U.S. patent application number 17/372173 was filed with the patent office on 2022-07-07 for modulation of apolipoprotein c-iii (apociii) expression in lipoprotein lipase deficient (lpld) populations.
This patent application is currently assigned to Ionis Pharmaceuticals, Inc.. The applicant listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to Veronica J. Alexander, Nicholas J. Viney, Joseph L. Witztum.
Application Number | 20220213472 17/372173 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220213472 |
Kind Code |
A1 |
Alexander; Veronica J. ; et
al. |
July 7, 2022 |
MODULATION OF APOLIPOPROTEIN C-III (APOCIII) EXPRESSION IN
LIPOPROTEIN LIPASE DEFICIENT (LPLD) POPULATIONS
Abstract
Provided herein are methods, compounds, and compositions for
reducing expression of ApoCIII mRNA and protein in a patient with
Fredrickson Type I dyslipidemia, FCS, LPLD. Also provided herein
are methods, compounds, and compositions for treating, preventing,
delaying, or ameliorating Fredrickson Type I dyslipidemia, FCS,
LPLD, in a patient. Further 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
a patient with Fredrickson Type I dyslipidemia, FCS, LPLD. Such
methods, compounds, and compositions are useful to treat, prevent,
delay, or ameliorate any one or more of pancreatitis,
cardiovascular disease or metabolic disorder, or a symptom
thereof.
Inventors: |
Alexander; Veronica J.; (San
Diego, CA) ; Viney; Nicholas J.; (Carlsbad, CA)
; Witztum; Joseph L.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
Carlsbad
CA
|
Appl. No.: |
17/372173 |
Filed: |
July 9, 2021 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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16284998 |
Feb 25, 2019 |
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17372173 |
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15415408 |
Jan 25, 2017 |
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16284998 |
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14768180 |
Aug 14, 2015 |
9593333 |
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PCT/US2014/016546 |
Feb 14, 2014 |
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15415408 |
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61880779 |
Sep 20, 2013 |
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61764969 |
Feb 14, 2013 |
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International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/7088 20060101 A61K031/7088; A61K 45/06
20060101 A61K045/06 |
Claims
1.-34. (canceled)
35. A method of treating familial chylomicronemia syndrome (FCS)
comprising administering to an affected individual a
therapeutically effective amount of a compound comprising a
modified oligonucleotide having nucleobase sequence SEQ ID NO: 3,
or a pharmaceutically acceptable salt thereof; wherein the modified
oligonucleotide comprises: (a) a gap segment consisting of 10
linked deoxynucleosides, (b) a 5' wing segment consisting of 5
linked nucleosides, and (c) a 3' wing segment consisting of 5
linked nucleosides, wherein the gap segment is positioned
immediately adjacent to and between the 5' wing segment and the 3'
wing segment, each nucleoside of each wing segment comprises a
2'-O-methyoxyethyl sugar, each cytosine is a 5'-methylcytosine, and
each internucleoside linkage is a phosphorothioate linkage; and
wherein fasting triglyceride levels are reduced by at least forty
percent (40%) from baseline level in the individual; thereby
treating familial chylomicronemia syndrome (FCS) in the affected
individual.
36. The method of claim 35 wherein the compound is parenterally
administered.
37. The method of claim 36, wherein the administration is
subcutaneous administration.
38. The method of claim 35, wherein the compound is in the form of
a sodium salt.
39. The method of claim 35, wherein the compound is administered as
a composition further comprising a pharmaceutically acceptable
carrier or diluent.
40. The method of claim 35, further comprising administering a
second agent or therapy.
41. The method of claim 40, wherein the second agent is selected
from an ApoCIII lowering agent, a non-HDL lipid lowering agent, an
LDL lowering agent, a TG lowering agent, a cholesterol lowering
agent, an HDL raising agent, fish oil, niacin, a fibrate, a statin,
DCCR (salt of diazoxide), a glucose-lowering agent or an
anti-diabetic agent.
42. The method of claim 40, wherein the second therapy is dietary
fat restriction.
43. The method of claim 35, wherein fasting triglyceride levels are
reduced by at least forty five percent (45%), at least fifty
percent (50%), at least sixty percent (60%), at least seventy
percent (70%), or at least eighty percent (80%) from baseline level
in the individual.
44. The method of claim 35, wherein fasting triglyceride levels are
reduced by at least seventy percent (70%) from baseline level in
the individual.
45. A method of treating familial chylomicronemia syndrome (FCS)
comprising administering to an affected individual a
therapeutically effective amount of a compound comprising a
modified oligonucleotide having nucleobase sequence SEQ ID NO: 3,
or a pharmaceutically acceptable salt thereof, wherein the modified
oligonucleotide comprises: (a) a gap segment consisting of 10
linked deoxynucleosides, (b) a 5' wing segment consisting of 5
linked nucleosides, and (c) a 3' wing segment consisting of 5
linked nucleosides, wherein the gap segment is positioned
immediately adjacent to and between the 5' wing segment and the 3'
wing segment, each nucleoside of each wing segment comprises a
2'-O-methyoxyethyl sugar, each cytosine is a 5'-methylcytosine, and
each internucleoside linkage is a phosphorothioate linkage; and
wherein fasting triglyceride levels are reduced to less than or
equal to seven hundred fifty mg per dL (.ltoreq.750 mg/dL) in the
individual; thereby treating familial chylomicronemia syndrome
(FCS) in the affected individual.
46. The method of claim 45 wherein the compound is parenterally
administered.
47. The method of claim 46, wherein the administration is
subcutaneous administration.
48. The method of claim 45, wherein the compound is in the form of
a sodium salt.
49. The method of claim 45, wherein the compound is administered as
a composition further comprising a pharmaceutically acceptable
carrier or diluent.
50. The method of claim 45, further comprising administering a
second agent or therapy.
51. The method of claim 40, wherein the second agent is selected
from an ApoCIII lowering agent, a non-HDL lipid lowering agent, an
LDL lowering agent, a TG lowering agent, a cholesterol lowering
agent, an HDL raising agent, fish oil, niacin, a fibrate, a statin,
DCCR (salt of diazoxide), a glucose-lowering agent or an
anti-diabetic agent.
52. The method of claim 50, wherein the second therapy is dietary
fat restriction.
53. The method of claim 45, wherein fasting triglyceride levels are
reduced to .ltoreq.750 mg/dL, .ltoreq.700 mg/dL, .ltoreq.650 mg/dL,
.ltoreq.600 mg/dL, .ltoreq.550 mg/dL, .ltoreq.500 mg/dL,
.ltoreq.450 mg/dL, .ltoreq.400 mg/dL, .ltoreq.350 mg/dL,
.ltoreq.300 mg/dL, .ltoreq.250 mg/dL, or .ltoreq.200 mg/dL in the
individual.
54. The method of claim 45, wherein fasting triglyceride levels are
reduced to .ltoreq.400 mg/dL in the individual.
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format.
[0002] The Sequence Listing is provided as a file entitled
BIOL0218USC3SEQ_ST25.txt, created on Jul. 7, 2021 which is 16 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
[0003] Provided herein are methods, compounds, and compositions for
reducing expression of Apolipoprotein C-III (ApoCIII) mRNA and
protein, reducing triglyceride levels and increasing high density
lipoprotein (HDL) levels or HDL activity in Fredrickson Type I
dyslipidemia patients. Also, provided herein are compounds and
compositions for use in treating Fredrickson Type I dyslipidemia or
associated disorders thereof.
BACKGROUND
[0004] 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
[0005] Apolipoprotein C-III (also called APOC3, APOC-III, ApoCIII,
and APO C-III) is a constituent of HDL and of triglyceride
(TG)-rich lipoproteins. Elevated ApoCIII is associated with
elevated 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 at., Atherosclerosis, 2003,
168:81-89; Mendivil et al., Circulation, 2011, 124:2065-2072;
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;
Davidsson et al., J. Lipid Res. 2005. 46: 1999-2006; Sacks et al.,
Circulation, 2000. 102: 1886-1892; Lee et al., Arterioscler Thromb
Vasc Biol, 2003. 23: 853-858). ApoCIII slows clearance of TG-rich
lipoproteins by inhibiting lipolysis, both through inhibition of
lipoprotein lipase (LPL) and by interfering with lipoprotein
binding to cell-surface glycosaminoglycan matrix (Shachter, Curr.
Opin. Lipidol, 2001, 12, 297-304). As ApoCIII inhibits LPL leading
to a decrease in lipolysis of TGs, it would be unexpected that
inhibition of ApoCIII would have a beneficial effect in LPL
deficient (LPLD) subjects.
[0006] LPLD is characterized by the inability of affected
individuals to produce functionally active LPL. LPL is mainly
produced in skeletal muscle, fat tissue, and heart muscle and has
multiple key functions, among which is the catabolism of TG-rich
lipoproteins (e.g. VLDL) and chylomicrons (CM). Off-loading TG from
CM (and VLDL) normally protects against excessive postprandial rise
in CM mass and TG. In LPLD, LPL is dysfunctional and more than 12
hours after meals hyperTG and chylomicronaemia are still present
and visible as lipemia.
[0007] The Fredrickson system is used to classify primary (genetic)
causes of dyslipidemia such as hypertriglyceridemia in patients.
Fredrickson Type I (also known as LPLD or Familial Chylomicronemia
Syndrome (FCS)) 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 (i.e.
LPLD). Patients have mutations that are either homozygous (having
the same mutation on each allele) or compound heterozygous (having
different mutations on each allele). 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.
[0008] Currently, Fredrickson Type I, FCS, LPLD, patients respond
minimally, or not at all, to TG-lowering drugs such as statins,
fibrates and nicotinic acid (Tremblay et al., J Clin Lipidol, 2011,
5:37-44; Brisson et al., Pharmacogenet Genom, 2010, 20:742-747).
Clinical management of Fredrickson Type I, FCS, LPLD, patients
generally consist of severe reduction in all dietary fat to much
less than 20% of caloric intake and the use of medium-chain TG,
which are absorbed via the portal system and therefore do not
directly enter into plasma. Such a life-long dietary regimen
presents significant compliance issues for patients. Even when
patients are compliant to the diet and are tightly followed in a
lipid clinic by a dietician and a medical team, TGs often do not
decrease below the threshold of increased pancreatitis risk.
Recently, a gene therapy product (Glybera.sup.R) has been approved
in Europe for treating adult LPLD patients suffering from severe or
multiple pancreatitis attacks despite dietary fat restrictions.
Patients treated with Glybera.sup.R require administration of an
immunosuppressive drug prior to and following Glybera.sup.R
treatment. Glybera.sup.R will only be offered through dedicated
centers with expertise in treating LPLD and by specially trained
doctors to ensure ongoing safety of the treatment
(http://www.uniqure.com/products/glybera/).
[0009] Accordingly, there is still a need to provide patients with
Fredrickson Type I dyslipidemia, FCS, LPLD, novel treatment
options. Antisense technology is emerging as an effective means for
reducing the expression of certain gene products and may prove to
be uniquely useful in a number of therapeutic, diagnostic, and
research applications for the modulation of ApoCIII. 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), WO 2004/093783 and WO
2012/149495, all incorporated-by-reference herein. An antisense
oligonucleotide targeting ApoCIII has been tested in a Phase I
clinical trial and was shown to be safe. Currently, an antisense
oligonucleotide targeting ApoCIII is in Phase II clinical trials to
assess its effectiveness in the treatment of diabetes or
hypertriglyceridemia.
SUMMARY OF THE INVENTION
[0010] Certain embodiments provide a method of treating,
preventing, delaying or ameliorating Fredrickson Type I
dyslipidemia, FCS, LPLD, comprising administering a therapeutically
effective amount of a compound comprising an ApoCIII specific
inhibitor to the animal. Certain embodiments provide an ApoCIII
specific inhibitor for use in treating, preventing, delaying or
ameliorating Fredrickson Type I dyslipidemia, FCS, LPLD.
[0011] Certain embodiments provide a method of reducing
triglyceride levels in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, comprising administering a therapeutically
effective amount of a compound comprising an ApoCIII specific
inhibitor to the animal.
[0012] Certain embodiments provide a method of increasing HDL
levels and/or improving the ratio of TG to HDL in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal.
[0013] Certain embodiments provide a method of preventing, delaying
or ameliorating a cardiovascular and/or metabolic disease,
disorder, condition, or symptom thereof, in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal.
[0014] Certain embodiments provide a method of preventing, delaying
or ameliorating pancreatitis, or symptom thereof, in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal.
[0015] In certain embodiments, the ApoCIII specific inhibitor is a
nucleic acid, peptide, antibody, small molecule or other agent
capable of inhibiting the expression of ApoCIII. In certain
embodiments, the nucleic acid is an antisense compound. In certain
embodiments, the antisense compound is an oligonucleotide targeting
ApoCIII. In certain embodiments, the oligonucleotide is a modified
oligonucleotide targeting ApoCIII. In certain embodiments, the
modified oligonucleotide has a nucleobase sequence comprising at
least 8 contiguous nucleobases of a nucleobase sequence of SEQ ID
NO: 3. In certain embodiments, the modified oligonucleotide
consists of the nucleobase sequence of SEQ ID NO: 3.
[0016] Certain embodiments provide a method of reducing
triglyceride levels in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, comprising administering to the animal a
therapeutically effective amount of a compound comprising a
modified oligonucleotide having the sequence of 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, and 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-methyoxyethyl sugar, wherein each cytosine
is a 5'-methylcytosine, and wherein each internucleoside linkage is
a phosphorothioate linkage.
[0017] Certain embodiments provide a method of increasing HDL
levels and/or improving the ratio of TG to HDL in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, by administering to the
animal a therapeutically effective amount of a compound comprising
a modified oligonucleotide having the sequence of 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, and 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-methyoxyethyl sugar, wherein each cytosine
is a 5'-methylcytosine, and wherein each internucleoside linkage is
a phosphorothioate linkage.
[0018] Certain embodiments provide a method of preventing, delaying
or ameliorating a cardiovascular and/or metabolic disease,
disorder, condition, or symptom thereof, in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, by administering to the
animal a therapeutically effective amount of a compound comprising
a modified oligonucleotide having the sequence of 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, and 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-methyoxyethyl sugar, wherein each cytosine
is a 5'-methylcytosine, and wherein each internucleoside linkage is
a phosphorothioate linkage.
[0019] Certain embodiments provide a method of preventing, delaying
or ameliorating pancreatitis or symptom thereof, in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, by administering to the
animal a therapeutically effective amount of a compound comprising
a modified oligonucleotide having the sequence of 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, and 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-methyoxyethyl sugar, wherein each cytosine
is a 5'-methylcytosine, and wherein each internucleoside linkage is
a phosphorothioate linkage.
[0020] In certain embodiments, the ApoCIII specific inhibitor is a
nucleic acid, peptide, antibody, small molecule or other agent
capable of inhibiting the expression of ApoCIII. In certain
embodiments, the nucleic acid is an antisense compound targeting
ApoCIII. In certain embodiments, the antisense compound is an
antisense oligonucleotide. In certain embodiments, the antisense
oligonucleotide is a modified oligonucleotide. In certain
embodiments, the modified oligonucleotide has a nucleobase sequence
comprising at least 8 contiguous nucleobases of ISIS 304801,
AGCTTCTTGTCCAGCTTTAT (SEQ ID NO: 3). In certain embodiments, the
modified oligonucleotide is at least 70%, at least 75%, 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, SEQ ID NO: 2 or SEQ ID
NO: 4.
DETAILED DESCRIPTION OF THE INVENTION
[0021] 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.
[0022] 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
[0023] 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.
[0024] Unless otherwise indicated, the following terms have the
following meanings:
[0025] "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.
[0026] "2'-O-methoxyethyl nucleotide" means a nucleotide comprising
a 2'-O-methoxyethyl modified sugar moiety.
[0027] "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.
[0028] "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.
[0029] "5-methylcytosine" means a cytosine modified with a methyl
group attached to the 5' position. A 5-methylcytosine is a modified
nucleobase.
[0030] "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%.
[0031] "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.
[0032] "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.
[0033] "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.
[0034] "Administering" means providing a pharmaceutical agent to an
individual, and includes, but is not limited to administering by a
medical professional and self-administering.
[0035] "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.
[0036] "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.
[0037] "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.
[0038] "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.
[0039] "Antisense compound" means an oligomeric compound that is
capable of undergoing hybridization to a target nucleic acid
through hydrogen bonding. Examples of antisense compounds include
single-stranded and double-stranded compounds, such as, antisense
oligonucleotides, siRNAs, shRNAs, ssRNAi and occupancy-based
compounds.
[0040] "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.
[0041] "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.
[0042] "ApoA5", "Apolipoprotein A-V" or "ApoA-V" means any nucleic
acid or protein sequence encoding ApoA5.
[0043] "ApoCII", "Apolipoprotein C-II" or "ApoC2" means any nucleic
acid or protein sequence encoding ApoCII. The ApoCII protein is a
component of chylomicrons and VLDL particles and activates LPL to
hydrolyze TGs.
[0044] "ApoCIII", "Apolipoprotein C-III" or "ApoC3" 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.
[0045] "ApoCIII specific inhibitor" refers to any agent capable of
specifically inhibiting the expression of ApoCIII mRNA and/or the
expression or activity of ApoCIII protein at the molecular level.
For example, ApoCIII specific inhibitors include nucleic acids
(including antisense compounds), peptides, antibodies, small
molecules, and other agents capable of inhibiting the expression of
ApoCIII mRNA and/or ApoCIII protein. In certain embodiments, the
nucleic acid is an antisense compound. In certain embodiments, the
antisense compound is a an oligonucleotide targeting ApoCIII. In
certain embodiments, the oligonucleotide targeting ApoCIII is a
modified oligonucleotide targeting ApoCIII. In certain embodiments,
the oligonucleotide targeting ApoCIII has a sequence as shown in
SEQ ID NO: 3 or another sequence, for example, such as those
disclosed in U.S. Pat. Nos. 7,598,227, 7,750,141, PCT Publication
WO 2004/093783 or WO 2012/149495, all incorporated-by-reference
herein. In certain embodiments, by specifically modulating ApoCIII
mRNA level and/or ApoCIII protein expression, ApoCIII specific
inhibitors may affect components of the lipogenic pathway.
Similarly, in certain embodiments, ApoCIII specific inhibitors may
affect other molecular processes in an animal.
[0046] "ApoCIII mRNA" means a mRNA encoding an ApoCIII protein.
[0047] "ApoCIII protein" means any protein sequence encoding
ApoCIII.
[0048] "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.
[0049] "Bicyclic sugar" means a furosyl ring modified by the
bridging of two non-geminal ring atoms. A bicyclic sugar is a
modified sugar.
[0050] "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.
[0051] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
[0052] "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.
[0053] "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.
[0054] "Chimeric antisense compound" means an antisense compound
that has at least two chemically distinct regions.
[0055] "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-esterified cholesterol present
in the plasma or serum.
[0056] "Cholesterol absorption inhibitor" means an agent that
inhibits the absorption of exogenous cholesterol obtained from
diet.
[0057] "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.
[0058] "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.
[0059] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other.
[0060] "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.
[0061] "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.
[0062] "Cure" means a method that restores health or a prescribed
treatment for an illness.
[0063] "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.
[0064] "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.
[0065] "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.
[0066] "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.
[0067] "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.
[0068] "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 or hypertriglyceridemia.
[0069] "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.
[0070] "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.
[0071] "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.
[0072] "Fibrates" are agonists of peroxisome proliferator-activated
receptor-.alpha. (PPAR-.alpha.), acting via transcription factors
regulating various steps in lipid and lipoprotein metabolism. By
interacting with PPAR-.alpha., fibrates recruit different cofactors
and regulate gene expression. As a consequence, fibrates are
effective in lowering fasting TG levels as well as post-prandial TG
and TRL remnant particles. Fibrates also have modest LDL-C lowering
and HDL-C raising effects. Reduction in the expression and levels
of ApoC-III is a consistent effect of PPAR-.alpha. agonists (Hertz
et al. J Biol Chem, 1995, 270(22):13470-13475). A 36% reduction in
plasma ApoC-III levels was reported with fenofibrate treatment in
the metabolic syndrome (Watts et al. Diabetes, 2003, 52:803-811).
However, fibrates have been ineffective in treating LPLD subjects
with hypertriglyceridemia.
[0073] The "Fredrickson" system is used to classify primary
(genetic) causes of dyslipidemia into several subgroups or types.
Dyslipidemia types that may be amenable to therapy with the
compounds disclosed herein include, but are not limited to,
Fredrickson Type I, FCS, LPLD.
[0074] "Fredrickson Type I" is also known as "Lipoprotein lipase
deficiency", "LPLD", "Familial Chylomicronemia Syndrome" or "FCS"
and exists in several forms: Type Ia (also known as Buerger-Gruestz
syndrome) is a lipoprotein lipase deficiency commonly due to a
deficiency of LPL or altered ApoC-II; Type Ib (also known as
familial apoprotein CII deficiency) is a condition caused by lack
of lipoprotein lipase activator apoprotein C-II; 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 sufficient functionally active LPL. Patients are either
homozygous for such mutations or compound heterozygous. Fredrickson
Type I can also be due to mutations in the GPIHBP1, APOA5, LMF1 or
other genes leading to dysfunctional LPL. Brunzell, In: Pagon R A,
Adam M P, Bird T D, Dolan C R, Fong C T, Stephens K, editors.
GeneReviews.TM. [Internet]. Seattle (Wash.): University of
Washington, Seattle; 1993-2013.1999 Oct. 12 [updated 2011 Dec. 15].
Further, Fredrickson Type I, in some instances, can be due to the
presence of LPL inhibitors (e.g., anti-LPL antibodies) in an
individual causing dysfunctional LPL. The prevalence of Fredrickson
Type I 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.
[0075] "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%.
[0076] "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.
[0077] "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.
[0078] "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.
[0079] "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.
[0080] "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."
[0081] "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.
[0082] "Genetic screening" means to screen for genotypic variations
or mutations in an animal. In some instances the mutation can lead
to a phenotypic change in the animal. In certain instances the
phenotypic change is, or leads to, a disease, disorder or condition
in the animal. For example, mutations in the LPL or ApoC-II genes
can lead to Fredrickson Type I dyslipidemia, FCS, LPLD. Genetic
screening can be done by any of the art known techniques, for
example, sequencing of the LPL or ApoC-II gene or mRNA to detect
mutations. The sequence of the animal being screened is compared to
the sequence of a normal animal to determine whether there is any
mutation in the sequence. Alternatively, for example,
identification of mutations in the LPL or ApoC-II gene or mRNA can
be performed using PCR amplification and gel or chip analysis.
[0083] "Glucose" is a monosaccharide used by cells as a source of
energy and inflammatory intermediate. "Plasma glucose" refers to
glucose present in the plasma.
[0084] "High density lipoprotein" or "HDL" refers to 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). 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 are
independent of triglyceride and LDL concentrations. In clinical
practice, a low plasma HDL 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).
[0085] "High density lipoprotein-Cholesterol" or "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.
[0086] "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.
[0087] "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.
[0088] "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).
[0089] "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).
[0090] "Hypertriglyceridemia" means a condition characterized by
elevated triglyceride levels. 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). 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).
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 syndrome (FCS), familial combined
hyperlipidemia and familial hypertriglyceridemia. 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. The same is true for high TG
levels (200-499 mg/dL) except that as plasma TG levels increase,
underlying genetic factors play an increasingly important etiologic
role. Very high TG levels (.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 levels exceed 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.
[0091] "Identifying" or "diagnosing" an animal with a named
disease, disorder or condition means identifying, by art known
methods, a subject prone to, or having, the named disease, disorder
or condition.
[0092] "Identifying" or "diagnosing" an animal with Fredrickson
Type 1 dyslipidemia means to identify a subject prone to, or
having, Fredrickson Type I (a, b or c) dyslipidemia, FCS, LPLD.
Identification of subjects with Fredrickson Type I, FCS, LPLD, can
done by an examination of the subject's medical history in
conjunction with any art known screening technique e.g., genetic
screening or screening for LPL inhibitors. For example, a patient
with a documented medical history of fasting TG above 750 mg/dL is
then screened for mutations in the LPL gene or genes affecting the
LPL such as ApoC2, ApoA5, GPIHBP1 or LMF1.
[0093] "Identifying" or "diagnosing" an animal with metabolic or
cardiovascular disease means identifying a subject prone to, or
having, a metabolic disease, a cardiovascular disease, or a
metabolic syndrome; or, identifying 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.
[0094] "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.
[0095] "Immediately adjacent" means there are no intervening
elements between the immediately adjacent elements, for example,
between regions, segments, nucleotides and/or nucleosides.
[0096] "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.
[0097] "Individual" or "subject" or "animal" means a human or
non-human animal selected for treatment or therapy.
[0098] "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.
[0099] "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.
[0100] "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.
[0101] "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.
[0102] "Internucleoside linkage" refers to the chemical bond
between nucleosides.
[0103] "Intravenous administration" means administration into a
vein.
[0104] "Linked nucleosides" means adjacent nucleosides which are
bonded together.
[0105] "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.
[0106] "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.
[0107] "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.
[0108] "Lipoprotein Lipase" or "LPL" refers to an enzyme that
hydrolyzes TGs found in lipoproteins, such as CM or VLDL, into free
fatty acids and monoacylglycerols. LPL requires apo C-II as a
cofactor to function in hydrolyzing TGs. LPL is mainly produced in
skeletal muscle, fat tissue, and heart muscle. Hydrolysis and
removal of TG from CM and VLDL normally protects against excessive
postprandial rise in CM mass and TG.
[0109] "Lipoprotein lipase deficient", "lipoprotein lipase
deficiency", "LPL deficiency" or "LPLD" is also known as
"Fredrickson's Type I dyslipidemia", "chylomicronemia", "Familial
Chylomicronemia Syndrome" or "FCS". Although subjects with LPLD
generally lack LPL or LPL activity necessary for effective
breakdown of fatty acids such as TGs, these subjects may still have
a minimal LPL activity or express a minimal level of LPL. In some
instances, a LPLD subject may express LPL or have LPL activity up
to about, or no more than, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% activity. In
other instances, the LPLD subject has no measurable LPL or LPL
activity. One embodiment of LPLD encompasses subjects with
"hyperlipoproteinemia type Ia" (also known as "Fredrickson's Type
Ia") and refers to the inability of the subjects to produce
sufficient functional lipoprotein lipase enzymes necessary for
effective breakdown of fatty acids such as TGs. The inability to
breakdown TGs leads to hypertriglyceridemia in the subject and,
often more than 12 hours after meals, hyperTG and chylomicronemia
are still present and visible as lipemia. Type Ia is commonly
caused by one or more mutations in the LPL gene. As disclosed
herein, LPLD also encompasses subjects that have dysfunctional
lipoprotein lipase such as those subjects with
"hyperlipoproteinemia type Ib" (also known as "Fredrickson's Type
Ib") and "hyperlipoproteinemia type Ic" (also known as
"Fredrickson's Type Ic"). Type Ib is caused by lack of lipoprotein
lipase activator apoprotein C-II. Type Ic is due to a circulating
inhibitor of lipoprotein lipase. As with Type Ia, Type Ib/Ic
subjects suffer from an inability to breakdown TGs leading to
hypertriglyceridemia and hyperTG and chylomicronemia are still
present and visible as lipemia often more than 12 hours after
meals. In certain embodiments, LPLD is associated with at least one
mutation in the LPL gene such as P207L, G188L or D9N or other
mutations that affect LPL (Brunzell, In: Pagon R A, Adam M P, Bird
T D, Dolan C R, Fong C T, Stephens K, editors. GeneReviews.TM.
[Internet]. Seattle (Wash.): University of Washington, Seattle;
1993-2013.1999 Oct. 12 [updated 2011 Dec. 15]).
[0110] "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.
[0111] "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.
[0112] "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.
[0113] "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).
[0114] "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.
[0115] "Mixed dyslipidemia" means a condition characterized by
elevated cholesterol and elevated triglycerides.
[0116] "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.
[0117] "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).
[0118] "Modified nucleoside" means a nucleoside having at least one
modified sugar moiety, and/or modified nucleobase.
[0119] "Modified nucleotide" means a nucleotide having at least one
modified sugar moiety, modified internucleoside linkage and/or
modified nucleobase.
[0120] "Modified oligonucleotide" means an oligonucleotide
comprising at least one modified nucleotide.
[0121] "Modified sugar" refers to a substitution or change from a
natural sugar. For example, a 2'-O-methoxyethyl modified sugar is a
modified sugar.
[0122] "Motif" means the pattern of chemically distinct regions in
an antisense compound.
[0123] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0124] "Natural sugar moiety" means a sugar found in DNA (2'-H) or
RNA (2'-OH).
[0125] "Nicotinic acid" or "niacin" has been reported to decrease
fatty acid influx to the liver and the secretion of VLDL by the
liver. This effect appears to be mediated in part by the effects on
hormone-sensitive lipase in the adipose tissue. Nicotinic acid has
key action sites in both liver and adipose tissue. In the liver,
nicotinic acid is reported to inhibit diacylglycerol
acyltransferase-2 (DGAT-2) that results in the decreased secretion
of VLDL particles from the liver, which is also reflected in
reductions of both IDL and LDL particles, in addition, nicotinic
acid raises HDL-C and apo A1 primarily by stimulating apo A1
production in the liver and has also been shown to reduce
VLDL-ApoCIII concentrations in patients with hyperlipidemia
(Wahlberg et al. Acta Med Scand 1988; 224:319-327). The effects of
nicotinic acid on lipolysis and fatty acid mobilization in
adipocytes are well established. However, nicotinic acid has not
been effective in treating LPLD subjects with
hypertriglyceridemia.
[0126] "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.
[0127] "Nucleobase" means a heterocyclic moiety capable of pairing
with a base of another nucleic acid.
[0128] "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.
[0129] "Nucleobase sequence" means the order of contiguous
nucleobases independent of any sugar, linkage, or nucleobase
modification.
[0130] "Nucleoside" means a nucleobase linked to a sugar.
[0131] "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.
[0132] "Nucleotide" means a nucleoside having a phosphate group
covalently linked to the sugar portion of the nucleoside.
[0133] "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).
[0134] "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.
[0135] "Oligonucleotide" means a polymer of linked nucleosides each
of which can be modified or unmodified, independent from one
another.
[0136] "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.
[0137] "Peptide" means a molecule formed by linking at least two
amino acids by amide bonds. Peptide refers to polypeptides and
proteins.
[0138] "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.
[0139] "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.
[0140] "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.
[0141] "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.
[0142] "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.
[0143] "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.
[0144] "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.
[0145] "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.
[0146] "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.
[0147] "Raise" means to increase in amount. For example, to raise
plasma HDL levels means to increase the amount of HDL in the
plasma.
[0148] "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.
[0149] "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.
[0150] "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.
[0151] "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.
[0152] "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, DGAT1 inhibitors,
cholesterol lowering agents, lipid lowering agents, glucose
lowering agents and anti-inflammatory agents.
[0153] "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.
[0154] "Shortened" or "truncated" versions of antisense
oligonucleotides or target nucleic acids taught herein have one,
two or more nucleosides deleted.
[0155] "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.
[0156] "Single-stranded oligonucleotide" means an oligonucleotide
which is not hybridized to a complementary strand.
[0157] "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.
[0158] "Statin" means an agent that inhibits the activity of
HMG-CoA reductase. Statins reduce synthesis of cholesterol in the
liver by competitively inhibiting HMG-CoA reductase activity. The
reduction in intracellular cholesterol concentration induces LDL
receptor expression on the hepatocyte cell surface, which results
in increased extraction of LDL-C from the blood and a decreased
concentration of circulating LDL-C and other apo-B containing
lipoproteins including TG-rich particles. Independent of their
effects on LDL-C and LDL receptor, statins lower the plasma
concentration and cellular mRNA levels of ApoC-III (Ooi et al.
Clinical Sci, 2008, 114:611-624). As statins have significant
effects on mortality as well as most cardiovascular disease outcome
parameters, these drugs are the first choice to reduce both total
cardiovascular disease risk and moderately elevated TG levels. More
potent statins (atorvastatin, rosuvastatin, and pitavastatin)
demonstrate a robust lowering of TG levels, especially at high
doses and in patients with elevated TG. However, statins have been
ineffective in treating LPLD subjects with
hypertriglyceridemia.
[0159] "Subcutaneous administration" means administration just
below the skin.
[0160] "Subject" means a human or non-human animal selected for
treatment or therapy.
[0161] "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.
[0162] "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.
[0163] "Target nucleic acid," "target RNA," and "target RNA
transcript" all refer to a nucleic acid capable of being targeted
by antisense compounds.
[0164] "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.
[0165] "Treat" refers to administering a compound of the invention
to effect an alteration or improvement of a disease, disorder, or
condition.
[0166] "Triglyceride" or "TG" means a lipid or neutral fat
consisting of glycerol combined with three fatty acid
molecules.
[0167] "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.
[0168] "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).
[0169] "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
[0170] Certain embodiments provide a method of reducing ApoCIII
levels in an animal with Fredrickson Type I dyslipidemia, FCS,
LPLD, comprising administering a therapeutically effective amount
of a compound comprising an ApoCIII specific inhibitor to the
animal. In certain embodiments, ApoCIII levels are reduced in the
liver, adipose tissue, heart, skeletal muscle or small
intestine.
[0171] Certain embodiments provide a method of treating,
preventing, delaying or ameliorating Fredrickson Type I
dyslipidemia, FCS, LPLD, in an animal comprising administering a
therapeutically effective amount of a compound comprising an
ApoCIII specific inhibitor to the animal. In certain embodiments, a
cardiovascular and/or metabolic disease or disorder, or symptom or
risk thereof, related to Fredrickson Type I dyslipidemia, FCS,
LPLD, is improved.
[0172] Certain embodiments provide a method of treating,
preventing, delaying or ameliorating pancreatitis in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal. In certain
embodiments, pancreatitis, or a symptom or risk thereof, is
improved.
[0173] Certain embodiments provide a method of reducing TG levels
in an animal with Fredrickson Type I dyslipidemia, FCS, LPLD,
comprising administering a therapeutically effective amount of a
compound comprising an ApoCIII specific inhibitor to the
animal.
[0174] In certain embodiments, the animal has a TG level of at
least .gtoreq.1200 mg/dL, .gtoreq.1100 mg/dL, .gtoreq.1000 mg/dL,
.gtoreq.900 mg/dL, .gtoreq.880 mg/dL, .gtoreq.850 mg/dL,
.gtoreq.800 mg/dL, .gtoreq.750 mg/dL, .gtoreq.700 mg/dL,
.gtoreq.650 mg/dL, .gtoreq.600 mg/dL, .gtoreq.550 mg/dL,
.gtoreq.500 mg/dL, .gtoreq.450 mg/dL, .gtoreq.440 mg/dL,
.gtoreq.400 mg/dL, .gtoreq.350 mg/dL, .gtoreq.300 mg/dL,
.gtoreq.250 mg/dL, .gtoreq.200 mg/dL, .gtoreq.150 mg/dL In certain
embodiments, the animal has a history of TG level .gtoreq.880
mg/dL, fasting TG level .gtoreq.750 mg/dL and/or TG level
.gtoreq.440 mg/dL after dieting.
[0175] In certain embodiments, the compound decreases TGs
(postprandial or fasting) by at least 90%, by at least 80%, by at
least 70%, by at least 60%, by at least 50%, by at least 45%, at
least 40%, by at least 35%, by at least 30%, by at least 25%, by at
least 20%, by at least 15%, by at least 10%, by at least 5% or by
at least 1% from the baseline TG level. In certain embodiments, the
TG (postprandial or fasting) level is .ltoreq.1900 mg/dL,
.ltoreq.1800 mg/dL, .ltoreq.1700 mg/dL, .ltoreq.1600 mg/dL,
.ltoreq.1500 mg/dL, .ltoreq.1400 mg/dL, .ltoreq.1300 mg/dL,
.ltoreq.1200 mg/dL, .ltoreq.1100 mg/dL, .ltoreq.1000 mg/dL,
.ltoreq.900 mg/dL, .ltoreq.800 mg/dL, .ltoreq.750 mg/dL,
.ltoreq.700 mg/dL, .ltoreq.650 mg/dL, .ltoreq.600 mg/dL,
.ltoreq.550 mg/dL, .ltoreq.500 mg/dL, .ltoreq.450 mg/dL,
.ltoreq.400 mg/dL, .ltoreq.350 mg/dL, .ltoreq.300 mg/dL,
.ltoreq.250 mg/dL, .ltoreq.200 mg/dL, .ltoreq.150 mg/dL or
.ltoreq.100 mg/dL.
[0176] Certain embodiments provide a method of increasing HDL
levels and/or improving the ratio of TG to HDL in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal. In certain
embodiments, the compound increases HDL (postprandial or fasting)
by at least 90%, by at least 80%, by at least 70%, by at least 60%,
by at least 50%, by at least 45%, at least 40%, by at least 35%, by
at least 30%, by at least 25%, by at least 20%, by at least 15%, by
at least 10%, by at least 5% or by at least 1% from the baseline
HDL level.
[0177] In certain embodiments, the compound decreases ApoCIII by
about 81%, decreases TG by about 69%, decreases VLDL ApoCIII by
about 80%, increases HDL by about 78%, decreases non-HDL-C by about
58% and/or decreases ApoB by about 13%.
[0178] Certain embodiments provide a method of preventing, delaying
or ameliorating a cardiovascular and/or metabolic disease,
disorder, condition, or symptom thereof, in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal. In certain
embodiments, the compound prevents, delays or ameliorates the
cardiovascular and/or metabolic disease, disorder, condition, or
symptom thereof, in the animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, by decreasing TG levels, increasing HDL
levels in the animal and/or improving the ratio of TG to HDL.
[0179] Certain embodiments provide a method of preventing, delaying
or ameliorating pancreatitis, or symptom thereof, in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal. In certain
embodiments, the compound prevents, delays or ameliorates
pancreatitis, or symptom thereof, in the animal with Fredrickson
Type I dyslipidemia, FCS, LPLD, by decreasing TG levels, 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 pancreatitis, or symptom thereof, in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal. In certain
embodiments, the compound prevents, delays or ameliorates the
pancreatitis, or symptom thereof, in the animal with Fredrickson
Type I dyslipidemia, FCS, LPLD, by decreasing TG levels, increasing
HDL levels in the animal and/or improving the ratio of TG to
HDL.
[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
with Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the animal. In certain
embodiments, the compound prevents, treats, ameliorates, delays the
onset, or reduces of the risk of the cardiovascular disease,
disorder or condition in the animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, by decreasing TG levels, increasing HDL
levels and/or improving the ratio of TG to HDL.
[0182] Certain embodiments provide a method of decreasing CETP,
VLDL, VLDL ApoCIII, cholesterol, chylomicrons and/or ApoB levels in
an animal with Fredrickson Type I dyslipidemia, FCS, LPLD,
comprising administering a therapeutically effective amount of a
compound comprising an ApoCIII specific inhibitor to the animal. In
certain embodiments, the ApoB is ApoB-48 or ApoB-100. In certain
embodiments, the amount of ApoB-48 reflects the amount of
chylomicrons in the animal. In certain embodiments, the cholesterol
is total cholesterol or non-HDL-cholesterol.
[0183] Certain embodiments provide a method of increasing ApoA1,
PON1, fat clearance, chylomicron-triglyceride (CM-TG) clearance
and/or HDL in an animal with Fredrickson Type I dyslipidemia, FCS,
LPLD, comprising administering a therapeutically effective amount
of a compound comprising an ApoCIII specific inhibitor to the
animal. Certain embodiments provide a method for improving the
ratio of TG to HDL in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD comprising administering a therapeutically
effective amount of a compound comprising an ApoCIII specific
inhibitor to the animal.
[0184] Certain embodiments provide a method for treating adult
patients with Fredrickson Type I dyslipidemia, FCS, LPLD suffering
from severe or multiple pancreatitis attacks comprising comprising
administering a therapeutically effective amount of a compound
comprising an ApoCIII specific inhibitor to the patient. In certain
embodiments, the patient suffers from pancreatitis despite dietary
fat restrictions.
[0185] Certain embodiments provide a method for identifying a
subject suffering from Fredrickson Type I dyslipidemia, FCS, LPLD,
comprising genetically screening the subject. Certain embodiments
provide a method for identifying a subject at risk for Fredrickson
Type I dyslipidemia, FCS, LPLD, comprising genetically screening
the subject. In certain embodiments the genetic screening is
performed by sequence analysis of the gene or RNA transcript
encoding LPL or ApoC-II. In certain embodiments, the subject is
genetically screened for at least one mutation in the LPL gene such
as P207L, G188L, D9N or other mutations that affect LPL (Brunzell,
In: Pagon R A, Adam M P, Bird T D, Dolan C R, Fong C T, Stephens K,
editors. GeneReviews.TM. [Internet]. Seattle (Wash.): University of
Washington, Seattle; 1993-2013.1999 Oct. 12 [updated 2011 Dec.
15]).
[0186] Certain embodiments provide a method for identifying a
subject suffering from Fredrickson Type I dyslipidemia, FCS, LPLD,
comprising screening the subject for the presence of LPL inhibiting
antibodies. Certain embodiments provide a method for identifying a
subject at risk for Fredrickson Type I dyslipidemia, FCS, LPLD,
comprising screening the subject for the presence of LPL inhibiting
antibodies.
[0187] In certain embodiments, the level of LPL expression in a
LPLD subject is undetectable. In certain embodiments, the level of
LPL in a LPLD subject is detectable. In certain embodiments, the
level of LPL in the LPLD subject is at most 25%, at most 24%, at
most 23%, at most 22%, at most 21%, at most 20%, at most 19%, at
most 18%, at most 17%, at most 16%, at most 15%, at most 14%, at
most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at
most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most
3%, at most 2% or at most 1% of the LPL level of a non-LPLD
subject.
[0188] In certain embodiments, the level of LPL activity in a LPLD
subject is undetectable. In certain embodiments, the level of LPL
activity in a LPLD subject is detectable. In certain embodiments,
the level of LPL activity in the LPLD subject is at most 25%, at
most 24%, at most 23%, at most 22%, at most 21%, at most 20%, at
most 19%, at most 18%, at most 17%, at most 16%, at most 15%, at
most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at
most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most
4%, at most 3%, at most 2% or at most 1% of the LPL activity level
of a non-LPLD subject. 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), GENBANK
Accession No. NT_033899.8 truncated from nucleotides 20262640 to
20266603 (incorporated herein as SEQ ID NO: 2), and GenBank
Accession No. NT_035088.1 truncated from nucleotides 6238608 to
U.S. Pat. No. 6,242,565 (incorporated herein as SEQ ID NO: 4).
[0189] In certain embodiments, the ApoCIII specific inhibitor is a
nucleic acid, peptide, antibody, small molecule or other agent
capable of inhibiting the expression of ApoCIII. In certain
embodiments, the nucleic acid is an antisense compound targeting
ApoCIII. In certain embodiments, the antisense compound is an
antisense oligonucleotide. In certain embodiments, the antisense
oligonucleotide is a modified oligonucleotide. In certain
embodiments, the modified oligonucleotide has a sequence
complementary to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4. In
certain embodiments, the modified oligonucleotide is at least 70%,
at least 75%, 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,
SEQ ID NO: 2 or SEQ ID NO: 4.
[0190] In certain embodiments, the modified oligonucleotide has a
nucleobase sequence comprising at least 8 contiguous nucleobases of
an antisense oligonucleotide complementary to an ApoCIII. In
certain 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 has a nucleobase sequence of ISIS 304801 (SEQ ID
NO: 3). In certain embodiments, the modified oligonucleotide
targeting ApoCIII has a sequence other than that of SEQ ID NO: 3.
In certain embodiments, the modified oligonucleotide has a
nucleobase sequence comprising at least 8 contiguous nucleobases of
a sequence selected from any sequence disclosed in U.S. Pat. Nos.
7,598,227, 7,750,141, PCT Publication WO 2004/093783 or PCT
Publication WO 2012/149495, all incorporated-by-reference herein.
In certain embodiments, the modified oligonucleotide has a sequence
selected from any sequence disclosed in U.S. Pat. Nos. 7,598,227,
7,750,141, PCT Publication WO 2004/093783 or PCT Publication WO
2012/149495, all incorporated-by-reference herein.
[0191] In certain embodiments, the modified oligonucleotide
consists of a single-stranded modified oligonucleotide.
[0192] In certain embodiments, the modified oligonucleotide
consists of 12-30 linked nucleosides.
[0193] In certain embodiments, the modified oligonucleotide
consists of 20 linked nucleosides. In certain embodiments, the
modified oligonucleotide consists of 20 linked nucleosides and the
nucleobase sequence of ISIS 304801 (SEQ ID NO: 3).
[0194] In certain embodiments, the compound comprises at least one
modified internucleoside linkage. In certain embodiments, the
internucleoside linkage is a phosphorothioate internucleoside
linkage. In certain embodiments, each internucleoside linkage is a
phosphorothioate internucleoside linkage.
[0195] 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.
[0196] In certain embodiments, the compound comprises at least one
nucleoside comprising a modified nucleobase. In certain
embodiments, the modified nucleobase is a 5-methylcytosine.
[0197] In certain embodiments, the compound comprises a modified
oligonucleotide comprising: (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.
[0198] In certain embodiments, the compound comprises a modified
oligonucleotide comprising: (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.
[0199] In certain embodiments, the compound comprises a modified
oligonucleotide comprising: (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.
[0200] Certain embodiments provide a method of reducing the risk of
a cardiovascular disease in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, 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 decreases TG
levels, increases HDL levels and/or improves the ratio of TG to
HDL. In certain embodiments, the ApoCIII nucleic acid is SEQ ID NO:
1, SEQ ID NO: 2 or SEQ ID NO: 4. In certain embodiments, the
modified oligonucleotide is at least 70%, least 75%, least 80%, at
least 85%, at least 90%, at least 95%, at least 98% or 100%
complementary to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4. In
certain embodiments, the modified oligonucleotide comprises at
least 8 contiguous nucleobases of an antisense oligonucleotide
targeting ApoCIII. In further embodiments, the modified
oligonucleotide comprises at least 8 contiguous nucleobases of the
nucleobase sequence of ISIS 304801 (SEQ ID NO: 3).
[0201] Certain embodiments provide a method of preventing,
treating, ameliorating, or reducing at least one symptom of a
cardiovascular disease in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, 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, SEQ
ID NO: 2 or SEQ ID NO: 4. In certain embodiments, the modified
oligonucleotide is at least 70%, least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 98% or 100% complementary
to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4. In further
embodiments, the modified oligonucleotide administered to the
animal prevents, treats, ameliorates or reduces at least one
symptom of the cardiovascular disease by decreasing TG levels,
increasing HDL levels and/or improving the ratio of TG to HDL. In
certain embodiments, the modified oligonucleotide comprises at
least 8 contiguous nucleobases of an antisense oligonucleotide
targeting ApoCIII. In further embodiments, the modified
oligonucleotide comprises at least 8 contiguous nucleobases of ISIS
304801 (SEQ ID NO: 3).
[0202] 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.
[0203] Certain embodiments provide a method of decreasing TG
levels, raising HDL levels and/or improving the ratio of TG to HDL
in an animal with Fredrickson Type I dyslipidemia, FCS, LPLD, by
administering to the animal a therapeutically effective amount of a
compound consisting of a modified oligonucleotide targeting
ApoCIII. Further embodiments provide a method of preventing,
treating, ameliorating or reducing at least one symptom of a
cardiovascular and/or metabolic disease, disorder, condition, or
symptom thereof, in the animal by administering to the animal a
compound consisting of a modified oligonucleotide targeting
ApoCIII, thereby decreasing TG levels, increasing the HDL levels
and/or improving the ratio of TG to HDL in the animal.
[0204] Certain embodiments provide a method of decreasing TG
levels, raising HDL levels and/or improving the ratio of TG to HDL
in an animal with Fredrickson Type I dyslipidemia, FCS, LPLD, by
administering to the animal a therapeutically effective amount of 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 and/or metabolic disease, disorder, condition, or
symptom thereof, in the animal by administering to the animal a
compound consisting of the nucleobase sequence of ISIS 304801 (SEQ
ID NO: 3), thereby decreasing TG levels, increasing the HDL levels
and/or improving the ratio of TG to HDL in the animal.
[0205] Certain embodiments provide a method of decreasing TG
levels, raising HDL levels and/or improving the ratio of TG to HDL
in an animal with Fredrickson Type I dyslipidemia, FCS, LPLD, by
administering to the animal a therapeutically effective amount of a
modified oligonucleotide having the sequence of ISIS 304801 (SEQ ID
NO: 3), 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.
[0206] Certain embodiments provide a method of preventing,
delaying, treating, ameliorating, or reducing at least one symptom
of a cardiovascular and/or metabolic disease, disorder, condition,
or symptom thereof, in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, by administering to the animal a
therapeutically effective amount of a modified oligonucleotide
targeting ApoCIII, 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.
[0207] Certain embodiments provide a method of preventing,
delaying, treating, ameliorating, or reducing at least one symptom
of a cardiovascular and/or metabolic disease, disorder, condition,
or symptom thereof, in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, by administering to the animal a
therapeutically effective amount of 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.
[0208] Certain embodiments provide a method of decreasing TG
levels, raising the HDL levels and/or improving the ratio of TG to
HDL in an animal with Fredrickson Type I dyslipidemia, FCS, LPLD,
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. In certain
embodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1, SEQ
ID NO: 2 or SEQ ID NO: 4. 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, SEQ ID NO: 2 or SEQ ID NO: 4.
[0209] Certain embodiments provide a method of preventing,
delaying, treating, ameliorating, or reducing at least one symptom
of a cardiovascular and/or metabolic disease, disorder, condition,
or symptom thereof, in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, by administering to the animal a compound
comprising a therapeutically effective amount of a modified
oligonucleotide consisting of 12 to 30 linked nucleosides, wherein
the modified oligonucleotide is complementary to an ApoCIII nucleic
acid, and decreases TG levels and/or raises the HDL levels in the
animal. In certain embodiments, the ApoCIII nucleic acid is either
SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 4. 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, SEQ ID NO: 2 or SEQ ID NO: 4.
[0210] In certain embodiments, the animal is human.
[0211] 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 hypertriglyceridemia or
chylomicronemia (e.g., FCS). In certain embodiments, the metabolic
disease is diabetes, obesity or metabolic syndrome.
[0212] In certain embodiments, the animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, is at risk for pancreatitis. In certain
embodiments, reducing ApoCIII levels in the liver and/or small
intestine prevents pancreatitis. In certain embodiments, reducing
TG levels, raising HDL levels and/or improving the ratio of TG to
HDL prevents pancreatitis.
[0213] In certain embodiments, reducing ApoCIII levels in the liver
and/or small intestine of an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, enhances clearance of postprandial TG. In
certain embodiments, raising HDL levels and/or improving the ratio
of TG to HDL enhance clearance of postprandial TG in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD. In certain embodiments,
reducing ApoCIII levels in the liver and/or small intestine lowers
postprandial triglyceride in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD. In certain embodiments, raising HDL levels
and/or improving the ratio of TG to HDL lowers postprandial TG.
[0214] In certain embodiments, reducing ApoCIII levels in the liver
and/or small intestine of an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD, improves the ratio of HDL to TG.
[0215] In certain embodiments, the compound is parenterally
administered. In further embodiments, the parenteral administration
is subcutaneous.
[0216] In certain embodiments, the compound is co-administered with
a second agent or therapy. In certain embodiments, the second agent
is an ApoCIII lowering agent, Apo C-II lowering agent, DGAT1
lowering agent, LPL raising agent, cholesterol lowering agent,
non-HDL lipid lowering agent, LDL lowering agent, TG lowering
agent, cholesterol lowering agent, HDL raising agent, fish oil,
niacin (nicotinic acid), fibrate, statin, DCCR (salt of diazoxide),
glucose-lowering agent or anti-diabetic agents. In certain
embodiments, the second therapy is dietary fat restriction.
[0217] In certain embodiments, the ApoCIII lowering agents include
an ApoCIII antisense oligonucleotide different from the first
agent, fibrate or an Apo B antisense oligonucleotide.
[0218] In certain embodiments, the DGAT1 lowering agent is
LCQ908.
[0219] In certain embodiments, the LPL raising agents include gene
therapy agents that raise the level of LPL (e.g., Glybera.sup.R,
normal copies of ApoC-II, GPIHBP1, APOA5, LMF1 or other genes that,
when mutated, can lead to dysfunctional LPL).
[0220] In certain embodiments, the glucose-lowering and/or
anti-diabetic agents include, but are not limited to, 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.
[0221] In certain embodiments, the cholesterol or lipid lowering
agents include, but are not limited to, 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. The
therapeutic lifestyle change can be dietary fat restriction.
[0222] In certain embodiments, the 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.
[0223] In certain embodiments, the compound and the second agent
are administered concomitantly or sequentially.
[0224] In certain embodiments, the compound is a salt form. In
further embodiments, the compound further comprises of a
pharmaceutically acceptable carrier or diluent.
[0225] Certain embodiments provide a compound comprising an ApoCIII
specific inhibitor for use in the preparation of a medicament for
treating, preventing, delaying or ameliorating Fredrickson Type I
dyslipidemia, FCS, LPLD.
[0226] Certain embodiments provide use of a compound comprising an
ApoCIII specific inhibitor in the preparation of a medicament for
decreasing ApoCIII levels in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD. In certain embodiments, ApoCIII levels are
decreased in the liver or small intestine.
[0227] Certain embodiments provide a use of a compound comprising
an ApoCIII specific inhibitor in the preparation of a medicament
for decreasing TG levels, increasing HDL levels and/or improving
the ratio of TG to HDL in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD.
[0228] Certain embodiments provide use of a compound comprising an
ApoCIII specific inhibitor in the preparation of a medicament for
preventing, treating, ameliorating or reducing at least one symptom
of a cardiovascular or metabolic disease by decreasing TG levels,
increasing HDL levels and/or improving the ratio of TG to HDL in an
animal with Fredrickson Type I dyslipidemia, FCS, LPLD.
[0229] Certain embodiments provide use of a compound comprising an
ApoCIII specific inhibitor in the preparation of a medicament for
treating an animal with Fredrickson Type I dyslipidemia, FCS, LPLD,
at risk for or having pancreatitis.
[0230] In certain embodiments, the ApoCIII specific inhibitor used
in the preparation of a medicament is a nucleic acid, peptide,
antibody, small molecule or other agent capable of inhibiting the
expression of ApoCIII. In certain embodiments, the nucleic acid is
an antisense compound. In certain embodiments, the antisense
compound is a modified oligonucleotide targeting ApoCIII. In
certain 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 70%, at least 75%, 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, SEQ ID NO: 2 or SEQ ID NO:
4.
[0231] Certain embodiments provide a compound comprising an ApoCIII
specific inhibitor for use in treating, preventing, delaying or
ameliorating Fredrickson Type I dyslipidemia, FCS, LPLD.
[0232] Certain embodiments provide use of a compound comprising an
ApoCIII specific inhibitor for decreasing ApoCIII levels in an
animal with Fredrickson Type I dyslipidemia, FCS, LPLD. In certain
embodiments, ApoCIII levels are decreased in the liver or small
intestine.
[0233] Certain embodiments provide a use of a compound comprising
an ApoCIII specific inhibitor for decreasing TG levels, increasing
HDL levels and/or improving the ratio of TG to HDL in an animal
with Fredrickson Type I dyslipidemia, FCS, LPLD.
[0234] Certain embodiments provide use of a compound comprising an
ApoCIII specific inhibitor for preventing, treating, ameliorating
or reducing at least one symptom of a cardiovascular disease by
decreasing TG levels, increasing HDL levels and/or improving the
ratio of TG to HDL in an animal with Fredrickson Type I
dyslipidemia, FCS, LPLD.
[0235] Certain embodiments provide use of a compound comprising an
ApoCIII specific inhibitor for treating an animal with Fredrickson
Type I dyslipidemia, FCS, LPLD, at risk for or having
pancreatitis.
[0236] In certain embodiments, the ApoCIII specific inhibitor used
is a nucleic acid, peptide, antibody, small molecule or other agent
capable of inhibiting the expression of ApoCIII. In certain
embodiments, the nucleic acid is an antisense compound. In certain
embodiments, the antisense compound is a modified oligonucleotide
targeting ApoCIII. In certain 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 70%, at least
75%, 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, SEQ ID
NO: 2 or SEQ ID NO: 4.
[0237] Certain embodiments provide a composition comprising an
ApoCIII specific inhibitor for use in: reducing TG levels in an
animal with Fredrickson Type I dyslipidemia, FCS, LPLD; increasing
HDL levels and/or improving the ratio of TG to HDL in an animal
with Fredrickson Type I dyslipidemia, FCS, LPLD; preventing,
delaying or ameliorating a cardiovascular and/or metabolic disease,
disorder, condition, or a symptom thereof, in an animal with
Fredrickson Type I dyslipidemia, FCS, LPLD; and/or preventing,
delaying or ameliorating pancreatitis, or a symptom thereof, in an
animal with Fredrickson Type I dyslipidemia, FCS, LPLD. In certain
embodiments, the ApoCIII specific inhibitor is a nucleic acid,
peptide, antibody, small molecule or other agent capable of
inhibiting the expression of ApoCIII. In certain embodiments, the
nucleic acid is an antisense compound. In certain embodiments, the
antisense compound is a modified oligonucleotide targeting ApoCIII.
In certain 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 70%, at least 75%, 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, SEQ ID NO: 2 or SEQ ID NO:
4.
[0238] Certain embodiments provide a composition to reduce TG
levels in an animal with Fredrickson Type I dyslipidemia, FCS,
LPLD; increase HDL levels and/or improving the ratio of TG to HDL
in an animal with Fredrickson Type I dyslipidemia, FCS, LPLD;
prevent, delay or ameliorate a cardiovascular and/or metabolic
disease, disorder, condition, or a symptom thereof, in an animal
with Fredrickson Type I dyslipidemia, FCS, LPLD; and/or prevent,
delay or ameliorate pancreatitis, or a symptom thereof, in an
animal with Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
an ApoCIII specific inhibitor. In certain embodiments, the ApoCIII
specific inhibitor is a nucleic acid, peptide, antibody, small
molecule or other agent capable of inhibiting the expression of
ApoCIII. In certain embodiments, the nucleic acid is an antisense
compound. In certain embodiments, the antisense compound is a
modified oligonucleotide targeting ApoCIII. In certain 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 70%,
at least 75%, 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,
SEQ ID NO: 2 or SEQ ID NO: 4.
Antisense Compounds
[0239] 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 it is capable of
undergoing hybridization to a target nucleic acid through hydrogen
bonding.
[0240] Antisense compounds provided herein refer to oligomeric
compounds capable of undergoing hybridization to a target nucleic
acid through hydrogen bonding. Examples of antisense compounds
include single-stranded and double-stranded compounds, such as,
antisense oligonucleotides, siRNAs, shRNAs, and miRNAs.
[0241] 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.
[0242] 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.
[0243] 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.
[0244] 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.
[0245] 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.
[0246] 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.
[0247] 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
[0248] 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.
[0249] 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 a RNA: DNA duplex.
[0250] 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.
[0251] 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.
[0252] In certain embodiments, antisense compounds targeted to an
ApoCIII nucleic acid possess a 5-10-5 gapmer motif.
[0253] 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
[0254] Nucleotide sequences that encode ApoCIII include, without
limitation, the following: GENBANK Accession No. NM_000040.1
(incorporated herein as SEQ ID NO: 1), GENBANK Accession No.
NT_033899.8 truncated from nucleotides 20262640 to 20266603
(incorporated herein as SEQ ID NO: 2) and GenBank Accession No.
NT_035088.1 truncated from nucleotides 6238608 to U.S. Pat. No.
6,242,565 (incorporated herein as SEQ ID NO: 4).
[0255] 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.
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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.
[0260] 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.
[0261] 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).
[0262] 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 level, decrease in LDL
level, or decrease in TG level are among phenotypic changes that
may be assayed for inhibition of ApoCIII expression. Other
phenotypic indications, e.g., symptoms associated with a
cardiovascular or metabolic 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
[0263] 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.
[0264] 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.
[0265] Methods of determining whether a sequence is specifically
hybridizable to a target nucleic acid are well known in the art
(Sambrook and Russell, Molecular Cloning: A Laboratory Manual,
3.sup.rd Ed., 2001, CSHL Press). In certain embodiments, the
antisense compounds provided herein are specifically hybridizable
with an ApoCIII nucleic acid.
Complementarity
[0266] 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).
[0267] 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).
[0268] 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.
[0269] 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).
[0270] 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.
[0271] 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.
[0272] 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.
[0273] 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.
[0274] 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
[0275] 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.
[0276] 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
[0277] 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.
[0278] 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.
[0279] 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
[0280] 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.
[0281] 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.
[0282] 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
[0283] 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).
[0284] 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.l)--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n)-
, where each R.sub.l, R.sub.m and R.sub.n is, independently, H or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl.
[0285] As used herein, "bicyclic nucleosides" refer to modified
nucleosides comprising a bicyclic sugar moiety. Examples of
bicyclic nucleic acids (BNAs) 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 BNA nucleosides wherein the bridge
comprises one of the formulas: 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
PCT/US2008/068922 published as WO/2009/006478, published Jan. 8,
2009); 4'-CH.sub.2--N(OCH.sub.3)-2' (and analogs thereof see
PCT/US2008/064591 published as 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--(=CH.sub.2)-2' (and analogs thereof see
PCT/US2008/066154 published as WO 2008/154401, published on Dec. 8,
2008).
[0286] Further bicyclic nucleosides have been reported in published
literature (see for example: Srivastava et al., J. Am. Chem. Soc.,
2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research,
2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs,
2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum
et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wahlestedt et
al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Singh et
al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron,
1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998,
8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;
U.S. Pat. Nos. 7,399,845; 7,053,207; 7,034,133; 6,794,499;
6,770,748; 6,670,461; 6,525,191; 6,268,490; U.S. Patent Publication
Nos.: US2008-0039618; US2007-0287831; US2004-0171570; U.S. patent
application Ser. Nos. 12/129,154; 61/099,844; 61/097,787;
61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574;
International applications WO 2007/134181; WO 2005/021570; WO
2004/106356; WO 94/14226; and PCT International Applications Nos.:
PCT/US2008/068922; PCT/US2008/066154; and PCT/US2008/064591). 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).
[0287] As used herein, "monocyclic 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.
[0288] 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.
[0289] 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' carbon atoms of the
pentofuranosyl sugar moiety including without limitation, bridges
comprising 1 or from 1 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.NR.sub.a)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--,
--Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x, and --N(R.sub.a)--;
wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; 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
[0290] 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.
[0291] 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.
[0292] In certain embodiments, bicyclic nucleosides are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-(CH.sub.2)--O-2' 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).
[0293] In certain embodiments, bicyclic nucleosides include those
having a 4' to 2' bridge wherein such bridges include without
limitation, .alpha.-L-4'-(CH.sub.2)--O-2',
.beta.-D-4'-CH.sub.2--O-2', 4'-(CH.sub.2).sub.2--O-2',
4'-CH.sub.2--O--N(R)-2', 4'-CH.sub.2--N(R)--O-2',
4'-CH(CH.sub.3)--O-2', 4'-CH.sub.2--S-2', 4'-CH.sub.2--N(R)-2',
4'-CH.sub.2--CH(CH.sub.3)-2', and 4'-(CH.sub.2).sub.3-2', wherein R
is H, a protecting group or C.sub.1-C.sub.12 alkyl.
[0294] In certain embodiment, bicyclic nucleosides have the
formula:
##STR00001##
wherein:
[0295] Bx is a heterocyclic base moiety;
[0296] -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;
[0297] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting
group; and
[0298] 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.
[0299] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00002##
wherein:
[0300] Bx is a heterocyclic base moiety;
[0301] 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;
[0302] 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 thiol.
[0303] 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.eC(.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.
[0304] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00003##
[0305] wherein:
[0306] Bx is a heterocyclic base moiety;
[0307] 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;
[0308] 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)--).
[0309] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00004##
wherein:
[0310] Bx is a heterocyclic base moiety;
[0311] 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;
[0312] 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;
[0313] 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;
[0314] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00005##
[0315] wherein:
[0316] Bx is a heterocyclic base moiety;
[0317] 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;
[0318] q.sub.a, q.sub.b, q.sub.e and q.sub.f of 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;
[0319] or q.sub.e and q.sub.f of together are
.dbd.C(q.sub.g)(q.sub.h);
[0320] 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.
[0321] The synthesis and preparation of adenine, cytosine, guanine,
5-methyl-cytosine, thymine and uracil bicyclic nucleosides having a
4'-CH.sub.2--O-2' bridge, along with their oligomerization, and
nucleic acid recognition properties have been described (Koshkin et
al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of bicyclic
nucleosides has also been described in WO 98/39352 and WO
99/14226.
[0322] Analogs of various bicyclic nucleosides that have 4' to 2'
bridging groups such as 4'-CH.sub.2--O-2' and 4'-CH.sub.2--S-2',
have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett.,
1998, 8, 2219-2222). Preparation of oligodeoxyribonucleotide
duplexes comprising bicyclic nucleosides for use as substrates for
nucleic acid polymerases has also been described (Wengel et al., WO
99/14226). Furthermore, synthesis of 2'-amino-BNA, a novel
conformationally 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.
[0323] In certain embodiments, bicyclic nucleosides have the
formula:
##STR00006##
wherein:
[0324] Bx is a heterocyclic base moiety;
[0325] 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;
[0326] 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
[0327] 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.
[0328] 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 (Frier 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).
[0329] 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, (F) methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA (also referred to as constrained ethyl
or cEt), (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, (J) propylene carbocyclic
(4'-(CH.sub.2).sub.3-2') BNA, and (K) vinyl BNA as depicted
below.
##STR00007## ##STR00008##
[0330] wherein Bx is the base moiety and R is, independently, H, a
protecting group, C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6
alkoxy.
[0331] As used herein, the term "modified tetrahydropyran
nucleoside" or "modified THP nucleoside" means a nucleoside having
a six-membered tetrahydropyran "sugar" substituted for the
pentofuranosyl residue in normal nucleosides and can be referred to
as 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) or fluoro HNA
(F-HNA) having a tetrahydropyranyl ring system as illustrated
below.
##STR00009##
[0332] In certain embodiment, sugar surrogates are selected having
the formula:
##STR00010##
wherein:
[0333] Bx is a heterocyclic base moiety;
[0334] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to the oligomeric compound or one of T.sub.3 and
T.sub.4 is an internucleoside linking group linking the
tetrahydropyran nucleoside analog to an oligomeric compound or
oligonucleotide and the other of T.sub.3 and T.sub.4 is H, a
hydroxyl protecting group, a linked conjugate group or a 5' or
3'-terminal group;
[0335] 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
[0336] one of R.sub.1 and R.sub.2 is hydrogen and the other is
selected from 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.
[0337] In certain embodiments, 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 are provided
wherein one of R.sub.1 and R.sub.2 is F. 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.
[0338] In certain embodiments, sugar surrogates comprise rings
having more than 5 atoms and more than one heteroatom. For example
nucleosides comprising morpholino sugar moieties and their use in
oligomeric compounds has been reported (see for example: Braasch et
al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos.
5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the
term "morpholino" means a sugar surrogate having the following
formula:
##STR00011##
In certain embodiments, morpholinos may be modified, for example by
adding or altering various substituent groups from the above
morpholino structure. Such sugar surrogates are referred to herein
as "modified morpholinos."
[0339] Combinations of modifications are also provided without
limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT
International Application WO 2008/101157 published on Aug. 21, 2008
for other disclosed 5', 2'-bis substituted nucleosides) and
replacement of the ribosyl ring oxygen atom with S and 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 bicyclic nucleic acid (see PCT
International Application WO 2007/134181, published on Nov. 22,
2007 wherein a 4'-CH.sub.2--O-2' bicyclic nucleoside is further
substituted at the 5' position with a 5'-methyl or a 5'-vinyl
group). The synthesis and preparation of carbocyclic bicyclic
nucleosides along with their oligomerization and biochemical
studies have also been described (see, e.g., Srivastava et al., J.
Am. Chem. Soc. 2007, 129(26), 8362-8379).
[0340] In certain embodiments, antisense compounds comprise one or
more modified cyclohexenyl nucleosides, which is a nucleoside
having a six-membered cyclohexenyl in place of the pentofuranosyl
residue in naturally occurring nucleosides. Modified cyclohexenyl
nucleosides include, but are not limited to those described in the
art (see for example commonly owned, published PCT Application WO
2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am.
Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron
Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem.
Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides
& Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al.,
Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al.,
Acta Crystallographica, Section F: Structural Biology and
Crystallization Communications, 2005, F61(6), 585-586; Gu et al.,
Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides,
2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68,
4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24),
4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et
al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),
785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published
PCT application, WO 06/047842; and Published PCT Application WO
01/049687; the text of each is incorporated by reference herein, in
their entirety). Certain modified cyclohexenyl nucleosides have
Formula X.
##STR00012##
[0341] wherein independently for each of said at least one
cyclohexenyl nucleoside analog of Formula X:
[0342] Bx is a heterocyclic base moiety;
[0343] T.sub.3 and T.sub.4 are each, independently, an
internucleoside linking group linking the cyclohexenyl nucleoside
analog to an antisense compound or one of T.sub.3 and T.sub.4 is an
internucleoside linking group linking the tetrahydropyran
nucleoside analog to an antisense compound and the other of T.sub.3
and T.sub.4 is H, a hydroxyl protecting group, a linked conjugate
group, or a 5'- or 3'-terminal group; and
[0344] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6,
q.sub.7, q.sub.8 and q.sub.9 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, substituted C.sub.2-C.sub.6 alkynyl or
other sugar substituent group.
[0345] Many other monocyclic, bicyclic and tricyclic ring systems
are known in the art and are suitable as sugar surrogates that can
be used to modify nucleosides for incorporation into oligomeric
compounds as provided herein (see for example review article:
Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854).
Such ring systems can undergo various additional substitutions to
further enhance their activity.
[0346] 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).
[0347] 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, 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.
[0348] As used herein, "2'-F" refers to a nucleoside comprising a
sugar comprising a fluoro group at the 2' position of the sugar
ring.
[0349] As used herein, "2'-OMe" or "2'-OCH.sub.3", "2'-O-methyl" or
"2'-methoxy" each refers to a nucleoside comprising a sugar
comprising an --OCH.sub.3 group at the 2' position of the sugar
ring.
[0350] 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.
[0351] Methods for the preparations of modified sugars are well
known to those skilled in the art. Some representative U.S. patents
that teach the preparation of such modified sugars include without
limitation, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044;
5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and
International Application PCT/US2005/019219, filed Jun. 2, 2005 and
published as WO 2005/121371 on Dec. 22, 2005, and each of which is
herein incorporated by reference in its entirety.
[0352] 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).
[0353] 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.
[0354] 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.
Modified Nucleobases
[0355] 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).
[0356] 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-aminoadenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine.
[0357] 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.
[0358] 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.
RNAi Compounds
[0359] In certain embodiments, antisense compounds are interfering
RNA compounds (RNAi), which include double-stranded RNA compounds
(also referred to as short-interfering RNA or siRNA) and
single-stranded RNAi compounds (or ssRNA). Such compounds work at
least in part through the RISC pathway to degrade and/or sequester
a target nucleic acid (thus, include microRNA/microRNA-mimic
compounds). In certain embodiments, antisense compounds comprise
modifications that make them particularly suited for such
mechanisms.
[0360] i. ssRNA Compounds
[0361] In certain embodiments, antisense compounds including those
particularly suited for use as single-stranded RNAi compounds
(ssRNA) comprise a modified 5'-terminal end. In certain such
embodiments, the 5'-terminal end comprises a modified phosphate
moiety. In certain embodiments, such modified phosphate is
stabilized (e.g., resistant to degradation/cleavage compared to
unmodified 5'-phosphate). In certain embodiments, such 5'-terminal
nucleosides stabilize the 5'-phosphorous moiety. Certain modified
5'-terminal nucleosides may be found in the art, for example in
WO/2011/139702.
[0362] In certain embodiments, the 5'-nucleoside of an ssRNA
compound has Formula IIc:
##STR00013##
wherein:
[0363] T.sub.1 is an optionally protected phosphorus moiety;
[0364] T.sub.2 is an internucleoside linking group linking the
compound of Formula IIc to the oligomeric compound;
[0365] A has one of the formulas:
##STR00014##
[0366] Q.sub.1 and Q.sub.2 are each, independently, H, halogen,
C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy,
C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6 alkynyl or
N(R.sub.3)(R.sub.4);
[0367] Q.sub.3 is O, S, N(R.sub.5) or C(R.sub.6)(R.sub.7);
[0368] each R.sub.3, R.sub.4 R.sub.5, R.sub.6 and R.sub.7 is,
independently, H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy;
[0369] M.sub.3 is O, S, NR.sub.14, C(R.sub.15)(R.sub.16),
C(R.sub.15)(R.sub.16)C(R.sub.17)(R.sub.18),
C(R.sub.15).dbd.C(R.sub.17), OC(R.sub.15)(R.sub.16) or
OC(R.sub.15)(Bx.sub.2);
[0370] R.sub.14 is H, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, 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;
[0371] R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are each,
independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, 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;
[0372] Bx.sub.1 is a heterocyclic base moiety;
[0373] or if Bx.sub.2 is present then Bx.sub.2 is a heterocyclic
base moiety and Bx.sub.1 is H, halogen, C.sub.1-C.sub.6 alkyl,
substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy,
substituted C.sub.1-C.sub.6 alkoxy, 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;
[0374] J.sub.4, J.sub.5, J.sub.6 and J.sub.7 are each,
independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, 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;
[0375] or J.sub.4 forms a bridge with one of J.sub.5 or J.sub.7
wherein said bridge comprises from 1 to 3 linked biradical groups
selected from O, S, NR.sub.19, C(R.sub.20)(R.sub.21),
C(R.sub.20).dbd.C(R.sub.21), C[.dbd.C(R.sub.20)(R.sub.21)] and
C(.dbd.O) and the other two of J.sub.5, J.sub.6 and J.sub.7 are
each, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, 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;
[0376] each R.sub.19, R.sub.20 and R.sub.21 is, independently, H,
C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy,
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;
[0377] G is H, OH, halogen or
O--[C(R.sub.8)(R.sub.9)].sub.n--[(C.dbd.O).sub.m--X.sub.1].sub.j--Z;
[0378] each R.sub.8 and R.sub.9 is, independently, H, halogen,
C.sub.1-C.sub.6 alkyl or substituted C.sub.1-C.sub.6 alkyl;
[0379] X.sub.1 is O, S or N(E.sub.1);
[0380] Z is 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, substituted
C.sub.2-C.sub.6 alkynyl or N(E.sub.2)(E.sub.3);
[0381] E.sub.1, E.sub.2 and E.sub.3 are each, independently, H,
C.sub.1-C.sub.6 alkyl or substituted C.sub.1-C.sub.6 alkyl;
[0382] n is from 1 to about 6;
[0383] m is 0 or 1;
[0384] j is 0 or 1;
[0385] each substituted group comprises one or more optionally
protected substituent groups independently selected from halogen,
OJ.sub.1, N(J.sub.1)(J.sub.2), =NJ.sub.1, SJ.sub.1, N.sub.3, CN,
OC(.dbd.X.sub.2)J.sub.1, OC(.dbd.X.sub.2)--N(J.sub.1)(J.sub.2) and
C(.dbd.X.sub.2)N(J.sub.1)(J.sub.2);
[0386] X.sub.2 is O, S or NJ.sub.3;
[0387] each J.sub.1, J.sub.2 and J.sub.3 is, independently, H or
C.sub.1-C.sub.6 alkyl;
[0388] when j is 1 then Z is other than halogen or
N(E.sub.2)(E.sub.3); and
[0389] wherein said oligomeric compound comprises from 8 to 40
monomeric subunits and is hybridizable to at least a portion of a
target nucleic acid.
[0390] In certain embodiments, M.sub.3 is O, CH.dbd.CH, OCH.sub.2
or OC(H)(Bx.sub.2). In certain embodiments, M.sub.3 is O.
[0391] In certain embodiments, J.sub.4, J.sub.5, J.sub.6 and
J.sub.7 are each H. In certain embodiments, J.sub.4 forms a bridge
with one of J.sub.5 or J.sub.7.
[0392] In certain embodiments, A has one of the formulas:
##STR00015##
wherein:
[0393] Q.sub.1 and Q.sub.2 are each, independently, H, halogen,
C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkoxy or substituted C.sub.1-C.sub.6 alkoxy. In
certain embodiments, Q.sub.1 and Q.sub.2 are each H. In certain
embodiments, Q.sub.1 and Q.sub.2 are each, independently, H or
halogen. In certain embodiments, Q.sub.1 and Q.sub.2 is H and the
other of Q.sub.1 and Q.sub.2 is F, CH.sub.3 or OCH.sub.3.
[0394] In certain embodiments, T.sub.1 has the formula:
##STR00016##
wherein:
[0395] R.sub.a and R.sub.c are each, independently, protected
hydroxyl, protected thiol, C.sub.1-C.sub.6 alkyl, substituted
C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted
C.sub.1-C.sub.6 alkoxy, protected amino or substituted amino;
and
[0396] R.sub.b is O or S. In certain embodiments, R.sub.b is O and
R.sub.a and R.sub.c are each, independently, OCH.sub.3,
OCH.sub.2CH.sub.3 or CH(CH.sub.3).sub.2.
[0397] In certain embodiments, G is halogen, OCH.sub.3, OCH.sub.2F,
OCHF.sub.2, OCF.sub.3, OCH.sub.2CH.sub.3, O(CH.sub.2).sub.2F,
OCH.sub.2CHF.sub.2, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2,
O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--SCH.sub.3,
O(CH.sub.2).sub.2--OCF.sub.3,
O(CH.sub.2).sub.3--N(R.sub.10)(R.sub.11),
O(CH.sub.2).sub.2--ON(R.sub.10)(R.sub.11),
O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(R.sub.10)(R.sub.11),
OCH.sub.2C(.dbd.O)--N(R.sub.10)(R.sub.11),
OCH.sub.2C(.dbd.O)--N(R.sub.12)--(CH.sub.2).sub.2--N(R.sub.10)(R.sub.11)
or
O(CH.sub.2).sub.2--N(R.sub.12)--C(.dbd.NR.sub.13)[N(R.sub.10)(R.sub.11-
)] wherein R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are each,
independently, H or C.sub.1-C.sub.6 alkyl. In certain embodiments,
G is halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3,
OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2,
O(CH.sub.2).sub.2--OCH.sub.3,
O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2,
OCH.sub.2C(.dbd.O)--N(H)CH.sub.3,
OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2 or
OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2. In certain embodiments, G is
F, OCH.sub.3 or O(CH.sub.2).sub.2--OCH.sub.3. In certain
embodiments, G is O(CH.sub.2).sub.2--OCH.sub.3.
[0398] In certain embodiments, the 5'-terminal nucleoside has
Formula lie:
##STR00017##
[0399] In certain embodiments, antisense compounds, including those
particularly suitable for ssRNA comprise one or more type of
modified sugar moieties and/or naturally occurring sugar moieties
arranged along an oligonucleotide or region thereof in a defined
pattern or sugar modification motif. Such motifs may include any of
the sugar modifications discussed herein and/or other known sugar
modifications.
[0400] In certain embodiments, the oligonucleotides comprise or
consist of a region having uniform sugar modifications. In certain
such embodiments, each nucleoside of the region comprises the same
RNA-like sugar modification. In certain embodiments, each
nucleoside of the region is a 2'-F nucleoside. In certain
embodiments, each nucleoside of the region is a 2'-OMe nucleoside.
In certain embodiments, each nucleoside of the region is a 2'-MOE
nucleoside. In certain embodiments, each nucleoside of the region
is a cEt nucleoside. In certain embodiments, each nucleoside of the
region is an LNA nucleoside. In certain embodiments, the uniform
region constitutes all or essentially all of the oligonucleotide.
In certain embodiments, the region constitutes the entire
oligonucleotide except for 1-4 terminal nucleosides.
[0401] In certain embodiments, oligonucleotides comprise one or
more regions of alternating sugar modifications, wherein the
nucleosides alternate between nucleotides having a sugar
modification of a first type and nucleotides having a sugar
modification of a second type. In certain embodiments, nucleosides
of both types are RNA-like nucleosides. In certain embodiments the
alternating nucleosides are selected from: 2'-OMe, 2'-F, 2'-MOE,
LNA, and cEt. In certain embodiments, the alternating modifications
are 2'-F and 2'-OMe. Such regions may be contiguous or may be
interrupted by differently modified nucleosides or conjugated
nucleosides.
[0402] In certain embodiments, the alternating region of
alternating modifications each consist of a single nucleoside
(i.e., the pattern is (AB).sub.xA.sub.y wherein A is a nucleoside
having a sugar modification of a first type and B is a nucleoside
having a sugar modification of a second type; x is 1-20 and y is 0
or 1). In certain embodiments, one or more alternating regions in
an alternating motif includes more than a single nucleoside of a
type. For example, oligonucleotides may include one or more regions
of any of the following nucleoside motifs:
AABBAA;
ABBABB;
AABAAB;
ABBABAABB;
ABABAA;
AABABAB;
ABABAA;
ABBAABBABABAA;
BABBAABBABABAA; or
ABABBAABBABABAA;
[0403] wherein A is a nucleoside of a first type and B is a
nucleoside of a second type. In certain embodiments, A and B are
each selected from 2'-F, 2'-OMe, BNA, and MOE.
[0404] In certain embodiments, oligonucleotides having such an
alternating motif also comprise a modified 5' terminal nucleoside,
such as those of formula IIc or IIe.
[0405] In certain embodiments, oligonucleotides comprise a region
having a 2-2-3 motif. Such regions comprises the following
motif:
-(A).sub.2-(B).sub.x(A).sub.2-(C).sub.y-(A).sub.3-
[0406] wherein: A is a first type of modified nucleoside;
[0407] B and C, are nucleosides that are differently modified than
A, however, B and C may have the same or different modifications as
one another;
[0408] x and y are from 1 to 15.
[0409] In certain embodiments, A is a 2'-OMe modified nucleoside.
In certain embodiments, B and C are both 2'-F modified nucleosides.
In certain embodiments, A is a 2'-OMe modified nucleoside and B and
C are both 2'-F modified nucleosides.
[0410] In certain embodiments, oligonucleosides have the following
sugar motif:
5'-(Q)-(AB).sub.xA.sub.y-(D).sub.z
wherein:
[0411] Q is a nucleoside comprising a stabilized phosphate moiety.
In certain embodiments, Q is a nucleoside having Formula IIc or
IIe;
[0412] A is a first type of modified nucleoside;
[0413] B is a second type of modified nucleoside;
[0414] D is a modified nucleoside comprising a modification
different from the nucleoside adjacent to it. Thus, if y is 0, then
D must be differently modified than B and if y is 1, then D must be
differently modified than A. In certain embodiments, D differs from
both A and B.
[0415] X is 5-15;
[0416] Y is 0 or 1;
[0417] Z is 0-4.
[0418] In certain embodiments, oligonucleosides have the following
sugar motif:
5'-(Q)-(A).sub.x(D).sub.z
wherein:
[0419] Q is a nucleoside comprising a stabilized phosphate moiety.
In certain embodiments, Q is a nucleoside having Formula IIc or
IIe;
[0420] A is a first type of modified nucleoside;
[0421] D is a modified nucleoside comprising a modification
different from A.
[0422] X is 11-30;
[0423] Z is 0-4.
[0424] In certain embodiments A, B, C, and D in the above motifs
are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA, and cEt. In certain
embodiments, D represents terminal nucleosides. In certain
embodiments, such terminal nucleosides are not designed to
hybridize to the target nucleic acid (though one or more might
hybridize by chance). In certain embodiments, the nucleobase of
each D nucleoside is adenine, regardless of the identity of the
nucleobase at the corresponding position of the target nucleic
acid. In certain embodiments the nucleobase of each D nucleoside is
thymine.
[0425] In certain embodiments, antisense compounds, including those
particularly suited for use as ssRNA comprise modified
internucleoside linkages arranged along the oligonucleotide or
region thereof in a defined pattern or modified internucleoside
linkage motif. In certain embodiments, oligonucleotides comprise a
region having an alternating internucleoside linkage motif. In
certain embodiments, oligonucleotides comprise a region of
uniformly modified internucleoside linkages. In certain such
embodiments, the oligonucleotide comprises a region that is
uniformly linked by phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide is uniformly linked by
phosphorothioate internucleoside linkages. In certain embodiments,
each internucleoside linkage of the oligonucleotide is selected
from phosphodiester and phosphorothioate. In certain embodiments,
each internucleoside linkage of the oligonucleotide is selected
from phosphodiester and phosphorothioate and at least one
internucleoside linkage is phosphorothioate.
[0426] In certain embodiments, the oligonucleotide comprises at
least 6 phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least 8
phosphorothioate internucleoside linkages. In certain embodiments,
the oligonucleotide comprises at least 10 phosphorothioate
internucleoside linkages. In certain embodiments, the
oligonucleotide comprises at least one block of at least 6
consecutive phosphorothioate internucleoside linkages. In certain
embodiments, the oligonucleotide comprises at least one block of at
least 8 consecutive phosphorothioate internucleoside linkages. In
certain embodiments, the oligonucleotide comprises at least one
block of at least 10 consecutive phosphorothioate internucleoside
linkages. In certain embodiments, the oligonucleotide comprises at
least one block of at least one 12 consecutive phosphorothioate
internucleoside linkages. In certain such embodiments, at least one
such block is located at the 3' end of the oligonucleotide. In
certain such embodiments, at least one such block is located within
3 nucleosides of the 3' end of the oligonucleotide.
[0427] Oligonucleotides having any of the various sugar motifs
described herein, may have any linkage motif. For example, the
oligonucleotides, including but not limited to those described
above, may have a linkage motif selected from non-limiting the
table below:
TABLE-US-00001 5' most linkage Central region 3'-region PS
Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS PS Alternating
PO/PS 8 PS
[0428] ii. siRNA Compounds
[0429] In certain embodiments, antisense compounds are
double-stranded RNAi compounds (siRNA). In such embodiments, one or
both strands may comprise any modification motif described above
for ssRNA. In certain embodiments, ssRNA compounds may be
unmodified RNA. In certain embodiments, siRNA compounds may
comprise unmodified RNA nucleosides, but modified internucleoside
linkages.
[0430] Several embodiments relate to double-stranded compositions
wherein each strand comprises a motif defined by the location of
one or more modified or unmodified nucleosides. In certain
embodiments, compositions are provided comprising a first and a
second oligomeric compound that are fully or at least partially
hybridized to form a duplex region and further comprising a region
that is complementary to and hybridizes to a nucleic acid target.
It is suitable that such a composition comprise a first oligomeric
compound that is an antisense strand having full or partial
complementarity to a nucleic acid target and a second oligomeric
compound that is a sense strand having one or more regions of
complementarity to and forming at least one duplex region with the
first oligomeric compound.
[0431] The compositions of several embodiments modulate gene
expression by hybridizing to a nucleic acid target resulting in
loss of its normal function. In some embodiments, the target
nucleic acid is ApoCIII. In certain embodiment, the degradation of
the targeted ApoCIII is facilitated by an activated RISC complex
that is formed with compositions of the invention.
[0432] Several embodiments are directed to double-stranded
compositions wherein one of the strands is useful in, for example,
influencing the preferential loading of the opposite strand into
the RISC (or cleavage) complex. The compositions are useful for
targeting selected nucleic acid molecules and modulating the
expression of one or more genes. In some embodiments, the
compositions of the present invention hybridize to a portion of a
target RNA resulting in loss of normal function of the target
RNA.
[0433] Certain embodiments are drawn to double-stranded
compositions wherein both the strands comprise a hemimer motif, a
fully modified motif, a positionally modified motif or an
alternating motif. Each strand of the compositions of the present
invention can be modified to fulfill a particular role in for
example the siRNA pathway. Using a different motif in each strand
or the same motif with different chemical modifications in each
strand permits targeting the antisense strand for the RISC complex
while inhibiting the incorporation of the sense strand. Within this
model, each strand can be independently modified such that it is
enhanced for its particular role. The antisense strand can be
modified at the 5'-end to enhance its role in one region of the
RISC while the 3'-end can be modified differentially to enhance its
role in a different region of the RISC.
[0434] The double-stranded oligonucleotide molecules can be a
double-stranded polynucleotide molecule comprising
self-complementary sense and antisense regions, wherein the
antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense region having
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The double-stranded oligonucleotide
molecules can be assembled from two separate oligonucleotides,
where one strand is the sense strand and the other is the antisense
strand, wherein the antisense and sense strands are
self-complementary (i.e. each strand comprises nucleotide sequence
that is complementary to nucleotide sequence in the other strand;
such as where the antisense strand and sense strand form a duplex
or double-stranded structure, for example wherein the
double-stranded region is about 15 to about 30, e.g., about 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base
pairs; the antisense strand comprises nucleotide sequence that is
complementary to nucleotide sequence in a target nucleic acid
molecule or a portion thereof and the sense strand comprises
nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof (e.g., about 15 to about 25 or more
nucleotides of the double-stranded oligonucleotide molecule are
complementary to the target nucleic acid or a portion thereof).
Alternatively, the double-stranded oligonucleotide is assembled
from a single oligonucleotide, where the self-complementary sense
and antisense regions of the siRNA are linked by means of a nucleic
acid based or non-nucleic acid-based linker(s).
[0435] The double-stranded oligonucleotide can be a polynucleotide
with a duplex, asymmetric duplex, hairpin or asymmetric hairpin
secondary structure, having self-complementary sense and antisense
regions, wherein the antisense region comprises nucleotide sequence
that is complementary to nucleotide sequence in a separate target
nucleic acid molecule or a portion thereof and the sense region
having nucleotide sequence corresponding to the target nucleic acid
sequence or a portion thereof. The double-stranded oligonucleotide
can be a circular single-stranded polynucleotide having two or more
loop structures and a stem comprising self-complementary sense and
antisense regions, wherein the antisense region comprises
nucleotide sequence that is complementary to nucleotide sequence in
a target nucleic acid molecule or a portion thereof and the sense
region having nucleotide sequence corresponding to the target
nucleic acid sequence or a portion thereof, and wherein the
circular polynucleotide can be processed either in vivo or in vitro
to generate an active siRNA molecule capable of mediating RNAi.
[0436] In certain embodiments, the double-stranded oligonucleotide
comprises separate sense and antisense sequences or regions,
wherein the sense and antisense regions are covalently linked by
nucleotide or non-nucleotide linkers molecules as is known in the
art, or are alternately non-covalently linked by ionic
interactions, hydrogen bonding, van der Waals interactions,
hydrophobic interactions, and/or stacking interactions. In certain
embodiments, the double-stranded oligonucleotide comprises
nucleotide sequence that is complementary to nucleotide sequence of
a target gene. In another embodiment, the double-stranded
oligonucleotide interacts with nucleotide sequence of a target gene
in a manner that causes inhibition of expression of the target
gene.
[0437] As used herein, double-stranded oligonucleotides need not be
limited to those molecules containing only RNA, but further
encompasses chemically modified nucleotides and non-nucleotides. In
certain embodiments, the short interfering nucleic acid molecules
lack 2'-hydroxy (2'-OH) containing nucleotides. In certain
embodiments short interfering nucleic acids optionally do not
include any ribonucleotides (e.g., nucleotides having a 2'-OH
group). Such double-stranded oligonucleotides that do not require
the presence of ribonucleotides within the molecule to support RNAi
can however have an attached linker or linkers or other attached or
associated groups, moieties, or chains containing one or more
nucleotides with 2'-OH groups. Optionally, double-stranded
oligonucleotides can comprise ribonucleotides at about 5, 10, 20,
30, 40, or 50% of the nucleotide positions. As used herein, the
term siRNA is meant to be equivalent to other terms used to
describe nucleic acid molecules that are capable of mediating
sequence specific RNAi, for example short interfering RNA (siRNA),
double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA
(shRNA), short interfering oligonucleotide, short interfering
nucleic acid, short interfering modified oligonucleotide,
chemically modified siRNA, post-transcriptional gene silencing RNA
(ptgsRNA), ssRNAi and others. In addition, as used herein, the term
RNAi is meant to be equivalent to other terms used to describe
sequence specific RNA interference, such as post transcriptional
gene silencing, translational inhibition, or epigenetics. For
example, double-stranded oligonucleotides can be used to
epigenetically silence genes at both the post-transcriptional level
and the pre-transcriptional level. In a non-limiting example,
epigenetic regulation of gene expression by siRNA molecules of the
invention can result from siRNA mediated modification of chromatin
structure or methylation pattern to alter gene expression (see, for
example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et
al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297,
1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,
2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,
2232-2237).
[0438] It is contemplated that compounds and compositions of
several embodiments provided herein can target ApoCIII by a
dsRNA-mediated gene silencing or RNAi mechanism, including, e.g.,
"hairpin" or stem-loop double-stranded RNA effector molecules in
which a single RNA strand with self-complementary sequences is
capable of assuming a double-stranded conformation, or duplex dsRNA
effector molecules comprising two separate strands of RNA. In
various embodiments, the dsRNA consists entirely of ribonucleotides
or consists of a mixture of ribonucleotides and deoxynucleotides,
such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364,
filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21,
1999. The dsRNA or dsRNA effector molecule may be a single molecule
with a region of self-complementarity such that nucleotides in one
segment of the molecule base pair with nucleotides in another
segment of the molecule. In various embodiments, a dsRNA that
consists of a single molecule consists entirely of ribonucleotides
or includes a region of ribonucleotides that is complementary to a
region of deoxyribonucleotides. Alternatively, the dsRNA may
include two different strands that have a region of complementarity
to each other.
[0439] In various embodiments, both strands consist entirely of
ribonucleotides, one strand consists entirely of ribonucleotides
and one strand consists entirely of deoxyribonucleotides, or one or
both strands contain a mixture of ribonucleotides and
deoxyribonucleotides. In certain embodiments, the regions of
complementarity are at least 70, 80, 90, 95, 98, or 100%
complementary to each other and to a target nucleic acid sequence.
In certain embodiments, the region of the dsRNA that is present in
a double-stranded conformation includes at least 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000
or 5000 nucleotides or includes all of the nucleotides in a cDNA or
other target nucleic acid sequence being represented in the dsRNA.
In some embodiments, the dsRNA does not contain any single stranded
regions, such as single stranded ends, or the dsRNA is a hairpin.
In other embodiments, the dsRNA has one or more single stranded
regions or overhangs. In certain embodiments, RNA/DNA hybrids
include a DNA strand or region that is an antisense strand or
region (e.g, has at least 70, 80, 90, 95, 98, or 100%
complementarity to a target nucleic acid) and an RNA strand or
region that is a sense strand or region (e.g, has at least 70, 80,
90, 95, 98, or 100% identity to a target nucleic acid), and vice
versa.
[0440] In various embodiments, the RNA/DNA hybrid is made in vitro
using enzymatic or chemical synthetic methods such as those
described herein or those described in WO 00/63364, filed Apr. 19,
2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other
embodiments, a DNA strand synthesized in vitro is complexed with an
RNA strand made in vivo or in vitro before, after, or concurrent
with the transformation of the DNA strand into the cell. In yet
other embodiments, the dsRNA is a single circular nucleic acid
containing a sense and an antisense region, or the dsRNA includes a
circular nucleic acid and either a second circular nucleic acid or
a linear nucleic acid (see, for example, WO 00/63364, filed Apr.
19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.)
Exemplary circular nucleic acids include lariat structures in which
the free 5' phosphoryl group of a nucleotide becomes linked to the
2' hydroxyl group of another nucleotide in a loop back fashion.
[0441] In other embodiments, the dsRNA includes one or more
modified nucleotides in which the 2' position in the sugar contains
a halogen (such as fluorine group) or contains an alkoxy group
(such as a methoxy group) which increases the half-life of the
dsRNA in vitro or in vivo compared to the corresponding dsRNA in
which the corresponding 2' position contains a hydrogen or an
hydroxyl group. In yet other embodiments, the dsRNA includes one or
more linkages between adjacent nucleotides other than a
naturally-occurring phosphodiester linkage. Examples of such
linkages include phosphoramide, phosphorothioate, and
phosphorodithioate linkages. The dsRNAs may also be chemically
modified nucleic acid molecules as taught in U.S. Pat. No.
6,673,661. In other embodiments, the dsRNA contains one or two
capped strands, as disclosed, for example, by WO 00/63364, filed
Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21,
1999.
[0442] In other embodiments, the dsRNA can be any of the at least
partially dsRNA molecules disclosed in WO 00/63364, as well as any
of the dsRNA molecules described in U.S. Provisional Application
60/399,998; and U.S. Provisional Application 60/419,532, and
PCT/US2003/033466, the teaching of which is hereby incorporated by
reference. Any of the dsRNAs may be expressed in vitro or in vivo
using the methods described herein or standard methods, such as
those described in WO 00/63364.
Compositions and Methods for Formulating Pharmaceutical
Compositions
[0443] Antisense compounds 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.
[0444] Antisense compounds 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.
[0445] 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.).
[0446] 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.
[0447] 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.
[0448] 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.
[0449] 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
[0450] 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.
[0451] 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
[0452] 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,
N.C.; 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
[0453] Described herein are methods for treatment of cells with
antisense oligonucleotides, which can be modified appropriately for
treatment with other antisense compounds.
[0454] In general, cells are treated with antisense
oligonucleotides when the cells reach approximately 60-80%
confluence in culture.
[0455] 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.
[0456] 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.
[0457] 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.
[0458] 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.
[0459] 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.
[0460] Another technique used to introduce antisense
oligonucleotides into cultured cells includes electroporation
(Sambrook and Russell in Molecular Cloning. A Laboratory Manual.
Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 2001).
[0461] 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 (Sambrook 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.
[0462] 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 (Sambrook 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
[0463] RNA analysis can be performed on total cellular RNA or
poly(A)+ mRNA. Methods of RNA isolation are well known in the art
(Sambrook 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
[0464] Inhibition of levels or expression of an ApoCIII nucleic
acid can be assayed in a variety of ways known in the art (Sambrook
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
[0465] 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.
[0466] 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.
[0467] 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.
[0468] 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.).
[0469] 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
[0470] 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 immunosorbent assay
(ELISA), quantitative protein assays, protein activity assays (for
example, caspase activity assays), immunohistochemistry,
immunocytochemistry or fluorescence-activated cell sorting (FACS)
(Sambrook 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
[0471] 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
[0472] Novel effects of ApoCIII inhibition in patients with
Fredrickson Type I dyslipidemia, FCS, LPLD, have been identified
and disclosed herein. The example disclosed hereinbelow disclose
surprising reductions in TG and increases in HDL among other
biomarkers in Fredrickson Type I dyslipidemia, FCS, LPLD, patients
who have little or no detectable LPL activity,
[0473] Without being bound by any particular theory, two potential
explanations for the surprising results are discussed. First,
inhibiting ApoCIII may activate residual LPL activity in the
Fredrickson Type I dyslipidemia, FCS, LPLD, patients. This is not a
very likely explanation as these patients have little to no
detectable LPL activity while ApoCIII inhibition has profoundly
affected TG and HDL levels. Second, and more likely, is that
ApoCIII inhibits clearance of TG particles mediated by
apoE-mediated receptors such as the low density lipoprotein
receptor-related protein 1 (LRP1) or Syndecan 1. Once ApoCIII is
removed from VLDL and chylomicron particles, they become more
amenable to uptake by the liver. Indeed, these receptor mediated
clearance mechanisms may significantly contribute to the clinically
observed phenotype (e.g., substantial TG lowering) observed in the
Fredrickson Type I dyslipidemia, FCS, LPLD, patients treated with
an ApoCIII inhibitor.
[0474] In certain embodiments, provided herein are methods of
treating a subject with Fredrickson Type I dyslipidemia, FCS, LPLD,
comprising administering one or more pharmaceutical compositions as
described herein. In certain embodiments, the pharmaceutical
composition comprises an antisense compound targeted to an
ApoCIII.
[0475] In certain embodiments, administration of an antisense
compound targeted to an ApoCIII nucleic acid to a subject with
Fredrickson Type I dyslipidemia, FCS, LPLD, 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.
[0476] In certain embodiments, the subject has a disease or
disorder related to Fredrickson Type I dyslipidemia, FCS, LPLD. In
certain embodiments the disease or disorder is a cardiovascular or
metabolic disease or disorder. In certain embodiments, the disease
is pancreatitis.
[0477] In certain embodiments, the cardiovascular disease include,
but are not limited to, 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 (e.g., FCS) or
hypertriglyceridemia. In certain embodiments, the disease is
pancreatitis caused by dyslipidemia.
[0478] In certain embodiments, the metabolic disease or disorder
include, but are not limited to, hyperglycemia, prediabetes,
diabetes (type I and type II), obesity, insulin resistance,
metabolic syndrome and diabetic dyslipidemia.
[0479] In certain embodiments, compounds targeted to ApoCIII as
described herein modulate physiological markers or phenotypes of
pancreatitis, a cardiovascular or a metabolic disease or disorder
in a subject with Fredrickson Type I dyslipidemia, FCS, LPLD. In
certain of the experiments, the compounds can increase or decrease
physiological markers or phenotypes compared to untreated animals.
In certain embodiments, the increase or decrease in physiological
markers or phenotypes is associated with inhibition of ApoCIII by
the compounds described herein.
[0480] In certain embodiments, physiological markers or phenotype
of a cardiovascular disease or disorder can be quantifiable. For
example, TG or HDL levels can be measured and quantified by, for
example, standard lipid tests. In certain embodiments,
physiological markers or phenotypes such as HDL can 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. In certain embodiments, physiological markers phenotypes
such as TG (postprandial or fasting) can 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
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.
[0481] In certain embodiments, physiological markers or phenotypes
of a metabolic disease or disorder can be quantifiable. For
example, glucose levels or insulin resistance can be measured and
quantified by standard tests known in the art. In certain
embodiments, physiological markers or phenotypes such as glucose
levels or insulin resistance can 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 certain
embodiments, physiological markers phenotypes such as insulin
sensitivity can 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.
[0482] Also, provided herein are methods for preventing, treating
or ameliorating a symptom associated with a disease or disorder in
a subject with Fredrickson Type I dyslipidemia, FCS, LPLD with a
compound described herein. In certain embodiments, provided is a
method for reducing the rate of onset of a symptom associated with
a disease associated with Fredrickson Type I dyslipidemia, FCS,
LPLD. In certain embodiments, provided is a method for reducing the
severity of a symptom associated with Fredrickson Type I
dyslipidemia, FCS, LPLD. In such embodiments, the methods comprise
administering to an individual with Fredrickson Type I dyslipidemia
a therapeutically effective amount of a compound targeted to an
ApoCIII nucleic acid. In certain embodiments the disease or
disorder is pancreatitis or a cardiovascular or metabolic disease
or disorder.
[0483] Cardiovascular diseases or disorders 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
in the methods described herein. In certain embodiments, the
symptom can 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.
[0484] Metabolic diseases or 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
in the methods described herein. In certain embodiments, the
symptom can 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.
[0485] Pancreatitis is characterized by numerous physical symptoms.
Any symptom known to one of skill in the art to be associated with
a pancreatitis can be prevented, treated, ameliorated or otherwise
modulated as set forth in the methods described herein. In certain
embodiments, the symptom can be any of, but not limited to,
abdominal pain, vomiting, nausea, and abdominal sensitivity to
pressure.
[0486] In certain embodiments, provided are methods of treating a
subject with Fredrickson Type I dyslipidemia, FCS, LPLD, comprising
administering a therapeutically effective amount of one or more
pharmaceutical compositions as described herein. 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 disease markers
associated with Fredrickson Type I dyslipidemia, FCS, LPLD, to
determine a subject's response to the antisense compound. A
subject's response to administration of the antisense compound is
used by a physician to determine the amount and duration of
therapeutic intervention.
[0487] In certain embodiments, pharmaceutical compositions
comprising an antisense compound targeted to ApoCIII are used for
the preparation of a medicament for treating a subject with
Fredrickson Type I dyslipidemia, FCS, LPLD.
Administration
[0488] 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.
[0489] 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.
[0490] 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.
[0491] 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.
[0492] 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.
[0493] 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
[0494] 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 Fredrickson Type I dyslipidemia, FCS, LPLD.
[0495] 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.
[0496] 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
[0497] 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. In certain
embodiments, the first agent is administered to a subject that has
failed or become non-responsive to a second agent. In certain
embodiments, the first agent is administered to a subject in
replacement of a second agent.
[0498] 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.
[0499] In certain embodiments, second agents include, but are not
limited to, ApoCIII lowering agent, DGAT1 inhibitor, LPL raising
agent, cholesterol lowering agent, non-HDL lipid lowering (e.g.,
LDL) agent, HDL raising agent, fish oil, niacin (nicotinic acid),
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.
[0500] Examples of ApoCIII lowering agents include an ApoCIII
antisense oligonucleotide different from the first agent, fibrate
or an Apo B antisense oligonucleotide.
[0501] An example of a DGAT1 inhibitor is LCQ908 (Novartis
Pharmaceuticals) currently being tested in a Phase 3 clinical trial
for treating Familial Chylomicronemia Syndrome (FCS).
[0502] LPL raising agents include gene therapy agents that raise
the level of LPL. Examples of such agents include copies of normal
genes that supplement the lack of the normal gene. For example,
Glybera.sup.R raises LPL levels by providing normal copies of the
LPL gene to supplement a lack of the normal LPL gene. In other
examples, the LPL raising agent includes normal copies of ApoC-II,
GPIHBP1, APOA5, LMF1 or other genes that, when mutated, can lead to
dysfunctional LPL. In certain embodiments, the combination of the
first agent (e.g., ApoCIII ASO) and the second agent (e.g.,
Glybera) provides an additive or synergistic effect. In certain
embodiments, the first agent (e.g., ApoCIII ASO) is administered to
a subject that has failed or become non-responsive to a second
agent (e.g., Glybera.sup.R).
[0503] 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.
[0504] 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. The
therapeutic lifestyle change can be dietary fat restriction.
[0505] 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
[0506] 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
Fredrickson Type I dyslipidemia, FCS, LPLD.
[0507] Subjects with Fredrickson Type I dyslipidemia, FCS, LPLD,
are at a significant risk of pancreatitis, cardiovascular and
metabolic disease. For these subjects, recurrent pancreatitis is
the most debilitating and potentially lethal complication; other
sequelae include increased tendency for atherosclerosis and
diabetes.
[0508] Fredrickson Type I, FCS, LPLD, subjects lack a significant
amount of functionally active LPL. ApoCIII plays an important role
in TG metabolism and is an independent risk factor for
cardiovascular disease in subjects with functional or partially
functional LPL. ApoCIII is currently in clinical trials to treat
non-Fredrickson Type I hypertriglyceridemia subjects. However, as
ApoCIII pathway is thought to work through the LPL pathway,
inhibition of ApoCIII has not been considered as a treatment option
for Fredrickson Type I, FCS, LPLD, subjects.
[0509] ApoCIII inhibition, as shown herein, unexpectedly decreases
TG levels and/or raises HDL levels in Fredrickson Type I
dyslipidemic, FCS, LPLD, subjects. The decrease in TG and/or
increase in HDL can, in turn, prevent, treat, delay or ameliorate a
disease, disorder, or symptom thereof, associated with Fredrickson
Type I dyslipidemia, FCS, LPLD.
Certain Compounds
[0510] We have previously disclosed compositions comprising
antisense compounds targeting ApoCIII and methods for inhibiting
ApoCIII by the antisense compounds in US 20040208856 (U.S. Pat. No.
7,598,227), US 20060264395 (U.S. Pat. No. 7,750,141), WO
2004/093783 and WO 2012/149495, all incorporated-by-reference
herein. In these applications, a series of antisense compounds was
designed to target different regions of the human ApoCIII RNA,
using published sequences (nucleotides 6238608 to 6242565 of
GenBank accession number NT_035088.1, representing a genomic
sequence, incorporated herein as SEQ ID NO: 4, and GenBank
accession number NM_000040.1, incorporated herein as SEQ ID NO: 1).
The compounds were chimeric oligonucleotides ("gapmers") 20
nucleotides in length, composed of a central "gap" region
consisting of ten 2'-deoxynucleotides, which is flanked on both
sides (5' and 3' directions) by five-nucleotide "wings". The wings
are composed of 2'-O-(2-methoxyethyl) nucleotides, also known as
(2'-MOE) nucleotides. The internucleoside (backbone) linkages are
phosphorothioate (P.dbd.S) throughout the oligonucleotide. All
cytosine residues are 5-methylcytosines.
[0511] The antisense compounds were analyzed for their effect on
human ApoCIII mRNA levels in HepG2 cells by quantitative real-time
PCR. Several compounds demonstrated at least 45% inhibition of
ApoCIII mRNA and are therefore preferred. Several compounds
demonstrated at least 50% inhibition of human ApoCIII mRNA and are
therefore preferred. Several compounds demonstrated at least 60%
inhibition of human ApoCIII mRNA and are therefore preferred.
Several compounds demonstrated at least 70% inhibition of human
ApoCIII mRNA and are therefore preferred. Several compounds
demonstrated at least 80% inhibition of human ApoCIII mRNA and are
therefore preferred. Several compounds demonstrated at least 90%
inhibition of human ApoCIII mRNA and are therefore preferred.
[0512] The target regions to which these preferred antisense
compounds are complementary are referred to as "preferred target
segments" and are therefore preferred for targeting by antisense
compounds.
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
[0513] 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: ISIS 304801 Clinical Trial
[0514] As described herein, an open label study was performed on
patients with Fredrickson Type I dyslipidemia, FCS, LPLD, to
evaluate the response to, and the pharmacodynamic effects of, the
Study Drug ISIS 304801. ISIS 304801 was previously disclosed in
U.S. Pat. No. 7,598,227 and has the sequence
5'-AGCTTCTTGTCCAGCTTTAT-3' (SEQ ID NO: 3) starting at position 508
on SEQ ID NO: 1 (GENBANK Accession No. NM 000040.1) or starting at
position 3139 on SEQ ID NO: 2 (GENBANK Accession NT 033899.8
truncated from nucleotides 20262640 to 20266603). ISIS 304801 has a
5-10-5 MOE gapmer motif comprising 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, wherein each
nucleoside of each wing segment comprises a 2'-O-methyoxyethyl
sugar, wherein each cytosine is a 5'-methylcytosine, and wherein
each internucleoside linkage is a phosphorothioate linkage. ISIS
304801 has been shown to be potent in inhibiting ApoC-III and
tolerable when administered to subjects.
[0515] Many of the patients recruited for this study have been
diagnosed with Fredrickson Type I dyslipidemia, FCS, LPLD.
Fredrickson Type I, FCS, LPLD, patients with a history of TG level
.gtoreq.880 mg/dL, fasting TG level .gtoreq.750 mg/dL during
screening for the study and/or TG level .gtoreq.440 mg/dL after
dieting but before the start of treatment are included in the
study.
[0516] To enlarge the study population, some patients suffering
from hyperTG but not diagnosed with Fredrickson Type I
dyslipidemia, FCS, LPLD, may be screened for Fredrickson Type I
dyslipidemia, FCS, LPLD. In an example, patients with hyperTG will
be identified through their medical history with a TG level
.gtoreq.880 mg/dL and/or by centrifugation of the lipids in their
blood for fasting TG level .gtoreq.750 mg/dL. The patients with
fasting TG level .gtoreq.750 mg/dL will be further screened for at
least one of the following parameters to confirm the diagnosis of
Fredrickson Type I dyslipidemia, FCS, LPLD:
[0517] (1) homozygous or compound heterozygous loss-of-function
mutations in genes such as LPL (e.g., P207L, G188L, D9N), ApoC2,
GPIHBP1, ApoC5 or LMF1 known to cause Fredrickson Type I
dyslipidemia, FCS, LPLD;
[0518] (2) LPL activity .ltoreq.20% of normal; and
[0519] (3) anti-LPL antibodies.
[0520] For each patient diagnosed with Fredrickson Type I
dyslipidemia, FCS, LPLD, the participation period consists of a
.ltoreq.8-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 35 weeks of study participation. Patients with a diet
controlled TG level .gtoreq.440 mg/dL are included in the study.
Concomitant medications and adverse events (AEs) are recorded
throughout all periods of the study.
[0521] Patients are 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 have
baseline measurements and are assessed for qualification of
enrollment into the treatment phase of the study.
[0522] Endpoints to evaluate include: the pharmacodynamic (PD)
effects of ISIS 304801 as measured by fasting lipoprotein, total
ApoC-III, TG, ApoC-II (total and associated with VLDL),
apolipoprotein B-100 (apoB-100 and/or apoB-48), 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-cholesterol (CM-C), chylomicron-triglyceride (CM-TG),
free fatty acids (FFA), and glycerol levels; the post-prandial
lipid, apolipoprotein and lipoprotein characteristics and kinetics,
and glucose levels; and, the safety, tolerability and
pharmacokinetics (PK) of ISIS 304801. Additional endpoints to be
evaluated may include a decrease in CETP or an increase in ApoA1,
PON1, fat clearance and triglyceride clearance, and an improvement
in the ratio of HDL to TG.
Study Drug and Treatment
[0523] A solution of the Study Drug ISIS 304801 (200 mg/mL, 1.0 mL)
contained in 2-mL stoppered glass vials is provided. Vials are for
single-use only. ISIS 304801 solution and placebo are prepared by a
pharmacist (or qualified delegate). A trained professional
administers 300 mg of the Study Drug as a single SC injection in
the abdomen, thigh, or outer area of the upper arm on each dosing
day.
[0524] Patients receive 13 doses of the 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). Patients 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 an extensive PK
group also visit the clinic on Day 2 and 86.+-.0 days relative to
Day 1 and 85, respectively, for a 24 hour blood draw. Patients
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 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.
[0525] Preceding each visit that includes a blood draw for PD
measurements (Days 8, 15, 29, 43, 57, 71, and 85), patients are
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 remain
fasted. Alcohol consumption is not allowed for 48 hrs preceding
these clinic visits.
[0526] Blood is collected after fasting and/or after a meal for
measurement of VLDL, ApoC-III and other PD markers on Days 8, 15,
29, 43, 57, 71, and 85 (prior to Study Drug administration).
[0527] Patients in the post-prandial assessment group 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 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 receive a standardized pre-cooked meal 9
hrs after consuming the liquid meal, after which they fast until
the 24 hour blood draw the following day.
[0528] In addition to trough sample collection, patients in the
extensive PK assessment group undergo serial blood sampling for 24
hrs after their first (Day 1-2) and last (Day 85-86) dose of Study
Drug. PK parameters such as area under the curve (AUC), trough
concentration (Cmin) and others will be assessed.
Post-Treatment Evaluation Period
[0529] Patients are followed until Study Day 176. During this time,
patients 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.
[0530] Blood samples for PK and PD analysis are 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 are performed at the various times
throughout the study.
[0531] Post-prandial assessments are done in a subset of patients
as described below.
Post-Prandial Meal, Sampling Schedule, and Assessment
[0532] Post-prandial assessment for lipoproteins metabolism are
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).
[0533] 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.) are
administered as previously described (Normand-Lauziere et al. 2010,
PLoS. One, 5: e10956). Plasma palmitate and glycerol appearance
rates are 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).
[0534] Blood samples are 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 is
given to the participants after the 9 hr blood draw. Blood is
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.
[0535] The following are measured at each time-point: [0536] Plasma
and CM fraction levels for 3H-tracer [0537] Plasma [U-13C]-K
palmitate and [1, 1, 2, 3, 3-2H]-glycerol appearance rates [0538]
Plasma and CM fraction levels for TG, TC, and apoB [0539] Plasma
and VLDL fraction levels for apo CIII, apo CII, and apo E [0540]
Plasma levels for glucose
[0541] 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.
Results
[0542] Results for three patients diagnosed with Fredrickson Type I
dyslipidemia, FCS, LPLD, recruited for this study are presented
below. Two patients are homozygous for the P207L null LPL gene
mutation and one patient is compound heterozygous for the P207L and
G188E null LPL gene mutations. All patients have LPL mass but no or
extremely low levels (.ltoreq.5%) of LPL activity. The patients had
a TG level .gtoreq.440 mg/dL after dieting but before the start of
treatment. Two of the patients had confirmed past history of acute
pancreatitis and one had been on gene therapy with Glybera.sup.R in
December 2007.
[0543] The data for percent change in fasting ApoCIII levels is
presented in the Table below. The results indicate that treatment
with ISIS 304801 reduced fasting levels of ApoC-III. `n.d.`
indicates that data was not yet collected for that particular time
point.
TABLE-US-00002 TABLE 1 Percent change in fasting ApoCIII levels
Patient 1 Patient 2 Patient 3 Day 1 0 0 0 Day 8 n.d. -23 -18 Day 15
n.d. -63 -44 Day 29 -47 -69 -61 Day 43 -58 -80 -77 Day 57 -60 -85
-85 Day 71 -66 -90 -84 Day 85 -71 -91 -84 Day 92 -71 -90 -81 Day 99
-62 -87 -78 Day 127 -61 -68 -75 Day 176 -14 -67 -39
[0544] Levels of fasting triglyceride levels were also measured.
The data for percent change, as well as absolute levels, of fasting
triglyceride levels, are presented in the Tables below. The results
indicate that treatment with ISIS 304801 reduced fasting levels of
triglycerides.
TABLE-US-00003 TABLE 2 Percent change in fasting triglyceride
levels Patient 1 Patient 2 Patient 3 Day 1 0 0 0 Day 8 -39 -8 -6
Day 15 -35 -57 -63 Day 29 -54 -40 -61 Day 43 -49 -63 -81 Day 57 -55
-68 -82 Day 71 -53 -76 -89 Day 85 -49 -88 -71 Day 92 -64 -84 -57
Day 99 -17 -62 -69 Day 127 -66 -43 -79 Day 176 -6 -58 -16
TABLE-US-00004 TABLE 3 Fasting triglyceride levels (mg/dL) Patient
1 Patient 2 Patient 3 Day 1 1406 2083 2043 Day 8 851 1918 1922 Day
15 911 892 751 Day 29 651 1260 804 Day 43 719 775 389 Day 57 633
667 368 Day 71 658 505 234 Day 85 723 251 595 Day 92 510 324 874
Day 99 1167 793 626 Day 127 485 1197 429 Day 176 1317 867 1706
[0545] Levels of fasting non-HDL cholesterol levels were also
measured. The data for percent change, as well as absolute levels,
of fasting non-HDL cholesterol levels, are presented in the Tables
below. The results indicate that treatment with ISIS 304801 reduced
fasting levels of non-HDL cholesterol.
TABLE-US-00005 TABLE 4 Percent change in fasting non-HDL
cholesterol levels Patient 1 Patient 2 Patient 3 Day 1 0 0 0 Day 8
-23 -24 -15 Day 15 -19 -60 -51 Day 29 -38 -49 -50 Day 43 -43 -64
-64 Day 57 -43 -65 -59 Day 71 -44 -71 -55 Day 85 -42 -74 -56 Day 92
-51 -75 -53 Day 99 -21 -60 -55 Day 127 -42 -47 -56 Day 176 -2 -57
-16
TABLE-US-00006 TABLE 5 Fasting non-HDL cholesterol levels (mg/dL)
Patient 1 Patient 2 Patient 3 Day 1 214 327 244 Day 8 165 250 207
Day 15 173 131 119 Day 29 133 167 123 Day 43 123 118 88 Day 57 122
116 99 Day 71 119 96 109 Day 85 125 85 107 Day 92 104 83 115 Day 99
169 131 110 Day 127 125 173 108 Day 176 210 139 206
[0546] Levels of ApoB-48, a measure of chylomicrons, were also
measured. The data for percent change, as well as absolute levels,
of ApoB-48 levels, are presented in the Tables below. The results
indicate that treatment with ISIS 304801 reduced fasting levels of
ApoB-48.
TABLE-US-00007 TABLE 6 Percent change in ApoB-48 levels Patient 1
Patient 2 Patient 3 Day 1 0 0 0 Day 8 30 21 31 Day 15 13 -71 -64
Day 29 -48 -10 -35 Day 43 -21 -71 -76 Day 57 -36 -69 -75 Day 71 -21
-84 -80 Day 85 21 -89 -50 Day 92 -36 -92 -29 Day 99 190 -13 -55 Day
127 -39 86 -42 Day 176 366 -28 28
TABLE-US-00008 TABLE 7 ApoB-48 levels (mg/dL) Patient 1 Patient 2
Patient 3 Day 1 1.68 3.40 2.16 Day 8 2.19 4.13 2.82 Day 15 1.89
1.00 0.78 Day 29 0.87 3.07 1.40 Day 43 1.32 0.99 0.51 Day 57 1.07
1.04 0.55 Day 71 1.32 0.53 0.43 Day 85 2.03 0.36 1.07 Day 92 1.07
0.28 1.53 Day 99 4.87 2.97 0.98 Day 127 1.03 6.34 1.26 Day 176 7.83
2.45 2.77
[0547] The overall lipid profile in fasting FCS patients was
measured at the end of treatment and compared to baseline. The data
are presented in the Tables below and indicates that treatment with
ISIS 304801 improved the overall lipid profile in the patients.
TABLE-US-00009 TABLE 8 Percent change (mean) in lipid profile %
ApoC-III -81 Triglycerides -69 HDL-C +78 VLDL ApoC-III -80 ApoB -13
Non-HDL-C -58 VLDL -65 Total cholestetol -53
TABLE-US-00010 TABLE 9 Individual patient profile End of Absolute
Mean Lipid Baseline treatment change % % parameter Patient #
(mg/dL) (mg/dL) (mg/dL) change change ApoC-III 1 19 6 -13 -71 -81 2
35 3 -32 -90 3 20 4 -16 -83 Tri- 1 1406 617 -790 -56 -69 glycerides
2 2083 288 -1796 -86 3 2043 735 -1309 -64 VLDL 1 12 5 -8 -64 -80
ApoC-III 2 33 3 -30 -92 3 17 2 15 86 HDL-C 1 16 24 8 50 +78 2 8 21
13 163 3 14 17 3 21 Non 1 214 115 -100 -47 -58 HDL-C 2 327 84 -243
-74 3 244 111 -133 -55 ApoB 1 109 57 -53 -48 -13 2 65 68 3 5 3 114
120 6 5
Safety Assessment
[0548] Treatment with ISIS 304801 did not have any issues of liver
enzyme elevations more than three times the ULN, abnormalities in
renal function, meaningful clinical changes in other laboratory
values, or relates SAEs or significant AEs.
[0549] Treatment was tolerated by all the patients with no flu-like
symptoms and infrequent mild site reactions, which was resolved
without treatment. There were no discontinuations due to injection
site reactions.
Sequence CWU 1
1
41533DNAHomo sapiensCDS(47)..(346) 1tgctcagttc atccctagag
gcagctgctc caggaacaga ggtgcc atg cag ccc 55 Met Gln Pro 1cgg gta
ctc ctt gtt gtt gcc ctc ctg gcg ctc ctg gcc tct gcc cga 103Arg Val
Leu Leu Val Val Ala Leu Leu Ala Leu Leu Ala Ser Ala Arg 5 10 15gct
tca gag gcc gag gat gcc tcc ctt ctc agc ttc atg cag ggt tac 151Ala
Ser Glu Ala Glu Asp Ala Ser Leu Leu Ser Phe Met Gln Gly Tyr20 25 30
35atg aag cac gcc acc aag acc gcc aag gat gca ctg agc agc gtg cag
199Met Lys His Ala Thr Lys Thr Ala Lys Asp Ala Leu Ser Ser Val Gln
40 45 50gag tcc cag gtg gcc cag cag gcc agg ggc tgg gtg acc gat ggc
ttc 247Glu Ser Gln Val Ala Gln Gln Ala Arg Gly Trp Val Thr Asp Gly
Phe 55 60 65agt tcc ctg aaa gac tac tgg agc acc gtt aag gac aag ttc
tct gag 295Ser Ser Leu Lys Asp Tyr Trp Ser Thr Val Lys Asp Lys Phe
Ser Glu 70 75 80ttc tgg gat ttg gac cct gag gtc aga cca act tca gcc
gtg gct gcc 343Phe Trp Asp Leu Asp Pro Glu Val Arg Pro Thr Ser Ala
Val Ala Ala 85 90 95tga gacctcaata ccccaagtcc acctgcctat ccatcctgcg
agctccttgg 396gtcctgcaat ctccagggct gcccctgtag gttgcttaaa
agggacagta ttctcagtgc 456tctcctaccc cacctcatgc ctggcccccc
tccaggcatg ctggcctccc aataaagctg 516gacaagaagc tgctatg
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
2043958DNAHomo sapiens 4ctactccagg 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 gctacatagg 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 cgtccatagg ccgatccacc ccactcagcc
ctgctctttc 1260ctcaggagct tcagaggccg aggatgcctc ccttctcagc
ttcatgcagg gctacatgaa 1320gcacgccacc aagaccgcca aggatgcact
gagcagcgtg caggagtccc aggtggccca 1380gcaggccagg tacacccgct
ggcctccctc cccatccccc ctgccagctg cctccattcc 1440cacccacccc
tgccctggtg agatcccaac aatggaatgg aggtgctcca gcctcccctg
1500ggcctgtgcc tcttcagcct cctctttcct cacagggcct ttgtcaggct
gctgcgggag 1560agatgacaga gttgagactg cattcctccc aggtccctcc
tttctcccca gagcagtcct 1620agggcgcgcc gttttagccc tcatttccat
tttcctttcc tttccctttc tttccctttc 1680tatttctttc tttctttctt
tctttctttc tttctttctt tctttctttc tttctttctt 1740tctttctttc
ctttctttct ttcttttctt ctttctttct ttcctttctt tctctttctt
1800tctttctttc tttccttttt ctttctttcc ctctcttcct ttctctcttt
ctttcttctt 1860cttttttttt taatggagtc tccctctgtc acccaggctg
gagtgcagtg gtgccatctc 1920ggctcactgc aacctccgtc tcccgggttc
aacccattct cctgcctcag cctcccaagt 1980agctgggatt acaggcacgc
gccaccacac ccagctaatt tttgtatttt tagcagagat 2040ggggtttcac
catgttggcc aggttggtct tgaattcctg acctcagggg atcctcctgc
2100ctcggcctcc caaagcgctg ggattacagg catgagccac tgcgcctggc
cccattttcc 2160ttttctgaag gtctggctag agcagtggtc ctcagccttt
ttggcaccag ggaccagttt 2220tgtggtggac aatttttcca tgggccagcg
gggatggttt tgggatgaag ctgttccacc 2280tcagatcatc aggcattaga
ttctcataag gagccctcca cctagatccc tggcatgtgc 2340agttcacaac
agggttcaca ctcctatgag aatgtaaggc cacttgatct gacaggaggc
2400ggagctcagg cggtattgct cactcaccca ccactcactt cgtgctgtgc
agcccggctc 2460ctaacagtcc atggaccagt acctatctat gacttggggg
ttggggaccc ctgggctagg 2520ggtttgcctt gggaggcccc acctgaccta
attcaagccc gtgagtgctt ctgctttgtt 2580ctaagacctg gggccagtgt
gagcagaagt gtgtccttcc tctcccatcc tgcccctgcc 2640catcagtact
ctcctctccc ctactccctt ctccacctca ccctgactgg cattagctgg
2700catagcagag gtgttcataa acattcttag tccccagaac cggctttggg
gtaggtgtta 2760ttttctcact ttgcagatga gaaaattgag gctcagagcg
attaggtgac ctgccccaga 2820tcacacaact aatcaatcct ccaatgactt
tccaaatgag aggctgcctc cctctgtcct 2880accctgctca gagccaccag
gttgtgcaac tccaggcggt gctgtttgca cagaaaacaa 2940tgacagcctt
gacctttcac atctccccac cctgtcactt tgtgcctcag gcccaggggc
3000ataaacatct gaggtgacct ggagatggca gggtttgact tgtgctgggg
ttcctgcaag 3060gatatctctt ctcccagggt ggcagctgtg ggggattcct
gcctgaggtc tcagggctgt 3120cgtccagtga agttgagagg gtggtgtggt
cctgactggt gtcgtccagt ggggacatgg 3180gtgtgggtcc catggttgcc
tacagaggag ttctcatgcc ctgctctgtt gcttcccctg 3240actgatttag
gggctgggtg accgatggct tcagttccct gaaagactac tggagcaccg
3300ttaaggacaa gttctctgag ttctgggatt tggaccctga ggtcagacca
acttcagccg 3360tggctgcctg agacctcaat accccaagtc cacctgccta
tccatcctgc cagctccttg 3420ggtcctgcaa tctccagggc tgcccctgta
ggttgcttaa aagggacagt attctcagtg 3480ctctcctacc ccacctcatg
cctggccccc ctccaggcat gctggcctcc caataaagct 3540ggacaagaag
ctgctatgag tgggccgtcg caagtgtgcc atctgtgtct gggcatggga
3600aagggccgag gctgttctgt gggtgggcac tggacagact ccaggtcagg
caggcatgga 3660ggccagcgct ctatccacct tctggtagct gggcagtctc
tgggcctcag tttcttcatc 3720tctaaggtag gaatcaccct ccgtaccctg
ccttccttga cagctttgtg cggaaggtca 3780aacaggacaa taagtttgct
gatactttga taaactgtta ggtgctgcac aacatgactt 3840gagtgtgtgc
cccatgccag ccactatgcc tggcacttaa gttgtcatca gagttgagac
3900tgtgtgtgtt tactcaaaac tgtggagctg acctccccta tccaggccac ctagccct
3958
* * * * *
References