which is complementary to the 5'-most nucleotide of a
particular antisense compound.
[0048] "5-methylcytosine" means a cytosine modified with a methyl
group attached to the 5' position. A 5-methylcytosine is a modified
nucleobase.
[0049] "About" means within .+-.10% of a value. For example, if it
is stated, "a marker may be increased by about 50%", it is implied
that the marker may be increased between 45%-55%.
[0050] "Active pharmaceutical agent" means the substance or
substances in a pharmaceutical composition that provide a
therapeutic benefit when administered to an individual. For
example, in certain embodiments an antisense oligonucleotide
targeted to ApoCIII is an active pharmaceutical agent.
[0051] "Active target region" or "target region" means a region to
which one or more active antisense compounds is targeted. "Active
antisense compounds" means antisense compounds that reduce target
nucleic acid levels or protein levels.
[0052] "Administered concomitantly" refers to the co-administration
of two agents in any manner in which the pharmacological effects of
both are manifest in the patient at the same time. Concomitant
administration does not require that both agents be administered in
a single pharmaceutical composition, in the same dosage form, or by
the same route of administration. The effects of both agents need
not manifest themselves at the same time. The effects need only be
overlapping for a period of time and need not be coextensive.
[0053] "Administering" means providing a pharmaceutical agent to an
individual, and includes, but is not limited to administering by a
medical professional and self-administering.
[0054] "Agent" means an active substance that can provide a
therapeutic benefit when administered to an animal. "First Agent"
means a therapeutic compound of the invention. For example, a first
agent can be an antisense oligonucleotide targeting ApoCIII.
"Second agent" means a second therapeutic compound of the invention
(e.g. a second antisense oligonucleotide targeting ApoCIII) and/or
a non-ApoCIII therapeutic compound.
[0055] "Amelioration" refers to a lessening of at least one
indicator, sign, or symptom of an associated disease, disorder, or
condition. The severity of indicators may be determined by
subjective or objective measures, which are known to those skilled
in the art.
[0056] "Animal" refers to a human or non-human animal, including,
but not limited to, mice, rats, rabbits, dogs, cats, pigs, and
non-human primates, including, but not limited to, monkeys and
chimpanzees.
[0057] "Antisense activity" means any detectable or measurable
activity attributable to the hybridization of an antisense compound
to its target nucleic acid. In certain embodiments, antisense
activity is a decrease in the amount or expression of a target
nucleic acid or protein encoded by such target nucleic acid.
[0058] "Antisense compound" means an oligomeric compound that is
capable of undergoing hybridization to a target nucleic acid
through hydrogen bonding. As used herein, the term "antisense
compound" encompasses pharmaceutically acceptable derivatives of
the compounds described herein.
[0059] "Antisense inhibition" means the reduction of target nucleic
acid levels or target protein levels in the presence of an
antisense compound complementary to a target nucleic acid compared
to target nucleic acid levels or target protein levels in the
absence of the antisense compound.
[0060] "Antisense oligonucleotide" means a single-stranded
oligonucleotide having a nucleobase sequence that permits
hybridization to a corresponding region or segment of a target
nucleic acid. As used herein, the term "antisense oligonucleotide"
encompasses pharmaceutically acceptable derivatives of the
compounds described herein.
[0061] "ApoCIII" means any nucleic acid or protein sequence
encoding ApoCIII. For example, in certain embodiments, an ApoCIII
includes a DNA sequence encoding ApoCIII, a RNA sequence
transcribed from DNA encoding ApoCIII (including genomic DNA
comprising introns and exons), a mRNA sequence encoding ApoCIII, or
a peptide sequence encoding ApoCIII.
[0062] "ApoCIII mRNA" means a mRNA encoding an ApoCIII protein.
[0063] "ApoCIII protein" means any protein sequence encoding
ApoCIII.
[0064] "Atherosclerosis" means a hardening of the arteries
affecting large and medium-sized arteries and is characterized by
the presence of fatty deposits. The fatty deposits are called
"atheromas" or "plaques," which consist mainly of cholesterol and
other fats, calcium and scar tissue, and damage the lining of
arteries.
[0065] "Bicyclic sugar" means a furosyl ring modified by the
bridging of two non-geminal ring atoms. A bicyclic sugar is a
modified sugar.
[0066] "Bicyclic nucleic acid" or "BNA" refers to a nucleoside or
nucleotide wherein the furanose portion of the nucleoside or
nucleotide includes a bridge connecting two carbon atoms on the
furanose ring, thereby forming a bicyclic ring system.
[0067] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
[0068] "Cardiovascular disease" or "cardiovascular disorder" refers
to a group of conditions related to the heart, blood vessels, or
the circulation. Examples of cardiovascular diseases include, but
are not limited to, aneurysm, angina, arrhythmia, atherosclerosis,
cerebrovascular disease (stroke), coronary heart disease,
hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia
and hypercholesterolemia.
[0069] "Chemically distinct region" refers to a region of an
antisense compound that is in some way chemically different than
another region of the same antisense compound. For example, a
region having 2'-O-methoxyethyl nucleotides is chemically distinct
from a region having nucleotides without 2'-O-methoxyethyl
modifications.
[0070] "Chimeric antisense compound" means an antisense compound
that has at least two chemically distinct regions.
[0071] "Cholesterol" is a sterol molecule found in the cell
membranes of all animal tissues. Cholesterol must be transported in
an animal's blood plasma by lipoproteins including very low density
lipoprotein (VLDL), intermediate density lipoprotein (IDL), low
density lipoprotein (LDL), and high density lipoprotein (HDL).
"Plasma cholesterol" refers to the sum of all lipoproteins (VDL,
IDL, LDL, HDL) esterified and/or non-estrified cholesterol present
in the plasma or serum.
[0072] "Cholesterol absorption inhibitor" means an agent that
inhibits the absorption of exogenous cholesterol obtained from
diet.
[0073] "Co-administration" means administration of two or more
agents to an individual. The two or more agents can be in a single
pharmaceutical composition, or can be in separate pharmaceutical
compositions. Each of the two or more agents can be administered
through the same or different routes of administration.
Co-administration encompasses parallel or sequential
administration.
[0074] "Complementarity" means the capacity for pairing between
nucleobases of a first nucleic acid and a second nucleic acid. In
certain embodiments, complementarity between the first and second
nucleic acid can be between two DNA strands, between two RNA
strands, or between a DNA and an RNA strand. In certain
embodiments, some of the nucleobases on one strand are matched to a
complementary hydrogen bonding base on the other strand. In certain
embodiments, all of the nucleobases on one strand are matched to a
complementary hydrogen bonding base on the other strand. In certain
embodiments, a first nucleic acid is an antisense compound and a
second nucleic acid is a target nucleic acid. In certain such
embodiments, an antisense oligonucleotide is a first nucleic acid
and a target nucleic acid is a second nucleic acid.
[0075] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other.
[0076] "Constrained ethyl" or "cEt" refers to a bicyclic nucleoside
having a furanosyl sugar that comprises a methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') bridge between the 4' and the 2' carbon
atoms.
[0077] "Cross-reactive" means an oligomeric compound targeting one
nucleic acid sequence can hybridize to a different nucleic acid
sequence. For example, in some instances an antisense
oligonucleotide targeting human ApoCIII can cross-react with a
murine ApoCIII. Whether an oligomeric compound cross-reacts with a
nucleic acid sequence other than its designated target depends on
the degree of complementarity the compound has with the non-target
nucleic acid sequence. The higher the complementarity between the
oligomeric compound and the non-target nucleic acid, the more
likely the oligomeric compound will cross-react with the nucleic
acid.
[0078] "Cure" means a method that restores health or a prescribed
treatment for an illness.
[0079] "Coronary heart disease (CHD)" means a narrowing of the
small blood vessels that supply blood and oxygen to the heart,
which is often a result of atherosclerosis.
[0080] "Deoxyribonucleotide" means a nucleotide having a hydrogen
at the 2' position of the sugar portion of the nucleotide.
Deoxyribonucleotides may be modified with any of a variety of
substituents.
[0081] "Diabetes mellitus" or "diabetes" is a syndrome
characterized by disordered metabolism and abnormally high blood
sugar (hyperglycemia) resulting from insufficient levels of insulin
or reduced insulin sensitivity. The characteristic symptoms are
excessive urine production (polyuria) due to high blood glucose
levels, excessive thirst and increased fluid intake (polydipsia)
attempting to compensate for increased urination, blurred vision
due to high blood glucose effects on the eye's optics, unexplained
weight loss, and lethargy.
[0082] "Diabetic dyslipidemia" or "type 2 diabetes with
dyslipidemia" means a condition characterized by Type 2 diabetes,
reduced HDL-C, elevated triglycerides, and elevated small, dense
LDL particles.
[0083] "Diluent" means an ingredient in a composition that lacks
pharmacological activity, but is pharmaceutically necessary or
desirable. For example, the diluent in an injected composition may
be a liquid, e.g. saline solution.
[0084] "Dyslipidemia" refers to a disorder of lipid and/or
lipoprotein metabolism, including lipid and/or lipoprotein
overproduction or deficiency. Dyslipidemias may be manifested by
elevation of lipids such as chylomicron, cholesterol and
triglycerides as well as lipoproteins such as low-density
lipoprotein (LDL) cholesterol. An example of a dyslipidemia is
chylomicronemia.
[0085] "Dosage unit" means a form in which a pharmaceutical agent
is provided, e.g. pill, tablet, or other dosage unit known in the
art. In certain embodiments, a dosage unit is a vial containing
lyophilized antisense oligonucleotide. In certain embodiments, a
dosage unit is a vial containing reconstituted antisense
oligonucleotide.
[0086] "Dose" means a specified quantity of a pharmaceutical agent
provided in a single administration, or in a specified time period.
In certain embodiments, a dose can be administered in one, two, or
more boluses, tablets, or injections. For example, in certain
embodiments where subcutaneous administration is desired, the
desired dose requires a volume not easily accommodated by a single
injection, therefore, two or more injections can be used to achieve
the desired dose. In certain embodiments, the pharmaceutical agent
is administered by infusion over an extended period of time or
continuously. Doses can be stated as the amount of pharmaceutical
agent per hour, day, week, or month. Doses can also be stated as
mg/kg or g/kg.
[0087] "Effective amount" or "therapeutically effective amount"
means the amount of active pharmaceutical agent sufficient to
effectuate a desired physiological outcome in an individual in need
of the agent. The effective amount can vary among individuals
depending on the health and physical condition of the individual to
be treated, the taxonomic group of the individuals to be treated,
the formulation of the composition, assessment of the individual's
medical condition, and other relevant factors.
[0088] The "Fredrickson" system is used to classify primary
(genetic) causes of dislipidemia into several subgroups or types.
Dislipidemia types that may be amenable to therapy with the
compounds disclosed herein include, but are not limited to,
Fredrickson Type II, IV and V.
[0089] "Fredrickson Type I" exists in several forms: Type 1a is a
lipoprotein lipase deficiency due to a deficiency of LPL or altered
apoC-II; Type Ib is a familial apoprotein CII deficiency, a
condition caused by a lack of lipoprotein lipase activator; and
Type Ic is a chylomicronemia due to circulating inhibitor of
lipoprotein lipase. Type I is a rare disorder that usually presents
in childhood. It is characterized by severe elevations in
chylomicrons and extremely elevated TG levels (always reaching well
above 1000 mg/dL and not infrequently rising as high as 10,000
mg/dL or more) with episodes of abdominal pain, recurrent acute
pancreatitis, eruptive cutaneous xanthomata, and
hepatosplenomegaly. Patients rarely develop atherosclerosis,
perhaps because their plasma lipoprotein particles are too large to
enter into the arterial intima (Nordestgaard et al., J Lipid Res,
1988, 29:1491-1500; Nordestgaard et al., Arteriosclerosis, 1988,
8:421-428). Type I is usually caused by mutations of either the LPL
gene, or of the gene's cofactor apoC-II, resulting in the inability
of affected individuals to produce functionally active LPL.
Patients are either homozygous for such mutations or compound
heterozygous. The prevalence is approximately 1 in 1,000,000 in the
general population and much higher in South Africa and Eastern
Quebec as a result of a founder effect. Patients respond minimally,
or not at all, to TG-lowering drugs (Tremblay et al., J Clin
Lipidol, 2011, 5:37-44; Brisson et al., Pharmacogenet Genom, 2010,
20:742-747) and hence restriction of dietary fat to 20 grams/day or
less is used to manage the symptoms of this rare disorder.
[0090] "Fredrickson Type II" is the most common form of primary
hyperlipidemia. It is further classified into Type IIa and Type
IIb, depending mainly on whether there is elevation in VLDL in
addition to LDL cholesterol (LDL-C). Type IIa (familial
hypercholesterolemia) may be sporadic (due to dietary factors),
polygenic, or truly familial as a result of a mutation in either
the LDL receptor gene on chromosome 19 (0.2% of the population) or
the apolipoprotein B (apoB) gene (0.2%). The familial form is
characterized by tendon xanthoma, xanthelasma and premature
cardiovascular disease. The incidence of this disease is about 1 in
500 for heterozygotes, and 1 in 1,000,000 for homozygotes. Type IIb
(also known as familial combined hyperlipoproteinemia) is a mixed
hyperlipidemia (high cholesterol and TG levels), caused by
elevations in LDL-C and in VLDL. The high VLDL levels are due to
overproduction of substrates, including TG, acetyl CoA, and an
increase in B-100 synthesis. They may also be caused by the
decreased clearance of LDL. Prevalence in the population is about
10%.
[0091] "Fredrickson Type III" (also known as
dysbetalipoproteinemia) is a remnant removal disease, or broad-beta
disease (Fern et al., J Clin Pathol, 2008, 61:1174-118). It is due
to cholesterol-rich VLDL (.beta.-VLDL). Typically, patients with
this condition have elevated plasma cholesterol and TG levels
because of impaired clearance of chylomicron and VLDL remnants
(e.g. IDL). The impaired clearance is due to a defect in
apolipoprotein E (apoE). Normally functioning apoE contained on the
remnants would enable binding to the LDL receptor and removal from
the circulation. Accumulation of the remnants in affected
individuals can result in xanthomatosis and premature coronary
and/or peripheral vascular disease. The most common cause for Type
III is the presence of apoE E2/E2 genotype. Its prevalence has been
estimated to be approximately 1 in 10,000.
[0092] "Fredrickson Type IV" (also known as familial
hypertriglyceridemia) is an autosomal dominant condition occurring
in approximately 1% of the population. TG levels are elevated as a
result of excess hepatic production of VLDL or heterozygous LPL
deficiency, but are almost always less than 1000 mg/dL. Serum
cholesterol levels are usually within normal limits. The disorder
is heterogeneous and the phenotype strongly influenced by
environmental factors, particularly carbohydrate and ethanol
consumption.
[0093] "Fredrickson Type V" has high VLDL and chylomicrons. It is
characterized by carriers of loss-of-function LPL gene variants
associated with LPL activity of at least 20% (i.e. partial LPL
deficiency as compared to Fredrickson Type I). These patients
present with lactescent plasma and severe hypertriglyceridemia
because of chylomicrons and VLDL. TG levels are invariably greater
than 1000 mg/dL and total cholesterol levels are always elevated.
The LDL-C level is usually low. It is also associated with
increased risk for acute pancreatitis, glucose intolerance and
hyperuricemia. Symptoms generally present in adulthood (>35
years) and, although the prevalence is relatively rare, it is much
more common than homozygous or compound heterozygous LPL deficient
patients.
[0094] "Fully complementary" or "100% complementary" means each
nucleobase of a nucleobase sequence of a first nucleic acid has a
complementary nucleobase in a second nucleobase sequence of a
second nucleic acid. In certain embodiments, a first nucleic acid
is an antisense compound and a second nucleic acid is a target
nucleic acid.
[0095] "Gapmer" means a chimeric antisense compound in which an
internal region having a plurality of nucleosides that support
RNase H cleavage is positioned between external regions having one
or more nucleosides, wherein the nucleosides comprising the
internal region are chemically distinct from the nucleoside or
nucleosides comprising the external regions. The internal region
may be referred to as a "gap" or "gap segment" and the external
regions may be referred to as "wings" or "wing segments."
[0096] "Gap-widened" means a chimeric antisense compound having a
gap segment of 12 or more contiguous 2'-deoxyribonucleosides
positioned between and immediately adjacent to 5' and 3' wing
segments having from one to six nucleosides.
[0097] "Glucose" is a monosaccharide used by cells as a source of
energy and inflammatory intermediate. "Plasma glucose" refers to
glucose present in the plasma.
[0098] "High density lipoprotein-C(HDL-C)" means cholesterol
associated with high density lipoprotein particles. Concentration
of HDL-C in serum (or plasma) is typically quantified in mg/dL or
nmol/L. "HDL-C" and "plasma HDL-C" mean HDL-C in serum and plasma,
respectively.
[0099] "HMG-CoA reductase inhibitor" means an agent that acts
through the inhibition of the enzyme HMG-CoA reductase, such as
atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin,
and simvastatin.
[0100] "Hybridization" means the annealing of complementary nucleic
acid molecules. In certain embodiments, complementary nucleic acid
molecules include an antisense compound and a target nucleic
acid.
[0101] "Hypercholesterolemia" means a condition characterized by
elevated cholesterol or circulating (plasma) cholesterol,
LDL-cholesterol and VLDL-cholesterol, as per the guidelines of the
Expert Panel Report of the National Cholesterol Educational Program
(NCEP) of Detection, Evaluation of Treatment of high cholesterol in
adults (see, Arch. Int. Med. (1988) 148, 36-39).
[0102] "Hyperlipidemia" or "hyperlipemia" is a condition
characterized by elevated serum lipids or circulating (plasma)
lipids. This condition manifests an abnormally high concentration
of fats. The lipid fractions in the circulating blood are
cholesterol, low density lipoproteins, very low density
lipoproteins, chylomicrons and triglycerides. The Fredrickson
classification of hyperlipidemias is based on the pattern of TG and
cholesterol-rich lipoprotein particles, as measured by
electrophoresis or ultracentrifugation and is commonly used to
characterize primary causes of hyperlipidemias such as
hypertriglyceridemia (Fredrickson and Lee, Circulation, 1965,
31:321-327; Fredrickson et al., New Eng J Med, 1967, 276 (1):
34-42).
[0103] "Hypertriglyceridemia" means a condition characterized by
elevated triglyceride levels. Its etiology includes primary (i.e.
genetic causes) and secondary (other underlying causes such as
diabetes, metabolic syndrome/insulin resistance, obesity, physical
inactivity, cigarette smoking, excess alcohol and a diet very high
in carbohydrates) factors or, most often, a combination of both
(Yuan et al. CMAJ, 2007, 176:1113-1120).
[0104] "Identifying" or "selecting an animal with metabolic or
cardiovascular disease" means identifying or selecting a subject
prone to or having been diagnosed with a metabolic disease, a
cardiovascular disease, or a metabolic syndrome; or, identifying or
selecting a subject having any symptom of a metabolic disease,
cardiovascular disease, or metabolic syndrome including, but not
limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia,
hypertriglyceridemia, hypertension increased insulin resistance,
decreased insulin sensitivity, above normal body weight, and/or
above normal body fat content or any combination thereof. Such
identification can be accomplished by any method, including but not
limited to, standard clinical tests or assessments, such as
measuring serum or circulating (plasma) cholesterol, measuring
serum or circulating (plasma) blood-glucose, measuring serum or
circulating (plasma) triglycerides, measuring blood-pressure,
measuring body fat content, measuring body weight, and the
like.
[0105] "Improved cardiovascular outcome" means a reduction in the
occurrence of adverse cardiovascular events, or the risk thereof.
Examples of adverse cardiovascular events include, without
limitation, death, reinfarction, stroke, cardiogenic shock,
pulmonary edema, cardiac arrest, and atrial dysrhythmia.
[0106] "Immediately adjacent" means there are no intervening
elements between the immediately adjacent elements, for example,
between regions, segments, nucleotides and/or nucleosides.
[0107] "Increasing HDL" or "raising HDL" means increasing the level
of HDL in an animal after administration of at least one compound
of the invention, compared to the HDL level in an animal not
administered any compound.
[0108] "Individual" or "subject" or "animal" means a human or
non-human animal selected for treatment or therapy.
[0109] "Induce", "inhibit", "potentiate", "elevate", "increase",
"decrease", "reduce" or the like denote quantitative differences
between two states. For example, "an amount effective to inhibit
the activity or expression of ApoCIII" means that the level of
activity or expression of ApoCIII in a treated sample will differ
from the level of ApoCIII activity or expression in an untreated
sample. Such terms are applied to, for example, levels of
expression, and levels of activity.
[0110] "Inhibiting the expression or activity" refers to a
reduction or blockade of the expression or activity of a RNA or
protein and does not necessarily indicate a total elimination of
expression or activity.
[0111] "Insulin resistance" is defined as the condition in which
normal amounts of insulin are inadequate to produce a normal
insulin response from fat, muscle and liver cells. Insulin
resistance in fat cells results in hydrolysis of stored
triglycerides, which elevates free fatty acids in the blood plasma.
Insulin resistance in muscle reduces glucose uptake whereas insulin
resistance in liver reduces glucose storage, with both effects
serving to elevate blood glucose. High plasma levels of insulin and
glucose due to insulin resistance often leads to metabolic syndrome
and type 2 diabetes.
[0112] "Insulin sensitivity" is a measure of how effectively an
individual processes glucose. An individual having high insulin
sensitivity effectively processes glucose whereas an individual
with low insulin sensitivity does not effectively process
glucose.
[0113] "Internucleoside linkage" refers to the chemical bond
between nucleosides.
[0114] "Intravenous administration" means administration into a
vein.
[0115] "Linked nucleosides" means adjacent nucleosides which are
bonded together.
[0116] "Lipid-lowering" means a reduction in one or more lipids in
a subject. "Lipid-raising" means an increase in a lipid (e.g., HDL)
in a subject. Lipid-lowering or lipid-raising can occur with one or
more doses over time.
[0117] "Lipid-lowering therapy" or "lipid lowering agent" means a
therapeutic regimen provided to a subject to reduce one or more
lipids in a subject. In certain embodiments, a lipid-lowering
therapy is provided to reduce one or more of CETP, ApoB, total
cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small
dense LDL particles, and Lp(a) in a subject. Examples of
lipid-lowering therapy include statins, fibrates, MTP
inhibitors.
[0118] "Lipoprotein", such as VLDL, LDL and HDL, refers to a group
of proteins found in the serum, plasma and lymph and are important
for lipid transport. The chemical composition of each lipoprotein
differs in that the HDL has a higher proportion of protein versus
lipid, whereas the VLDL has a lower proportion of protein versus
lipid.
[0119] "Low density lipoprotein-cholesterol (LDL-C)" means
cholesterol carried in low density lipoprotein particles.
Concentration of LDL-C in serum (or plasma) is typically quantified
in mg/dL or nmol/L. "Serum LDL-C" and "plasma LDL-C" mean LDL-C in
the serum and plasma, respectively.
[0120] "Major risk factors" refers to factors that contribute to a
high risk for a particular disease or condition. In certain
embodiments, major risk factors for coronary heart disease include,
without limitation, cigarette smoking, hypertension, low HDL-C,
family history of coronary heart disease, age, and other factors
disclosed herein.
[0121] "Metabolic disorder" or "metabolic disease" refers to a
condition characterized by an alteration or disturbance in
metabolic function. "Metabolic" and "metabolism" are terms well
known in the art and generally include the whole range of
biochemical processes that occur within a living organism.
Metabolic disorders include, but are not limited to, hyperglycemia,
prediabetes, diabetes (type 1 and type 2), obesity, insulin
resistance, metabolic syndrome and dyslipidemia due to type 2
diabetes.
[0122] "Metabolic syndrome" means a condition characterized by a
clustering of lipid and non-lipid cardiovascular risk factors of
metabolic origin. In certain embodiments, metabolic syndrome is
identified by the presence of any 3 of the following factors: waist
circumference of greater than 102 cm in men or greater than 88 cm
in women; serum triglyceride of at least 150 mg/dL; HDL-C less than
40 mg/dL in men or less than 50 mg/dL in women; blood pressure of
at least 130/85 mmHg; and fasting glucose of at least 110 mg/dL.
These determinants can be readily measured in clinical practice
(JAMA, 2001, 285: 2486-2497).
[0123] "Mismatch" or "non-complementary nucleobase" refers to the
case when a nucleobase of a first nucleic acid is not capable of
pairing with the corresponding nucleobase of a second or target
nucleic acid.
[0124] "Mixed dyslipidemia" means a condition characterized by
elevated cholesterol and elevated triglycerides.
[0125] "Modified internucleoside linkage" refers to a substitution
or any change from a naturally occurring internucleoside bond. For
example, a phosphorothioate linkage is a modified internucleoside
linkage.
[0126] "Modified nucleobase" refers to any nucleobase other than
adenine, cytosine, guanine, thymidine, or uracil. For example,
5-methylcytosine is a modified nucleobase. An "unmodified
nucleobase" means the purine bases adenine (A) and guanine (G), and
the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
[0127] "Modified nucleoside" means a nucleoside having at least one
modified sugar moiety, and/or modified nucleobase.
[0128] "Modified nucleotide" means a nucleotide having at least one
modified sugar moiety, modified internucleoside linkage and/or
modified nucleobase.
[0129] "Modified oligonucleotide" means an oligonucleotide
comprising at least one modified nucleotide.
[0130] "Modified sugar" refers to a substitution or change from a
natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a
modified sugar.
[0131] "Motif" means the pattern of chemically distinct regions in
an antisense compound.
[0132] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0133] "Natural sugar moiety" means a sugar found in DNA (2'-H) or
RNA (2'-OH).
[0134] "Nucleic acid" refers to molecules composed of monomeric
nucleotides. A nucleic acid includes ribonucleic acids (RNA),
deoxyribonucleic acids (DNA), single-stranded nucleic acids
(ssDNA), double-stranded nucleic acids (dsDNA), small interfering
ribonucleic acids (siRNA), and microRNAs (miRNA). A nucleic acid
may also comprise a combination of these elements in a single
molecule.
[0135] "Nucleobase" means a heterocyclic moiety capable of pairing
with a base of another nucleic acid.
[0136] "Nucleobase complementarity" refers to a nucleobase that is
capable of base pairing with another nucleobase. For example, in
DNA, adenine (A) is complementary to thymine (T). For example, in
RNA, adenine (A) is complementary to uracil (U). In certain
embodiments, complementary nucleobase refers to a nucleobase of an
antisense compound that is capable of base pairing with a
nucleobase of its target nucleic acid. For example, if a nucleobase
at a certain position of an antisense compound is capable of
hydrogen bonding with a nucleobase at a certain position of a
target nucleic acid, then the oligonucleotide and the target
nucleic acid are considered to be complementary at that nucleobase
pair.
[0137] "Nucleobase sequence" means the order of contiguous
nucleobases independent of any sugar, linkage, or nucleobase
modification.
[0138] "Nucleoside" means a nucleobase linked to a sugar.
[0139] "Nucleoside mimetic" includes those structures used to
replace the sugar or the sugar and the base, and not necessarily
the linkage at one or more positions of an oligomeric compound; for
example nucleoside mimetics having morpholino, cyclohexenyl,
cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics
such as non-furanose sugar units.
[0140] "Nucleotide" means a nucleoside having a phosphate group
covalently linked to the sugar portion of the nucleoside.
[0141] "Nucleotide mimetic" includes those structures used to
replace the nucleoside and the linkage at one or more positions of
an oligomeric compound such as for example peptide nucleic acids or
morpholinos (morpholinos linked by --N(H)--C(.dbd.O)--O-- or other
non-phosphodiester linkage).
[0142] "Oligomeric compound" or "oligomer" means a polymer of
linked monomeric subunits which is capable of hybridizing to a
region of a nucleic acid molecule. In certain embodiments,
oligomeric compounds are oligonucleosides. In certain embodiments,
oligomeric compounds are oligonucleotides. In certain embodiments,
oligomeric compounds are antisense compounds. In certain
embodiments, oligomeric compounds are antisense oligonucleotides.
In certain embodiments, oligomeric compounds are chimeric
oligonucleotides.
[0143] "Oligonucleotide" means a polymer of linked nucleosides each
of which can be modified or unmodified, independent from one
another.
[0144] "Parenteral administration" means administration through
injection or infusion. Parenteral administration includes
subcutaneous administration, intravenous administration,
intramuscular administration, intraarterial administration,
intraperitoneal administration, or intracranial administration,
e.g. intrathecal or intracerebroventricular administration.
Administration can be continuous, chronic, short or
intermittent.
[0145] "Peptide" means a molecule formed by linking at least two
amino acids by amide bonds. Peptide refers to polypeptides and
proteins.
[0146] "Pharmaceutical agent" means a substance that provides a
therapeutic benefit when administered to an individual. For
example, in certain embodiments, an antisense oligonucleotide
targeted to ApoCIII is pharmaceutical agent.
[0147] "Pharmaceutical composition" or "composition" means a
mixture of substances suitable for administering to an individual.
For example, a pharmaceutical composition may comprise one or more
active agents and a pharmaceutical carrier, such as a sterile
aqueous solution.
[0148] "Pharmaceutically acceptable carrier" means a medium or
diluent that does not interfere with the structure of the compound.
Certain of such carriers enable pharmaceutical compositions to be
formulated as, for example, tablets, pills, dragees, capsules,
liquids, gels, syrups, slurries, suspension and lozenges for the
oral ingestion by a subject. Certain of such carriers enable
pharmaceutical compositions to be formulated for injection,
infusion or topical administration. For example, a pharmaceutically
acceptable carrier can be a sterile aqueous solution.
[0149] "Pharmaceutically acceptable derivative" or "salts"
encompasses derivatives of the compounds described herein such as
solvates, hydrates, esters, prodrugs, polymorphs, isomers,
isotopically labelled variants, pharmaceutically acceptable salts
and other derivatives known in the art.
[0150] "Pharmaceutically acceptable salts" means physiologically
and pharmaceutically acceptable salts of antisense compounds, i.e.,
salts that retain the desired biological activity of the parent
compound and do not impart undesired toxicological effects thereto.
The term "pharmaceutically acceptable salt" or "salt" includes a
salt prepared from pharmaceutically acceptable non-toxic acids or
bases, including inorganic or organic acids and bases.
"Pharmaceutically acceptable salts" of the compounds described
herein may be prepared by methods well-known in the art. For a
review of pharmaceutically acceptable salts, see Stahl and Wermuth,
Handbook of Pharmaceutical Salts: Properties, Selection and Use
(Wiley-VCH, Weinheim, Germany, 2002). Sodium salts of antisense
oligonucleotides are useful and are well accepted for therapeutic
administration to humans. Accordingly, in one embodiment the
compounds described herein are in the form of a sodium salt.
[0151] "Phosphorothioate linkage" means a linkage between
nucleosides where the phosphodiester bond is modified by replacing
one of the non-bridging oxygen atoms with a sulfur atom. A
phosphorothioate linkage is a modified internucleoside linkage.
[0152] "Portion" means a defined number of contiguous (i.e. linked)
nucleobases of a nucleic acid. In certain embodiments, a portion is
a defined number of contiguous nucleobases of a target nucleic
acid. In certain embodiments, a portion is a defined number of
contiguous nucleobases of an antisense compound.
[0153] "Prevent" refers to delaying or forestalling the onset or
development of a disease, disorder, or condition for a period of
time from minutes to indefinitely. Prevent also means reducing risk
of developing a disease, disorder, or condition.
[0154] "Prodrug" means a therapeutic agent that is prepared in an
inactive form that is converted to an active form (i.e., a drug)
within the body or cells thereof by the action of endogenous
enzymes or other chemicals or conditions.
[0155] "Raise" means to increase in amount. For example, to raise
plasma HDL levels means to increase the amount of HDL in the
plasma.
[0156] "Ratio of TG to HDL" means the TG levels relative to HDL
levels. The occurrence of high TG and/or low HDL has been linked to
cardiovascular disease incidence, outcomes and mortality.
"Improving the ratio of TG to HDL" means to decrease TG and/or
raise HDL levels.
[0157] "Reduce" means to bring down to a smaller extent, size,
amount, or number. For example, to reduce plasma triglyceride
levels means to bring down the amount of triglyceride in the
plasma.
[0158] "Region" or "target region" is defined as a portion of the
target nucleic acid having at least one identifiable structure,
function, or characteristic. For example, a target region may
encompass a 3' UTR, a 5' UTR, an exon, an intron, an exon/intron
junction, a coding region, a translation initiation region,
translation termination region, or other defined nucleic acid
region. The structurally defined regions for ApoCIII can be
obtained by accession number from sequence databases such as NCBI
and such information is incorporated herein by reference. In
certain embodiments, a target region may encompass the sequence
from a 5' target site of one target segment within the target
region to a 3' target site of another target segment within the
target region.
[0159] "Ribonucleotide" means a nucleotide having a hydroxy at the
2' position of the sugar portion of the nucleotide. Ribonucleotides
can be modified with any of a variety of substituents.
[0160] "Second agent" or "second therapeutic agent" means an agent
that can be used in combination with a "first agent". A second
therapeutic agent can include, but is not limited to, an siRNA or
antisense oligonucleotide including antisense oligonucleotides
targeting ApoCIII. A second agent can also include anti-ApoCIII
antibodies, ApoCIII peptide inhibitors, cholesterol lowering
agents, lipid lowering agents, glucose lowering agents and
anti-inflammatory agents.
[0161] "Segments" are defined as smaller, sub-portions of regions
within a nucleic acid. For example, a "target segment" means the
sequence of nucleotides of a target nucleic acid to which one or
more antisense compounds is targeted. "5' target site" refers to
the 5'-most nucleotide of a target segment. "3' target site" refers
to the 3'-most nucleotide of a target segment.
[0162] "Shortened" or "truncated" versions of antisense
oligonucleotides or target nucleic acids taught herein have one,
two or more nucleosides deleted.
[0163] "Side effects" means physiological responses attributable to
a treatment other than the desired effects. In certain embodiments,
side effects include injection site reactions, liver function test
abnormalities, renal function abnormalities, liver toxicity, renal
toxicity, central nervous system abnormalities, myopathies, and
malaise. For example, increased aminotransferase levels in serum
may indicate liver toxicity or liver function abnormality. For
example, increased bilirubin may indicate liver toxicity or liver
function abnormality.
[0164] "Single-stranded oligonucleotide" means an oligonucleotide
which is not hybridized to a complementary strand.
[0165] "Specifically hybridizable" refers to an antisense compound
having a sufficient degree of complementarity to a target nucleic
acid to induce a desired effect, while exhibiting minimal or no
effects on non-target nucleic acids under conditions in which
specific binding is desired, i.e. under physiological conditions in
the case of in vivo assays and therapeutic treatments.
[0166] "Statin" means an agent that inhibits the activity of
HMG-CoA reductase.
[0167] "Subcutaneous administration" means administration just
below the skin.
[0168] "Subject" means a human or non-human animal selected for
treatment or therapy.
[0169] "Symptom of cardiovascular disease or disorder" means a
phenomenon that arises from and accompanies the cardiovascular
disease or disorder and serves as an indication of it. For example,
angina; chest pain; shortness of breath; palpitations; weakness;
dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia;
atrial fibrillation; swelling in the lower extremities; cyanosis;
fatigue; fainting; numbness of the face; numbness of the limbs;
claudication or cramping of muscles; bloating of the abdomen; or
fever are symptoms of cardiovascular disease or disorder.
[0170] "Targeting" or "targeted" means the process of design and
selection of an antisense compound that will specifically hybridize
to a target nucleic acid and induce a desired effect.
[0171] "Target nucleic acid," "target RNA," and "target RNA
transcript" all refer to a nucleic acid capable of being targeted
by antisense compounds.
[0172] "Therapeutic lifestyle change" means dietary and lifestyle
changes intended to lower fat/adipose tissue mass and/or
cholesterol. Such change can reduce the risk of developing heart
disease, and may includes recommendations for dietary intake of
total daily calories, total fat, saturated fat, polyunsaturated
fat, monounsaturated fat, carbohydrate, protein, cholesterol,
insoluble fiber, as well as recommendations for physical
activity.
[0173] "Treat" refers to administering a compound of the invention
to effect an alteration or improvement of a disease, disorder, or
condition.
[0174] "Triglyceride" or "TG" means a lipid or neutral fat
consisting of glycerol combined with three fatty acid
molecules.
[0175] "Type 2 diabetes," (also known as "type 2 diabetes
mellitus", "diabetes mellitus, type 2", "non-insulin-dependent
diabetes (NIDDM)", "obesity related diabetes", or "adult-onset
diabetes") is a metabolic disorder that is primarily characterized
by insulin resistance, relative insulin deficiency, and
hyperglycemia. "Unmodified nucleotide" means a nucleotide composed
of naturally occurring nucleobases, sugar moieties, and
internucleoside linkages. In certain embodiments, an unmodified
nucleotide is an RNA nucleotide (i.e. .beta.-D-ribonucleosides) or
a DNA nucleotide (i.e. .beta.-D-deoxyribonucleoside).
[0176] "Wing segment" means one or a plurality of nucleosides
modified to impart to an oligonucleotide properties such as
enhanced inhibitory activity, increased binding affinity for a
target nucleic acid, or resistance to degradation by in vivo
nucleases.
Certain Embodiments
[0177] Certain embodiments provide a method of reducing ApoCIII
levels in an animal by administering a compound to the animal
targeting ApoCIII, thereby decreasing ApoCIII levels. In certain
embodiments, ApoCIII levels are reduced in the liver or small
intestine.
[0178] Certain embodiments provide a method of increasing HDL
levels and/or improving the ratio of TG to HDL in an animal by
administering a compound to the animal wherein the compound targets
ApoCIII and increases HDL levels and/or improves the ratio of TG to
HDL.
[0179] Certain embodiments provide a method of preventing, delaying
or ameliorating a cardiovascular disease, disorder, condition, or
symptom thereof, in an animal comprising administering a compound
targeting ApoCIII to the animal, wherein the compound administered
to the animal prevents, treats or ameliorates the cardiovascular
disease, disorder, condition or symptom in the animal by increasing
HDL levels in the animal and/or improving the ratio of TG to
HDL.
[0180] Certain embodiments provide a method of preventing, delaying
or ameliorating a pancreatitis in an animal comprising
administering a compound targeting ApoCIII to the animal, wherein
the compound administered to the animal prevents, treats or
ameliorates the pancreatitis in the animal by increasing HDL levels
in the animal and/or improving the ratio of TG to HDL. In certain
embodiments, the pancreatitis is acute pancreatitis.
[0181] Certain embodiments provide a method of preventing,
treating, ameliorating, delaying the onset, or reducing the risk
of, a cardiovascular disease, disorder or condition in an animal,
comprising of administering to the animal a compound that targets
ApoCIII, wherein the compound prevents, treats, ameliorates, delays
the onset, or reduces of the risk of the cardiovascular disease,
disorder or condition in the animal by increasing HDL levels in the
animal and/or improving the ratio of TG to HDL.
[0182] Certain embodiments provide a method of decreasing CETP
levels by administering a compound targeting ApoCIII to an
animal.
[0183] Certain embodiments provide a method of increasing ApoA1,
PON1, fat clearance, chylomicron triglyceride clearance and/or HDL
by administering a compound targeting ApoCIII to an animal. Certain
embodiments provide a method for improving the ratio of TG to
HDL.
[0184] In certain embodiments, the ApoCIII nucleic acid is any of
the sequences set forth in GENBANK Accession No. NM_000040.1
(incorporated herein as SEQ ID NO: 1), and GENBANK Accession No.
NT_033899.8 truncated from nucleotides 20262640 to 20266603
(incorporated herein as SEQ ID NO: 2).
[0185] In certain embodiments, the compound targeting ApoCIII is a
modified oligonucleotide. In further embodiments, the modified
oligonucleotide has a nucleobase sequence comprising at least 8
contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain
embodiments, the modified oligonucleotide is at least 80%, at least
85%, at least 90%, at least 95%, at least 98% or at least 100%
complementary to SEQ ID NO: 1 or SEQ ID NO: 2.
[0186] In certain embodiments, the modified oligonucleotide
consists of a single-stranded modified oligonucleotide.
[0187] In certain embodiments, the modified oligonucleotide
consists of 12-30 linked nucleosides.
[0188] In certain embodiments, the modified oligonucleotide
consists of 20 linked nucleosides.
[0189] In certain embodiments, the compound comprises at least one
modified internucleoside linkage. In certain embodiments, the
internucleoside linkage is a phosphorothioate internucleoside
linkage.
[0190] In certain embodiments, the compound comprises at least one
nucleoside comprising a modified sugar. In certain embodiments, the
at least one modified sugar is a bicyclic sugar. In certain
embodiments, the at least one modified sugar comprises a
2'-O-methoxyethyl.
[0191] In certain embodiments, the compound comprises at least one
nucleoside comprising a modified nucleobase. In certain
embodiments, the modified nucleobase is a 5-methylcytosine.
[0192] In certain embodiments, the compound comprising modified
oligonucleotide comprises: (i) a gap segment consisting of linked
deoxynucleosides; (ii) a 5' wing segment consisting of linked
nucleosides; (iii) a 3' wing segment consisting of linked
nucleosides, wherein the gap segment is positioned immediately
adjacent to and between the 5' wing segment and the 3' wing segment
and wherein each nucleoside of each wing segment comprises a
modified sugar.
[0193] In certain embodiments, the compound comprising modified
oligonucleotide comprises: (i) a gap segment consisting of 8-12
linked deoxynucleosides; (ii) a 5' wing segment consisting of 1-5
linked nucleosides; (iii) a 3' wing segment consisting of 1-5
linked nucleosides, wherein the gap segment is positioned
immediately adjacent to and between the 5' wing segment and the 3'
wing segment, wherein each nucleoside of each wing segment
comprises a 2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage.
[0194] In certain embodiments, the compound comprising modified
oligonucleotide comprises: (i) a gap segment consisting of ten
linked deoxynucleosides; (ii) a 5' wing segment consisting of five
linked nucleosides; (iii) a 3' wing segment consisting of five
linked nucleosides, wherein the gap segment is positioned
immediately adjacent to and between the 5' wing segment and the 3'
wing segment, wherein each nucleoside of each wing segment
comprises a 2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage.
[0195] In certain embodiments, the modified oligonucleotide has a
nucleobase sequence comprising at least 8 contiguous nucleobases of
a nucleobase sequence of ISIS 304801 (SEQ ID NO: 3).
[0196] Certain embodiments provide a method of reducing the risk of
a cardiovascular disease in an animal by administering to the
animal a therapeutically effective amount of a compound comprising
a modified oligonucleotide consisting of 12 to 30 linked
nucleosides, wherein the modified oligonucleotide is complementary
to an ApoCIII nucleic acid and wherein the modified oligonucleotide
increases HDL levels and/or improves the ratio of TG to HDL. In
certain embodiments, the ApoCIII nucleic acid is either SEQ ID NO:
1 or SEQ ID NO: 2. In certain embodiments, the modified
oligonucleotide is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98% or at least 100% complementary to SEQ ID
NO: 1 or SEQ ID NO: 2. In further embodiments the modified
nucleotide comprises at least 8 contiguous nucleobases of the
nucleobase sequence of ISIS 304801 (SEQ ID NO: 3).
[0197] Certain embodiments provide a method of preventing,
treating, ameliorating, or reducing at least one symptom of a
cardiovascular disease in an animal, comprising administering to
the animal a therapeutically effective amount of a compound
comprising a modified oligonucleotide consisting of 12 to 30 linked
nucleosides and is complementary to an ApoCIII nucleic acid. In
certain embodiments, the ApoCIII nucleic acid is either SEQ ID NO:
1 or SEQ ID NO: 2. In certain embodiments, the modified
oligonucleotide is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98% or at least 100% complementary to SEQ ID
NO: 1 or SEQ ID NO: 2. In further embodiments, the compound
administered to the animal prevents, treats, ameliorates or reduces
at least one symptom of the cardiovascular disease by increasing
HDL levels and/or improving the ratio of TG to HDL. In further
embodiments, the modified oligonucleotide comprises at least 8
contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3).
[0198] In further embodiments, symptoms of a cardiovascular disease
include, but are not limited to, angina; chest pain; shortness of
breath; palpitations; weakness; dizziness; nausea; sweating;
tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling
in the lower extremities; cyanosis; fatigue; fainting; numbness of
the face; numbness of the limbs; claudication or cramping of
muscles; bloating of the abdomen; or fever.
[0199] Certain embodiments provide a method of raising HDL levels
and/or improving the ratio of TG to HDL in an animal by
administering to the animal a compound consisting of the nucleobase
sequence of ISIS 304801 (SEQ ID NO: 3). Further embodiments provide
a method of preventing, treating, ameliorating or reducing at least
one symptom of a cardiovascular disease in an animal by
administering to the animal a compound consisting of the nucleobase
sequence of ISIS 304801 (SEQ ID NO: 3), thereby increasing the HDL
levels and/or improving the ratio of TG to HDL in the animal.
[0200] Certain embodiments provide a method of raising HDL levels
and/or improving the ratio of TG to HDL in an animal by
administering to the animal a modified oligonucleotide having the
sequence of ISIS 304801, wherein the modified oligonucleotide
comprises: (i) a gap segment consisting of ten linked
deoxynucleosides; (ii) a 5' wing segment consisting of five linked
nucleosides; (iii) a 3' wing segment consisting of five linked
nucleosides, wherein the gap segment is positioned immediately
adjacent to and between the 5' wing segment and the 3' wing
segment, wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage.
[0201] Certain embodiments provide a method of preventing,
treating, ameliorating, or reducing at least one symptom of a
cardiovascular disease in an animal by administering to the animal
a modified oligonucleotide having the sequence of ISIS 304801 (SEQ
ID NO: 3), wherein the modified oligonucleotide of the compound
comprises: (i) a gap segment consisting of ten linked
deoxynucleosides; (ii) a 5' wing segment consisting of five linked
nucleosides; (iii) a 3' wing segment consisting of five linked
nucleosides, wherein the gap segment is positioned immediately
adjacent to and between the 5' wing segment and the 3' wing
segment, wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar, wherein each cytosine is a
5-methylcytosine, and wherein each internucleoside linkage is a
phosphorothioate linkage.
[0202] Certain embodiments provide a method of raising the HDL
levels and/or improving the ratio of TG to HDL in an animal by
administering to the animal a compound comprising a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, wherein
the modified oligonucleotide is complementary to an ApoCIII nucleic
acid. In certain embodiments, the ApoCIII nucleic acid is either
SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified
oligonucleotide is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98% or at least 100% complementary to SEQ ID
NO: 1 or SEQ ID NO: 2.
[0203] Certain embodiments provide a method of preventing,
treating, ameliorating or reducing at least one symptom of a
cardiovascular disease in an animal by administering to the animal
a compound comprising a modified oligonucleotide consisting of 12
to 30 linked nucleosides, wherein the modified oligonucleotide is
complementary to an ApoCIII nucleic acid, and raises the HDL levels
in the animal. In certain embodiments, the ApoCIII nucleic acid is
either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the
modified oligonucleotide is at least 80%, at least 85%, at least
90%, at least 95%, at least 98% or at least 100% complementary to
SEQ ID NO: 1 or SEQ ID NO: 2.
[0204] Certain embodiments provide a method of decreasing CETP
levels by administering a compound targeting ApoCIII to an animal.
In certain embodiments, the compound comprises a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, wherein
the modified oligonucleotide is complementary to an ApoCIII nucleic
acid. In certain embodiments, the ApoCIII nucleic acid is either
SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified
oligonucleotide is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98% or at least 100% complementary to SEQ ID
NO: 1 or SEQ ID NO: 2. In certain embodiments, the compound
comprises a modified oligonucleotide consisting of 12 to 30 linked
nucleosides and has a nucleobase sequence comprising at least 8
contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain
embodiments, the compound consists of the nucleobases of ISIS
304801 (SEQ ID NO: 3).
[0205] Certain embodiments provide a method of increasing ApoA1,
PON1, fat clearance, chylomicron triglyceride clearance and/or HDL
by administering a compound targeting ApoCIII to an animal. In
certain embodiments, the compound comprises a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, wherein
the modified oligonucleotide is complementary to an ApoCIII nucleic
acid. In certain embodiments, the ApoCIII nucleic acid is either
SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified
oligonucleotide is at least 80%, at least 85%, at least 90%, at
least 95%, at least 98% or at least 100% complementary to SEQ ID
NO: 1 or SEQ ID NO: 2. In certain embodiments, the compound
comprises a modified oligonucleotide consisting of 12 to 30 linked
nucleosides and has a nucleobase sequence comprising at least 8
contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain
embodiments, the compound consists of the nucleobases of ISIS
304801 (SEQ ID NO: 3).
[0206] In certain embodiments, the animal is human.
[0207] In certain embodiments, the cardiovascular disease is
aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular
disease, coronary heart disease, hypertension, dyslipidemia,
hyperlipidemia, hypertriglyceridemia or hypercholesterolemia. In
certain embodiments, the dyslipidemia is chylomicronemia.
[0208] In certain embodiments, the animal is at risk for
pancreatitis. In certain embodiments, reducing ApoCIII levels in
the liver and/or small intestine prevents pancreatitis. In certain
embodiments, raising HDL levels and/or improving the ratio of TG to
HDL prevents pancreatitis.
[0209] In certain embodiments, reducing ApoCIII levels in the liver
and/or small intestine enhance clearance of postprandial
triglyceride. In certain embodiments, raising HDL levels and/or
improving the ratio of TG to HDL enhance clearance of postprandial
triglyceride. In certain embodiments, reducing ApoCIII levels in
the liver and/or small intestine lowers postprandial triglyceride.
In certain embodiments, raising HDL levels and/or improving the
ratio of TG to HDL lowers postprandial triglyceride.
[0210] In certain embodiments, reducing ApoCIII levels in the liver
and/or small intestine improves the ratio of HDL to TG.
[0211] In certain embodiments, the compound is parenterally
administered. In further embodiments, the parenteral administration
is subcutaneous.
[0212] In certain embodiments, the compound is co-administered with
a second agent. In certain embodiments, the second agent is a
glucose-lowering agent. In certain embodiments, the second agent is
an LDL, TG or cholesterol lowering agent.
[0213] In certain embodiments, the compound and the second agent
are administered concomitantly or sequentially.
[0214] In certain embodiments, the compound is a salt form. In
further embodiments, the compound further comprises of a
pharmaceutically acceptable carrier or diluent.
[0215] Certain embodiments provide use of a compound targeted to
ApoCIII in the preparation of a medicament for decreasing ApoCIII
levels in an animal. Certain embodiments provide use of a compound
targeted to ApoCIII for decreasing ApoCIII levels in an animal. In
certain embodiments, ApoCIII levels are decreased in the liver or
small intestine. Certain embodiments provide use of a compound
targeted to ApoCIII in the preparation of a medicament for
preventing, treating, ameliorating or reducing at least one symptom
of a cardiovascular disease by increasing HDL levels and/or
improving the ratio of TG to HDL. Certain embodiments provide use
of a compound targeted to ApoCIII for preventing, treating,
ameliorating or reducing at least one symptom of a cardiovascular
disease by increasing HDL levels and/or improving the ratio of TG
to HDL. Certain embodiments provide a use of a compound targeted to
ApoCIII for increasing HDL levels and/or improving the ratio of TG
to HDL in an animal. Certain embodiments provide a use of a
compound targeted to ApoCIII for the preparation of a medicament
for increasing HDL levels and/or improving the ratio of TG to HDL
in an animal. In certain embodiments, the compound is at least 80%,
at least 85%, at least 90%, at least 95%, at least 98% or at least
100% complementary to an ApoCIII nucleic acid sequence. In certain
embodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ
ID NO: 2. In certain embodiments, the compound is a modified
oligonucleotide. In certain embodiments, the modified
oligonucleotide has a nucleobase sequence comprising at least 8
nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments,
the modified oligonucleotide has the nucleobase sequence of ISIS
304801 (SEQ ID NO: 3). Certain embodiments provide use of a
compound targeted to ApoCIII in the preparation of a medicament for
treating an animal with pancreatitis or at risk for pancreatitis.
Certain embodiments provide use of a compound targeted to ApoCIII
for treating an animal with pancreatitis or at risk for
pancreatitis. Certain embodiments provide use of a compound
targeted to ApoCIII in the preparation of a medicament for reducing
ApoCIII levels in the liver and/or small intestine. Certain
embodiments provide use of a compound targeted to ApoCIII for
reducing ApoCIII levels in the liver and/or small intestine.
Certain embodiments provide use of a compound targeted to ApoCIII
in the preparation of a medicament for preventing pancreatitis.
Certain embodiments provide use of a compound targeted to ApoCIII
for preventing pancreatitis.
[0216] Certain embodiments provide use of a compound targeted to
ApoCIII in the preparation of a medicament for reducing ApoCIII
levels in the liver and/or small intestine in an animal with
hypertriglyceridemia. Certain embodiments provide use of a compound
targeted to ApoCIII for reducing ApoCIII levels in the liver and/or
small intestine in an animal with hypertriglyceridemia. Certain
embodiments provide use of a compound targeted to ApoCIII in the
preparation of a medicament for enhancing clearance of postprandial
triglyceride. Certain embodiments provide use of a compound
targeted to ApoCIII for enhancing clearance of postprandial
triglyceride. Certain embodiments provide use of a compound
targeted to ApoCIII in the preparation of a medicament for lowering
postprandial triglyceride. Certain embodiments provide use of a
compound targeted to ApoCIII for lowering postprandial
triglyceride.
Antisense Compounds
[0217] Oligomeric compounds include, but are not limited to,
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics, antisense compounds, antisense
oligonucleotides, and siRNAs. An oligomeric compound may be
"antisense" to a target nucleic acid, meaning that is capable of
undergoing hybridization to a target nucleic acid through hydrogen
bonding.
[0218] In certain embodiments, an antisense compound has a
nucleobase sequence that, when written in the 5' to 3' direction,
comprises the reverse complement of the target segment of a target
nucleic acid to which it is targeted. In certain such embodiments,
an antisense oligonucleotide has a nucleobase sequence that, when
written in the 5' to 3' direction, comprises the reverse complement
of the target segment of a target nucleic acid to which it is
targeted.
[0219] In certain embodiments, an antisense compound targeted to an
ApoCIII nucleic acid is 12 to 30 nucleotides in length. In other
words, antisense compounds are from 12 to 30 linked nucleobases. In
other embodiments, the antisense compound comprises a modified
oligonucleotide consisting of 8 to 80, 10 to 80, 12 to 50, 15 to
30, 18 to 24, 19 to 22, or 20 linked nucleobases. In certain such
embodiments, the antisense compound comprises a modified
oligonucleotide consisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked
nucleobases in length, or a range defined by any two of the above
values. In some embodiments, the antisense compound is an antisense
oligonucleotide.
[0220] In certain embodiments, the antisense compound comprises a
shortened or truncated modified oligonucleotide. The shortened or
truncated modified oligonucleotide can have one or more nucleosides
deleted from the 5' end (5' truncation), one or more nucleosides
deleted from the 3' end (3' truncation) or one or more nucleosides
deleted from the central portion. Alternatively, the deleted
nucleosides may be dispersed throughout the modified
oligonucleotide, for example, in an antisense compound having one
nucleoside deleted from the 5' end and one nucleoside deleted from
the 3' end.
[0221] When a single additional nucleoside is present in a
lengthened oligonucleotide, the additional nucleoside may be
located at the central portion, 5' or 3' end of the
oligonucleotide. When two or more additional nucleosides are
present, the added nucleosides may be adjacent to each other, for
example, in an oligonucleotide having two nucleosides added to the
central portion, to the 5' end (5' addition), or alternatively to
the 3' end (3' addition), of the oligonucleotide. Alternatively,
the added nucleosides may be dispersed throughout the antisense
compound, for example, in an oligonucleotide having one nucleoside
added to the 5' end and one subunit added to the 3' end.
[0222] It is possible to increase or decrease the length of an
antisense compound, such as an antisense oligonucleotide, and/or
introduce mismatch bases without eliminating activity. For example,
in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a
series of antisense oligonucleotides 13-25 nucleobases in length
were tested for their ability to induce cleavage of a target RNA in
an oocyte injection model. Antisense oligonucleotides 25
nucleobases in length with 8 or 11 mismatch bases near the ends of
the antisense oligonucleotides were able to direct specific
cleavage of the target mRNA, albeit to a lesser extent than the
antisense oligonucleotides that contained no mismatches. Similarly,
target specific cleavage was achieved using 13 nucleobase antisense
oligonucleotides, including those with 1 or 3 mismatches.
[0223] Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March
2001) demonstrated the ability of an oligonucleotide having 100%
complementarity to the bcl-2 mRNA and having 3 mismatches to the
bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in
vitro and in vivo. Furthermore, this oligonucleotide demonstrated
potent anti-tumor activity in vivo.
[0224] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988)
tested a series of tandem 14 nucleobase antisense oligonucleotides,
and 28 and 42 nucleobase antisense oligonucleotides comprised of
the sequence of two or three of the tandem antisense
oligonucleotides, respectively, for their ability to arrest
translation of human DHFR in a rabbit reticulocyte assay. Each of
the three 14 nucleobase antisense oligonucleotides alone was able
to inhibit translation, albeit at a more modest level than the 28
or 42 nucleobase antisense oligonucleotides.
Antisense Compound Motifs
[0225] In certain embodiments, antisense compounds targeted to an
ApoCIII nucleic acid have chemically modified subunits arranged in
patterns, or motifs, to confer to the antisense compounds
properties such as enhanced inhibitory activity, increased binding
affinity for a target nucleic acid, or resistance to degradation by
in vivo nucleases.
[0226] Chimeric antisense compounds typically contain at least one
region modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, increased binding affinity
for the target nucleic acid, and/or increased inhibitory activity.
A second region of a chimeric antisense compound may optionally
serve as a substrate for the cellular endonuclease RNase H, which
cleaves the RNA strand of an RNA: DNA duplex.
[0227] Antisense compounds having a gapmer motif are considered
chimeric antisense compounds. In a gapmer an internal region having
a plurality of nucleotides that supports RNase H cleavage is
positioned between external regions having a plurality of
nucleotides that are chemically distinct from the nucleosides of
the internal region. In the case of an antisense oligonucleotide
having a gapmer motif, the gap segment generally serves as the
substrate for endonuclease cleavage, while the wing segments
comprise modified nucleosides. In certain embodiments, the regions
of a gapmer are differentiated by the types of sugar moieties
comprising each distinct region. The types of sugar moieties that
are used to differentiate the regions of a gapmer may in some
embodiments include .beta.-D-ribonucleosides,
.beta.-D-deoxyribonucleosides, 2'-modified nucleosides (such
2'-modified nucleosides may include 2'-MOE, and 2'-O--CH.sub.3,
among others), and bicyclic sugar modified nucleosides (such
bicyclic sugar modified nucleosides may include those having a
4'-(CH.sub.2)n-O-2' bridge, where n=1 or n=2). Preferably, each
distinct region comprises uniform sugar moieties. The wing-gap-wing
motif is frequently described as "X-Y-Z", where "X" represents the
length of the 5' wing region, "Y" represents the length of the gap
region, and "Z" represents the length of the 3' wing region. As
used herein, a gapmer described as "X-Y-Z" has a configuration such
that the gap segment is positioned immediately adjacent to each of
the 5' wing segment and the 3' wing segment. Thus, no intervening
nucleotides exist between the 5' wing segment and gap segment, or
the gap segment and the 3' wing segment. Any of the antisense
compounds described herein can have a gapmer motif. In some
embodiments, X and Z are the same; in other embodiments they are
different. In a preferred embodiment, Y is between 8 and 15
nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30 or more nucleotides. Thus, gapmers include, but are not
limited to, for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3,
2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 6-8-6,
5-8-5, 1-8-1, 2-6-2, 2-13-2, 1-8-2, 2-8-3, 3-10-2, 1-18-2 or
2-18-2.
[0228] In certain embodiments, the antisense compound as a
"wingmer" motif, having a wing-gap or gap-wing configuration, i.e.
an X-Y or Y-Z configuration as described above for the gapmer
configuration. Thus, wingmer configurations include, but are not
limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1,
10-3, 2-10, 1-10, 8-2, 2-13 or 5-13.
[0229] In certain embodiments, antisense compounds targeted to an
ApoCIII nucleic acid possess a 5-10-5 gapmer motif.
[0230] In certain embodiments, an antisense compound targeted to an
ApoCIII nucleic acid has a gap-widened motif.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
[0231] Nucleotide sequences that encode ApoCIII include, without
limitation, the following: GENBANK Accession No. NM_000040.1
(incorporated herein as SEQ ID NO: 1), and GENBANK Accession No.
NT_033899.8 truncated from nucleotides 20262640 to 20266603
(incorporated herein as SEQ ID NO: 2).
[0232] It is understood that the sequence set forth in each SEQ ID
NO in the Examples contained herein is independent of any
modification to a sugar moiety, an internucleoside linkage, or a
nucleobase. As such, antisense compounds defined by a SEQ ID NO may
comprise, independently, one or more modifications to a sugar
moiety, an internucleoside linkage, or a nucleobase. Antisense
compounds described by Isis Number (Isis No) indicate a combination
of nucleobase sequence and motif.
[0233] In certain embodiments, a target region is a structurally
defined region of the target nucleic acid. For example, a target
region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an
exon/intron junction, a coding region, a translation initiation
region, translation termination region, or other defined nucleic
acid region. The structurally defined regions for ApoCIII can be
obtained by accession number from sequence databases such as NCBI
and such information is incorporated herein by reference. In
certain embodiments, a target region may encompass the sequence
from a 5' target site of one target segment within the target
region to a 3' target site of another target segment within the
target region.
[0234] In certain embodiments, a "target segment" is a smaller,
sub-portion of a target region within a nucleic acid. For example,
a target segment can be the sequence of nucleotides of a target
nucleic acid to which one or more antisense compounds are targeted.
"5' target site" refers to the 5'-most nucleotide of a target
segment. "3' target site" refers to the 3'-most nucleotide of a
target segment.
[0235] A target region may contain one or more target segments.
Multiple target segments within a target region may be overlapping.
Alternatively, they may be non-overlapping. In certain embodiments,
target segments within a target region are separated by no more
than about 300 nucleotides. In certain embodiments, target segments
within a target region are separated by a number of nucleotides
that is, is about, is no more than, is no more than about, 250,
200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on
the target nucleic acid, or is a range defined by any two of the
preceding values. In certain embodiments, target segments within a
target region are separated by no more than, or no more than about,
5 nucleotides on the target nucleic acid. In certain embodiments,
target segments are contiguous. Contemplated are target regions
defined by a range having a starting nucleic acid that is any of
the 5' target sites or 3' target sites listed, herein.
[0236] Targeting includes determination of at least one target
segment to which an antisense compound hybridizes, such that a
desired effect occurs. In certain embodiments, the desired effect
is a reduction in mRNA target nucleic acid levels. In certain
embodiments, the desired effect is reduction of levels of protein
encoded by the target nucleic acid or a phenotypic change
associated with the target nucleic acid.
[0237] Suitable target segments may be found within a 5' UTR, a
coding region, a 3' UTR, an intron, an exon, or an exon/intron
junction. Target segments containing a start codon or a stop codon
are also suitable target segments. A suitable target segment may
specifically exclude a certain structurally defined region such as
the start codon or stop codon.
[0238] The determination of suitable target segments may include a
comparison of the sequence of a target nucleic acid to other
sequences throughout the genome. For example, the BLAST algorithm
may be used to identify regions of similarity amongst different
nucleic acids. This comparison can prevent the selection of
antisense compound sequences that may hybridize in a non-specific
manner to sequences other than a selected target nucleic acid
(i.e., non-target or off-target sequences).
[0239] There can be variation in activity (e.g., as defined by
percent reduction of target nucleic acid levels) of the antisense
compounds within an active target region. In certain embodiments,
reductions in ApoCIII mRNA levels are indicative of inhibition of
ApoCIII expression. Reductions in levels of an ApoCIII protein can
be indicative of inhibition of target mRNA expression. Further,
phenotypic changes can be indicative of inhibition of ApoCIII
expression. For example, an increase in HDL levels, decrease in LDL
levels, or decrease in triglyceride levels, are among phenotypic
changes that may be assayed for inhibition of ApoCIII expression.
Other phenotypic indications, e.g., symptoms associated with a
cardiovascular disease, may also be assessed; for example, angina;
chest pain; shortness of breath; palpitations; weakness; dizziness;
nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial
fibrillation; swelling in the lower extremities; cyanosis; fatigue;
fainting; numbness of the face; numbness of the limbs; claudication
or cramping of muscles; bloating of the abdomen; or fever.
Hybridization
[0240] In some embodiments, hybridization occurs between an
antisense compound disclosed herein and an ApoCIII nucleic acid.
The most common mechanism of hybridization involves hydrogen
bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding) between complementary nucleobases of the nucleic
acid molecules.
[0241] Hybridization can occur under varying conditions. Stringent
conditions are sequence-dependent and are determined by the nature
and composition of the nucleic acid molecules to be hybridized.
[0242] Methods of determining whether a sequence is specifically
hybridizable to a target nucleic acid are well known in the art
(Sambrooke and Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Ed., 2001). In certain embodiments, the antisense
compounds provided herein are specifically hybridizable with an
ApoCIII nucleic acid.
Complementarity
[0243] An antisense compound and a target nucleic acid are
complementary to each other when a sufficient number of nucleobases
of the antisense compound can hydrogen bond with the corresponding
nucleobases of the target nucleic acid, such that a desired effect
will occur (e.g., antisense inhibition of a target nucleic acid,
such as an ApoCIII nucleic acid).
[0244] An antisense compound may hybridize over one or more
segments of an ApoCIII nucleic acid such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure, mismatch or hairpin structure).
[0245] In certain embodiments, the antisense compounds provided
herein, or a specified portion thereof, are, or are at least, 70%,
75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% complementary to an ApoCIII nucleic
acid, a target region, target segment, or specified portion
thereof. Percent complementarity of an antisense compound with a
target nucleic acid can be determined using routine methods.
[0246] For example, an antisense compound in which 18 of 20
nucleobases of the antisense compound are complementary to a target
region, and would therefore specifically hybridize, would represent
90 percent complementarity. In this example, the remaining
non-complementary nucleobases may be clustered or interspersed with
complementary nucleobases and need not be contiguous to each other
or to complementary nucleobases. As such, an antisense compound
which is 18 nucleobases in length having 4 (four) non-complementary
nucleobases which are flanked by two regions of complete
complementarity with the target nucleic acid would have 77.8%
overall complementarity with the target nucleic acid and would thus
fall within the scope of the present invention. Percent
complementarity of an antisense compound with a region of a target
nucleic acid can be determined routinely using BLAST programs
(basic local alignment search tools) and PowerBLAST programs known
in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410;
Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology,
sequence identity or complementarity, can be determined by, for
example, the Gap program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, Madison Wis.), using default settings, which uses the
algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2,
482-489).
[0247] In certain embodiments, the antisense compounds provided
herein, or specified portions thereof, are fully complementary
(i.e. 100% complementary) to a target nucleic acid, or specified
portion thereof. For example, an antisense compound may be fully
complementary to an ApoCIII nucleic acid, or a target region, or a
target segment or target sequence thereof. As used herein, "fully
complementary" means each nucleobase of an antisense compound is
capable of precise base pairing with the corresponding nucleobases
of a target nucleic acid. For example, a 20 nucleobase antisense
compound is fully complementary to a target sequence that is 400
nucleobases long, so long as there is a corresponding 20 nucleobase
portion of the target nucleic acid that is fully complementary to
the antisense compound. Fully complementary can also be used in
reference to a specified portion of the first and/or the second
nucleic acid. For example, a 20 nucleobase portion of a 30
nucleobase antisense compound can be "fully complementary" to a
target sequence that is 400 nucleobases long. The 20 nucleobase
portion of the 30 nucleobase oligonucleotide is fully complementary
to the target sequence if the target sequence has a corresponding
20 nucleobase portion wherein each nucleobase is complementary to
the 20 nucleobase portion of the antisense compound. At the same
time, the entire 30 nucleobase antisense compound may or may not be
fully complementary to the target sequence, depending on whether
the remaining 10 nucleobases of the antisense compound are also
complementary to the target sequence.
[0248] The location of a non-complementary nucleobase(s) can be at
the 5' end or 3' end of the antisense compound. Alternatively, the
non-complementary nucleobase(s) can be at an internal position of
the antisense compound. When two or more non-complementary
nucleobases are present, they can be contiguous (i.e. linked) or
non-contiguous. In one embodiment, a non-complementary nucleobase
is located in the wing segment of a gapmer antisense
oligonucleotide.
[0249] In certain embodiments, antisense compounds that are, or are
up to, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length
comprise no more than 4, no more than 3, no more than 2, or no more
than 1 non-complementary nucleobase(s) relative to a target nucleic
acid, such as an ApoCIII nucleic acid, or specified portion
thereof.
[0250] In certain embodiments, antisense compounds that are, or are
up to, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, or 30 nucleobases in length comprise no more than 6, no
more than 5, no more than 4, no more than 3, no more than 2, or no
more than 1 non-complementary nucleobase(s) relative to a target
nucleic acid, such as an ApoCIII nucleic acid, or specified portion
thereof.
[0251] The antisense compounds provided herein also include those
which are complementary to a portion of a target nucleic acid. As
used herein, "portion" refers to a defined number of contiguous
(i.e. linked) nucleobases within a region or segment of a target
nucleic acid. A "portion" can also refer to a defined number of
contiguous nucleobases of an antisense compound. In certain
embodiments, the antisense compounds are complementary to at least
an 8 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 10 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 12 nucleobase portion of a target segment. In certain
embodiments, the antisense compounds are complementary to at least
a 15 nucleobase portion of a target segment. Also contemplated are
antisense compounds that are complementary to at least a 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a
target segment, or a range defined by any two of these values.
Identity
[0252] The antisense compounds provided herein may also have a
defined percent identity to a particular nucleotide sequence, SEQ
ID NO, or sequence of a compound represented by a specific Isis
number, or portion thereof. As used herein, an antisense compound
is identical to the sequence disclosed herein if it has the same
nucleobase pairing ability. For example, a RNA which contains
uracil in place of thymidine in a disclosed DNA sequence would be
considered identical to the DNA sequence since both uracil and
thymidine pair with adenine. Shortened and lengthened versions of
the antisense compounds described herein as well as compounds
having non-identical bases relative to the antisense compounds
provided herein also are contemplated. The non-identical bases may
be adjacent to each other or dispersed throughout the antisense
compound. Percent identity of an antisense compound is calculated
according to the number of bases that have identical base pairing
relative to the sequence to which it is being compared.
[0253] In certain embodiments, the antisense compounds, or portions
thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99% or 100% identical to one or more of the antisense compounds or
SEQ ID NOs, or a portion thereof, disclosed herein.
Modifications
[0254] A nucleoside is a base-sugar combination. The nucleobase
(also known as base) portion of the nucleoside is normally a
heterocyclic base moiety. Nucleotides are nucleosides that further
include a phosphate group covalently linked to the sugar portion of
the nucleoside. For those nucleosides that include a pentofuranosyl
sugar, the phosphate group can be linked to the 2', 3' or 5'
hydroxyl moiety of the sugar. Oligonucleotides are formed through
the covalent linkage of adjacent nucleosides to one another, to
form a linear polymeric oligonucleotide. Within the oligonucleotide
structure, the phosphate groups are commonly referred to as forming
the internucleoside linkages of the oligonucleotide.
[0255] Modifications to antisense compounds encompass substitutions
or changes to internucleoside linkages, sugar moieties, or
nucleobases. Modified antisense compounds are often preferred over
native forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced affinity for nucleic acid
target, increased stability in the presence of nucleases, or
increased inhibitory activity.
[0256] Chemically modified nucleosides can also be employed to
increase the binding affinity of a shortened or truncated antisense
oligonucleotide for its target nucleic acid. Consequently,
comparable results can often be obtained with shorter antisense
compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
[0257] The naturally occurring internucleoside linkage of RNA and
DNA is a 3' to 5' phosphodiester linkage. Antisense compounds
having one or more modified, i.e. non-naturally occurring,
internucleoside linkages are often selected over antisense
compounds having naturally occurring internucleoside linkages
because of desirable properties such as, for example, enhanced
cellular uptake, enhanced affinity for target nucleic acids, and
increased stability in the presence of nucleases.
[0258] Oligonucleotides having modified internucleoside linkages
include internucleoside linkages that retain a phosphorus atom as
well as internucleoside linkages that do not have a phosphorus
atom. Representative phosphorus containing internucleoside linkages
include, but are not limited to, phosphodiesters, phosphotriesters,
methylphosphonates, phosphoramidate, and phosphorothioates. Methods
of preparation of phosphorous-containing and
non-phosphorous-containing linkages are well known.
[0259] In certain embodiments, antisense compounds targeted to an
ApoCIII nucleic acid comprise one or more modified internucleoside
linkages. In certain embodiments, the modified internucleoside
linkages are phosphorothioate linkages. In certain embodiments,
each internucleoside linkage of an antisense compound is a
phosphorothioate internucleoside linkage.
Modified Sugar Moieties
[0260] Antisense compounds of the invention can optionally contain
one or more nucleosides wherein the sugar group has been modified.
Such sugar modified nucleosides may impart enhanced nuclease
stability, increased binding affinity, or some other beneficial
biological property to the antisense compounds. In certain
embodiments, nucleosides comprise chemically modified ribofuranose
ring moieties. Examples of chemically modified ribofuranose rings
include without limitation, addition of substitutent groups
(including 5' and 2' substituent groups, bridging of non-geminal
ring atoms to form bicyclic nucleic acids (BNA), replacement of the
ribosyl ring oxygen atom with S, N(R), or C(R.sub.1)(R.sub.2) (R,
R.sub.1 and R.sub.2 are each independently H, C.sub.1-C.sub.12
alkyl or a protecting group) and combinations thereof. Examples of
chemically modified sugars include 2'-F-5'-methyl substituted
nucleoside (see PCT International Application WO 2008/101157
Published on Aug. 21, 2008 for other disclosed 5',2'-bis
substituted nucleosides) or replacement of the ribosyl ring oxygen
atom with S with further substitution at the 2'-position (see
published U.S. Patent Application US2005-0130923, published on Jun.
16, 2005) or alternatively 5'-substitution of a BNA (see PCT
International Application WO 2007/134181 Published on Nov. 22, 2007
wherein LNA is substituted with for example a 5'-methyl or a
5'-vinyl group).
[0261] Examples of nucleosides having modified sugar moieties
include without limitation nucleosides comprising 5'-vinyl,
5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH.sub.3, 2'-OCH.sub.2CH.sub.3,
2'-OCH.sub.2CH.sub.2F and 2'-O(CH.sub.2).sub.2OCH.sub.3 substituent
groups. The substituent at the 2' position can also be selected
from allyl, amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl,
OCF.sub.3, OCH.sub.2F, O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n),
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), and
O--CH.sub.2--C(.dbd.O)--N(R.sub.1)--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n)-
, where each R.sub.1, R.sub.m and R.sub.n is, independently, H or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl.
[0262] As used herein, "bicyclic nucleosides" refer to modified
nucleosides comprising a bicyclic sugar moiety. Examples of
bicyclic nucleosides include without limitation nucleosides
comprising a bridge between the 4' and the 2' ribosyl ring atoms.
In certain embodiments, antisense compounds provided herein include
one or more bicyclic nucleosides comprising a 4' to 2' bridge.
Examples of such 4' to 2' bridged bicyclic nucleosides, include but
are not limited to one of the formulae: 4'-(CH.sub.2)--O-2' (LNA);
4'-(CH.sub.2)--S-2'; 4'-(CH.sub.2).sub.2--O-2' (ENA);
4'-CH(CH.sub.3)--O-2' and 4'-CH(CH.sub.2OCH.sub.3)--O-2' (and
analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15,
2008); 4'-C(CH.sub.3)(CH.sub.3)--O-2' (and analogs thereof see
published International Application WO/2009/006478, published Jan.
8, 2009); 4'-CH.sub.2--N(OCH.sub.3)-2' (and analogs thereof see
published International Application WO/2008/150729, published Dec.
11, 2008); 4'-CH.sub.2--O--N(CH.sub.3)-2' (see published U.S.
Patent Application US2004-0171570, published Sep. 2, 2004);
4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or
a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23,
2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see Chattopadhyaya et al.,
J. Org. Chem., 2009, 74, 118-134); and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2' (and analogs thereof see published
International Application WO 2008/154401, published on Dec. 8,
2008).
[0263] Further reports related to bicyclic nucleosides can also be
found in published literature (see for example: Singh et al., Chem.
Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,
3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000,
97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,
2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;
Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379;
Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561;
Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr.
Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490;
6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207;
7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No.
US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos. 60/989,574;
61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and
61/099,844; Published PCT International applications WO
1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO
2008/150729; WO 2008/154401; and WO 2009/006478. Each of the
foregoing bicyclic nucleosides can be prepared having one or more
stereochemical sugar configurations including for example
.alpha.-L-ribofuranose and .beta.-D-ribofuranose (see PCT
international application PCT/DK98/00393, published on Mar. 25,
1999 as WO 99/14226).
[0264] In certain embodiments, bicyclic sugar moieties of BNA
nucleosides include, but are not limited to, compounds having at
least one bridge between the 4' and the 2' position of the
pentofuranosyl sugar moiety wherein such bridges independently
comprises 1 or from 2 to 4 linked groups independently selected
from --[C(R.sub.a)(R.sub.b)].sub.n--,
--C(R.sub.a).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--, --C(.dbd.O)--,
--C(.dbd.NR.sub.a)--, --C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--,
--S(.dbd.O).sub.x--, and --N(R.sub.a)--;
[0265] wherein:
[0266] x is 0, 1, or 2;
[0267] n is 1, 2, 3, or 4;
[0268] each R.sub.a and R.sub.b is, independently, H, a protecting
group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted
C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle
radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7
alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical,
halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1,
acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
[0269] each J.sub.1 and J.sub.2 is, independently, H,
C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl,
C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl,
C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl
(C(.dbd.O)--H), substituted acyl, a heterocycle radical, a
substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl,
substituted C.sub.1-C.sub.12 aminoalkyl or a protecting group.
[0270] In certain embodiments, the bridge of a bicyclic sugar
moiety is --[C(R.sub.a)(R.sub.b)].sub.n--,
--[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O--
or --C(R.sub.aR.sub.b)--O--N(R)--. In certain embodiments, the
bridge is
4'-CH.sub.2-2',4'-(CH.sub.2).sub.2-2',4'-(CH.sub.2).sub.3-2',4'-CH.sub.2--
-O-2',4'-(CH.sub.2).sub.2--O-2',4'-CH.sub.2--O--N(R)-2' and
4'-CH.sub.2--N(R)--O-2'- wherein each R is, independently, H, a
protecting group or C.sub.1-C.sub.12 alkyl.
[0271] In certain embodiments, bicyclic nucleosides are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L
configuration or in the .beta.-D configuration. Previously,
.alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') BNA's have been
incorporated into antisense oligonucleotides that showed antisense
activity (Frieden et al., Nucleic Acids Research, 2003, 21,
6365-6372).
[0272] In certain embodiments, bicyclic nucleosides include, but
are not limited to, (A) .alpha.-L-methyleneoxy (4'-CH.sub.2--O-2')
BNA, (B) .beta.-D-methyleneoxy (4'-CH.sub.2--O-2') BNA, (C)
ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) aminooxy
(4'-CH.sub.2--O--N(R)-2') BNA, (E) oxyamino
(4'-CH.sub.2--N(R)--O-2') BNA, and (F) methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA, (G) methylene-thio (4'-CH.sub.2--S-2')
BNA, (H) methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl
carbocyclic (4'-CH.sub.2--CH(CH.sub.3)-2') BNA, and (J) propylene
carbocyclic (4'-(CH.sub.2).sub.3-2') BNA as depicted below.
##STR00001## ##STR00002##
wherein Bx is the base moiety and R is independently H, a
protecting group or C.sub.1-C.sub.12 alkyl.
[0273] In certain embodiments, bicyclic nucleosides are provided
having Formula I:
##STR00003##
wherein:
[0274] Bx is a heterocyclic base moiety;
[0275] -Q.sub.a-Q.sub.b-Q.sub.c- is
--CH.sub.2--N(R.sub.c)--CH.sub.2--,
--C(.dbd.O)--N(R.sub.c)--CH.sub.2--, --CH.sub.2--O--N(R.sub.c)--,
--CH.sub.2--N(R.sub.c)--O-- or --N(R.sub.c)--O--CH.sub.2;
[0276] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting
group; and
[0277] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium.
[0278] In certain embodiments, bicyclic nucleosides are provided
having Formula II:
##STR00004##
wherein:
[0279] Bx is a heterocyclic base moiety;
[0280] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0281] Z.sub.a is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl, acyl, substituted acyl, substituted amide, thiol or
substituted thio.
[0282] In one embodiment, each of the substituted groups is,
independently, mono or poly substituted with substituent groups
independently selected from halogen, oxo, hydroxyl, OJ.sub.c,
NJ.sub.cJ.sub.d, SJ.sub.c, N.sub.3, OC(.dbd.X)J.sub.c, and
NJ.sub.cC(.dbd.X)NJ.sub.cJ.sub.d, wherein each J.sub.c, J.sub.d and
J.sub.e is, independently, H, C.sub.1-C.sub.6 alkyl, or substituted
C.sub.1-C.sub.6 alkyl and X is O or NJ.sub.c.
[0283] In certain embodiments, bicyclic nucleosides are provided
having Formula III:
##STR00005##
wherein:
[0284] Bx is a heterocyclic base moiety;
[0285] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0286] Z.sub.b is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl,
substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6
alkynyl or substituted acyl (C(.dbd.O)--).
[0287] In certain embodiments, bicyclic nucleosides are provided
having Formula IV:
##STR00006##
wherein:
[0288] Bx is a heterocyclic base moiety;
[0289] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0290] R.sub.d is C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl;
[0291] each q.sub.a, q.sub.b, q.sub.c and q.sub.d is,
independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, substituted
C.sub.1-C.sub.6 alkoxyl, acyl, substituted acyl, C.sub.1-C.sub.6
aminoalkyl or substituted C.sub.1-C.sub.6 aminoalkyl;
[0292] In certain embodiments, bicyclic nucleosides are provided
having Formula V:
##STR00007##
wherein:
[0293] Bx is a heterocyclic base moiety;
[0294] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0295] q.sub.a, q.sub.b, q.sub.e and q.sub.f are each,
independently, hydrogen, halogen, C.sub.1-C.sub.12 alkyl,
substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl,
substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl,
substituted C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxy,
substituted C.sub.1-C.sub.12 alkoxy, OJ.sub.j, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j,
C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j,
O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k,
N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;
[0296] or q.sub.e and q.sub.f together are
.dbd.C(q.sub.g)(q.sub.h);
[0297] q.sub.g and q.sub.h are each, independently, H, halogen,
C.sub.1-C.sub.12 alkyl or substituted C.sub.1-C.sub.12 alkyl.
[0298] The synthesis and preparation of the methyleneoxy
(4'-CH.sub.2--O-2') BNA monomers adenine, cytosine, guanine,
5-methyl-cytosine, thymine and uracil, along with their
oligomerization, and nucleic acid recognition properties have been
described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs
and preparation thereof are also described in WO 98/39352 and WO
99/14226.
[0299] Analogs of methyleneoxy (4'-CH.sub.2--O-2') BNA and
2'-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med.
Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside
analogs comprising oligodeoxyribonucleotide duplexes as substrates
for nucleic acid polymerases has also been described (Wengel et
al., WO 99/14226). Furthermore, synthesis of 2'-amino-BNA, a novel
comformationally restricted high-affinity oligonucleotide analog
has been described in the art (Singh et al., J. Org. Chem., 1998,
63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's
have been prepared and the thermal stability of their duplexes with
complementary RNA and DNA strands has been previously reported.
[0300] In certain embodiments, bicyclic nucleosides are provided
having Formula VI:
##STR00008##
wherein:
[0301] Bx is a heterocyclic base moiety;
[0302] T.sub.a and T.sub.b are each, independently H, a hydroxyl
protecting group, a conjugate group, a reactive phosphorus group, a
phosphorus moiety or a covalent attachment to a support medium;
[0303] each q.sub.i, q.sub.j, q.sub.k and q.sub.l is,
independently, H, halogen, C.sub.1-C.sub.12 alkyl, substituted
C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted
C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted
C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxyl, substituted
C.sub.1-C.sub.12 alkoxyl, OJ.sub.j, SJ.sub.j, SOJ.sub.j,
SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j,
C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j,
O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k,
N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;
and
[0304] q.sub.i and q.sub.j or q.sub.l and q.sub.k together are
.dbd.C(q.sub.g)(q.sub.h), wherein q.sub.g and q.sub.h are each,
independently, H, halogen, C.sub.1-C.sub.12 alkyl or substituted
C.sub.1-C.sub.12 alkyl.
[0305] One carbocyclic bicyclic nucleoside having a
4'-(CH.sub.2).sub.3-2' bridge and the alkenyl analog bridge
4'-CH.dbd.CH--CH.sub.2-2' have been described (Freier et al.,
Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,
J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation
of carbocyclic bicyclic nucleosides along with their
oligomerization and biochemical studies have also been described
(Srivastava et al., J. Am. Chem. Soc., 2007, 129(26),
8362-8379).
[0306] As used herein, "4'-2' bicyclic nucleoside" or "4' to 2'
bicyclic nucleoside" refers to a bicyclic nucleoside comprising a
furanose ring comprising a bridge connecting two carbon atoms of
the furanose ring connects the 2' carbon atom and the 4' carbon
atom of the sugar ring.
[0307] As used herein, "monocylic nucleosides" refer to nucleosides
comprising modified sugar moieties that are not bicyclic sugar
moieties. In certain embodiments, the sugar moiety, or sugar moiety
analogue, of a nucleoside may be modified or substituted at any
position.
[0308] As used herein, "2'-modified sugar" means a furanosyl sugar
modified at the 2' position. In certain embodiments, such
modifications include substituents selected from: a halide,
including, but not limited to substituted and unsubstituted alkoxy,
substituted and unsubstituted thioalkyl, substituted and
unsubstituted amino alkyl, substituted and unsubstituted alkyl,
substituted and unsubstituted allyl, and substituted and
unsubstituted alkynyl. In certain embodiments, 2' modifications are
selected from substituents including, but not limited to:
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nF,
O(CH.sub.2).sub.nONH.sub.2, OCH.sub.2C(.dbd.O)N(H)CH.sub.3, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m
are from 1 to about 10. Other 2'-substituent groups can also be
selected from: C.sub.1-C.sub.12 alkyl, substituted alkyl, alkenyl,
alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3,
OCN, Cl, Br, CN, F, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving
pharmacokinetic properties, or a group for improving the
pharmacodynamic properties of an antisense compound, and other
substituents having similar properties. In certain embodiments,
modified nucleosides comprise a 2'-MOE side chain (Baker et al., J.
Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have
been described as having improved binding affinity compared to
unmodified nucleosides and to other modified nucleosides, such as
2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having
the 2'-MOE substituent also have been shown to be antisense
inhibitors of gene expression with promising features for in vivo
use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al.,
Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans.,
1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,
1997, 16, 917-926).
[0309] As used herein, a "modified tetrahydropyran nucleoside" or
"modified THP nucleoside" means a nucleoside having a six-membered
tetrahydropyran "sugar" substituted in for the pentofuranosyl
residue in normal nucleosides (a sugar surrogate). Modified THP
nucleosides include, but are not limited to, what is referred to in
the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA),
manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002,
10, 841-854), fluoro HNA (F-HNA) or those compounds having Formula
VII:
##STR00009##
wherein independently for each of said at least one tetrahydropyran
nucleoside analog of Formula VII:
[0310] Bx is a heterocyclic base moiety;
[0311] T.sub.a and T.sub.b are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the antisense compound or one of T.sub.a and
T.sub.b is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to the antisense compound and the
other of T.sub.a and T.sub.b is H, a hydroxyl protecting group, a
linked conjugate group or a 5' or 3'-terminal group;
[0312] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and
q.sub.7 are each independently, H, C.sub.1-C.sub.6 alkyl,
substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or
substituted C.sub.2-C.sub.6 alkynyl; and each of R.sub.1 and
R.sub.2 is selected from hydrogen, hydroxyl, halogen, substituted
or unsubstituted alkoxy, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3,
OC(.dbd.X)J.sub.1, OC(.dbd.X)NJ.sub.1J.sub.2,
NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2 and CN, wherein X is O, S or
NJ.sub.1 and each J.sub.1, J.sub.2 and J.sub.3 is, independently, H
or C.sub.1-C.sub.6 alkyl.
[0313] In certain embodiments, the modified THP nucleosides of
Formula VII are provided wherein q.sub.1, q.sub.2, q.sub.3,
q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain
embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4,
q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP
nucleosides of Formula VII are provided wherein one of R.sub.1 and
R.sub.2 is fluoro. In certain embodiments, R.sub.1 is fluoro and
R.sub.2 is H; R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is
methoxyethoxy and R.sub.2 is H.
[0314] As used herein, "2'-modified" or "2'-substituted" refers to
a nucleoside comprising a sugar comprising a substituent at the 2'
position other than H or OH. 2'-modified nucleosides, include, but
are not limited to, bicyclic nucleosides wherein the bridge
connecting two carbon atoms of the sugar ring connects the 2'
carbon and another carbon of the sugar ring; and nucleosides with
non-bridging 2' substituents, such as allyl, amino, azido, thio,
O-allyl, O--C.sub.1-C.sub.10 alkyl, --OCF.sub.3,
O--(CH.sub.2).sub.2--O-CH.sub.3, 2'-O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl. 2'-modified nucleosides may further
comprise other modifications, for example at other positions of the
sugar and/or at the nucleobase.
[0315] As used herein, "2'-F" refers to a nucleoside comprising a
sugar comprising a fluoro group at the 2' position.
[0316] As used herein, "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl"
each refers to a nucleoside comprising a sugar comprising an
--OCH.sub.3 group at the 2' position of the sugar ring.
[0317] As used herein, "MOE" or "2'-MOE" or
"2'-OCH.sub.2CH.sub.2OCH.sub.3" or "2'-O-methoxyethyl" each refers
to a nucleoside comprising a sugar comprising a
--OCH.sub.2CH.sub.2OCH.sub.3 group at the 2' position of the sugar
ring.
[0318] As used herein, "oligonucleotide" refers to a compound
comprising a plurality of linked nucleosides. In certain
embodiments, one or more of the plurality of nucleosides is
modified. In certain embodiments, an oligonucleotide comprises one
or more ribonucleosides (RNA) and/or deoxyribonucleosides
(DNA).
[0319] Many other bicyclo and tricyclo sugar surrogate ring systems
are also known in the art that can be used to modify nucleosides
for incorporation into antisense compounds (see for example review
article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854).
[0320] Such ring systems can undergo various additional
substitutions to enhance activity.
[0321] Methods for the preparations of modified sugars are well
known to those skilled in the art.
[0322] In nucleotides having modified sugar moieties, the
nucleobase moieties (natural, modified or a combination thereof)
are maintained for hybridization with an appropriate nucleic acid
target.
[0323] In certain embodiments, antisense compounds comprise one or
more nucleosides having modified sugar moieties. In certain
embodiments, the modified sugar moiety is 2'-MOE. In certain
embodiments, the 2'-MOE modified nucleosides are arranged in a
gapmer motif. In certain embodiments, the modified sugar moiety is
a bicyclic nucleoside having a (4'-CH(CH.sub.3)--O-2') bridging
group. In certain embodiments, the (4'-CH(CH.sub.3)--O-2') modified
nucleosides are arranged throughout the wings of a gapmer motif. In
certain embodiments, the modified sugar moiety is a cEt. In certain
embodiments, the cEt modified nucleotides are arranged throughout
the wings of a gapmer motif.
Modified Nucleobases
[0324] Nucleobase (or base) modifications or substitutions are
structurally distinguishable from, yet functionally interchangeable
with, naturally occurring or synthetic unmodified nucleobases. Both
natural and modified nucleobases are capable of participating in
hydrogen bonding. Such nucleobase modifications may impart nuclease
stability, binding affinity or some other beneficial biological
property to antisense compounds. Modified nucleobases include
synthetic and natural nucleobases such as, for example,
5-methylcytosine (5-me-C). Certain nucleobase substitutions,
including 5-methylcytosine substitutions, are particularly useful
for increasing the binding affinity of an antisense compound for a
target nucleic acid. For example, 5-methylcytosine substitutions
have been shown to increase nucleic acid duplex stability by
0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B.,
eds., Antisense Research and Applications, CRC Press, Boca Raton,
1993, pp. 276-278).
[0325] Additional modified nucleobases include 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,
5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine and other
alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine.
[0326] Heterocyclic base moieties may include those in which the
purine or pyrimidine base is replaced with other heterocycles, for
example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and
2-pyridone. Nucleobases that are particularly useful for increasing
the binding affinity of antisense compounds include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted
purines, including 2 aminopropyladenine, 5-propynyluracil and
5-propynylcytosine.
[0327] In certain embodiments, antisense compounds targeted to an
ApoCIII nucleic acid comprise one or more modified nucleobases. In
certain embodiments, gap-widened antisense oligonucleotides
targeted to an ApoCIII nucleic acid comprise one or more modified
nucleobases. In certain embodiments, the modified nucleobase is
5-methylcytosine. In certain embodiments, each cytosine is a
5-methylcytosine.
Compositions and Methods for Formulating Pharmaceutical
Compositions
[0328] Antisense oligonucleotides may be admixed with
pharmaceutically acceptable active or inert substance for the
preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical
compositions are dependent upon a number of criteria, including,
but not limited to, route of administration, extent of disease, or
dose to be administered.
[0329] Antisense compound targeted to an ApoCIII nucleic acid can
be utilized in pharmaceutical compositions by combining the
antisense compound with a suitable pharmaceutically acceptable
diluent or carrier.
[0330] In certain embodiments, the "pharmaceutical carrier" or
"excipient" is a pharmaceutically acceptable solvent, suspending
agent or any other pharmacologically inert vehicle for delivering
one or more nucleic acids to an animal. The excipient can be liquid
or solid and can be selected, with the planned manner of
administration in mind, so as to provide for the desired bulk,
consistency, etc., when combined with a nucleic acid and the other
components of a given pharmaceutical composition. Typical
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrants
(e.g., starch, sodium starch glycolate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc.).
[0331] Pharmaceutically acceptable organic or inorganic excipients,
which do not deleteriously react with nucleic acids, suitable for
parenteral or non-parenteral administration can also be used to
formulate the compositions of the present invention. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the
like.
[0332] A pharmaceutically acceptable diluent includes
phosphate-buffered saline (PBS). PBS is a diluent suitable for use
in compositions to be delivered parenterally. Accordingly, in one
embodiment, employed in the methods described herein is a
pharmaceutical composition comprising an antisense compound
targeted to an ApoCIII nucleic acid and a pharmaceutically
acceptable diluent. In certain embodiments, the pharmaceutically
acceptable diluent is PBS. In certain embodiments, the antisense
compound is an antisense oligonucleotide.
[0333] Pharmaceutical compositions comprising antisense compounds
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters, or an oligonucleotide which, upon administration to
an animal, including a human, is capable of providing (directly or
indirectly) the biologically active metabolite or residue thereof.
Accordingly, for example, the disclosure is also drawn to
pharmaceutically acceptable salts of antisense compounds, prodrugs,
pharmaceutically acceptable salts of such prodrugs, and other
bioequivalents. Suitable pharmaceutically acceptable salts include,
but are not limited to, sodium and potassium salts.
[0334] A prodrug can include the incorporation of additional
nucleosides at one or both ends of an antisense compound which are
cleaved by endogenous nucleases within the body, to form the active
antisense compound.
Conjugated Antisense Compounds
[0335] Antisense compounds may be covalently linked to one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the resulting antisense
oligonucleotides. Typical conjugate groups include cholesterol
moieties and lipid moieties. Additional conjugate groups include
carbohydrates, phospholipids, biotin, phenazine, folate,
phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,
coumarins, and dyes.
[0336] Antisense compounds can also be modified to have one or more
stabilizing groups that are generally attached to one or both
termini of antisense compounds to enhance properties such as, for
example, nuclease stability. Included in stabilizing groups are cap
structures. These terminal modifications protect the antisense
compound from exonuclease degradation, and can help in delivery
and/or localization within a cell. The cap can be present at the
5'-terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be
present on both termini. Cap structures are well known in the art
and include, for example, inverted deoxy abasic caps. Further 3'
and 5'-stabilizing groups that can be used to cap one or both ends
of an antisense compound to impart nuclease stability include those
disclosed in WO 03/004602 published on Jan. 16, 2003.
Cell Culture and Antisense Compounds Treatment
[0337] The effects of antisense compounds on the level, activity or
expression of ApoCIII nucleic acids or proteins can be tested in
vitro in a variety of cell types. Cell types used for such analyses
are available from commercial vendors (e.g. American Type Culture
Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park,
NC; Clonetics Corporation, Walkersville, Md.) and cells are
cultured according to the vendor's instructions using commercially
available reagents (e.g. Invitrogen Life Technologies, Carlsbad,
Calif.). Illustrative cell types include, but are not limited to,
HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells,
primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2
cells.
In Vitro Testing of Antisense Oligonucleotides
[0338] Described herein are methods for treatment of cells with
antisense oligonucleotides, which can be modified appropriately for
treatment with other antisense compounds.
[0339] In general, cells are treated with antisense
oligonucleotides when the cells reach approximately 60-80%
confluence in culture.
[0340] One reagent commonly used to introduce antisense
oligonucleotides into cultured cells includes the cationic lipid
transfection reagent LIPOFECTIN.RTM. (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN.RTM.
in OPTI-MEM.RTM. 1 (Invitrogen, Carlsbad, Calif.) to achieve the
desired final concentration of antisense oligonucleotide and a
LIPOFECTIN.RTM. concentration that typically ranges 2 to 12 ug/mL
per 100 nM antisense oligonucleotide.
[0341] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes LIPOFECTAMINE 2000.RTM. (Invitrogen,
Carlsbad, Calif.). Antisense oligonucleotide is mixed with
LIPOFECTAMINE 2000.RTM. in OPTI-MEM.RTM. 1 reduced serum medium
(Invitrogen, Carlsbad, Calif.) to achieve the desired concentration
of antisense oligonucleotide and a LIPOFECTAMINE.RTM. concentration
that typically ranges 2 to 12 ug/mL per 100 nM antisense
oligonucleotide.
[0342] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes Cytofectin.RTM. (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotide is mixed with Cytofectin.RTM. in
OPTI-MEM.RTM. 1 reduced serum medium (Invitrogen, Carlsbad, Calif.)
to achieve the desired concentration of antisense oligonucleotide
and a Cytofectin.RTM. concentration that typically ranges 2 to 12
ug/mL per 100 nM antisense oligonucleotide.
[0343] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes Oligofectamine.TM. (Invitrogen Life
Technologies, Carlsbad, Calif.). Antisense oligonucleotide is mixed
with Oligofectamine.TM. in Opti-MEM.TM.-1 reduced serum medium
(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the
desired concentration of oligonucleotide with an Oligofectamine.TM.
to oligonucleotide ratio of approximately 0.2 to 0.8 .mu.L per 100
nM.
[0344] Another reagent used to introduce antisense oligonucleotides
into cultured cells includes FuGENE 6 (Roche Diagnostics Corp.,
Indianapolis, Ind.). Antisense oligomeric compound was mixed with
FuGENE 6 in 1 mL of serum-free RPMI to achieve the desired
concentration of oligonucleotide with a FuGENE 6 to oligomeric
compound ratio of 1 to 4 .mu.L of FuGENE 6 per 100 nM.
[0345] Another technique used to introduce antisense
oligonucleotides into cultured cells includes electroporation
(Sambrooke and Russell in Molecular Cloning. A Laboratory Manual.
Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 2001).
[0346] Cells are treated with antisense oligonucleotides by routine
methods. Cells are typically harvested 16-24 hours after antisense
oligonucleotide treatment, at which time RNA or protein levels of
target nucleic acids are measured by methods known in the art and
described herein (Sambrooke and Russell in Molecular Cloning. A
Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. 2001). In general, when treatments
are performed in multiple replicates, the data are presented as the
average of the replicate treatments.
[0347] The concentration of antisense oligonucleotide used varies
from cell line to cell line. Methods to determine the optimal
antisense oligonucleotide concentration for a particular cell line
are well known in the art (Sambrooke and Russell in Molecular
Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. 2001). Antisense
oligonucleotides are typically used at concentrations ranging from
1 nM to 300 nM when transfected with LIPOFECTAMINE2000.RTM.
(Invitrogen, Carlsbad, Calif.), Lipofectin.RTM. (Invitrogen,
Carlsbad, Calif.) or Cytofectin.TM. (Genlantis, San Diego, Calif.).
Antisense oligonucleotides are used at higher concentrations
ranging from 625 to 20,000 nM when transfected using
electroporation.
RNA Isolation
[0348] RNA analysis can be performed on total cellular RNA or
poly(A)+mRNA. Methods of RNA isolation are well known in the art
(Sambrooke and Russell in Molecular Cloning. A Laboratory Manual.
Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 2001). RNA is prepared using methods well known in the
art, for example, using the TRIZOL.RTM. Reagent (Invitrogen,
Carlsbad, Calif.) according to the manufacturer's recommended
protocols.
Analysis of Inhibition of Target Levels or Expression
[0349] Inhibition of levels or expression of an ApoCIII nucleic
acid can be assayed in a variety of ways known in the art
(Sambrooke and Russell in Molecular Cloning. A Laboratory Manual.
Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 2001). For example, target nucleic acid levels can be
quantitated by, e.g., Northern blot analysis, competitive
polymerase chain reaction (PCR), or quantitative real-time PCR. RNA
analysis can be performed on total cellular RNA or poly(A)+mRNA.
Methods of RNA isolation are well known in the art. Northern blot
analysis is also routine in the art. Quantitative real-time PCR can
be conveniently accomplished using the commercially available ABI
PRISM.RTM. 7600, 7700, or 7900 Sequence Detection System, available
from PE-Applied Biosystems, Foster City, Calif. and used according
to manufacturer's instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
[0350] Quantitation of target RNA levels may be accomplished by
quantitative real-time PCR using the ABI PRISM.RTM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. Methods of
quantitative real-time PCR are well known in the art.
[0351] Prior to real-time PCR, the isolated RNA is subjected to a
reverse transcriptase (RT) reaction, which produces complementary
DNA (cDNA) that is then used as the substrate for the real-time PCR
amplification. The RT and real-time PCR reactions are performed
sequentially in the same sample well. RT and real-time PCR reagents
are obtained from Invitrogen (Carlsbad, Calif.). RT and
real-time-PCR reactions are carried out by methods well known to
those skilled in the art.
[0352] Gene (or RNA) target quantities obtained by real time PCR
can be normalized using either the expression level of a gene whose
expression is constant, such as cyclophilin A, or by quantifying
total RNA using RIBOGREEN.RTM. (Invitrogen, Inc. Carlsbad, Calif.).
Cyclophilin A expression is quantified by real time PCR, by being
run simultaneously with the target, multiplexing, or separately.
Total RNA is quantified using RIBOGREEN.RTM. RNA quantification
reagent (Invitrogen, Inc. Carlsbad, Calif.). Methods of RNA
quantification by RIBOGREEN.RTM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR.RTM.
4000 instrument (PE Applied Biosystems, Foster City, Calif.) is
used to measure RIBOGREEN.RTM. fluorescence.
[0353] Probes and primers are designed to hybridize to an ApoCIII
nucleic acid. Methods for designing real-time PCR probes and
primers are well known in the art, and may include the use of
software such as PRIMER EXPRESS.RTM. Software (Applied Biosystems,
Foster City, Calif.).
[0354] Gene target quantities obtained by RT, real-time PCR can use
either the expression level of GAPDH or Cyclophilin A, genes whose
expression are constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH or
Cyclophilin A expression can be quantified by RT, real-time PCR, by
being run simultaneously with the target, multiplexing, or
separately. Total RNA was quantified using RiboGreen.TM. RNA
quantification reagent (Molecular Probes, Inc. Eugene, Oreg.).
Analysis of Protein Levels
[0355] Antisense inhibition of ApoCIII nucleic acids can be
assessed by measuring ApoCIII protein levels. Protein levels of
ApoCIII can be evaluated or quantitated in a variety of ways well
known in the art, such as immunoprecipitation, Western blot
analysis (immunoblotting), enzyme-linked immunosorbant assay
(ELISA), quantitative protein assays, protein activity assays (for
example, caspase activity assays), immunohistochemistry,
immunocytochemistry or fluorescence-activated cell sorting (FACS)
(Sambrooke and Russell in Molecular Cloning. A Laboratory Manual.
Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 2001). Antibodies directed to a target can be
identified and obtained from a variety of sources, such as the MSRS
catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or
can be prepared via conventional monoclonal or polyclonal antibody
generation methods well known in the art. Antibodies useful for the
detection of human and mouse ApoCIII are commercially
available.
In Vivo Testing of Antisense Compounds
[0356] Antisense compounds, for example, antisense
oligonucleotides, are tested in animals to assess their ability to
inhibit expression of ApoCIII and produce phenotypic changes.
Testing can be performed in normal animals, or in experimental
disease models. For administration to animals, antisense
oligonucleotides are formulated in a pharmaceutically acceptable
diluent, such as phosphate-buffered saline. Administration includes
parenteral routes of administration. Calculation of antisense
oligonucleotide dosage and dosing frequency depends upon factors
such as route of administration and animal body weight. Following a
period of treatment with antisense oligonucleotides, RNA is
isolated from tissue and changes in ApoCIII nucleic acid expression
are measured. Changes in ApoCIII protein levels are also
measured.
Certain Indications
[0357] In certain embodiments, provided herein are methods of
treating an individual comprising administering one or more
pharmaceutical compositions as described herein. In certain
embodiments, the individual has a cardiovascular disease or a
metabolic disorder.
[0358] In certain embodiments, the cardiovascular disease is
aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular
disease, coronary heart disease, hypertension, dyslipidemia,
hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, stroke
and the like. In certain embodiments, the dyslipidemia is
chylomicronemia.
[0359] As shown in the examples below, compounds targeted to
ApoCIII as described herein have been shown to modulate
physiological markers or phenotypes of a cardiovascular disease. In
certain of the experiments, the compounds increased HDL levels, and
decreased LDL and triglyceride levels compared to untreated
animals. In certain embodiments, the increase in HDL levels and
decrease in LDL and triglyceride levels was associated with an
inhibition of ApoCIII by the compounds.
[0360] In certain embodiments, physiological markers of a
cardiovascular disease may be quantifiable. For example, HDL levels
may be measured and quantified by, for example, standard lipid
tests. For such markers, in certain embodiments, the marker may be
increased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of
these values.
[0361] Also, provided herein are methods for preventing, treating
or ameliorating a symptom associated with a cardiovascular disease
in a subject in need thereof. In certain embodiments, provided is a
method for reducing the rate of onset of a symptom associated with
a cardiovascular disease. In certain embodiments, provided is a
method for reducing the severity of a symptom associated with a
cardiovascular disease. In such embodiments, the methods comprise
administering to an individual in need thereof a therapeutically
effective amount of a compound targeted to an ApoCIII nucleic
acid.
[0362] Cardiovascular diseases are characterized by numerous
physical symptoms. Any symptom known to one of skill in the art to
be associated with a cardiovascular disease can be prevented,
treated, ameliorated or otherwise modulated as set forth above in
the methods described above. In certain embodiments, the symptom
may be any of, but not limited to, angina, chest pain, shortness of
breath, palpitations, weakness, dizziness, nausea, sweating,
tachycardia, bradycardia, arrhythmia, atrial fibrillation, swelling
in the lower extremities, cyanosis, fatigue, fainting, numbness of
the face, numbness of the limbs, claudication or cramping of
muscles, bloating of the abdomen or fever.
[0363] In certain embodiments, the symptom is angina. In certain
embodiments, the symptom is chest pain. In certain embodiments, the
symptom is shortness of breath. In certain embodiments, the symptom
is palpitations. In certain embodiments, the symptom is weakness.
In certain embodiments, the symptom is dizziness. In certain
embodiments, the symptom is nausea. In certain embodiments, the
symptom is sweating. In certain embodiments, the symptom is
tachycardia. In certain embodiments, the symptom is bradycardia. In
certain embodiments, the symptom is arrhythmia. In certain
embodiments, the symptom is atrial fibrillation. In certain
embodiments, the symptom is swelling in the lower extremities. In
certain embodiments, the symptom is cyanosis. In certain
embodiments, the symptom is fatigue. In certain embodiments, the
symptom is fainting. In certain embodiments, the symptom is
numbness of the face. In certain embodiments, the symptom is
numbness of the limbs. In certain embodiments, the symptom is
claudication or cramping of muscles. In certain embodiments, the
symptom is bloating of the abdomen. In certain embodiments, the
symptom is impaired short-term fever.
[0364] In certain embodiments, the metabolic disorders include, but
are not limited to, hyperglycemia, prediabetes, diabetes (type I
and type II), obesity, insulin resistance, metabolic syndrome and
diabetic dyslipidemia.
[0365] In certain embodiments, compounds targeted to ApoCIII as
described herein modulate physiological markers or phenotypes of a
metabolic disorder. In certain embodiments, physiological markers
of a metabolic disorder may be quantifiable. For example, glucose
levels or insulin resistance can be measured and quantified by
standard tests known in the art. For such markers, in certain
embodiments, the marker may be decreased by about 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%,
or a range defined by any two of these values. In another example,
insulin sensitivity or HDL levels can be measured and quantified by
standard tests known in the art. For such markers, in certain
embodiments, the marker may be increase by about 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a
range defined by any two of these values.
[0366] Also, provided herein are methods for preventing, treating
or ameliorating a symptom associated with a metabolic disorder in a
subject in need thereof. In certain embodiments, provided is a
method for reducing the rate of onset of a symptom associated with
a metabolic disorder. In certain embodiments, provided is a method
for reducing the severity of a symptom associated with a metabolic
disorder. In such embodiments, the methods comprise administering
to an individual in need thereof a therapeutically effective amount
of a compound targeted to an ApoCIII nucleic acid.
[0367] Metabolic disorders are characterized by numerous physical
symptoms. Any symptom known to one of skill in the art to be
associated with a metabolic disorder can be prevented, treated,
ameliorated or otherwise modulated as set forth above in the
methods described above. In certain embodiments, the symptom may be
any of, but not limited to, excessive urine production (polyuria),
excessive thirst and increased fluid intake (polydipsia), blurred
vision, unexplained weight loss and lethargy.
[0368] In certain embodiments, provided are methods of treating an
individual comprising administering a therapeutically effective
amount of one or more pharmaceutical compositions as described
herein. In certain embodiments, the individual has a cardiovascular
disease. In certain embodiments, administration of a
therapeutically effective amount of an antisense compound targeted
to an ApoCIII nucleic acid is accompanied by monitoring of ApoCIII
levels or markers of cardiovascular disease, diabetes or other
disease process associated with the expression of ApoCIII, to
determine an individual's response to administration of the
antisense compound. An individual's response to administration of
the antisense compound is used by a physician to determine the
amount and duration of therapeutic intervention.
[0369] In certain embodiments, administration of an antisense
compound targeted to an ApoCIII nucleic acid results in reduction
of ApoCIII expression by at least about 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined
by any two of these values. In certain embodiments, ApoCIII
expression is reduced to .ltoreq.50 mg/L, .ltoreq.60 mg/L,
.ltoreq.70 mg/L, .ltoreq.80 mg/L, .ltoreq.90 mg/L, .ltoreq.100
mg/L, .ltoreq.110 mg/L, .ltoreq.120 mg/L, .ltoreq.130 mg/L,
.ltoreq.140 mg/L, .ltoreq.150 mg/L, .ltoreq.160 mg/L, .ltoreq.170
mg/L, .ltoreq.180 mg/L, .ltoreq.190 mg/L or .ltoreq.200 mg/L.
[0370] In certain embodiments, administration of an antisense
compound targeted to an ApoCIII nucleic acid results in increase in
HDL levels by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any
two of these values.
[0371] In certain embodiments, administration of an antisense
compound targeted to an ApoCIII nucleic acid results in reduction
of TG (postprandial or fasting) levels by at least about 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 99%, or a range defined by any two of these values. In
certain embodiments, TG (postprandial or fasting) is reduced to
.ltoreq.100 mg/dL, .ltoreq.110 mg/dL, .ltoreq.120 mg/dL,
.ltoreq.130 mg/dL, .ltoreq.140 mg/dL, .ltoreq.150 mg/dL,
.ltoreq.160 mg/dL, .ltoreq.170 mg/dL, .ltoreq.180 mg/dL,
.ltoreq.190 mg/dL, .ltoreq.200 mg/dL, .ltoreq.210 mg/dL,
.ltoreq.220 mg/dL, .ltoreq.230 mg/dL, .ltoreq.240 mg/dL,
.ltoreq.250 mg/dL, .ltoreq.260 mg/dL, .ltoreq.270 mg/dL,
.ltoreq.280 mg/dL, .ltoreq.290 mg/dL, .ltoreq.300 mg/dL,
.ltoreq.350 mg/dL, .ltoreq.400 mg/dL, .ltoreq.450 mg/dL,
.ltoreq.500 mg/dL, .ltoreq.550 mg/dL, .ltoreq.600 mg/dL,
.ltoreq.650 mg/dL, .ltoreq.700 mg/dL, .ltoreq.750 mg/dL,
.ltoreq.800 mg/dL, .ltoreq.850 mg/dL, .ltoreq.900 mg/dL,
.ltoreq.950 mg/dL, .ltoreq.1000 mg/dL, .ltoreq.1100 mg/dL,
.ltoreq.1200 mg/dL, .ltoreq.1300 mg/dL, .ltoreq.1400 mg/dL,
.ltoreq.1500 mg/dL, .ltoreq.1600 mg/dL, .ltoreq.1700 mg/dL,
.ltoreq.1800 mg/dL or .ltoreq.1900 mg/dL.
[0372] In certain embodiments, pharmaceutical compositions
comprising an antisense compound targeted to ApoCIII are used for
the preparation of a medicament for treating a patient suffering or
susceptible to a cardiovascular disease.
Administration
[0373] The compounds or pharmaceutical compositions of the present
invention can be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration can be oral or parenteral.
[0374] In certain embodiments, the compounds and compositions as
described herein are administered parenterally. Parenteral
administration includes intravenous, intra-arterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion.
[0375] In certain embodiments, parenteral administration is by
infusion. Infusion can be chronic or continuous or short or
intermittent. In certain embodiments, infused pharmaceutical agents
are delivered with a pump. In certain embodiments, the infusion is
intravenous.
[0376] In certain embodiments, parenteral administration is by
injection. The injection can be delivered with a syringe or a pump.
In certain embodiments, the injection is a bolus injection. In
certain embodiments, the injection is administered directly to a
tissue or organ. In certain embodiments, parenteral administration
is subcutaneous.
[0377] In certain embodiments, formulations for parenteral
administration can include sterile aqueous solutions which can also
contain buffers, diluents and other suitable additives such as, but
not limited to, penetration enhancers, carrier compounds and other
pharmaceutically acceptable carriers or excipients.
[0378] In certain embodiments, formulations for oral administration
of the compounds or compositions of the invention can include, but
is not limited to, pharmaceutical carriers, excipients, powders or
granules, microparticulates, nanoparticulates, suspensions or
solutions in water or non-aqueous media, capsules, gel capsules,
sachets, tablets or minitablets. Thickeners, flavoring agents,
diluents, emulsifiers, dispersing aids or binders can be desirable.
In certain embodiments, oral formulations are those in which
compounds of the invention are administered in conjunction with one
or more penetration enhancers, surfactants and chelators.
Dosing
[0379] In certain embodiments, pharmaceutical compositions are
administered according to a dosing regimen (e.g., dose, dose
frequency, and duration) wherein the dosing regimen can be selected
to achieve a desired effect. The desired effect can be, for
example, reduction of ApoCIII or the prevention, reduction,
amelioration or slowing the progression of a disease or condition
associated with ApoCIII.
[0380] In certain embodiments, the variables of the dosing regimen
are adjusted to result in a desired concentration of pharmaceutical
composition in a subject. "Concentration of pharmaceutical
composition" as used with regard to dose regimen can refer to the
compound, oligonucleotide, or active ingredient of the
pharmaceutical composition. For example, in certain embodiments,
dose and dose frequency are adjusted to provide a tissue
concentration or plasma concentration of a pharmaceutical
composition at an amount sufficient to achieve a desired
effect.
[0381] Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting
from several days to several months, or until a cure is effected or
a diminution of the disease state is achieved. Dosing is also
dependent on drug potency and metabolism. In certain embodiments,
dosage is from 0.01 .mu.g to 100 mg per kg of body weight, or
within a range of 0.001 mg-1000 mg dosing, and may be given once or
more daily, weekly, monthly or yearly, or even once every 2 to 20
years. Following successful treatment, it may be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the oligonucleotide is administered
in maintenance doses, ranging from 0.01 .mu.g to 100 mg per kg of
body weight, once or more daily, to once every 20 years or ranging
from 0.001 mg to 1000 mg dosing.
Certain Combination Therapies
[0382] In certain embodiments, a first agent comprising the
compound described herein is co-administered with one or more
secondary agents. In certain embodiments, such second agents are
designed to treat the same disease, disorder, or condition as the
first agent described herein. In certain embodiments, such second
agents are designed to treat a different disease, disorder, or
condition as the first agent described herein. In certain
embodiments, a first agent is designed to treat an undesired side
effect of a second agent. In certain embodiments, second agents are
co-administered with the first agent to treat an undesired effect
of the first agent. In certain embodiments, such second agents are
designed to treat an undesired side effect of one or more
pharmaceutical compositions as described herein. In certain
embodiments, second agents are co-administered with the first agent
to produce a combinational effect. In certain embodiments, second
agents are co-administered with the first agent to produce a
synergistic effect. In certain embodiments, the co-administration
of the first and second agents permits use of lower dosages than
would be required to achieve a therapeutic or prophylactic effect
if the agents were administered as independent therapy.
[0383] In certain embodiments, one or more compositions described
herein and one or more other pharmaceutical agents are administered
at the same time. In certain embodiments, one or more compositions
of the invention and one or more other pharmaceutical agents are
administered at different times. In certain embodiments, one or
more compositions described herein and one or more other
pharmaceutical agents are prepared together in a single
formulation. In certain embodiments, one or more compositions
described herein and one or more other pharmaceutical agents are
prepared separately.
[0384] In certain embodiments, second agents include, but are not
limited to, ApoCIII lowering agent, cholesterol lowering agent,
non-HDL lipid lowering (e.g., LDL) agent, HDL raising agent, fish
oil, niacin, fibrate, statin, DCCR (salt of diazoxide),
glucose-lowering agent and/or anti-diabetic agents. In certain
embodiments, the first agent is administered in combination with
the maximally tolerated dose of the second agent. In certain
embodiments, the first agent is administered to a subject that
fails to respond to a maximally tolerated dose of the second
agent.
[0385] Examples of ApoCIII lowering agents include an ApoCIII
antisense oligonucleotide different from the first agent, niacin or
an Apo B antisense oligonucleotide.
[0386] Examples of glucose-lowering and/or anti-diabetic agents
include, but is not limited to, a therapeutic lifestyle change,
PPAR agonist, a dipeptidyl peptidase (IV) inhibitor, a GLP-1
analog, insulin or an insulin analog, an insulin secretagogue, a
SGLT2 inhibitor, a human amylin analog, a biguanide, an
alpha-glucosidase inhibitor, metformin, sulfonylurea,
rosiglitazone, meglitinide, thiazolidinedione, alpha-glucosidase
inhibitor and the like. The sulfonylurea can be acetohexamide,
chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide,
a glyburide, or a gliclazide. The meglitinide can be nateglinide or
repaglinide. The thiazolidinedione can be pioglitazone or
rosiglitazone. The alpha-glucosidase can be acarbose or
miglitol.
[0387] The cholesterol or lipid lowering therapy can include, but
is not limited to, a therapeutic lifestyle change, statins, bile
acids sequestrants, nicotinic acid and fibrates. The statins can be
atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin
and simvastatin and the like. The bile acid sequestrants can be
colesevelam, cholestyramine, colestipol and the like. The fibrates
can be gemfibrozil, fenofibrate, clofibrate and the like.
[0388] HDL increasing agents include cholesteryl ester transfer
protein (CETP) inhibiting drugs (such as Torcetrapib), peroxisome
proliferation activated receptor agonists, Apo-A1, Pioglitazone and
the like.
Certain Treatment Populations
[0389] Certain subjects with high TG levels are at a significant
risk of cardiovascular and metabolic disease. In many subjects with
high TG (e.g., hypertriglyceridemia), current treatments cannot
reduce their TG levels to safe levels. ApoCIII plays an important
role in TG metabolism and is an independent risk factor for
cardiovascular disease. ApoCIII inhibition, as shown herein,
significantly decreases TG levels which can ameliorate
cardiovascular or metabolic disease, or the risk thereof.
[0390] Borderline high TG levels (150-199 mg/dL) are commonly found
in the general population and are a common component of the
metabolic syndrome/insulin resistance states. High plasma TG level
of .gtoreq.200 mg/dL is a common clinical trait associated with an
increased risk of cardiovascular disease (Hegele et al., Hum Mol
Genet 2009, 18:4189-4194; Hegele and Pollex, Mol Cell Biochem,
2009, 326:35-43). Very high TG levels (.gtoreq.500 and .ltoreq.2000
mg/dL) are most often associated with elevated chylomicron levels
as well, and are accompanied by increasing risk for acute
pancreatitis.
[0391] In certain embodiments, the compounds, compositions and
methods disclosed herein are used to treat subjects with a TG level
between 100-200 mg/dL, 100-300 mg/dL, 100-400 mg/dL, 100-500 mg/dL,
200-500 mg/dL, 300-500 mg/dL, 400-500 mg/dL, 500-1000 mg/dL,
600-1000 mg/dL, 700-1000 mg/dL, 800-1000 mg/dL, 900-1000 mg/dL,
500-1500 mg/dL, 1000-1500 mg/dL, 100-2000 mg/dL, 150-2000 mg/dL,
200-2000 mg/dL, 300-2000 mg/dL, 400-2000 mg/dL, 500-2000 mg/dL,
600-2000 mg/dL, 700-2000 mg/dL, 800-2000 mg/dL, 900-2000 mg/dL,
1000-2000 mg/dL, 1100-2000 mg/dL, 1200-2000 mg/dL, 1300-2000 mg/dL,
1400-2000 mg/dL, or 1500-2000 mg/dL. In certain embodiments,
treatment with the compounds disclosed herein is indicated for a
subject with a TG level of .gtoreq.100 mg/dL, .gtoreq.110 mg/dL,
.gtoreq.120 mg/dL, .gtoreq.130 mg/dL, .gtoreq.140 mg/dL,
.gtoreq.150 mg/dL, .gtoreq.160 mg/dL, .gtoreq.170 mg/dL,
.gtoreq.180 mg/dL, .gtoreq.190 mg/dL, .gtoreq.200 mg/dL,
.gtoreq.300 mg/dL, .gtoreq.400 mg/dL, .gtoreq.500 mg/dL,
.gtoreq.600 mg/dL, .gtoreq.700 mg/dL, .gtoreq.800 mg/dL,
.gtoreq.900 mg/dL, .gtoreq.1000 mg/dL, .gtoreq.1100 mg/dL,
.gtoreq.1200 mg/dL, .gtoreq.1300 mg/dL, .gtoreq.1400 mg/dL,
.gtoreq.1500 mg/dL, .gtoreq.1600 mg/dL, .gtoreq.1700 mg/dL,
.gtoreq.1800 mg/dL, .gtoreq.1900 mg/dL, .gtoreq.2000 mg/dL,
.gtoreq.2100 mg/dL, .gtoreq.2200 mg/dL, .gtoreq.2300 mg/dL,
.gtoreq.2400 mg/dL or .gtoreq.2500 mg/dL.
[0392] Some types of hypertriglyceridemia can be characterized by
the Fredrickson classification system or by the classification
system described by Tremblay (Tremblay et al., J Clin Lipidol,
2011, 5:37-44). In certain embodiments, the compounds, compositions
and methods described herein are useful in treating subjects with,
or at risk for, Fredrickson Type II, IV or V
hypertriglyceridemia.
[0393] Fredrickson Type IIb (also known as familial combined
hyperlipoproteinemia) is a mixed hyperlipidemia (high cholesterol
and TG levels), caused by elevations in LDL-C and in VLDL. The high
VLDL levels are due to overproduction of substrates, including TG,
acetyl CoA, and an increase in B-100 synthesis. They may also be
caused by the decreased clearance of LDL. Prevalence in the
population is about 10%.
[0394] Fredrickson Type IV (also known as familial
hypertriglyceridemia) is an autosomal dominant condition occurring
in approximately 1% of the population. TG levels are elevated as a
result of excess hepatic production of VLDL or heterozygous LPL
deficiency, but are almost always less than 1000 mg/dL. Serum
cholesterol levels are usually within normal limits. The disorder
is heterogeneous and the phenotype strongly influenced by
environmental factors, particularly carbohydrate and ethanol
consumption. In certain embodiments, the compounds, compositions
and methods described herein are useful in treating subjects with a
TG level .gtoreq.200 mg/dL and heterozygous LPL deficiency or VLDL
overproduction. In certain embodiments, the compounds, compositions
and methods described herein are useful in treating subjects with a
TG level .gtoreq.500 mg/dL and heterozygous LPL deficiency or VLDL
overproduction.
[0395] Fredrickson Type V has high VLDL and chylomicrons. It is
characterized by carriers of loss-of-function LPL gene variants
associated with LPL activity of at least 20% (i.e. partial LPL
deficiency). These subjects present with lactescent plasma and
severe hypertriglyceridemia because of chylomicrons and VLDL. TG
levels are invariably greater than 1000 mg/dL and total cholesterol
levels are always elevated. The LDL-C level is usually low. It is
also associated with increased risk for acute pancreatitis, glucose
intolerance and hyperuricemia. Symptoms generally present in
adulthood (>35 years) and, although the prevalence is relatively
rare, it is much more common than homozygous or compound
heterozygous LPL deficient subjects. In certain embodiments, the
compounds, compositions and methods described herein are useful in
treating subjects with .gtoreq.1000 mg/dL TG. In certain
embodiments, the compounds, compositions and methods described
herein are useful in treating subjects with, or at risk for,
pancreatitis associated with high TG levels in a subject. In
certain embodiments, the compounds, compositions and methods
described herein are useful in treating subjects with, or at risk
for, cardiovascular or metabolic disease associated with high TG
levels in a subject. In certain embodiments, the cardiovascular
disease is aneurysm, angina, arrhythmia, atherosclerosis,
cerebrovascular disease, coronary heart disease, hypertension,
dyslipidemia, hyperlipidemia, hypertriglyceridemia,
hypercholesterolemia, stroke and the like. In certain embodiments,
the dyslipidemia is chylomicronemia. In certain embodiments, the
metabolic diseases or disorders include, but are not limited to,
hyperglycemia, prediabetes, diabetes (type I and type II), obesity,
insulin resistance, metabolic syndrome and diabetic
dyslipidemia.
[0396] In certain embodiments, treatment with the compounds
disclosed herein is indicated for a human animal with a genetic
defect that increases ApoCIII levels and/or triglyceride levels. In
certain embodiments, the genetic defect is an allelic variant or
polymorphism that increases ApoCIII expression. In certain
embodiments, the polymorphism are T (at position 74) to A, C (at
position -641) to A, G (at position -630) to A, T (at position
-625) to deletion, C (at position -482) to T, T (at position -455)
to C, C (at position 1100) to T, C (at position 3175) to G, T (at
position 3206) to G, C (at position 3238) to G, and the like. In
certain embodiments, the genetic defect is a heterozygous LPL
deficiency.
[0397] In certain embodiments, treatment with the compounds
disclosed herein is indicated for a human animal with elevated
ApoCIII levels. In certain embodiments, the elevated ApoCIII level
is .gtoreq.50 mg/L, .gtoreq.60 mg/L, .gtoreq.70 mg/L, .gtoreq.80
mg/L, .gtoreq.90 mg/L, .gtoreq.100 mg/L, .gtoreq.110 mg/L,
.gtoreq.120 mg/L, .gtoreq.130 mg/L, .gtoreq.140 mg/L, .gtoreq.150
mg/L, .gtoreq.160 mg/L, .gtoreq.170 mg/L, .gtoreq.180 mg/L,
.gtoreq.190 mg/L, .gtoreq.200 mg/L, .gtoreq.300 mg/L, .gtoreq.400
mg/L or .gtoreq.500 mg/L.
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
[0398] While certain compounds, compositions and methods described
herein have been described with specificity in accordance with
certain embodiments, the following examples serve only to
illustrate the compounds described herein and are not intended to
limit the same. Each of the references recited in the present
application is incorporated herein by reference in its
entirety.
Example 1: Effect of In Vivo Antisense Inhibition of Human ApoCIII
in huApoCIII Transgenic Mice
[0399] Transgenic mice with the human ApoCIII transgene utilized in
the study were the progeny of huApoCIII transgenic F1 hybrids
(Jackson Laboratories, CA) and C57BL/6 mice. ISIS 304801
(previously disclosed in U.S. Pat. No. 7,598,227) with a start site
of 508 on SEQ ID NO: 1 (GENBANK Accession No. NM_000040.1) and a
start site of 3139 on SEQ ID NO: 2 (GENBANK Accession NT_033899.8
truncated from nucleotides 20263040 to 20266203), with the sequence
5'-AGCTTCTTGTCCAGCTTTAT-3' (SEQ ID NO: 3) and a 5-10-5 MOE gapmer
motif was utilized in this assay. Another ISIS antisense
oligonucleotide, `Compound X`, with a 5-10-5 MOE gapmer motif,
targeting another region of SEQ ID NO: 1 or SEQ ID NO: 2, was also
included in this assay. Another ISIS antisense oligonucleotide,
`Compound Y`, with a 5-10-5 MOE gapmer motif, targeting a rodent
ApoCIII sequence (GenBank Accession No. NM_023114.3; SEQ ID NO: 5)
was also included in this assay.
Treatment
[0400] Human ApoCIII transgenic mice were maintained on a 12-hour
light/dark cycle and fed ad libitum Teklad lab chow. Animals were
acclimated for at least 7 days in the research facility before
initiation of the experiment. Antisense oligonucleotides (ASOs)
were prepared in PBS and sterilized by filtering through a 0.2
micron filter. Oligonucleotides were dissolved in 0.9% PBS for
injection.
[0401] Male and female mice were assayed separately. The male mice
were divided into three treatment groups consisting of 5 mice each.
Two such groups received subcutaneous injections of ISIS 304801 or
Compound X at a dose of 37.5 mg/kg twice a week for 2 weeks. One
group of mice received subcutaneous injections of PBS twice a week
for 2 weeks. The female mice were divided into four treatment
groups consisting of 4-5 mice each. Three such groups received
subcutaneous injections of ISIS 304801, Compounds X or Y at a dose
of 37.5 mg/kg twice a week for 2 weeks. One group of mice received
subcutaneous injections of PBS twice a week for 2 weeks. Prior to
the treatment as well as after the last dose, blood was withdrawn
from each mouse and plasma samples analyzed. Two days following the
final dose, the mice were euthanized, organs harvested and analyses
done.
Cholesterol and Triglyceride Levels
[0402] Plasma triglycerides and cholesterol were extracted by the
method of Bligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J.
Biochem. Physiol. 37: 911-917, 1959) and measured with a
commercially available triglyceride kit (DCL Triglyceride Reagent;
Diagnostic Chemicals Ltd.).
[0403] The results of the triglyceride analyses in males and
females are presented in Tables 1 and 2, and are expressed in
mg/dL. As observed, triglyceride levels in all the treatment groups
were significantly lowered compared to that in the control
groups.
[0404] For measuring the different fractions of cholesterol (HDL,
LDL and VLDL), the plasma samples from the female groups were
analyzed by HPLC and are presented in Table 3. As observed,
antisense inhibition of ApoCIII significantly decreased VLDL and
also significantly increased levels of HDL. An increase in HDL and
a decrease in VLDL levels is a cardiovascular beneficial effect of
antisense inhibition of ApoCIII and can be beneficial to animals
with, or at risk or, dyslipidemic diseases.
TABLE-US-00001 TABLE 1 Effect of antisense oligonucleotide
treatment on triglyceride levels (mg/dL) in female transgenic mice
% Week 0 Week 2 change PBS 2144 2533 +21 Compound X 2385 677 -72
Compound Y 2632 1644 -37 ISIS 304801 2390 542 -75
TABLE-US-00002 TABLE 2 Effect of antisense oligonucleotide
treatment on triglyceride levels (mg/dL) in male transgenic mice %
Week 0 Week 2 Change PBS 6191 7073 +14 ISIS 304801 6588 780 -88
Compound X 5464 861 -84
TABLE-US-00003 TABLE 3 Effect of antisense oligonucleotide
treatment on plasma cholesterol fractions (% total cholesterol) in
female transgenic mice VLDL (%) LDL (%) HDL (%) PBS 77 .+-. 2.6 4
.+-. 1.0 19 .+-. 1.9 ISIS 304801 41 .+-. 0.6 7 .+-. 0.3 52 .+-. 0.5
Compound X 46 .+-. 5.1 7 .+-. 1.0 48 .+-. 5.8
Example 2: Dose-Dependent Antisense Inhibition of Human ApoCIII in
huApoCIII Transgenic Mice
[0405] ISIS 304801 and Compound X were further studied in a
dose-dependent study using human ApoCIII transgenic mice.
Treatment
[0406] Human ApoCIII transgenic mice were maintained on a 12-hour
light/dark cycle and fed ad libitum Teklad lab chow. Animals were
acclimated for at least 7 days in the research facility before
initiation of the experiment. Antisense oligonucleotides (ASOs)
were prepared in PBS and sterilized by filtering through a 0.2
micron filter. Oligonucleotides were dissolved in 0.9% PBS for
injection.
[0407] Female mice were divided into nine treatment groups
consisting of 3 mice each. Eight such groups received subcutaneous
injections of ISIS 304801 or compound X at a dose of 1.5
mg/kg/week, 5 mg/kg/week, 15 mg/kg/week, or 50 mg/kg/week for 2
weeks. One group of mice received subcutaneous injections of PBS
for 2 weeks. Prior to the treatment as well as after the last dose,
blood was withdrawn from each mouse and plasma samples analyzed.
Two days following the final dose, the mice were euthanized, organs
harvested and analyses done.
Cholesterol and Triglyceride Levels
[0408] Plasma triglycerides and cholesterol were extracted by the
method of Bligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J.
Biochem. Physiol. 37: 911-917, 1959) and measured with a
commercially available triglyceride kit (DCL Triglyceride Reagent,
Diagnostic Chemicals Ltd.).
[0409] The results of the cholesterol and triglyceride analyses in
the mice are presented in Tables 4 and 5, and are expressed in
mg/dL. As observed, HDL levels in mice treated with higher doses of
ISIS 304801 were significantly elevated, indicating the beneficial
effect of inhibition of ApoCIII by the oligonucleotides. LDL and
triglyceride levels in the high dose treatment groups were lowered
compared to that in the control groups. An increase in HDL and a
decrease in LDL and TG levels is a cardiovascular beneficial effect
of antisense inhibition of ApoCIII and can be beneficial to animals
with, or at risk of, dyslipidemic diseases.
TABLE-US-00004 TABLE 4 Effect of antisense oligonucleotide
treatment on cholesterol and triglyceride levels (mg/dL) in
transgenic mice Dose Total (mg/kg/wk) Cholesterol Triglycerides PBS
-- 124 1017 ISIS 304801 50.0 105 417 15.0 116 593 5.0 101 871 1.5
125 1092 Compound X 50.0 90 496 15.0 127 1168 5.0 166 1506 1.5 168
1518
TABLE-US-00005 TABLE 5 Effect of antisense oligonucleotide
treatment on HDL and LDL cholesterol levels (mg/dL) in transgenic
mice Dose (mg/kg/wk) HDL LDL PBS -- 40 .+-. 8 42 .+-. 8 ISIS 304801
50.0 62 .+-. 19 28 .+-. 7 15.0 60 .+-. 9 34 .+-. 7 5.0 44 .+-. 3 30
.+-. 13 1.5 39 .+-. 2 40 .+-. 2 Compound X 50.0 46 .+-. 10 25 .+-.
3 15.0 37 .+-. 7 40 .+-. 2 5.0 40 .+-. 10 47 .+-. 5 1.5 45 .+-. 7
44 .+-. 6
Example 3: Effect of Antisense Inhibition of ApoCIII in CETP
Transgenic LDL Receptor Null Mice
[0410] Compound Y was further studied in a human CETP transgenic
LDLr.sup.-/- mouse model to examine the effects of a mouse ApoCIII
antisense inhibitor on plasma lipids and lipoprotein metabolism in
hyperlipidemic mice.
Treatment
[0411] Human CETP transgenic LDLr.sup.-/- transgenic mice were
maintained on a 12-hour light/dark cycle and fed ad libitum a
western diet (42% calories from fat, 0.2% cholesterol). Animals
were acclimated to this diet for 10 days in the research facility
before initiation of the experiment. Antisense oligonucleotides
(ASOs) were prepared in PBS and sterilized by filtering through a
0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for
injection.
[0412] Eight week old male mice were divided into three treatment
groups. One such group of 6 mice received subcutaneous injections
of compound Y at a dose of 12.5 mg/kg/week for 4 weeks. One group
of 4 mice received subcutaneous injections of the control
oligonucleotide ISIS 141923 (SEQ ID NO: 4) at a dose of 12.5
mg/kg/week for 4 weeks. One group of 5 mice received subcutaneous
injections of PBS for 4 weeks. Plasma samples were taken at prior
to the start of dosing, and at 2 and 4 weeks of treatment.
Cholesterol and Triglyceride Levels
[0413] Plasma triglycerides and cholesterol were extracted by the
method of Bligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J.
Biochem. Physiol. 37: 911-917, 1959) and measured with a
commercially available triglyceride kit (DCL Triglyceride Reagent;
Diagnostic Chemicals Ltd.).
[0414] The results of the cholesterol and triglyceride analyses in
the mice are presented in Tables 6 to 7, and are expressed in
mg/dL. Cholesterol and triglyceride levels in the treatment group
were significantly lowered compared to those in the control group.
A decrease in cholesterol and TG levels is a cardiovascular
beneficial effect of antisense inhibition of ApoCIII and can be
beneficial to animals with, or at risk of, dyslipidemic
diseases.
TABLE-US-00006 TABLE 6 Effect of antisense oligonucleotide
treatment on cholesterol levels (mg/dL) in transgenic mice Week PBS
Compound Y 0 1851 1747 2 2035 878 4 2359 686
TABLE-US-00007 TABLE 7 Effect of antisense oligonucleotide
treatment on triglyceride levels (mg/dL) in transgenic mice Week
PBS Compound Y 0 297 451 2 420 150 4 496 86
Inhibition of CETP Protein Levels and Activity
[0415] Plasma CETP protein levels were measured using a commercial
ELISA kit (ALPCO, Cat#47-CETHU-E01). CETP protein activity was
measured using a fluorometric assay kit (Roar Biomedical, Inc. Cat#
RB-CETP). As presented in Table 8, treatment with antisense
oligonucleotide reduced CETP protein expression and activity. CETP
(cholesteryl ester transfer protein) facilitates the exchange of
triglycerides and cholesterol esters between high density
lipoproteins (HDL) and apoB-containing lipoproteins, such as very
low density lipoproteins (VLDL), LDL and chylomicrons. A decrease
in CETP is associated with increased HDL levels and decreased LDL
levels (Barter P. J. et al. Artherioscler. Thromb. Vasc. Biol. 23:
160-167, 2003). Therefore, inhibition of CETP protein levels and
activity is a cardiovascular beneficial effect of antisense
inhibition of ApoCIII and can be beneficial to animals with, or at
risk of, dyslipidemic diseases. The control oligonucleotide did not
have any significant effect on CETP, as expected.
TABLE-US-00008 TABLE 8 Percent inhibition of CETP protein in
transgenic mice Level Activity Compound Y 24 24 ISIS 141923 0 3
Increase of apoA1 Protein Levels and Paraoxanase-1 (PON1)
Activity
[0416] Plasma ApoA1 protein levels were measured by ELISA. PON1
protein activity was measured using a EnzChek.RTM. Paroxanase
fluorometric assay kit (Invitrogen, Cat# E33702). As presented in
Tables 9 and 10, treatment with antisense oligonucleotide enhanced
ApoA1 protein expression and increased PON1 protein activity. ApoA1
and PON1 are major protein components of HDL in plasma (Aviram, M
and Rosenblat, M. Curr. Opin. Lipidol. 16: 393-399, 2005).
Therefore, enhancement of protein level and activity of these two
protein components is a cardiovascular beneficial effect of
antisense inhibition of ApoCIII and can be beneficial to animals
with, or at risk of, dyslipidemic diseases. The control
oligonucleotide did not have any effect on either protein, as
expected.
TABLE-US-00009 TABLE 9 Percent increase in APOA1 protein levels in
transgenic mice mg/dL PBS 65 Compound Y 211 ISIS 141923 106
TABLE-US-00010 TABLE 10 Percent increase in PON1 protein activity
in transgenic mice Minutes PBS Compound Y ISIS 141923 15 0.2 0.5
0.2 30 0.8 1.1 0.8 60 2.3 3.5 2.3 120 4.9 7.5 4.8 180 7.7 11.6
7.6
Example 4: Effect of Antisense Inhibition of ApoCIII on HDL
Cholesterol Clearance in CETP Transgenic LDL Receptor Null Mice
[0417] Compound Y was further studied in a human CETP transgenic
LDLr.sup.-/- mouse model to examine the effects of an ApoCIII
antisense inhibitor on HDL cholesterol clearance and metabolism in
hyperlipidemic mice.
Treatment
[0418] Human CETP transgenic LDLr.sup.-/- mice were maintained on a
12-hour light/dark cycle and fed ad libitum a western diet (42%
calories from fat, 0.2% cholesterol). Animals were acclimated to
this diet for 10 days in the research facility before initiation of
the experiment. Antisense oligonucleotides (ASOs) were prepared in
PBS and sterilized by filtering through a 0.2 micron filter.
Oligonucleotides were dissolved in 0.9% PBS for injection.
[0419] Eight week old male mice were divided into three treatment
groups. One group of 6 mice received subcutaneous injections of
compound Y at a dose of 15 mg/kg/week for 6 weeks. One group of 4
mice received subcutaneous injections of the control
oligonucleotide ISIS 141923 at a dose of 15 mg/kg/week for 6 weeks.
One group of 5 mice received subcutaneous injections of PBS for 6
weeks.
Cholesterol and Triglyceride Levels
[0420] Plasma triglycerides and cholesterol were extracted by the
method of Bligh and Dyer (Bligh, E. G. and Dyer, W. J. Can. J.
Biochem. Physiol. 37: 911-917, 1959) and measured with a
commercially available triglyceride kit (DCL Triglyceride Reagent;
Diagnostic Chemicals Ltd.).
[0421] The results of the cholesterol and triglyceride analyses in
the mice are presented in Table 11, and are expressed in mg/dL.
Cholesterol and triglyceride levels in the treatment group was
significantly lowered compared to that in the control group. A
decrease in cholesterol and TG levels is a cardiovascular
beneficial effect of antisense inhibition of ApoCIII and can be
beneficial to animals with, or at risk of, dyslipidemic
diseases.
TABLE-US-00011 TABLE 11 Effect of antisense oligonucleotide
treatment on cholesterol and triglyceide levels (mg/dL) in
transgenic mice Total cholesterol Triglycerides PBS 2188 641
Compound Y 1402 170
HDL Clearance
[0422] Mice from all groups were injected via tail vein with
1.times.10.sup.6 dpm of .sup.3H-cholesteryl ether
(.sup.3H-CEth)-labeled HDL. The radiolabeled cholesteryl ether is
structurally similar to cholesterol but it will be trapped in
tissues that take it up. Therefore, the clearance of the
radiolabeled cholesteryl ether from plasma and it's accumulation in
the liver can be used to evaluate effects on reverse cholesterol
transport. Plasma samples were collected at 5 min, 1.5 hrs, 3 hrs,
6 hrs and 24 hrs post-injection and the radioactivity was counted
using a liquid scintillation counter. At 24 hours, the mice were
sacrificed and liver were harvested. The liver samples were
extracted in 2:1 Chloroform/Methanol and the extract was blown down
under nitrogen gas, solubilized in scintillation cocktail and
counted using the same liquid scintillation counter.
[0423] The decrease in radiolabel, as presented in Table 12 is
associated with the clearance of HDL-Ceth from the plasma. The
results indicate that treatment with Compound Y lead to enhanced
rate of HDL cholesterol clearance from the plasma. This was
associated with the greater accumulation of radiolabeled
cholesteryl etherin the liver of Compound Y-treated mice, as
presented in Table 13. Therefore, the data indicates that
inhibition of ApoCIII in these transgenic mice improves reverse
cholesterol transport and, therefore, would have a beneficial
effect on patients with cardiovascular disease such as patients
with a dyslipidemic disease.
TABLE-US-00012 TABLE 12 Effect of antisense oligonucleotide
treatment on plasma HDL cholesterol (% of count at 0 hrs) in
transgenic mice 1.5 hr 3 hr 6 hr 24 hr PBS 87 79 64 38 ISIS 141923
84 82 69 39 Compound Y 78 71 57 25
TABLE-US-00013 TABLE 13 Effect of antisense oligonucleotide
treatment on hepatic uptake of radiolabeled CEth in transgenic mice
% Increase (dpm/g liver tissue) ISIS 141923 0 Compound Y 14
Example 5: Comparison of the Effect of Antisense Inhibition of
Human ApoCIII in C57BL/6 Mice with an ApoCIII Knockout Mouse
Model
[0424] ApoCIII knockout mice were obtained from Jackson
Laboratories (stock number 002057) and were compared to ApoCIII
antisense oligonucleotide treated C57BL/6 mice. Compound Z, with a
5-10-5 MOE gapmer motif and targeting a rodent ApoCIII sequence
(GenBank Accession No. NM_023114.3; SEQ ID NO: 5) was used in this
study.
Antisense Oligonucleotide Treatment
[0425] C57BL/6 mice were maintained on a 12-hour light/dark cycle
and fed a high fat diet (Harland Teklad lab chow #88137) for one
week. Antisense oligonucleotides (ASOs) were prepared in PBS and
sterilized by filtering through a 0.2 micron filter.
Oligonucleotides were dissolved in 0.9% PBS for injection. The mice
were randomized based on total plasma cholesterol and triglyceride
levels into groups of 6-8 mice each. Three groups of C57BL/6 mice
received weekly intraperitoneal injections of Compound Z at doses
of 3.1 mg/kg, 6.3 mg/kg, or 12.5 mg/kg for a period of 6 weeks. A
group of C57BL/6 mice received weekly intraperitoneal injections of
PBS for a period of 6 weeks. The PBS group served as a control to
which the oligonucleotide-treated groups and the ApoCIII knockout
mice were compared.
[0426] Two days after the final dose, the mice were sacrificed and
organs harvested. Similar groups of mice fed a normal murine chow
were also tested.
Liver Triglycerides
[0427] Liver triglycerides were with an Olympus clinical analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). The data is presented in
Table 14 and demonstrates that mice treated with an ApoCIII
antisense oligonucleotide have a different phenotype than ApoCIII
knockout mice. The high dose ApoCIII antisense oligonucleotide
treated mice had liver triglyceride levels similar to that of the
PBS control. Liver triglyceride levels in the ApoCIII knockout mice
were significantly higher than in C57BL/6 mice treated with an
ApoCIII antisense oligonucleotide or the PBS control. Therefore,
antisense inhibition of ApoCIII had the beneficial effect of
lowering the risk of liver steatosis compared to the ApoCIII
knockout mouse model.
TABLE-US-00014 TABLE 14 Liver triglyceride levels (mg/g liver
tissue) Dose High-fat (mg/kg) diet fed PBS -- 33 Compound Z 3.1 44
6.3 47 12.5 33 ApoCIII KO -- 60
Example 6: Effect of In Vivo Antisense Inhibition of ApoCIII in
C57BL/6 Mice
[0428] The effect of antisense inhibition of ApoCIII on plasma
lipid levels and fat clearance was evaluated.
Treatment
[0429] Male C57/BL6 mice were maintained on a 12-hour light/dark
cycle and fed ad libitum a western diet (Harland Tekland 88137).
Animals were acclimated for at least 7 days in the research
facility before initiation of the experiment. Antisense
oligonucleotides (ASOs) were prepared in PBS and sterilized by
filtering through a 0.2 micron filter. Oligonucleotides were
dissolved in 0.9% PBS for injection.
[0430] Groups of 7-8 mice each received intraperitoneal injections
of Compound Z at a dose of 12.5 mg/kg/wk for 6 weeks. Another group
of mice received intraperitoneal injections of control
oligonucleotide ISIS 141923 at a dose of 12.5 mg/kg/wk for 6 weeks.
A third group of mice received intraperitoneal injections of PBS
for 6 weeks. Two days after the final dose, the mice were fasted
for 4 hours, sacrificed and plasma and tissues were collected.
Inhibition of ApoCIII mRNA
[0431] Total RNA was extracted from the liver and small intestine
and ApoCIII mRNA was quantitated by RT-PCR using an ApoCIII primer
probe set and normalized to cyclophilin. The results are presented
in Table 15, expressed as percent inhibition of ApoCIII mRNA
compared to the PBS control. ISIS 141923 did not cause any
reduction in ApoCIII mRNA levels, as expected. The data
demonstrated the significant inhibition of ApoCIII mRNA in the
liver and small intestine by Compound Z compared to the PBS
control.
[0432] Inhibition of intestinal ApoCIII expression could be
important in the prevention of chylomicronemia (Chait et al., 1992,
Adv Intern Med. 1992, 37:249-73), a dyslipidemic state caused by
improper clearance of chylomicron triglyceride. Severe forms of
chylomicronemia can lead to pancreatitis, a life-threatening
condition. By inhibiting intestinal ApoCIII, inhibition of
lipoprotein lipase would be reduced, and chylomicron triglyceride
clearance would be enhanced, thereby preventing pancreatitis. In
addition, inhibition of intestinal ApoCIII would enhance clearance
of postprandial triglyceride, thereby lowering post prandial TG a
known risk factor for coronary heart disease.
TABLE-US-00015 TABLE 15 Percent inhibition of ApoCIII mRNA relative
to the PBS control % inhibition Liver 74 Small Intestine 13
Cholesterol and Triglyceride Levels
[0433] Plasma cholesterol were extracted by the method of Bligh and
Dyer (Bligh, E. G. and Dyer, W. J. Can. J. Biochem. Physiol. 37:
911-917, 1959) and measured with an Olympus clinical analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). HDL and non-HDL
cholesterol were individually measured by HPLC. Triglyceride levels
were measured with the use of a commercially available triglyceride
kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.,
Charlottetown, Canada). The results are presented in Table 16 and
are expressed in mg/dL. Treatment with Compound Z resulted in
significant reduction of total cholesterol, non-HDL cholesterol and
plasma triglyceride levels compared to the PBS control. A decrease
in total cholesterol, non-HDL cholesterol and TG levels is a
cardiovascular beneficial effect of antisense inhibition of ApoCIII
and can be beneficial to animals with, or at risk of, dyslipidemic
diseases.
TABLE-US-00016 TABLE 16 Plasma cholesterol and triglyceride levels
(mg/dL) in C56BL/6 mice Dose Total HDL LDL VLDL Treatment
(mg/kg/wk) cholesterol cholesterol cholesterol cholesterol
Triglycerides PBS -- 93 70 20 2.9 84 ISIS 141923 12.5 97 78 19 2.1
82 Compound Z 12.5 95 75 17 3.1 70
Fat Clearance
[0434] Plasma samples were collected at 30 min, 1 hr, 2 hrs, 3 hrs,
and 4 hrs post-injection and plasma total lipid content was
measured with an Olympus clinical analyzer (Hitachi Olympus AU400e,
Melville, N.Y.). The lipid level in the plasma, as presented in
Table 17 was an inverse indicator of lipid clearance from the
plasma. The results indicate that treatment with Compound Z lead to
enhanced rate of fat clearance from the plasma.
[0435] Therefore, the data indicates that inhibition of ApoCIII in
these transgenic mice improves reverse cholesterol transport and,
would have a beneficial effect on patients with cardiovascular
disease.
TABLE-US-00017 TABLE 17 Effect of antisense oligonucleotide
treatment on plasma lipid (mg/dL) in C57BL/6 mice Area under the 0
hr 0.5 hr 1 hr 2 hr 3 hr 4 hr curve PBS 86 80 64 122 118 90 23631
ISIS 141923 115 142 124 236 225 150 43677 Compound Z 66 55 96 156
151 101 28371
Example 7: Effect of In Vivo Antisense Inhibition of ApoCIII in
C57BL/6 Mice
[0436] The effect of antisense inhibition of ApoCIII on ApoCIII
expression levels and fat clearance was evaluated.
Treatment
[0437] Male C57/BL6 mice were maintained on a 12-hour light/dark
cycle and fed ad libitum a western diet (Harland Tekland 88137).
Animals were acclimated for at least 7 days in the research
facility before initiation of the experiment. Antisense
oligonucleotides (ASOs) were prepared in PBS and sterilized by
filtering through a 0.2 micron filter. Oligonucleotides were
dissolved in 0.9% PBS for injection.
[0438] Groups of 5 mice each received intraperitoneal injections of
an ApoCIII targeting antisense oligonucleotide, Compound Z, at a
dose of 12.5 mg/kg/wk for 6 weeks. Another group of mice received
intraperitoneal injections of control oligonucleotide ISIS 141923
at a dose of 12.5 mg/kg/wk for 6 weeks. Two days after the final
dose, the mice were fasted overnight, and a bolus of 200 .mu.L of
olive oil was administered by oral gavage. Following the bolus,
plasma triglyceride levels were measured at regular intervals for 4
hours. The mice were sacrificed and plasma and tissues were
collected.
Inhibition of ApoCIII mRNA
[0439] Total RNA was extracted from the liver and small intestine
and ApoCIII mRNA was quantitated by RT-PCR using an ApoCIII primer
probe set and normalized to cyclophilin. The results are presented
in Table 18, expressed as percent inhibition of ApoCIII mRNA
compared to the oligonucleotide control. The data demonstrated the
significant inhibition of ApoCIII mRNA in the liver and small
intestine by Compound Z compared to the oligonucleotide
control.
[0440] As noted elsewhere herein, inhibition of intestinal ApoCIII
expression could be important in the prevention of chylomicronemia
(Chait et al., 1992, Adv Intern Med. 1992, 37:249-73), a
dyslipidemic state caused by improper clearance of chylomicron
triglyceride. Severe forms of chylomicronemia can lead to
pancreatitis, a life-threatening condition. By inhibiting
intestinal ApoCIII, inhibition of lipoprotein lipase would be
reduced, and chylomicron triglyceride clearance would be enhanced,
thereby preventing pancreatitis. In addition, inhibition of
intestinal ApoCIII would enhance clearance of postprandial
triglyceride, thereby lowering post prandial TG a known risk factor
for coronary heart disease.
TABLE-US-00018 TABLE 18 Percent inhibition of ApoCIII mRNA relative
to the control oligonucleotide treated C57/BL/6 mice Strain of mice
% inhibition C57BL/6 Liver 74 Small Intestine 60
Fat Clearance
[0441] Plasma samples were collected at 0 min, 30 min, 60 min, 120
min, 180 min, and 240 min post-injection and plasma triglyceride
concentrations was measured with an Olympus clinical analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). The results indicate that
treatment with Compound Z lead to enhanced rate of triglyceride
clearance from the plasma.
[0442] This study can be compared to fat bolus clinical studies in
which patients expressing high apo-CIII levels showed increased
postprandial TG concentrations (Petersen K. F. et al., N Engl J Med
2010; 362: 1082-1089).
TABLE-US-00019 TABLE 19 Effect of antisense oligonucleotide
treatment on postprandial plasma TG (mg/dL) in C57BL/6 mice Strain
of 30 120 240 mice 0 min min 60 min min 180 min min C57BL/6 ISIS
141923 115 142 124 236 225 150 Compound Z 66 55 96 156 151 101
Example 8: Effect of In Vivo Antisense Inhibition of ApoCIII in
C57BL/6 Mice
[0443] The effect of antisense inhibition of ApoCIII on fat
clearance was evaluated.
Treatment
[0444] Male C57/BL6 mice were maintained on a 12-hour light/dark
cycle and fed ad libitum a western diet (Harland Tekland 88137).
Animals were acclimated for at least 7 days in the research
facility before initiation of the experiment. Antisense
oligonucleotides (ASOs) were prepared in PBS and sterilized by
filtering through a 0.2 micron filter. Oligonucleotides were
dissolved in 0.9% PBS for injection.
[0445] Groups of 6 mice each received intraperitoneal injections of
an ApoCIII targeting antisense oligonucleotide, Compound Y or
Compound Z, at a dose of 12.5 mg/kg/wk, 6.3 mg/kg/week or 3.1
mg/kg/week for 6 weeks. Another group of mice received
intraperitoneal injections of control oligonucleotide ISIS 141923
at a dose of 12.5 mg/kg/wk for 6 weeks. Another group of mice
received intraperitoneal injections of PBS for 6 weeks. Two days
after the final dose, the mice were fasted overnight, and a bolus
of 200 .mu.L of olive oil was administered by oral gavage.
Following the bolus, plasma triglyceride levels were measured at
regular intervals for 4 hours.
Fat Clearance
[0446] Plasma samples were collected at 0 min, 30 min, 60 min, 120
min, 180 min, and 240 min post-injection and plasma triglyceride
concentrations were measured with an Olympus clinical analyzer
(Hitachi Olympus AU400e, Melville, N.Y.). The results indicate that
treatment with Compound Y and Compound Z lead to enhanced rate of
fat clearance from the plasma. N.d. indicates that the data set was
not calculated.
[0447] This study can be compared to fat bolus clinical studies in
which patients expressing high apo-CIII levels showed increased
postprandial TG concentrations (Petersen K. F. et al., N Engl J Med
2010; 362: 1082-1089)
TABLE-US-00020 TABLE 20 Effect of antisense oligonucleotide
treatment on postprandial plasma TG (mg/dL) in C57BL/6 mice Dose 30
60 120 180 240 (mg/kg/wk) 0 min min min min min min PBS -- 79 104
118 126 113 116 ISIS 141923 12.5 75 100 116 150 138 133 Compound Y
12.5 79 74 103 117 120 96 6.3 64 70 81 94 120 112 3.1 91 85 106 139
164 133 Compound Z 12.5 73 65 84 118 94 76 6.3 70 73 89 117 120 89
3.1 86 98 143 137 152 128
Example 9: Effect of ISIS Antisense Oligonucleotides Targeting
Human ApoCIII in Monkey Model of Hypertriglyceridemia
[0448] Rhesus monkeys maintained on a high fructose diet were
treated with ISIS 304801. Antisense oligonucleotide efficacy and
tolerability, as well as the pharmacological effect were
evaluated.
Treatment
[0449] The monkeys were 2-4 years old and weighed between 2 and 5
kg. The monkeys were assigned to six groups of five randomly
assigned male rhesus monkeys each. About 60 g of diet (Certified
Primate Diet #5048, PMI Nutrition International, Inc.) was provided
to each monkey in Groups 1-4 twice daily. An appropriate fructose
supplement (i.e. approximately 15% Kool Aid.RTM. mixture) was
supplied in the morning for 16 weeks prior to antisense
oligonucleotide dosing. To confirm sufficient triglyceride level
elevations, blood samples for serum chemistry were collected from
all animals 1-2 weeks prior to dosing.
[0450] The groups of monkeys were injected subcutaneously with ISIS
oligonucleotide or PBS using a stainless steel dosing needle and
syringe of appropriate size into one of 4 sites on the back of the
monkeys; each site being used per dose in a clock-wise manner. Some
of the groups were dosed three times a week for the first week
(Days 1, 3, and 5) as loading doses, and subsequently twice a week
for weeks 2-12, with 5 mg/kg, 10 mg/kg, or 20 mg/kg of ISIS 304801.
Two control groups of 5 rhesus monkeys each were injected with PBS
subcutaneously three times a week for the first week (Days 1, 3,
and 5), and subsequently twice a week for weeks 2-12. The dosing
chart is shown in Table 21. Monkeys of Groups 1-4 were sacrificed
on Day 86.
[0451] An additional high fat challenge was administered to monkeys
of Groups 5 and 6 in the form of a whipping cream milk shake. The
milk shake was standardized to consist of 782 calories per m.sup.2
of body surface with 77.6% of calories from fat, 19.2% from
carbohydrate, and 3.1% from protein. Monkeys of Groups 5 and 6 were
fasted overnight and the milk shake was administered once on day 84
via a gastric tube. Blood was drawn just prior to (time=0 hours)
and 1, 2, 3, 4, and 6 hours after ingestion of the fat load to
assess the triglyceride excursion. The monkeys were rested and
remained otherwise fasting during the 6 hours post-fat challenge.
Monkeys of this group were sacrificed on Day 87.
TABLE-US-00021 TABLE 21 Groups of rhesus monkeys on a high fructose
diet Weekly Dose No. of Group Test Article (mg/kg/week) Sex Animals
Toxicology groups 1 PBS 0 Male 5 2 ISIS 304801 10 Male 5 3 ISIS
304801 20 Male 5 4 ISIS 304801 40 Male 5 High Fat Challenge Test
groups 5 PBS 0 Male 5 6 ISIS 304801 40 Male 5
Hepatic Target Reduction
RNA Analysis
[0452] Approximately 150 mg of liver was collected from Groups 1-4
for ApoCIII mRNA analysis at sacrifice. The liver was divided into
2 pieces and soaked in two tubes containing RLT buffer with 1%
beta-mercaptoethanol. The tissues were homogenized and ApoCIII
expression was quantified by RT-PCR analysis. As shown in Table 22,
treatment with ISIS 304801 resulted in significant reduction of
ApoCIII mRNA in comparison to the PBS control. The
TABLE-US-00022 TABLE 22 Percent Inhibition of ApoCIII mRNA in the
rhesus monkey liver relative to the PBS control Dose Groups
(mg/kg/week) % inhibition 2 10 68 3 20 78 4 40 83
Protein Analysis
[0453] Approximately 1.5 mL of blood was collected from all study
animals in Groups 1-4 and placed in tubes containing K.sub.2-EDTA
and then centrifuged for plasma separation. ApoCIII protein levels
were quantified on a clinical analyzer using a commercially
available turbidometric assay (Kamiya Biomedical Co., Seattle,
Wash.). As shown in Table 23, treatment with ISIS 304801 resulted
in significant reduction of ApoCIII protein levels in comparison to
the PBS control. The kinetics of ApoCIII protein level reduction
was also analyzed and is presented in Table 24.
TABLE-US-00023 TABLE 23 Percent Inhibition of ApoCIII plasma
protein levels in the rhesus monkey relative to the PBS control
Dose Groups (mg/kg/week) % inhibition 2 10 74 3 20 72 4 40 89
TABLE-US-00024 TABLE 24 ApoCIII plasma protein levels (mg/dL) on
different days in the rhesus monkey relative to the PBS control
Dose Day Day Day Groups (mg/kg/wk) Day -7 16 30 86 1 -- 4.0 5.5 3.8
5.7 2 10 4.8 3.2 0.2 1.5 3 20 4.5 3.8 0.9 1.6 4 40 5.2 2.9 0.0
0.6
Lipoprotein Particle Analysis
[0454] To establish the kinetics of plasma ApoCIII suppression,
plasma samples were collected 7 days before the commencement of
dosing, as well as on days 16, 30 and 86 of the dosing period. The
samples were subjected to NMR lipoprotein particle analysis
(Liposcience, Raleigh, N.C.). Since there were no significant
differences in ApoCIII lowering between the treatment groups
(Groups 2-4), the analyses is presented only of Group 2 (treatment
group receiving 10 mg/kg/week). The data is presented in Tables 25
and 26.
[0455] Statistically significant mean changes from the baseline
were observed in total plasma triglycerides (TG) and in VLDL and
chylomicron TG of the treatment group at day 30. At the same time,
the control monkeys demonstrated mean increases in the same
parameters. Sustained treatment with ISIS 304801 in these
fructose-fed monkeys led to time-dependent increases in HDL
cholesterol particle numbers by approximately 8 .mu.mol/L (Tables
27) and did not produce elevations in LDL cholesterol in these
studies (Table 28). There were no significant changes in LDL
cholesterol particle quantity over the 12 week treatment period,
relative to the PBS control group.
[0456] At the time of sacrifice, livers were extracted using the
Bligh and Dyer extraction method (Bligh E G and Dyer W. J. Can J
Biochem Physiol 1959; 37: 911-917) and quantified using a Wako
colorimentric TG assay. Antisense inhibition of ApoCIII did not
increase hepatic TG accumulation in any of the treatment groups
relative to the PBS control group (Table 29).
TABLE-US-00025 TABLE 25 Change from baseline plasma TGmg/dL) on
different days in the rhesus monkey Dose Day Day Day (mg/kg/wk) Day
-7 16 30 86 PBS -- 0 18 23 27 ISIS 10 0 -22 -32 -27 304801
TABLE-US-00026 TABLE 26 Change from baseline VLDL and chylomicron
TG (mg/dL) on different days in the rhesus monkey Dose Day Day Day
(mg/kg/wk) Day -7 16 30 86 PBS -- 0 17 22 28 ISIS 10 0 -22 -31 -26
304801
TABLE-US-00027 TABLE 27 Change from baseline HDL cholesterol
particles (.mu.mol/L) on different days in the rhesus monkey Dose
Day Day Day (mg/kg/wk) Day -7 16 30 86 PBS -- 0.3 -5.6 -3.5 -8.1
ISIS 10 0.0 0.5 9.7 8.0 304801
TABLE-US-00028 TABLE 28 Total LDL cholesterol particles (nmol/L) on
different days in the rhesus monkey Dose Day Day Day (mg/kg/wk) Day
-7 16 30 86 PBS -- 982 928 1005 1184 ISIS 10 1007 938 781 910
304801
TABLE-US-00029 TABLE 29 Hepatic triglyceride content in PBS control
and ISIS 304801 cohorts after 12 weeks in HTG rhesus monkeys Liver
TG (ug/mg) Average PBS 9 10 mg/kg/wk 16 20 mg/kg/wk 18 40 mg/kg/wk
6
Post-Prandial Plasma TG Clearance
[0457] At 10 weeks, the post-prandial plasma TG levels in monkeys
from the 10 mg/kg/week group (Group 2) were measured at 0 hr, 1 hr,
2 hr, 3 hr, and 4 hr after providing a meal to the monkeys. As
shown in Tables 30 and 31, post-prandial plasma TG clearance was
significantly increased, as shown by the 38% decrease in
post-prandial TG area under the curve (AUC) in monkeys of the 10
mg/kg/week group.
[0458] Post-prandial TG clearance was also assessed in Groups 5 and
6 (the PBS control and 40 mg/kg/week group after a fat challenge)
at 12 weeks. The data is presented in Table 32, and also indicates
a significant decrease in post-prandial TG area under the curve
(AUC) in monkeys of that group compared to the control.
TABLE-US-00030 TABLE 30 Plasma TG (mg/dL) in the rhesus monkey Dose
(mg/kg/wk) 0 hr 1 hr 2 hr 3 hr 4 hr PBS -- 167 169 146 147 131 ISIS
10 105 87 95 102 80 304801
TABLE-US-00031 TABLE 31 Post-prandial TG area under the curve (AUC)
in the rhesus monkey AUC PBS 610 ISIS 304801 376
TABLE-US-00032 TABLE 32 Post-prandial TG area under the curve (AUC)
in the rhesus monkey after fat challenge AUC PBS 613 ISIS 304801
405
[0459] Monkeys in the 10 mg/kg/wk group had lower fasting plasma TG
levels than the PBS group at 10 weeks. Results in non-human
primates demonstrate that antisense inhibition of ApoCIII represent
an attractive therapeutic strategy for reducing plasma TG and VLDL
in dyslipidemic individuals, and treatment can concurrently raise
HDL-C levels with no adverse effects on LDL-C.
Example 10: ISIS 304801 Phase I Clinical Trial
[0460] In a double-blind, single and multiple ascending-dose (SAD
and MAD) Phase 1 study, healthy subjects, aged 18 to 55 years, were
randomly assigned in a 3:1 ratio to receive ISIS 304801 or placebo
(normal saline).
[0461] SAD subjects were administered a single subcutaneous (SC)
injection of 50, 100, 200, or 400 mg (n=4/cohort) at the Study
Center. The subjects returned to the Study Center for an outpatient
visit on Days 4 and 8 (.+-.24 hour window) for blood sampling and
for clinical evaluation. The subjects were followed until Day 15
when they were evaluated by a telephone interview.
[0462] MAD subjects were administered multiple SC injections at 50,
100, 200, and 400 mg at the Study Center. The subjects received a
loading regimen of 3 doses the first week (Days 1, 3 and 5)
followed by once weekly dosing for 3 weeks (Days 8, 15 and 22). The
subjects were followed for 8 weeks after their last dose of study
drug. The subjects returned to the Study Center for an outpatient
visit on Days 29, 36 and 50 (.+-.24 hour window) for safety and
clinical laboratory evaluations and for blood sampling for PK
analysis. The subjects were followed until Day 78 (.+-.7 day
window) when they were evaluated by a telephone interview.
[0463] The MAD subjects stayed at the Study Center from days -1 to
6 and days 22 to 23, where they were provided the diet shown in
Table 33. The subjects fasted for at least 12 hours before blood
samples were taken for evaluation at Days 5, 8, 15, 22, 23, 29, 36,
43 and 50 (.+-.24 hour window).
TABLE-US-00033 TABLE 33 Study Center Patient Diet Serving Study Day
Portion Food Description Day -1 525 ml. Thai noodles with Beef
& mixed [Admit] vegetable, broccoli, bean sprout, green &
red peppers 375 ml. Fresh fruit salad 300 ml. Orange juice 1 Horse
shoe cake 250 ml. 2% Milk Day 1 1 Butter croissant 2 Eggs,
Scrambled 250 ml. Sliced peaches with syrup 300 ml. Orange juice 75
g. Grilled Chicken Breast [Sandwich] on Kaiser bun 1.5 cup Cream of
Mushroom Soup 1-pkg. Crackers Small Salad On side Condiments 355
ml. Ginger Ale 75 g. Roast Beef 250 ml. Mashed Potatoes 375 ml.
Mixed Vegetables On side Gravy On side Garlic bread 300 ml. Apple
juice 1 Carrot muffin 250 ml. 2% Milk Day 2 3 Pancakes On side
Syrup 1 Banana 300 ml. Orange juice 9'' Mesquite Chicken, Chicken,
bacon, cheddar, tomato, red onion, & lettuce on whole wheat
bread On side Ranch dressing Cup Broccoli & cheese soup 1-pkg.
Crackers 355 ml. Ginger ale 375 g. Beef Stir-fry & mixed
vegetables, broccoli, carrots, celery, & onions 150 g. Steamed
rice 1-pkg. Raisin 300 ml. Apple juice 250 ml. Fresh fruit salad
300 ml. Cranberry juice Day 3 Med. Cheese & vegetable omelet on
whole wheat bagel toasted 300 ml. Grape juice 9'' Black Angus
Steak, mozzarella, cheddar, sauteed onion & mushrooms on Cheese
bread On side Honey Bourbon Mustard, Zesty Grille Sauce Cup Chicken
noodle soup 1-pkg. Crackers 355 ml. Ginger ale 400 g. BBQ Chicken
Breast, Vegetables, cooked mixed, cauliflower, carrots, red &
green peppers, green beans 1.5 cups Flavored rice 1-pkg. Grapes 300
ml. Apple juice 1 Raisin Oatmeal cookie 250 ml. 2% milk Day 4 1
Cheese Croissant 250 ml. Sliced Peaches in syrup 300 ml. Orange
juice 2 Flatbread Sammie, with Chicken, bacon, cheddar, tomato
& romaine lettuce On side Buttermilk ranch dressing Cup
Broccoli & Cheese soup 1-pkg. Crackers 355 ml. Ginger ale 425
g. Beef Stew, tender Beef cubes, with carrots, onions, &
potatoes 325 g. Thai salad, with romaine lettuce, pasta, &
dressings 1 Italian bread 250 ml. Sliced Pears in syrup 300 ml.
Apple juice 1 Blueberry muffin 250 ml. 2% Milk Day 5 375 ml. Corn
Flakes Med. Banana 250 ml. 2% Milk 300 ml. Apple juice 6 pcs.
Chicken wings 250 ml. Fried rice 250 ml. Mixed cooked veggies,
green beans, carrots, & red peppers with sundried tomato sauce
355 ml. 7-up 6'' Pizza Pepperoni lovers Pizza a double helping of
deli style pepperoni & 100% Pizza Mozzarella Small Salad 355
ml. Ginger Ale 1 Almond Croissant 250 ml. 2% Milk Day 6 45 g. Bagel
with Cream Cheese [Discharge] 300 ml. Orange juice Day 22 450 g.
Breakfast Omelets Spinach & Feta cheese 2 slices Brown toast
300 ml. Orange juice 9'' Zesty Grille Steak Prime Rib Steak,
mozzarella, cheddar, mushroom, sauteed onion, on Cheese bread On
side Honey Bourbon Mustard, Zesty Grille Sauce Cup Broccoli &
Cheese soup 1-pkg. Crackers 355 ml. Ginger Ale 400 g. Herb Grilled
Chicken Breast with Vegetables 1.5 cups Flavored rice Small Fresh
fruit salad 300 ml. Apple juice 1 Raisin Oatmeal cookie 250 ml. 2%
Milk Day 23 45 g. Butter croissant [Discharge] 300 ml. Orange
juice
Results
[0464] Overall, ISIS 304801 demonstrated a good safety profile and
was well tolerated in all subjects with no clinically meaningful
elevations of transaminase enzymes and no significant adverse
events.
[0465] The baseline characteristics of MAD cohorts are shown in
Table 34. MAD subjects showed dose-dependent sustained reductions
in total apoC-III and TG levels expressed as a percentage change
from baseline in Tables 35-36.
TABLE-US-00034 TABLE 34 Baseline Characteristics of MAD Cohorts
Placebo 50 mg 100 mg 200 mg 400 mg (n = 4) (n = 3) (n = 3) (n = 3)
(n = 3) Gender (M:F) 3:1 3:0 3:0 3:0 3:0 Age (yrs) 43.0 40.0 40.0
43.0 40.0 BMI (kg/m2) 27.7 24.0 27.3 28.0 27.5 Lipids &
Lipoproteins, mg/dL Apo CIII 6.3 10.4 9.5 11.6 8.7 Triglycerides 97
124 94 195 89 Total Cholesterol 195 157 196 185 181 HDL-C 45 42 46
43 62 Non-HDL-C 136 118 150 149 126 LDL-C 112 93 131 95 102
Per-protocol population. Values presented are the median.
TABLE-US-00035 TABLE 35 Dose-Dependent Prolonged Reduction in Serum
ApoCIII: Median % change in ApoCIII from Baseline Study Day Placebo
50 mg 100 mg 200 mg 400 mg Day 5 38.6 33.4 -4.5 -10.8 -42.6 Day 8
31.9 17.8 -5.2 -36.0 -78.6 Day 15 0.0 -22.1 -24.0 -54.1 -79.8 Day
22 11.7 -20.9 -21.0 -61.7 -86.4 Day 23 15.8 -15.6 -11.9 -58.7 -79.0
Day 29 -11.0 -19.7 -17.3 -70.5 -77.5 Day 36 1.9 -26.6 -32.1 -57.3
-69.5 Day 50 16.4 21.9 -3.9 -63.0 -78.6
TABLE-US-00036 TABLE 36 Dose-Dependent Reductions in Triglycerides:
Median % Change in Triglycerides from Baseline Study Day Placebo 50
mg 100 mg 200 mg 400 mg Day 5 78.5 50.0 15.9 -15.6 -7.7 Day 8 34.9
17.7 -24.5 -31.8 -38.5 Day 15 21.2 -27.4 -12.8 -50.8 -46.2 Day 22
12.2 -18.3 -9.8 -25.1 -53.8 Day 23 51.8 -4.0 1.1 -41.0 -44.2 Day 29
28.5 -19.5 -25.0 -43.1 -43.8 Day 36 15.4 -33.1 -34.8 -17.9 -36.5
Day 50 33.1 48.8 -23.9 -48.2 -48.1
[0466] Median percent change from baseline values in the 50, 100,
200 and 400 mg multiple-dose groups showed reductions of total
apoC-III of 20, 17, 71, and 78% and of TG of 20, 25, 43, and 44%,
respectively, one week (Day 29) after the last dose. Reductions
were sustained for at least four weeks after the last dose in the
higher dose groups.
[0467] TG levels spiked at Day 5 and 23 for the placebo group,
coinciding with the subjects' overnight stays at the Study Center.
It is thought that the diet provided by the Study Center led to the
surge in TG levels in the subjects staying overnight at the Study
Center. ISIS 304801 decreased the TG spike in a dose-dependent
manner. In a manner, the results shown herein, indicate a
postprandial effect on TG (although TG levels were assessed after a
12 hour fast) by ISIS 304801 as a diet induced surge in TG was
decreased in a dose-dependent manner by ISIS 304801.
[0468] LDL-C values did not change (data not shown) while HDL-C
values tended to increase in a treatment-dependent manner as shown
in Table 37.
TABLE-US-00037 TABLE 37 No Deleterious Effects on HDL-C Study Day
Placebo 50 mg 100 mg 200 mg 400 mg Day 5 -5.9 7.7 -3.1 -3.2 -16.1
Day 8 2.1 2.4 0.0 -11.1 -2.9 Day 15 -2.8 4.8 10.9 4.7 -9.7 Day 22
-0.6 4.8 8.7 11.6 -2.0 Day 23 0.7 7.1 12.5 19.4 -1.6 Day 29 2.1
19.0 0.0 13.9 8.0 Day 36 0.0 23.8 8.8 13.9 1.6 Day 50 -4.8 16.7 5.9
25.0 14.5
Example 11: ISIS 304801 Phase II Clinical Trial
[0469] A randomized, double-blind, placebo-controlled, dose
response study is planned to evaluate the dose/response
pharmacodynamic effects of ISIS 304801 vs. placebo on fasting
apoC-III associated with VLDL levels. Additional endpoints to
evaluate include: the pharmacodynamic effects of ISIS 304801 vs.
placebo on fasting total apoC-III, TG, apoC-II (total and
associated with VLDL), apolipoprotein B-100 (apoB-100),
apolipoprotein A-1 (apoA-1), apolipoprotein A-2 (apoA-2),
apolipoprotein E (apoE), total cholesterol (TC), low-density
lipoprotein-cholesterol (LDL-C), LDL-TG, VLDL-C, VLDL-TG,
non-high-density lipoprotein-cholesterol (non-HDL-C), non-HDL-TG,
HDL-C, HDL-TG, chylomicron-C(CM-C), CM-TG, free fatty acids (FFA),
and glycerol levels; the post-prandial lipid, apolipoprotein and
lipoprotein characteristics and kinetics, and glucose levels in a
subset of the patients in the study and assess more extensive PK in
another subset of the patients (will not be the same patients as
those undergoing the post-prandial assessment); and, the safety,
tolerability and PK of ISIS 304801.
[0470] For each patient, the participation period consists of a
.ltoreq.5-week screening period, (which includes a 4-week tight
diet control run-in qualification period), a 1-week study
qualification/baseline assessment period, a 13-week treatment
period, and a post-treatment evaluation period of 13 weeks, for a
total of 32 weeks of study participation. Concomitant medications
and adverse events (AEs) will be recorded throughout all periods of
the study.
[0471] Patients will be at least 18 years of age and have fasting
TG.gtoreq.500 mg/dL at screening and fasting TG.gtoreq.300 mg/dL
and .ltoreq.2000 mg/dL after 4-weeks of tight diet control
run-in.
[0472] Seventy two (72) patients are planned for this study. There
will be 24 patients planned per dose cohort (100, 200, 300 mg) with
18 ISIS 304801 (active) and 6 placebo patients per cohort. Eligible
patients will be enrolled equally (1:1) into a non-extensive
PK/post-prandial group (Group 1) or an extensive PK/post-prandial
group (Group 2). Patients in Group 2 will be randomized equally
(1:1) to an extensive PK group (Group 2a) or a post-prandial
assessment group (Group 2b). Group 2a patients will be randomized
equally (1:1:1) to 1 of the 3 dose cohorts (100, 200, 300 mg) and,
within each dose cohort, 5:1 to receive active or placebo. Group 2b
patients will be randomized equally (1:1) to 1 of 2 dose cohorts
(200, 300 mg) and, within each dose cohort, 2:1 to receive active
or placebo. Group 1 patients will be randomized to dose cohort and
treatment in a manner that achieves an overall study randomization
of 1:1:1 to dose cohort (100, 200, 300 mg) and 3:1 to treatment
(active, placebo).
[0473] Patients will be placed on a tightly controlled diet (after
screening procedures are performed) for the duration of study
participation. After 28 days on the controlled diet, patients will
have baseline measurements and be assessed for qualification of
enrollment into the treatment phase of the study. Patients who meet
the enrollment criteria following diet run-in will be enrolled
equally (1:1) into a non-extensive PK/post-prandial group (Group 1)
or into an extensive PK/post-prandial group (Group 2) and
randomized within their Group assignment.
Study Drug and Treatment
[0474] A solution of ISIS 304801 (200 mg/mL, 1.0 mL) contained in
2-mL stoppered glass vials will be provided.
[0475] The placebo for this study will be 0.9% sterile saline.
ISIS 304801 solution and placebo will be prepared by an unblinded
pharmacist (or qualified delegate). Vials are for single-use only.
A trained professional, blinded to the identity of the drug, will
administer the Study Drug. The Study Drug will be administered as a
SC injection in the abdomen, thigh, or outer area of the upper arm
on each dosing day. Doses of 100 and 200 mg will be administered as
a single SC injection. Doses of 300 mg will be administered as two
equal volume noncontiguous SC injections.
[0476] Patients will receive 13 doses of Study Drug administered by
SC injection once a week for 13 weeks (Days 1, 8, 15, 22, 29, 36,
43, 50, 57, 64, 71, 78, and 85).
[0477] Patients will complete the treatment visits on Day 1.+-.0
days and on Day 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85
within .+-.1 day. Patients in the extensive PK group will also
visit the clinic on Day 2 and 86.+-.0 days relative to Day 1 and
85, respectively, for the 24 hour blood draw. Patients will
complete the follow-up visits on Day 92 and 99 within .+-.1 day,
Day 127 within .+-.3 days, and Day 176 within .+-.5 days of the
scheduled visit date. Patients in the post-prandial assessment
group will also visit the clinic on Day 103 within .+-.2 days and
on the day following the Day 103 visit for the 24 hour blood
draw.
[0478] Preceding each visit that includes a blood draw for
pharmacodynamic measurements (Days 8, 15, 29, 43, 57, 71, and 85),
patients will be provided a standardized pre-cooked meal for the
dinner on the evening prior to their visit (to ensure equal
moderation of fat intake, per patient and per time point) after
which they will remain fasted. Alcohol consumption will not be
allowed for 48 hrs preceding these clinic visits.
[0479] Blood will be collected for measurement of VLDL apoC-III and
other pharmacodynamic markers on Days 8, 15, 29, 43, 57, 71, and 85
(prior to Study Drug administration).
[0480] Patients in the post-prandial assessment group will consume
standardized pre cooked meals (lunches and dinners (provided) and
instructions for breakfasts and snacks) for the 2 days prior to the
post-prandial evaluations. On each of the post-prandial evaluation
days, following the blood draws, patients will consume a
standardized liquid meal, which represents about a third of the
daily caloric requirements, with a stable radioisotope tracer,
followed by serial blood sampling. Patients will receive a
standardized pre-cooked meal 9 hrs after consuming the liquid meal,
after which they will fast until the 24 hour blood draw the
following day.
[0481] In addition to trough sample collection, patients in the
extensive PK assessment group will undergo serial blood sampling
for 24 hrs after their first (Day 1-2) and last (Day 85-86) dose of
Study Drug.
Post-Treatment Evaluation Period
[0482] Patients will be followed until Study Day 176. During this
time, patients will return to the study center for outpatient
clinic visits on Study Days 92, 99, 127, and 176 (and Day 103 for
patients in the post-prandial assessment group) for safety and
clinical laboratory evaluations (blood draws), diet counseling and
monitoring, concomitant medication usage recording, and AE event
collection.
[0483] Blood samples for PK and PD analysis will be collected
periodically throughout the post-treatment evaluation period.
Laboratory measurements of serum chemistry, urinalysis,
coagulation, complement, hematology, immune function, thyroid
function, and full lipid panel will be performed at the various
times throughout the study.
[0484] Post-prandial assessments will be done in a subset of
patients as described below.
Post-Prandial Meal, Sampling Schedule, and Assessment
[0485] Post-prandial assessment for lipoproteins metabolism will be
performed using a radiolabelled meal supplemented with a labeled
tracer, 3H-palmitate (300 .mu.Ci, Perkin Elmer Inc., Woodbridge,
ON, Canada), sonicated into the liquid meal. Palmitate is a fatty
acid that is a common constituent of any diet. The 3H-palmitate
tracer emits weak radioactivity, equivalent to an X-ray. Since
dietary palmitate is incorporated into chylomicrons as they are
formed in the enterocytes of the gut, this enables monitoring the
appearance and clearance of newly-formed chylomicrons from
circulation. The methodology to be applied for studying
post-prandial kinetics of chylomicrons appearance and clearance is
well-established (Mittendorfer et al. 2003, Diabetes, 52:
1641-1648; Bickerton et al. 2007; Normand-Lauziere et al. 2010,
PLoS. One, 5: e10956).
[0486] A liquid meal (similar to a milkshake) containing a small
amount (300 .mu.Ci) of radiolabelled fatty acids (3H-palmitate)
will be provided. The liquid meal will provide about a third of the
daily caloric requirements. From 1 hr prior to 9 hrs after the
ingestion of the meal, a constant infusion of [U-13C]-K palmitate
(0.01 .mu.mol/kg/min in 100 ml 25% human serum albumin; Cambridge
Isotopes Laboratories Inc., Andover, Mass.) and a primed (1.6
.mu.mol/kg) continuous (0.05 .mu.mol/kg/min) infusion of
[1,1,2,3,3-2H]-glycerol (Cambridge Isotopes Laboratories Inc.) will
be administered as previously described (Normand-Lauziere et al.
2010, PLoS. One, 5: e10956). Plasma palmitate and glycerol
appearance rates will be calculated using Steele's non-steady state
equation assuming a volume of distribution of 90 ml/kg and 230
ml/kg, respectively (Gastaldelli et al. 1999, J Appl. Physiol, 87:
1813-1822).
[0487] Blood samples will be drawn at intervals before and after
the ingestion of the radiolabelled meal on days prior to and after
the Treatment phase as noted in the table below. A standardized
meal will be given to the participants after the 9 hr blood draw.
Blood will be collected in tubes containing Na2 EDTA and Orlistat
(30 .mu.g/ml, Roche, Mississauga, Canada) to prevent in vitro
triacylglycerol lipolysis and separate samples will be collected in
NaF tubes for plasma glucose determination.
[0488] The following will be measured at each time-point: [0489]
Plasma and CM fraction levels for 3H-tracer [0490] Plasma [U-13C]-K
palmitate and [1, 1, 2, 3, 3-2H]-glycerol appearance rates [0491]
Plasma and CM fraction levels for TG, TC, and apoB [0492] Plasma
and VLDL fraction levels for apo CIII, apo CII, and apo E [0493]
Plasma levels for glucose
[0494] Plasma samples may also be used for profiling of drug
binding proteins, bioanalytical method validation purposes,
stability and metabolite assessments, or to assess other actions of
ISIS 304801 with plasma constituents.
Sequence CWU 1
1
51533DNAHomo sapiens 1tgctcagttc atccctagag gcagctgctc caggaacaga
ggtgccatgc agccccgggt 60actccttgtt gttgccctcc tggcgctcct ggcctctgcc
cgagcttcag aggccgagga 120tgcctccctt ctcagcttca tgcagggtta
catgaagcac gccaccaaga ccgccaagga 180tgcactgagc agcgtgcagg
agtcccaggt ggcccagcag gccaggggct gggtgaccga 240tggcttcagt
tccctgaaag actactggag caccgttaag gacaagttct ctgagttctg
300ggatttggac cctgaggtca gaccaacttc agccgtggct gcctgagacc
tcaatacccc 360aagtccacct gcctatccat cctgcgagct ccttgggtcc
tgcaatctcc agggctgccc 420ctgtaggttg cttaaaaggg acagtattct
cagtgctctc ctaccccacc tcatgcctgg 480cccccctcca ggcatgctgg
cctcccaata aagctggaca agaagctgct atg 53323964DNAHomo sapiens
2ctactccagg ctgtgttcag ggcttggggc tggtggaggg aggggcctga aattccagtg
60tgaaaggctg agatgggccc gaggcccctg gcctatgtcc aagccatttc ccctctcacc
120agcctctccc tggggagcca gtcagctagg aaggaatgag ggctccccag
gcccaccccc 180agttcctgag ctcatctggg ctgcagggct ggcgggacag
cagcgtggac tcagtctcct 240agggatttcc caactctccc gcccgcttgc
tgcatctgga caccctgcct caggccctca 300tctccactgg tcagcaggtg
acctttgccc agcgccctgg gtcctcagtg cctgctgccc 360tggagatgat
ataaaacagg tcagaaccct cctgcctgtc tgctcagttc atccctagag
420gcagctgctc caggtaatgc cctctgggga ggggaaagag gaggggagga
ggatgaagag 480gggcaagagg agctccctgc ccagcccagc cagcaagcct
ggagaagcac ttgctagagc 540taaggaagcc tcggagctgg acgggtgccc
cccacccctc atcataacct gaagaacatg 600gaggcccggg aggggtgtca
cttgcccaaa gctacacagg gggtggggct ggaagtggct 660ccaagtgcag
gttcccccct cattcttcag gcttagggct ggaggaagcc ttagacagcc
720cagtcctacc ccagacaggg aaactgaggc ctggagaggg ccagaaatca
cccaaagaca 780cacagcatgt tggctggact ggacggagat cagtccagac
cgcaggtgcc ttgatgttca 840gtctggtggg ttttctgctc catcccaccc
acctcccttt gggcctcgat ccctcgcccc 900tcaccagtcc cccttctgag
agcccgtatt agcagggagc cggcccctac tccttctggc 960agacccagct
aaggttctac cttaggggcc acgccacctc cccagggagg ggtccagagg
1020catggggacc tggggtgccc ctcacaggac acttccttgc aggaacagag
gtgccatgca 1080gccccgggta ctccttgttg ttgccctcct ggcgctcctg
gcctctgccc gtaagcactt 1140ggtgggactg ggctgggggc agggtggagg
caacttgggg atcccagtcc caatgggtgg 1200tcaagcagga gcccagggct
cgtccagagg ccgatccacc ccactcagcc ctgctctttc 1260ctcaggagct
tcagaggccg aggatgcctc ccttctcagc ttcatgcagg gttacatgaa
1320gcacgccacc aagaccgcca aggatgcact gagcagcgtg caggagtccc
aggtggccca 1380gcaggccagg tacacccgct ggcctccctc cccatccccc
ctgccagctg cctccattcc 1440cacccgcccc tgccctggtg agatcccaac
aatggaatgg aggtgctcca gcctcccctg 1500ggcctgtgcc tcttcagcct
cctctttcct cacagggcct ttgtcaggct gctgcgggag 1560agatgacaga
gttgagactg cattcctccc aggtccctcc tttctccccg gagcagtcct
1620agggcgtgcc gttttagccc tcatttccat tttcctttcc tttccctttc
tttctctttc 1680tatttctttc tttctttctt tctttctttc tttctttctt
tctttctttc tttctttctt 1740tctttctttc ctttctttct ttcctttctt
tctttccttt ctttctttct ttcctttctt 1800tctctttctt tctttctttc
ctttttcttt ctttccctct cttcctttct ctctttcttt 1860cttcttcttt
tttttttaat ggagtctccc tctgtcacct aggctggagt gcagtggtgc
1920catctcggct cactgcaacc tccgtctccc gggttcaacc cattctcctg
cctcagcctc 1980ccaagtagct gggattacag gcacgcgcca ccacacccag
ctaatttttg tatttttagc 2040agagatgggg tttcaccatg ttggccaggt
tggtcttgaa ttcctgacct caggggatcc 2100tcctgcctcg gcctcccaaa
gtgctgggat tacaggcatg agccactgcg cctggcccca 2160ttttcctttt
ctgaaggtct ggctagagca gtggtcctca gcctttttgg caccagggac
2220cagttttgtg gtggacaatt tttccatggg ccagcgggga tggttttggg
atgaagctgt 2280tccacctcag atcatcaggc attagattct cataaggagc
cctccaccta gatccctggc 2340atgtgcagtt cacaataggg ttcacactcc
tatgagaatg taaggccact tgatctgaca 2400ggaggcggag ctcaggcggt
attgctcact cacccaccac tcacttcgtg ctgtgcagcc 2460cggctcctaa
cagtccatgg accagtacct atctatgact tgggggttgg ggacccctgg
2520gctaggggtt tgccttggga ggccccacct gacccaattc aagcccgtga
gtgcttctgc 2580tttgttctaa gacctggggc cagtgtgagc agaagtgtgt
ccttcctctc ccatcctgcc 2640cctgcccatc agtactctcc tctcccctac
tcccttctcc acctcaccct gactggcatt 2700agctggcata gcagaggtgt
tcataaacat tcttagtccc cagaaccggc tttggggtag 2760gtgttatttt
ctcactttgc agatgagaaa attgaggctc agagcgatta ggtgacctgc
2820cccagatcac acaactaatc aatcctccaa tgactttcca aatgagaggc
tgcctccctc 2880tgtcctaccc tgctcagagc caccaggttg tgcaactcca
ggcggtgctg tttgcacaga 2940aaacaatgac agccttgacc tttcacatct
ccccaccctg tcactttgtg cctcaggccc 3000aggggcataa acatctgagg
tgacctggag atggcagggt ttgacttgtg ctggggttcc 3060tgcaaggata
tctcttctcc cagggtggca gctgtggggg attcctgcct gaggtctcag
3120ggctgtcgtc cagtgaagtt gagagggtgg tgtggtcctg actggtgtcg
tccagtgggg 3180acatgggtgt gggtcccatg gttgcctaca gaggagttct
catgccctgc tctgttgctt 3240cccctgactg atttaggggc tgggtgaccg
atggcttcag ttccctgaaa gactactgga 3300gcaccgttaa ggacaagttc
tctgagttct gggatttgga ccctgaggtc agaccaactt 3360cagccgtggc
tgcctgagac ctcaataccc caagtccacc tgcctatcca tcctgcgagc
3420tccttgggtc ctgcaatctc cagggctgcc cctgtaggtt gcttaaaagg
gacagtattc 3480tcagtgctct cctaccccac ctcatgcctg gcccccctcc
aggcatgctg gcctcccaat 3540aaagctggac aagaagctgc tatgagtggg
ccgtcgcaag tgtgccatct gtgtctgggc 3600atgggaaagg gccgaggctg
ttctgtgggt gggcactgga cagactccag gtcaggcagg 3660catggaggcc
agcgctctat ccaccttctg gtagctgggc agtctctggg cctcagtttc
3720ttcatctcta aggtaggaat caccctccgt accctgcctt ccttgacagc
tttgtgcgga 3780aggtcaaaca ggacaataag tttgctgata ctttgataaa
ctgttaggtg ctgcacaaca 3840tgacttgagt gtgtgcccca tgccagccac
tatgcctggc acttaagttg tcatcagagt 3900tgagactgtg tgtgtttact
caaaactgtg gagctgacct cccctatcca ggccccctag 3960ccct
3964320DNAArtificial SequenceSynthetic oligonucleotide 3agcttcttgt
ccagctttat 20420DNAArtificial SequenceSynthetic oligonucleotide
4ccttccctga aggttcctcc 205524DNAMus musculus 5ctgctcagtt ttatccctag
aagcagctag ctactccagg tacgtaggtg ccatgcagcc 60ccggacgctc ctcactgtgg
ccctcttggc tctcctggca tctgcccgag ctgaagaggt 120agagggatcc
ttgctgctgg gctctgtaca gggctacatg gaacaagcct ccaagacggt
180ccaggatgcg ctaagtagcg tgcaggagtc cgatatagct gtggtggcca
ggggctggat 240ggacaatcac ttcagatccc tgaaaggcta ctggagcaag
tttactgaca agttcaccgg 300cttctgggat tctaaccctg aggaccaacc
aactccagct attgagtcgt gagacttctg 360tgttgcagat gtgcctgttc
ctccatcctg ctgcccccct ccaggcctgc caggtggccc 420ctgaaggttg
ctttaagggg aaagtatgtt ctcatgtctt cacccctccc tagatctcac
480ctaaacatgc tgtccctaat aaagctggat aagaagctgc tgtt 524
* * * * *