NZ616779B2 - Modulation of apolipoprotein ciii (apociii) expression - Google Patents

Modulation of apolipoprotein ciii (apociii) expression Download PDF

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NZ616779B2
NZ616779B2 NZ616779A NZ61677912A NZ616779B2 NZ 616779 B2 NZ616779 B2 NZ 616779B2 NZ 616779 A NZ616779 A NZ 616779A NZ 61677912 A NZ61677912 A NZ 61677912A NZ 616779 B2 NZ616779 B2 NZ 616779B2
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apociii
animal
certain embodiments
compound
levels
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NZ616779A
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NZ616779A (en
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Thomas A Bell
Rosanne M Crooke
Kenneth W Dobie
Mark J Graham
Richard Lee
Adam Mullick
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Ionis Pharmaceuticals Inc
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Priority claimed from PCT/US2012/035694 external-priority patent/WO2012149495A1/en
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Publication of NZ616779B2 publication Critical patent/NZ616779B2/en

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Abstract

Disclosed is a use of a compound targeting ApoCIII in the manufacture of a medicament for a) treating, preventing, delaying or ameliorating pancreatitis in an animal; b) increasing HDL levels and/or improving the ratio of TG to HDL in an animal thereby preventing, delaying or ameliorating a cardiovascular disease, disorder, condition or symptom thereof or onset of a cardiovascular disease, disorder, condition or symptom thereof in an animal; c) increasing HDL levels and/or improving the ratio of TG to HDL in an animal thereby reducing the risk of a cardiovascular disease, disorder, condition or symptom in an animal; and/or d) treating, preventing, delaying or ameliorating chylomicronemia, wherein the compound is capable of inhibiting apolipoprotein C-III. rdiovascular disease, disorder, condition or symptom thereof or onset of a cardiovascular disease, disorder, condition or symptom thereof in an animal; c) increasing HDL levels and/or improving the ratio of TG to HDL in an animal thereby reducing the risk of a cardiovascular disease, disorder, condition or symptom in an animal; and/or d) treating, preventing, delaying or ameliorating chylomicronemia, wherein the compound is capable of inhibiting apolipoprotein C-III.

Description

MODULATION OF APOLIPOPROTEIN CIII (APOCIII) EXPRESSION Sequence Listing The t application is being filed along with a Sequence Listing in electronic format.
The Sequence Listing is provided as a file entitled 30WOSEQ.TXT, created on April 27, 2012 which is 8 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its ty.
Field of the Invention Provided herein are methods, compounds, and itions for reducing expression of Apolipoprotein CIII (ApoCIII) mRNA and n, and increasing HDL or HDL ty in an animal. Also, provided herein are methods, compounds, and compositions for an ApoCIII inhibitor for reducing ApoCIII related diseases or conditions in an animal.
Background Lipoproteins are globular, micelle-like particles that consist of a non-polar core of acylglycerols and teryl 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 oteins (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 Lipoprotein particles undergo continuous metabolic processing and have variable properties and compositions. Lipoprotein densities increase without increasing particle diameter because the density of their outer gs is less than that of the inner core. The protein components of lipoproteins are known as apolipoproteins. At least nine apolipoproteins are distributed in significant amounts among the various human oteins. oprotein C-III (ApoCIII) is a constituent ofHDL and of triglyceride (TG)-rich lipoproteins. ed ApoCIII is associated with riglyceridemia. Accordingly, ApoCIIIhas a role in hypertriglyceridemia, a risk factor for ry artery disease (Davidsson et al., J. Lipid Res. 2005. 46: 1999-2006). ApoCIII slows clearance of triglyceride-rich lipoproteins by WO 49495 inhibiting lipolysis, both through inhibition of lipoprotein lipase and by interfering with lipoprotein binding to cell-surface glycosaminoglycan matrix (Shachter, Curr. Opin. l, 2001, 12, 297—304).
The gene encoding human apolipoprotein C-III (also called APOC3, APOC-III, ApoCIII, and APO C-III) was cloned in 1984 by three research groups (Levy-Wilson et al, DNA, 1984, 3, 359-364; Protter et al, DNA, 1984, 3, 449-456; Sharpe et al, Nucleic Acids Res, 1984, 12, 3917- 3932). The coding sequence is interrupted by three s (Protter et al, DNA, 1984, 3, 449- 456). The human ApoCIII gene is located approximately 2.6 kb to the 3’ direction of the apolipoprotein A-l gene and these two genes are convergently transcribed (Karathanasis, Proc.
Natl. Acad. Sci. U. S. A., 1985, 82, 6374-6378). Also cloned was a variant of human apolipoprotein C-III with a Thr74 to Ala74 mutation from a patient with unusually high level of serum apolipoprotein C-III. As the Thr74 is O-glycosylated, the Ala74 mutant therefore resulted in increased levels of serum I g the carbohydrate moiety (Maeda et al, J. Lipid Res, 1987, 28, 1405-1409). Other variants or polymorphisms that modulated Apo CIII sion were later identif1ed. Some of the polymorphsims elevated ApoCIII. Elevated ApoCIII levels were associated with elevated triglyceride (TG) levels and diseases such as cardiovascular disease, metabolic syndrome, obesity and diabetes (Chan et al, lntJ Clin Pract, 2008, 62:799-809; Onat et at, Atherosclerosis, 2003, 168:81-89; Mendivil et al, ation, 2011, 124:2065-2072).
Five polymorphisms have been identified in the promoter region of the gene: C (at position -641 of the gene) to A, G (at position -630 of the gene) to A, T (at position -625 of the gene) to deletion, C (at position -482 of the gene) to T and T (at position -455 of the gene) to C.
All of these polymorphisms are in linkage disequilibrium with the SstI rphism in the 3’ untranslated region. The SstI polymorphic site distinguishes the S1 and S2 alleles and the S2 allele has been ated with elevated plasma triglyceride levels rman et al, Proc.
Natl. Acad. Sci. U. S. A., 1993, 90, 4562-4566). The ApoCIII promoter is downregulated by insulin and this polymorphic site hes the insulin regulation. Thus the potential overexpression of ApoCIII resulting from the loss of insulin regulation may be a contributing factor to the development of hypertriglyceridemia associated with the S2 allele (Li et al, J. Clin.
Invest, 1995, 96, 2601-2605). The T (at position -455 of the gene) to C polymorphism has been ated with an increased risk of coronary artery disease (Olivieri et al, J. LipidRes, 2002, 43, 1450-1457). Other polymorphisms in the human ApoCIII gene that have been associated with elevated ApoCIII and/or triglyceride expression include: C (at position 1100) to T, C (at position 3175) to G, T (at position 3206) to G, C (at ons 323 8) to G, etc. (Tilly ei al, J. Lipid Res., 2003, 44:430-436; Waterworth el al, Arterioscler Thromb Vasc Biol, 2000, 20:2663-2669; Petersen el al., NEngl JMed, 2010, 362: 1082-1089).
In addition to insulin, other regulators of ApoCIII gene eXpression have been identified.
A response element for the nuclear orphan receptor rev-erb alpha has been located at positions - 23 to -18 of the gene, in the ApoCIII promoter region. Rev-erb alpha decreases ApoCIII promoter activity (Raspe el al, J. Lipid Res., 2002, 43, 179). The I promoter region from positions —86 to —74 of the gene is recognized by two nuclear s CIIIbl and CIIIB2 (Ogami el al, J. Biol Chem, 1991, 266, 9640-9646). ApoCIII expression is also upregulated by retinoids acting via the retinoid X receptor, and alterations in retinoid X receptor abundance affects I transcription (Vu-Dac ei al, J. Clin. , 1998, 102, 625-632).
Specificity protein 1 (Sp1) and hepatocyte r factor-4 (HNF-4) have been shown to work synergistically to transactivate the apolipoprotein C-III promoter via the HNF-4 binding site (Kardassis el al, Biochemistry, 2002, 41, 1217-1228). HNF-4 also works in ction with SMAD3-SMAD4 to transactivate the ApoCIII promoter (Kardassis ei al, J. Biol. Chem, 2000, 275, 41405-41414).
Transgenic and knockout mice have further defined the role of ApoCIII in sis.
OvereXpression of ApoCIII in transgenic mice leads to riglyceridemia and impaired clearance of VLDL-triglycerides (de Silva ei al, J. Biol. Chem, 1994, 269, 2324-2335; Ito ei al, Science, 1990, 249, 790-793). Knockout mice with a total absence of the ApoCIII protein eXhibited significantly reduced plasma cholesterol and triglyceride levels compared with wild- type mice and were protected from postprandial hypertriglyceridemia (Maeda ei al, J. Biol.
Chem, 1994, 269, 23610-23616).
Total plasma ApoCIII levels have been identified as a major determinant of serum cerides, and epidemiological studies have demonstrated that ApoCIII and ApoB oteins that have ApoCIII as a component independently predict coronary heart disease (Sacks et al., Circulation. 2000. 102: 1886-1892; Lee et al., Arterioscler Thromb Vasc Biol. 2003. 23.‘ 8537 858). Studies also demonstrate that ApoCIII is a key determinant in the clearance of triglyceride-rich oteins and its remnants in hypertriglyceridaemic states, including visceral obesity, n resistance and the metabolic syndrome (Mauger et al., J. Lipid Res. 2006. 47: 1212-1218; Chan et al., Clin. Chem. 2002. 278-283; Ooi et al., Clin. Sci. 2008. 114: 611-624).
WO 49495 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, 2178: 81-844; Tremblay et al. 2011, J Clin Lipidol, 5: 37-44). Examples of metabolic disease include, but are not limited to, diabetes, lic syndrome/insulin resistance, and genetic disorders such as familial chylomicronemia, familial combined hyperlipidemia and familial hypertriglyceridemia.
Hypertriglyceridemia is the consequence of increased production and/or reduced or delayed lism of triglyceride ich lipoproteins: VLDL and, to a lesser extent, chylomicrons (CM). Borderline high TGs (150-199 mg/dL) are commonly found in the l population and are a common ent of the metabolic syndrome/insulin ance states.
The same is true for high TGs (200-499 mg/dL) except that as plasma TG levels se, underlying genetic factors play an increasingly important etiologic role. Very high TGs (2500 mg/dL) are most often associated with elevated CM levels as well, and are accompanied by increasing risk for acute pancreatitis. The risk of pancreatitis is considered clinically significant if TG exceeds 880 mg/dL (>10 mmol) and the European Atherosclerosis Society/European Society of Cardiology (EAS/ESC) 2011 guidelines state that actions to prevent acute pancreatitis are mandatory (Catapano et al. 2011, Atherosclerosis, 2178: 81-844). According to the EAS/ESC 2011 guidelines, riglyceridemia is the cause of approximately 10% of all cases of pancreatitis, and development of pancreatitis can occur at TG levels n 440-880 mg/dL.
Based on evidence from clinical studies trating that elevated TG levels are an independent risk factor for atherosclerotic CVD, the guidelines from both the National Cholesterol Education Program Adult ent Panel III (NCEP 2002, ation, 106: 3143-421) and the American Diabetes Association (ADA 2008, Diabetes Care, 31: $12-$54.) recommend a target TG level of less than 150 mg/dL to reduce cardiovascular risk.
ApoCIII-knockout mice had normal intestinal lipid absorption and hepatic VLDL triacylglycerol secretion, but a rapid clearance of VLDL triacylglycerols and VLDL cholesteryl esters from plasma that may explain the observed hypolipidaemia (Gerritsen et al., J. Lipid Res. 2005. 46: 1466-1473; Jong et al., J. Lipid Res. 2001. 42: 1578-1585). VLDL particles with I have been cited to play a major role in identifying the high risk of coronary heart disease in hypertriglyceridemia (Campos et al., J. Lipid Res. 2001. 42: 1239-1249). A genome-wide association study found a naturally occurring ApoCIII null mutation in ter Amish people demonstrated a favorable lipid profile and apparent cardioprotection, with no obvious detrimental effects n el al., Science. 2008. 322: 1702-1705). The mutation carriers are observed to have lower fasting and postprandial serum triglycerides and LDL-cholesterol, and higher levels of HDL-cholesterol.
The HDL class of lipoproteins ses a heterogeneous and polydisperse population of particles that are the most dense and smallest of size (Havel and Kane. In, The Metabolic & Molecular Bases of Inherited Disease. 8th n. McGraw-Hill, New York, 2001 :2705—16).
HDL is a macromolecular compleX of lipids (cholesterol, cerides and olipids) and proteins (apolipoproteins (apo) and enzymes). The surface ofHDL ns 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, JLipidRes, 2010, 51 :2032-2057) Epidemiological studies have indicated that increased levels ofHDL t t cardiovascular disease or coronary heart disease (Gordon et al., Am. J. Med. 1977. 62: 707-714).
These effects of HDL-cholesterol are ndent of triglyceride and LDL-cholesterol concentrations. In clinical practice, a low plasma HDL-cholesterol is more commonly ated 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).
Currently, there are no known direct therapeutic agents that affect the function of ApoCIII. The hypolipidemic effect of the fibrate class of drugs has been postulated to occur via a mechanism where peroxisome proliferator activated or (PPAR) mediates the displacement ofHNF-4 from the apolipoprotein C-III promoter, ing in transcriptional suppression of apolipoprotein C-III (Hertz el al., J. Biol. Chem, 1995, 270, 13475). The statin class of hypolipidemic drugs also lower triglyceride levels via an unknown mechanism, which results in increases in lipoprotein lipase mRNA and a decrease in plasma levels of oprotein C-III (Schoonjans el al., FEBS Lett, 1999, 452, 160-164). Consequently, there remains a long felt need for additional agents capable of effectively inhibiting apolipoprotein C-III function.
Antisense technology is emerging as an effective means for reducing the expression of certain gene products and may therefore prove to be uniquely useful in a number of therapeutic, stic, and research applications for the modulation of ApoCIII.
We have previously disclosed compositions and method for inhibiting ApoCIII by antisense compounds in US 20040208856 (US Patent 7,598,227), US 20060264395 (US Patent 7,750,141), and 783. In the present application, we disclose the cted result that antisense inhibition of ApoCIII resulted in the elevation ofHDL levels and decrease in postprandial ceride levels. This result will be useful, for example, to treat, prevent, delay, decrease or ameliorate any one or more diseases, such as cardiovascular disease (e.g., coronary heart disease or atherogenic es). For e, elevated postprandial (non-fasting) triglyceride levels have been identified as a significant risk factor for cardiovascular diseases (Bansal el al., JAA/IA, 2007, 9-16; Nordestgaard el al., , 2007, 298:299-308), Also, in the present application, inhibition of ApoCIII expression unexpectedly results in increased icron clearance and is ore important in the prevention of chylomicronemia (Chait et al., 1992, Adv Intern Med. 1992, 37 :249-73), a dyslipidemic state caused by improper clearance of chylomicron triglyceride. Severe forms of chylomicronemia can lead to pancreatitis, a life- threatening condition. By inhibiting intestinal ApoCIII, inhibition of lipoprotein lipase would be reduced, and icron triglyceride clearance would be enhanced, thereby preventing pancreatitis. y of the Invention Provided herein are methods of increasing HDL levels by stering to an animal a compound targeting ApoCIII.
Certain embodiments provide a method of preventing, treating, ameliorating, delaying the onset of or ng the risk of a cardiovascular disease, disorder or condition in an animal comprising administering a compound targeting ApoCIII to the animal. The compound administered to the animal prevents, treats, rates, delays the onset of, or reduces the risk of, the cardiovascular disease, disorder or condition by increasing HDL levels in the animal.
Certain embodiments provide a method of reducing the risk for a cardiovascular disease in an animal comprising administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified ucleotide is complementary to an ApoCIII nucleic acid. In certain embodiments, the ApoCIII c acid is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides, and having a nucleobase sequence comprising at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In further embodiments, the compound administered to the animal reduces the risk for a cardiovascular disease, by increasing HDL levels.
Certain embodiments provide a method of preventing, ng, ameliorating or reducing at least one symptom of a cardiovascular disease in an animal, sing administering to the animal a therapeutically effective amount of a compound comprising a d oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to an ApoCIII nucleic acid. In certain embodiments, the ApoCIII nucleic acid is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the nd comprises a modified oligonucleotide ting of 12 to 30 linked nucleosides, and having a nucleobase sequence comprising at least 8 uous nucleobases of ISIS 304801 (SEQ ID NO: 3). In further embodiments, the compound administered to the animal ts, treats, ameliorates or reduces at least one symptom of the cardiovascular disease in the animal, by increasing HDL levels in the animal. n embodiments provide a method of raising HDL levels in an animal by administering to the animal a compound consisting of ISIS 304801 (SEQ ID NO: 3) to raise the HDL levels in the animal.
Certain embodiments e a method of preventing, treating, rating or reducing at least one symptom of a cardiovascular disease in an animal by administering to the animal a 2O compound ting of ISIS 304801 (SEQ ID NO: 3) to prevent, treat, ameliorate or reduce at least one symptom of the cardiovascular disease in the animal, by increasing HDL levels in the animal.
Certain ments provide a method of raising HDL levels in an animal by administering to the animal a modified oligonucleotide, having the sequence of SEQ ID NO: 3 (ISIS 304801) wherein the modified oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a 5’ wing segment consisting of 5 linked nucleosides; a 3’ wing segment consisting 5 linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5’ wing segment and the 3’ wing segment and wherein each nucleoside of each wing segment comprises a 2’-O-methyoxyethyl sugar, wherein each cytosine is a 5’-methylcytosine, and wherein each internucleoside linkage is a phosphorothioate e, wherein the modified oligonucleotide raises the HDL levels in the animal.
Certain embodiments provide a method of preventing, treating, ameliorating or reducing at least one symptom of a cardiovascular disease in an animal by stering to the animal a modified oligonucleotide, having the sequence of ISIS 304801 (SEQ ID NO: 3) wherein the modified oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a ’ wing segment consisting of 5 linked nucleosides; a 3’ wing segment ting 5 linked nucleosides; n the gap segment is positioned immediately nt to and between the 5’ wing segment and the 3’ wing segment and 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 e, wherein the modified oligonucleotide prevents, treats, ameliorates or reduces at least one symptom in the animal with the vascular e by raising the HDL levels in the animal. n embodiments provide a method of g HDL levels in an animal by administering to the animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is mentary to an ApoCIII nucleic acid, as shown in SEQ ID NO: 1 or SEQ ID NO: 2, to raise the HDL levels in the animal.
Certain ments provide a method of preventing, treating, ameliorating or reducing at least one m of a cardiovascular disease in an animal by administering to the animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to an ApoCIII nucleic acid, as shown in 2O SEQ ID NO: 1 or SEQ ID NO: 2, to preventing, treating, ameliorating or reducing at least one symptom of the cardiovascular disease in the animal, by raising the HDL levels of the animal.
Certain embodiments provide a method of decreasing CETP levels by administering a compound targeting ApoCIII to an animal. In certain embodiments, the compound comprises a modified oligonucleotide ting of 12 to 30 linked nucleosides, and having a nucleobase sequence complementary to an ApoCIII nucleic acid. In certain embodiments, the nucleobase sequence comprises at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, the compound consists of the bases of ISIS 304801 (SEQ ID NO: 3).
Certain embodiments provide a method of increasing ApoAl, PONl, fat clearance, icron triglyceride clearance, post prandial triglyceride clearance or HDL by administering a nd targeting ApoCIII to an . In certain embodiments, the compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides, and having a nucleobase sequence complementary to an ApoCIII nucleic acid. In certain embodiments, the nucleobase WO 49495 sequence comprises at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, the compound ts of the nucleobases of ISIS 304801 (SEQ ID NO: 3).
Certain embodiments provide a method of ting, delaying or rating pancreatitis comprising: (a) selecting an animal with, or at risk of, pancreatitis, and (b) administering a compound targeting ApoCIII to the animal, wherein the pancreatitis is prevented, delayed or ameliorated.
Certain ments provide a method of preventing, delaying or ameliorating pancreatitis sing: (a) selecting an animal with, or at risk of, pancreatiis, and (b) administering a compound targeting ApoCIII to the animal, thereby increasing chylomicron clearance, wherein the pancreatitis is prevented, delayed or ameliorated.
In n embodiments, the animal has, or is at risk for, hypertriglyceridemia. In certain embodiments, the riglyceridemia is Fredrickson Type II, IV or V. In certain embodiments, the animal has a genetic defect leading to hypertriglyceridemia. In certain ments, the genetic defect is a heterozygous LPL deficiency or an ApoCIII polymorphism. In certain embodiments, the animal has a ceride level 2 500 mg/dL and a heterozygous LPL deficiency.
In certain embodiments, the animal has a ceride level n 100-200 mg/dL, 100- 300 mg/dL, 100-400 mg/dL, 100-500 mg/dL, 0 mg/dL, 300-500 mg/dL, 400-500 mg/dL, 500-1000 mg/dL, 600-1000 mg/dL, 700-1000 mg/dL, 800-1000 mg/dL, 900-1000 mg/dL, 500- 1500 mg/dL, 1000-1500 mg/dL, 100-2000 mg/dL, 150-2000 mg/dL, 200-2000 mg/dL, 300-2000 mg/dL, 400-2000 mg/dL, 500-2000 mg/dL, 600-2000 mg/dL, 700-2000 mg/dL, 800-2000 mg/dL7 900-2000 mg/dL, 1000-2000 mg/dL, 1100-2000 mg/dL, 1200-2000 mg/dL, 1300-2000 mg/dL, 1400-2000 mg/dL, or 1500-2000 mg/dL.
In certain embodiments, increased chylomicron clearance enhances clearance of postprandial triglycerides and/or decreases postprandial triglycerides.
Certain embodiments provide a use of a compound targeted to ApoCIII for preventing, treating, ameliorating or reducing at least one symptom of a cardiovascular disease, by increasing HDL levels.
Certain embodiments provide a use of a compound targeted to ApoCIII for increasing HDL levels in an .
Certain embodiments provide a use of a compound targeted to ApoCIII for the preparation of a medicament for increasing HDL levels in an animal.
Certain embodiments e a use of a compound targeted to ApoCIII for the preparation of a medicament for ing the ratio of TG to HDL.
Detailed Description of the Invention It is to be understood that both the foregoing l 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 r” 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.
The section headings used herein are for organizational purposes only and are not to be construed as ng the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, s, patent ations, 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 Unless specific definitions are provided, the lature 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, ations, 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 ed to throughout in the disclosure herein are orated by reference for the portions of the document discussed herein, as well as in their ty.
Unless ise indicated, the following terms have the following meanings: “2’-O-methoxyethyl” (also 2’-MOE, 2’-O(CH2)2-OCH3 and 2’-O-(2-methoxyethyl)) refers to an O-methoxy-ethyl modification of the 2’ position of a fiirosyl ring. A 2 yethyl modified sugar is a modified sugar. “2’-O-methoxyethyl tide” means a nucleotide comprising a 2’-O-methoxyethyl modified sugar moiety. “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. “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. “5-methylcytosine” means a cytosine modified with a methyl group attached to the 5’ position. A 5-methylcytosine is a modified nucleobase.
“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%-5 5%.
“Active ceutical agent” means the nce or substances in a pharmaceutical composition that provide a eutic benefit when administered to an dual. For example, in n embodiments an antisense oligonucleotide ed to ApoCIII is an active pharmaceutical agent.
“Active target region” or “target ” 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.
“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 s 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.
“Administering” means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering. ” 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 nse oligonucleotide targeting ApoCIII. “Second agent” means a second therapeutic nd of the invention (e. g. a second antisense oligonucleotide targeting ApoCIII) and/or a non-ApoCIII therapeutic compound.
“Amelioration” refers to a ing of at least one indicator, sign, or symptom of an associated e, disorder, or condition. The severity of indicators may be determined by subjective or objective es, which are known to those skilled in the art. l” 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.
“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 se in the amount or expression of a target nucleic acid or protein d by such target nucleic acid. ense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. As used herein, the term “antisense compound” asses pharmaceutically acceptable derivatives of the compounds described herein.
“Antisense inhibition” means the reduction of target nucleic acid levels or target n 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.
“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a ponding region or segment of a target nucleic acid.
As used , the term “antisense oligonucleotide” encompasses pharmaceutically acceptable derivatives of the compounds described herein.
“ApoCIII” means any nucleic acid or protein sequence encoding ApoCIII. For example, in n embodiments, an ApoCIII includes a DNA sequence encoding ApoCIII, a RNA sequence transcribed from DNA encoding ApoCIII (including genomic DNA comprising introns and , a mRNA sequence encoding ApoCIII, or a peptide ce encoding ApoCIII.
“ApoCIII mRNA” means a mRNA encoding an ApoCIII protein.
“ApoCIII protein” means any protein sequence encoding ApoCIII.
“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 es,” which consist mainly of cholesterol and other fats, m and scar tissue, and damage the lining of arteries.
“Bicyclic sugar” means a filrosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar.
“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotide n the furanose n of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.
“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
“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, ovascular disease e), coronary heart disease, hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia and hypercholesterolemia.
“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 tides is chemically distinct from a region having nucleotides without 2’-O-methoxyethyl ations.
“Chimeric antisense nd” means an antisense compound that has at least two chemically ct regions.
“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 y lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL). “Plasma cholesterol” refers to the sum of all lipoproteins (VDL, IDL, LDL, HDL) esterified and/or non-estrified cholesterol present in the plasma or serum.
“Cholesterol absorption inhibitor” means an agent that inhibits the absorption of exogenous cholesterol obtained from diet.
“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 asses parallel or tial administration.
“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. In certain embodiments, mentarity 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 n 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.
“Contiguous nucleobases” means bases immediately adjacent to each other.
“Constrained ethyl” or “cEt” refers to a bicyclic nucleoside having a fiiranosyl sugar that ses a methyl(methyleneoxy) (4’-CH(CH3)-O-2’) bridge n the 4’ and the 2’ carbon atoms. -reactive” means an oligomeric compound ing one nucleic acid sequence can hybridize to a different c 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 c acid, the more likely the oligomeric compound will cross-react with the nucleic acid.
“Cure” means a method that restores health or a ibed treatment for an illness. ary 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.
“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2’ on of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of sub stituents.
“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 , unexplained weight loss, and lethargy. tic dyslipidemia” or “type 2 diabetes with dyslipidemia” means a condition characterized by Type 2 es, reduced HDL-C, elevated triglycerides, and elevated small, dense LDL particles.
“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For e, the diluent in an injected composition may be a liquid, e. g. saline solution.
“Dyslipidemia” refers to a disorder of lipid and/or lipoprotein metabolism, including lipid and/or lipoprotein overproduction or deficiency. idemias may be manifested by elevation of lipids such as icron, cholesterol and triglycerides as well as lipoproteins such as low- density lipoprotein (LDL) cholesterol. An example of a dyslipidemia is chylomicronemia.
“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 lized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial containing tituted antisense oligonucleotide.
“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 stered 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 e the 2O desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or uously. 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.
“Effective amount” or “therapeutically effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological e 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.
The “Fredrickson” system is used to classify primary ic) causes of dislipidemia into several subgroups or types. Dislipidemia types that may be amenable to therapy with the compounds disclosed herein e, but are not limited to, Fredrickson Type II, IV and V.
“Fredrickson Type 1” exists in several forms: Type 1a is a lipoprotein lipase deficiency due to a deficiency ofLPL or altered apoC-II; Type 1b is a al apoprotein CII ncy, a condition caused by a lack of lipoprotein lipase activator; and Type 1c 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 icrons and extremely elevated TG levels (always reaching well above 1000 mg/dL and not uently 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 p atherosclerosis, s 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 or apoC-II, resulting in the inability of affected individuals to produce filnctionally active LPL. Patients are either homozygous for such mutations or compound heterozygous. The prevalence is approximately 1 in 1,000,000 in the general population and much higher in South Africa and Eastern Quebec as a result of a founder effect. Patients respond minimally, or not at all, to TG- lowering drugs (Tremblay et al., J Clin Lipidol, 2011, 5:37-44; Brisson et al., Pharmacogenet Genom, 2010, 20:742-747) and hence restriction of dietary fat to 20 grams/day or less is used to manage the symptoms of this rare disorder.
“Fredrickson Type II” is the most common form of primary hyperlipidemia. It is further classified into Type 11a and Type IIb, depending mainly on whether there is elevation in VLDL in addition to LDL cholesterol (LDL-C). Type IIa (familial holesterolemia) may be sporadic (due to dietary factors), polygenic, or truly al 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 sis. They may also be caused by the decreased clearance of LDL. ence in the tion is about 10%.
“Fredrickson Type III” (also known as dysbetalipoproteinemia) is a remnant l disease, or broad-beta disease (Fern el al., J Clin Pathol, 2008, 61:1174-118). It is due to cholesterol-rich VLDL (B-VLDL). Typically, patients with this condition have elevated plasma cholesterol and TG levels because of impaired clearance of chylomicron and VLDL ts (e. g. IDL). The impaired nce is due to a defect in apolipoprotein E (apoE). Normally functioning apoE contained on the remnants would enable binding to the LDL or and removal from the circulation. lation of the remnants in affected individuals can result in xanthomatosis and premature coronary and/or peripheral ar 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.
“Fredrickson Type IV” (also known as familial hypertriglyceridemia) is an autosomal dominant condition occurring in approximately 1% of the tion. 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 geneous and the phenotype strongly influenced by environmental factors, ularly carbohydrate and ethanol consumption.
“Fredrickson Type V” has high VLDL and chylomicrons. It is characterized by carriers of loss-of-fiinction LPL gene ts associated with LPL activity of at least 20% (i.e. partial LPL deficiency as compared to Fredrickson Type I). These ts present with lactescent plasma and severe hypertriglyceridemia because of chylomicrons and VLDL. TG levels are invariably r 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 (> 35years) and, gh the prevalence is relatively rare, it is much more common than homozygous or compound heterozygous LPL deficient ts.
“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.
“Gapmer” means a chimeric nse compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned n 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.” “Gap-widened” means a chimeric nse 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.
“Glucose” is a monosaccharide used by cells as a source of energy and inflammatory intermediate. a glucose” refers to glucose present in the plasma.
“High density lipoprotein-C (HDL-C)” means terol associated with high density lipoprotein particles. Concentration 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, tively.
“HMG—CoA reductase inhibitor” means an agent that acts through the tion of the enzyme HlVlG—CoA reductase, such as atorvastatin, rosuvastatin, fluvastatin, lovastatin, pravastatin, and simvastatin.
“Hybridization” means the annealing of complementary c acid molecules. In certain embodiments, complementary c acid molecules include an antisense compound and a target nucleic acid.
“Hypercholesterolemia” means a condition characterized by elevated cholesterol or circulating (plasma) cholesterol, LDL-cholesterol and VLDL-cholesterol, as per the guidelines of 2O 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).
“Hyperlipidemia” or “hyperlipemia” is a condition terized 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, icrons and triglycerides. The Fredrickson classification of hyperlipidemias is based on the pattern of TG and cholesterol-rich lipoprotein les, as measured by electrophoresis or ultracentrifugation and is commonly used to terize y causes of hyperlipidemias such as hypertriglyceridemia (Fredrickson and Lee, Circulation, 1965, 31:321-327; ckson et al., New Eng J Med, 1967, 276 (1): 34—42).
“Hypertriglyceridemia” means a condition characterized by elevated triglyceride levels.
Its etiology includes primary (i.e. genetic causes) and secondary (other underlying causes such as diabetes, metabolic syndrome/insulin resistance, obesity, physical vity, cigarette smoking, excess alcohol and a diet very high in carbohydrates) factors or, most often, a combination of both (Yuan er a]. CMAJ, 2007, 176: l 1 13-1 120). ifying” or “selecting an animal with metabolic or cardiovascular disease” means identifying or selecting a subject prone to or having been diagnosed with a metabolic disease, a cardiovascular disease, or a metabolic syndrome; or, identifying or selecting a subject having any symptom of a lic disease, cardiovascular disease, or metabolic syndrome including, but not limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, ension increased insulin resistance, decreased n sensitivity, above normal body weight, and/or above normal body fat content or any combination thereof Such identif1cation can be accomplished by any , ing 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 ating (plasma) triglycerides, ing blood-pressure, measuring body fat content, measuring body weight, and the like.
“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, ary edema, cardiac arrest, and atrial dysrhythmia.
“Immediately adjacent” means there are no intervening ts between the immediately adjacent elements, for e, between regions, segments, nucleotides and/or sides. asing HDL” or “raising HDL” means increasing the level ofHDL 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.
“Individual” or “subject” or “animal” means a human or non-human animal selected for ent or therapy.
“Induce”, “inhibit”, “potentiate77 (Lelevate77 (L 77 (L 7 7 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.
“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.
“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 cerides, which es 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.
“Insulin sensitivity” is a measure of how effectively an individual processes glucose. An individual having high n sensitivity ively processes glucose whereas an individual with low insulin sensitivity does not effectively process glucose.
“Internucleoside e” refers to the al bond between nucleosides.
“Intravenous administration” means administration into a vein.
“Linked nucleosides” means adjacent nucleosides which are bonded together.
“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 raising can occur with one or more doses over time.
“Lipid-lowering therapy” or “lipid lowering agent” means a eutic 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, L-C, triglycerides, small dense LDL particles, and Lp(a) in a t. Examples of lipid-lowering therapy include statins, fibrates, MTP inhibitors.
“Lipoprotein”, such as VLDL, LDL and HDL, refers to a group of ns 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 tion of protein versus lipid, s the VLDL has a lower proportion of protein versus lipid.
“Low density lipoprotein-cholesterol (LDL-C)” means cholesterol carried in low density lipoprotein particles. Concentration ofLDL-C in serum (or plasma) is typically fied in mg/dL or nmol/L. “Serum LDL-C” and “plasma LDL-C” mean LDL-C in the serum and plasma, respectively.
“Major risk factors” refers to factors that contribute to a high risk for a particular disease or condition. In certain embodiments, major risk s for ry heart disease include, without limitation, cigarette smoking, hypertension, low HDL-C, family history of coronary heart disease, age, and other factors disclosed herein.
“Metabolic disorder” or “metabolic e” refers to a ion characterized by an alteration or disturbance in metabolic fiinction. “Metabolic” and “metabolism” are terms well known in the art and generally include the whole range of mical processes that occur within a living organism. Metabolic disorders include, but are not limited to, lycemia, prediabetes, diabetes (type 1 and type 2), y, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes.
“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 re of at least 130/85 mmHg; and fasting e of at least 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).
“Mismatch” or omplementary base” 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.
“Mixed dyslipidemia” means a condition characterized by elevated cholesterol and elevated cerides.
“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.
“Modified nucleobase” refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. For e, 5-methylcytosine is a modified nucleobase. An “unmodified base” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
“Modified nucleoside” means a nucleoside having at least one modified sugar moiety, and/or modified nucleobase.
“Modified nucleotide” means a nucleotide having at least one modified sugar moiety, modified internucleoside linkage and/or modified nucleobase.
“Modified oligonucleotide” means an oligonucleotide comprising at least one modified tide.
“Modified sugar” refers to a substitution or change from a natural sugar. For example, a ethoxyethyl modified sugar is a modified sugar.
“Motif’ means the n of chemically ct regions in an antisense compound.
“Naturally occurring internucleoside linkage” means a 3' to 5' phosphodiester linkage.
“Natural sugar moiety” means a sugar found in DNA (2’-H) or RNA (2’-OH).
“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 ering ribonucleic acids (siRNA), and microRNAs ). A nucleic acid may also comprise a combination of these elements in a single molecule.
“Nucleobase” means a cyclic moiety capable of pairing with a base of another nucleic acid.
“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, mentary base 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 base at a certain on 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 c acid are considered to be mentary at that nucleobase pair.
“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
“Nucleoside” means a nucleobase linked to a sugar.
“Nucleoside mimetic” includes those ures 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-filranose sugar units.
“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
“Nucleotide mimetic” includes those structures used to e 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(=O)-O- or other non-phosphodiester linkage).
“Oligomeric compound” or “oligomer” means a polymer of linked ric subunits which is capable of izing to a region of a nucleic acid molecule. In certain embodiments, oligomeric compounds are oligonucleosides. In certain embodiments, oligomeric nds are oligonucleotides. In certain embodiments, oligomeric compounds are antisense compounds. In certain ments, oligomeric compounds are antisense oligonucleotides. In certain embodiments, oligomeric compounds are chimeric oligonucleotides.
“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent from one r.
“Parenteral administration” means stration through injection or infiision. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or ranial administration, e. g. intrathecal or intracerebroventricular administration. Administration can be continuous, chronic, short or intermittent.
“Peptide” means a le formed by linking at least two amino acids by amide bonds.
Peptide refers to ptides and proteins.
“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.
“Pharmaceutical composition” or sition” 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.
“Pharmaceutically able carrier” means a medium or diluent that does not interfere with the structure of the compound. Certain of such rs enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, es, s, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject. Certain of such carriers enable WO 49495 pharmaceutical compositions to be formulated for injection, infusion or topical administration.
For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution.
“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.
“Pharmaceutically acceptable salts” means physiologically and ceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent nd 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 bed herein may be ed by methods well-known in the art. For a review of pharmaceutically acceptable salts, see Stahl and h, Handbook of Pharmaceutical Salts: Properties, Selection and Use -VCH, Weinheim, Germany, 2002). Sodium salts of antisense oligonucleotides are useful and are well accepted for eutic administration to humans. Accordingly, in one embodiment the compounds described herein are in the form of a sodium salt.
“Phosphorothioate linkage” means a linkage between sides where the phosphodiester bond is d by replacing one of the non-bridging oxygen atoms with a sulfur 2O atom. A phosphorothioate linkage is a modified ucleoside e.
“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.
“Prevent” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to itely. Prevent also means reducing risk of developing a disease, disorder, or condition.
“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 f by the action of endogenous enzymes or other chemicals or conditions.
“Raise” means to increase in amount. For example, to raise plasma HDL levels means to increase the amount ofHDL in the plasma.
“Ratio of TG to HDL” means the TG levels relative to HDL . The occurrence of high TG and/or low HDL has been linked to cardiovascular disease nce, outcomes and mortality. ving the ratio of TG to HDL” means to se TG and/or raise HDL levels.
“Reduce” means to bring down to a r extent, size, amount, or number. For example, to reduce plasma triglyceride levels means to bring down the amount of triglyceride in the plasma.
“Region” or “target ” is defined as a portion of the target nucleic acid having at least one identifiable structure, fiinction, 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 tion , 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.
“Ribonucleotide” means a nucleotide having a hydroxy at the 2’ position of the sugar portion of the tide. Ribonucleotides can be modified with any of a variety of substituents.
“Second agent” or “second therapeutic agent” means an agent that can be used in combination with a “first agent”. A second therapeutic agent can include, but is not limited to, an siRNA or antisense oligonucleotide including antisense oligonucleotides targeting ApoCIII. A second agent can also include anti-ApoCIII antibodies, ApoCIII peptide inhibitors, cholesterol lowering agents, lipid ng agents, glucose lowering agents and anti-inflammatory agents.
“Segments” are defined as smaller, sub-portions of regions within a nucleic acid. For example, a “target t” 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 tide of a target segment. “3’ target site” refers to the t nucleotide of a target segment.
“Shortened” or “truncated” versions of antisense oligonucleotides or target nucleic acids taught herein have one, two or more nucleosides d.
“Side effects” means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include ion 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.
“Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand.
“Specifically hybridizable” refers to an antisense compound having a ient 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 d, i.e. under physiological conditions in the case of in vivo assays and therapeutic treatments.
“Statin” means an agent that inhibits the activity of HlVlG—CoA reductase.
“Subcutaneous administration” means administration just below the skin.
“Subject” means a human or non-human animal ed for treatment or therapy.
“Symptom of cardiovascular disease or disorder” means a phenomenon that arises from and accompanies the vascular disease or disorder and serves as an indication of it. For example, angina; chest pain; shortness of breath; ations; ss; dizziness; nausea; sweating; tachycardia; bradycardia; arrhythmia; atrial fibrillation; swelling in the lower extremities; is; fatigue; fainting; numbness of the face; numbness of the limbs; claudication or cramping of muscles; bloating of the abdomen; or fever are ms of cardiovascular disease or disorder.
“Targeting” or “targeted” means the process of design and selection of an antisense compound that will specif1cally hybridize to a target nucleic acid and induce a desired effect.
“Target c acid,” “target RNA,” and “target RNA transcript” all refer to a nucleic acid capable of being targeted by antisense compounds.
“Therapeutic lifestyle ” 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, ydrate, protein, cholesterol, insoluble fiber, as well as recommendations for physical activity.
“Treat” refers to administering a compound of the invention to effect an alteration or improvement of a disease, disorder, or condition.
“Triglyceride” or “TG” means a lipid or neutral fat consisting of ol ed with three fatty acid molecules.
“Type 2 diabetes,” (also known as “type 2 diabetes mellitus”, tes mellitus, type 2”, “non-insulin-dependent diabetes (NIDDM)”, “obesity d diabetes”, or “adult-onset diabetes”) is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia.
“Unmodified tide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain ments, an unmodified nucleotide is an RNA tide (i.e. B-D-ribonucleosides) or a DNA nucleotide (i.e. oxyribonucleoside).
“Wing segment” means one or a ity 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 Certain embodiments provide a method of reducing ApoCIII levels in an animal by administering a compound to the animal targeting I, thereby decreasing ApoCIII levels. In certain embodiments, ApoCIII levels are d in the liver or small intestine.
Certain embodiments provide a method of increasing HDL levels and/or improving the ratio of TG to HDL in an animal by administering a compound to the animal wherein the compound targets ApoCIII and increases HDL levels and/or improves the ratio of TG to HDL. 2O Certain ments provide a method of preventing, delaying or ameliorating a cardiovascular disease, disorder, condition, or symptom thereof, in an animal comprising administering a compound targeting ApoCIII to the animal, wherein the compound administered to the animal prevents, treats or ameliorates the cardiovascular disease, disorder, condition or m in the animal by increasing HDL levels in the animal and/or ing the ratio of TG to HDL.
Certain ments provide a method of preventing, delaying or ameliorating a pancreatitis in an animal comprising administering a compound targeting ApoCIII to the animal, wherein the compound administered to the animal prevents, treats or ameliorates the atitis in the animal by increasing HDL levels in the animal and/or improving the ratio of TG to HDL.
In certain embodiments, the pancreatitis is acute pancreatitis.
Certain embodiments provide a method of preventing, treating, ameliorating, ng the onset, or reducing the risk of, a cardiovascular disease, disorder or condition in an animal, comprising of administering to the animal a nd that targets ApoCIII, wherein the compound prevents, treats, ameliorates, delays the onset, or reduces of the risk of the cardiovascular disease, er or ion in the animal by increasing HDL levels in the animal and/or improving the ratio of TG to HDL.
Certain embodiments provide a method of decreasing CETP levels by administering a compound targeting ApoCIII to an .
Certain embodiments provide a method of increasing ApoAl, PONl, fat clearance, chylomicron ceride clearance and/or HDL by administering a compound targeting ApoCIII to an animal. Certain embodiments provide a method for improving the ratio of TG to HDL.
In certain embodiments, the ApoCIII nucleic acid is any of the sequences set forth in GERBANK Accession No. NM_000040.1 (incorporated herein as SEQ ID NO: 1), and GERBANK ion No. NT_O33899.8 truncated from nucleotides 20262640 to 20266603 porated herein as SEQ ID NO: 2).
In certain embodiments, the compound targeting ApoCIII is a d oligonucleotide.
In further embodiments, the modified oligonucleotide has a base sequence comprising at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, the modified ucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2.
In certain embodiments, the modified oligonucleotide consists of a single-stranded modified oligonucleotide.
In certain embodiments, the modified oligonucleotide consists of 12-30 linked nucleosides.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.
In certain embodiments, the compound comprises at least one modified internucleoside linkage. In certain embodiments, the internucleoside linkage is a phosphorothioate internucleoside linkage.
In n embodiments, the compound comprises at least one nucleoside comprising a modified sugar. In certain ments, the at least one modified sugar is a ic sugar. In certain embodiments, the at least one d sugar comprises a 2’-O-methoxyethyl.
In certain embodiments, the compound comprises at least one nucleoside comprising a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.
In certain embodiments, the compound comprising modified oligonucleotide comprises: (i) a gap segment consisting of linked deoxynucleosides; (ii) a 5’ wing segment consisting of linked nucleosides, (iii) a 3’ wing segment consisting of linked nucleosides, n 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.
In certain embodiments, the compound comprising modified oligonucleotide comprises: (i) a gap segment consisting of 8-12 linked deoxynucleosides; (ii) a 5’ wing segment ting of l-5 linked nucleosides, (iii) a 3’ wing segment consisting of l-5 linked nucleosides, wherein the gap segment is positioned ately adjacent to and between the 5’ wing segment and the 3’ wing segment, wherein each nucleoside of each wing segment comprises a ethoxyethyl sugar, wherein each cytosine is a 5’-methylcytosine, and wherein each internucleoside linkage is a phosphorothioate linkage.
In certain embodiments, the compound comprising modified oligonucleotide comprises: (i) a gap segment consisting often linked ucleosides; (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 oned immediately adjacent to and between the 5’ wing segment and the 3’ wing segment, wherein each side of each wing segment comprises a 2’-O-methoxyethyl sugar, wherein each ne is a 5’-methylcytosine, and wherein each internucleoside e is a orothioate linkage.
In n embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 8 contiguous bases of a nucleobase sequence of ISIS 304801 (SEQ ID NO: 3).
Certain embodiments provide a method of reducing the risk of a cardiovascular disease in an animal by administering to the animal a eutically effective amount of a compound sing a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to an ApoCIII nucleic acid and wherein the modified oligonucleotide increases HDL levels and/or improves the ratio of TG to HDL. In certain embodiments, the ApoCIII c acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2. In r embodiments the modified nucleotide comprises at least 8 contiguous nucleobases of the nucleobase sequence of ISIS 304801 (SEQ ID NO: 3). 2012/035694 Certain embodiments e a method of preventing, treating, ameliorating, or reducing at least one m of a cardiovascular disease in an animal, comprising administering to the animal a therapeutically effective amount of a nd sing a modified oligonucleotide consisting of 12 to 30 linked nucleosides and is complementary to an ApoCIII nucleic acid. In certain embodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% mentary to SEQ ID NO: 1 or SEQ ID NO: 2. In further embodiments, the compound administered to the animal prevents, treats, ameliorates or reduces at least one m of the vascular disease by increasing HDL levels and/or improving the ratio of TG to HDL. In further embodiments, the modified oligonucleotide comprises at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3).
In further embodiments, symptoms of a vascular disease include, but are not d to, angina; chest pain; ess of breath; palpitations; weakness; dizziness; nausea; sweating; tachycardia; ardia; arrhythmia; atrial fibrillation; swelling in the lower extremities; is; fatigue; fainting; numbness of the face; numbness of the limbs; claudication or cramping of muscles; bloating of the abdomen; or fever.
Certain embodiments provide a method of raising HDL levels and/or improving the ratio of TG to HDL in an animal by administering to the animal a compound consisting of the nucleobase sequence of ISIS 304801 (SEQ ID NO: 3). Further embodiments provide a method of preventing, treating, ameliorating or reducing at least one symptom of a cardiovascular disease in an animal by administering to the animal a compound consisting of the nucleobase sequence of ISIS 304801 (SEQ ID NO: 3), thereby increasing the HDL levels and/or improving the ratio of TG to HDL in the animal.
Certain embodiments provide a method of raising HDL levels and/or improving the ratio of TG to HDL in an animal by administering to the animal a modified oligonucleotide having the sequence of ISIS 304801, wherein the modified oligonucleotide comprises: (i) a gap segment consisting often 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 t 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 ucleoside linkage is a phosphorothioate linkage.
Certain embodiments provide a method of preventing, treating, ameliorating, or ng at least one symptom of a cardiovascular e in an animal by administering to the animal a modified oligonucleotide having the sequence of ISIS 304801 (SEQ ID NO: 3), n the modified oligonucleotide of the compound comprises: (i) a gap segment consisting often 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, n each ne is a 5’-methylcytosine, and wherein each internucleoside linkage is a phosphorothioate linkage.
Certain embodiments provide a method of raising the HDL levels and/or improving the ratio of TG to HDL in an animal by administering to the animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to an ApoCIII nucleic acid. In certain embodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2.
Certain embodiments provide a method of preventing, treating, ameliorating or reducing at least one symptom of a cardiovascular disease in an animal by administering to the animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, n the modified ucleotide is complementary to an ApoCIII nucleic acid, and raises the HDL levels in the animal. In certain ments, the ApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified ucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2.
Certain embodiments provide a method of decreasing CETP levels by administering a compound ing ApoCIII to an animal. In certain embodiments, the compound comprises a d oligonucleotide consisting of 12 to 30 linked sides, wherein the modified oligonucleotide is complementary to an ApoCIII nucleic acid. In certain embodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the compound comprises a modified oligonucleotide ting of 12 to 30 linked nucleosides and has a nucleobase sequence comprising at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, the compound consists of the nucleobases of ISIS 304801 (SEQ ID NO: 3).
Certain embodiments provide a method of increasing ApoAl, PONl, fat clearance, chylomicron triglyceride clearance and/or HDL by administering a compound targeting I to an animal. In certain ments, the compound ses a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the d oligonucleotide is complementary to an ApoCIII nucleic acid. In certain embodiments, the ApoCIII nucleic acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% complementary to SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the compound comprises a modified oligonucleotide ting of 12 to 30 linked nucleosides and has a nucleobase sequence sing at least 8 contiguous nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, the compound consists of the nucleobases of ISIS 304801 (SEQ ID NO: 3).
In certain embodiments, the animal is human.
In certain embodiments, the cardiovascular disease is aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary heart disease, hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia or hypercholesterolemia. In certain embodiments, the dyslipidemia is chylomicronemia.
In certain embodiments, the animal is at risk for pancreatitis. In certain ments, reducing ApoCIII levels in the liver and/or small intestine prevents pancreatitis. In certain embodiments, raising HDL levels and/or improving the ratio of TG to HDL prevents pancreatitis.
In certain embodiments, reducing ApoCIII levels in the liver and/or small intestine e clearance of postprandial triglyceride. In certain embodiments, raising HDL levels and/or ing the ratio of TG to HDL enhance clearance of andial triglyceride. In certain embodiments, reducing ApoCIII levels in the liver and/or small intestine lowers andial triglyceride. In certain ments, g HDL levels and/or improving the ratio of TG to HDL lowers postprandial triglyceride.
In certain embodiments, reducing ApoCIII levels in the liver and/or small intestine improves the ratio ofHDL to TG.
In certain ments, the nd is parenterally administered. In r embodiments, the parenteral administration is subcutaneous.
In certain embodiments, the compound is co-administered with a second agent. In certain embodiments, the second agent is a e-lowering agent. In certain embodiments, the second agent is an LDL, TG or cholesterol lowering agent.
In certain ments, the compound and the second agent are administered concomitantly or sequentially.
In certain embodiments, the compound is a salt form. In further embodiments, the compound further comprises of a pharmaceutically acceptable carrier or diluent.
Certain embodiments provide use of a compound targeted to ApoCIII in the preparation of a medicament for decreasing I levels in an animal. Certain embodiments provide use of a compound targeted to ApoCIII for decreasing ApoCIII levels in an . In certain embodiments, ApoCIII levels are decreased in the liver or small intestine. Certain embodiments e use of a compound targeted to ApoCIII in the preparation of a medicament for for preventing, treating, ameliorating or reducing at least one symptom of a cardiovascular e by increasing HDL levels and/or improving the ratio of TG to HDL. Certain embodiments provide use of a compound targeted to ApoCIII for preventing, treating, ameliorating or reducing at least one symptom of a cardiovascular e by increasing HDL levels and/or improving the ratio of TG to HDL. Certain embodiments provide a use of a compound ed to ApoCIII for increasing HDL levels and/or improving the ratio of TG to HDL in an animal. Certain embodiments provide a use of a compound targeted to ApoCIII for the preparation of a ment for sing HDL levels and/or improving the ratio of TG to HDL in an animal. In certain embodiments, the compound is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 100% complementary to an ApoCIII nucleic acid sequence. In certain embodiments, the ApoCIII c acid is either SEQ ID NO: 1 or SEQ ID NO: 2. In certain embodiments, the compound is a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 8 nucleobases of ISIS 304801 (SEQ ID NO: 3). In certain embodiments, the modified oligonucleotide has the nucleobase ce of ISIS 304801 (SEQ ID NO: 3).Certain embodiments provide use of a compound targeted to ApoCIII in the preparation of a medicament for treating an animal with pancreatitis or at risk for pancreatitis. Certain ments provide use of a compound targeted to ApoCIII for treating an animal with pancreatitis or at risk for pancreatitis. Certain embodiments provide use of a compound targeted to ApoCIII in the preparation of a medicament for reducing ApoCIII levels in the liver and/or small intestine. Certain embodiments provide use of a compound targeted to ApoCIII for reducing I levels in the liver and/or small intestine. Certain embodiments provide use of a compound targeted to ApoCIII in the preparation of a medicament for preventing atitis. Certain embodiments provide use of a nd targeted to ApoCIII for preventing pancreatitis.
Certain embodiments provide use of a compound targeted to ApoCIII in the preparation of a medicament for reducing ApoCIII levels in the liver and/or small intestine in an animal with hypertriglyceridemia. Certain embodiments provide use of a compound targeted to ApoCIII for reducing ApoCIII levels in the liver and/or small intestine in an animal with hypertriglyceridemia. Certain embodiments provide use of a compound targeted to ApoCIII in the ation of a ment for enhancing clearance of postprandial triglyceride. Certain embodiments provide use of a compound targeted to ApoCIII for enhancing clearance of postprandial triglyceride. Certain embodiments provide use of a compound targeted to ApoCIII in the preparation of a medicament for lowering postprandial triglyceride. Certain embodiments provide use of a compound targeted to ApoCIII for ng andial triglyceride.
Antisense Compounds Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, 2O oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric nd may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase ce that, when n 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 ucleotide 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 ed.
In n embodiments, an antisense compound ed 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 ucleotide 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 nse oligonucleotide.
In certain embodiments, the antisense nd comprises a shortened or truncated modified oligonucleotide. The shortened or truncated modified oligonucleotide can have one or more nucleosides d 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 sed 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.
When a single additional side 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 ucleotide 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 hout the nse compound, for example, in an oligonucleotide having one nucleoside added to the 5’ end and one subunit added to the 3’ end.
It is possible to increase or decrease the length of an antisense nd, such as an nse 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 ucleotides 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.
Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an ucleotide 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 trated potent anti-tumor activity in viva.
Maher and Dolnick (Nuc. Acid. Res. l6:334l-3358,l988) tested a series oftandem 14 nucleobase antisense oligonucleotides, and 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense ucleotides, respectively, for their ability to arrest ation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 base antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.
Antisense CompoundMotifs 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 ed inhibitory activity, sed binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
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 nse compound may optionally serve as a substrate for the ar endonuclease RNase H, which cleaves the RNA strand of an RNA: DNA dupleX. 2O Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of tides that supports RNase H cleavage is positioned between al regions having a plurality of nucleotides that are chemically ct from the sides 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 ts 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 es that are used to differentiate the regions of a gapmer may in some embodiments include B-D-ribonucleosides, [3-D- deoxyribonucleosides, 2'—modified nucleosides (such 2’-modified nucleosides may include 2’- MOE, and 2’-O-CH3, among ), and bicyclic sugar modified nucleosides (such bicyclic sugar modified sides may include those having a 4’-(CH2)n-O-2’ bridge, where n=1 or n=2). Preferably, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is ntly 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 n 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 , 44, 412—3, 4—124, 314—3, 213—5, 22, 11, 310—3, 210—2, 11,22, 66, 55, 11, 22, 2132, 12, 23, 3102, 12 or 22.
In certain embodiments, the antisense compound as a “wingmer” motif, haVing a wing- gap or ng 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 -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.
In certain embodiments, antisense compounds targeted to an I nucleic acid possess a 55 gapmer motif 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 Nucleotide sequences that encode I include, without limitation, the following: GERBANK Accession No. NM_000040.1 (incorporated herein as SEQ ID NO: 1), and K Accession No. NT_O33899.8 truncated from nucleotides 20262640 to 20266603 (incorporated herein as SEQ ID NO: 2).
It is understood that the sequence set forth in each SEQ ID NO in the es ned herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, nse nds defined by a SEQ ID NO may se, 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 2012/035694 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 ntron junction, a coding region, a translation initiation region, translation ation region, or other defined nucleic acid region. The structurally defined regions for I 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 ce 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.
In certain embodiments, a “target segment” is a smaller, sub-portion of a target region within a nucleic acid. For example, a target t 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.
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 ments, 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 ts 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.
Targeting includes determination of at least one target segment to which an nse 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 n encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid. le 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.
The ination 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 rity 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).
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, ions in ApoCIII mRNA levels are indicative of inhibition of ApoCIII expression.
Reductions in levels of an ApoCIII n can be indicative of inhibition of target mRNA sion. Further, phenotypic changes can be indicative of inhibition of I expression.
For example, an increase in HDL levels, decrease in LDL levels, or se in triglyceride levels, are among phenotypic changes that may be assayed for inhibition of ApoCIII expression.
Other phenotypic indications, e. g., symptoms associated with a cardiovascular disease, may also be assessed; for example, angina; chest pain; ess of breath; palpitations; weakness; dizziness; ; sweating; ardia; bradycardia; arrhythmia; atrial fibrillation; swelling in the lower extremities; cyanosis; fatigue; fainting; numbness of the face; numbness of the limbs; claudication or cramping of s; bloating of the abdomen; or fever.
Hybridization In some embodiments, hybridization occurs between an antisense compound disclosed herein and an ApoCIII c 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. ization can occur under g conditions. Stringent conditions are sequence- dependent and are determined by the nature and ition of the nucleic acid molecules to be hybridized.
Methods of determining whether a ce is specifically hybridizable to a target nucleic acid are well known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed, 2001). In certain ments, the antisense compounds ed herein are specifically izable with an ApoCIII nucleic acid.
Complementarity 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 bases 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).
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).
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 s.
For e, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically ize, would represent 90 percent complementarity. In this example, the ing mplementary nucleobases may be clustered or interspersed with mentary 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) mplementary 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 ms (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol, 1990, 215, 403 410; Zhang and , 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).
In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fillly complementary (i.e. 100% complementary) to a target c acid, or specified portion thereof. For example, an antisense compound may be fully complementary to an ApoCIII c acid, or a target region, or a target segment or target sequence thereof. As used , “fully complementary” means each nucleobase of an antisense nd is capable of precise base pairing with the ponding nucleobases of a target nucleic acid. For example, a 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 fillly complementary to the antisense compound. Fully complementary can also be used in nce to a specified portion of the first and /or the second nucleic acid. For example, a nucleobase portion of a 30 nucleobase antisense compound can be “fiilly 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 n each nucleobase is complementary to the 20 base 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.
The location of a non-complementary nucleobase(s) can be at the 5’ end or 3’ end of the 2O antisense compound. Alternatively, the non-complementary base(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 ucleotide.
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 In certain embodiments, nse 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 I c acid, or specified portion thereof The antisense compounds provided herein also e 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 base portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 base n of a target t. 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 The antisense nds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or ce 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 nds having non-identical bases ve to the antisense nds 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 g relative to the sequence to which it is being ed.
In certain embodiments, the nse 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 A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base . Nucleotides are nucleosides that further e a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofiiranosyl sugar, the phosphate group can be linked to the 2', 3' or ' hydroxyl moiety of the sugar. Oligonucleotides are formed through the nt linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the ate groups are commonly ed to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside es, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable ties such as, for example, enhanced cellular , enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides can also be employed to increase the binding affinity of a ned 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 Inlernucleosz'de Linkages The naturally ing internucleoside linkage ofRNA and DNA is a 3' to 5' phosphodiester linkage. Antisense compounds having one or more modified, i.e. turally occurring, internucleoside linkages are often selected over nse compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, ed cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages e internucleoside linkages that retain a phosphorus atom as well as internucleoside es 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- ning linkages are well known.
In certain embodiments, antisense nds targeted to an ApoCIII nucleic acid comprise one or more modified internucleoside linkages. In certain ments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a orothioate internucleoside linkage.
Modified Sugar Moielies Antisense nds 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 g affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise ally modified ribofiiranose ring moieties. es of chemically modified ribofiiranose 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(R1)(R2) (R, R1 and R2 are each independently H, C1- C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2'—F-5'—methyl substituted nucleoside (see PCT International Application WC 2008/101 157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further tution at the 2'—position (see published US. Patent Application US2005-0130923, published on June 16, 2005) or alternatively stitution of a BNA (see PCT International Application WC 2007/134181 hed on 11/22/07 wherein LNA is tuted with for example a 5'-methyl or a 5'-vinyl group).
Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'—S, 2'-F, 3, 2’-OCH2CH3, 2’- OCH2CH2F and 2'—O(CH2)2OCH3 substituent groups. The substituent at the 2’ position can also be ed from allyl, amino, azido, thio, O-allyl, O-Cl-Clo alkyl, OCF3, OCHzF, 2SCH3, O(CH2)2-O-N(Rm)(Rn), O-CH2-C(=O)-N(Rm)(Rn), and O-CH2-C(=0)-N(R1)-(CH2)2-N(Rm)(Rn), where each R1, RIn and RH is, independently, H or substituted or unsubstituted C1-C10 alkyl.
As used herein, lic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, antisense nds provided herein include one or more bicyclic nucleosides comprising a 4’ to 2’ bridge. Examples of such 4’ to 2’ bridged bicyclic nucleosides, include but are not d to one of the formulae: 4'—(CH2)—O-2' (LNA); 4'—(CH2)-S-2'; 4'—(CH2)2-O-2' (ENA); CH3)- O-2' and 4'—CH(CH20CH3)-O-2' (and analogs thereof see US. Patent 845, issued on July , 2008); 4'—C(CH3)(CH3)-O-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'-CH2-N(OCH3)-2' (and analogs thereof see published International Application WO/2008/150729, published er 11, 2008); 4'-CH2-O- N(CH3)-2' (see published US. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH2-N(R)-O-2', wherein R is H, C1-C12 alkyl, or a protecting group (see US. Patent 7,427,672, issued on September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see padhyaya el al., J.
Org. Chem, 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof see published International Application WC 2008/154401, published on December 8, 2008).
Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh el al., Chem. Commun, 1998, 4, 455-456; Koshkin el 61]., edron, 1998, 54, 3607-3630; tedt el 61]., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 638; Kumar el al., . Med. Chem. Left, 1998, 8, 2219-2222; Singh el al., J. Org. Chem, 1998, 63, 10039; SriVastava el al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi el 61]., Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch el 61]., Chem. Biol, 2001, 8, l-7; and Drum el 61]., Curr. Opinion Mol. Ther., 2001, 3, 239-243; US. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; US. Patent Publication No. US2008-00396l8; US2009-001228l; US. Patent Serial Nos. 60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and 61/099,844; hed PCT International applications ; WC 2004/1063 56; WO 2005/021570; ; ; ; and WO 06478.
Each of the foregoing bicyclic nucleosides can be prepared haVing one or more stereochemical sugar configurations including for example bofuranose and B-D-ribofuranose (see PCT international application 98/00393, published on March 25, 1999 as W0 99/14226).
In certain embodiments, bicyclic sugar moieties ofBNA nucleosides include, but are not limited to, nds haVing at least one bridge between the 4' and the 2’ position of the pentofilranosyl sugar moiety n such bridges independently ses 1 or from 2 to 4 linked groups independently selected from -[C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=O)-, -C(= a): -C(=S)-a , -Si(Ra)2-, -S(=0)x-. and -N(Ra)-; wherein: X is 0, l, or 2; n is l, 2, 3, or 4; each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)—H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and each J1 and J2 is, ndently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, tuted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted cycle radical, C1-C12 aminoalkyl, substituted C1-C12 lkyl or a protecting group.
In certain embodiments, the bridge of a bicyclic sugar moiety is -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-, -C(RaRb)-N(R)-O- or —C(RaRb)-O-N(R)-. In certain embodiments, the bridge is 4'—CH2-2', 4'—(CH2)2-2', 4'—(CH2)3-2', -O-2', 4'—(CH2)2-O-2', 4'-CH2-O-N(R)-2' and 4'—CH2- N(R)—O-2'— wherein each R is, independently, H, a protecting group or C1-C12 alkyl.
In certain ments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4’-2’ methylene-oxy bridge, may be in the d-L configuration or in the [3-D ration. Previously, d-L-methyleneoxy (4’-CH2-O-2’) BNA's have been incorporated into antisense oligonucleotides that showed antisense actiVity (Frieden el al., Nucleic Acids Research, 2003, 21 , 63 65-63 72).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) d-L- methyleneoxy (4’-CH2-O-2’) BNA BNA , (B) thyleneoxy (4’-CH2-O-2’) , (C) ethyleneoxy (4’-(CH2)2-O-2’) BNA , (D) xy (4’-CH2-O-N(R)-2’) BNA, (E) oxyamino (4’-CH2-N(R)-O-2’) BNA, and (F) methyl(methyleneoxy) (4’-CH(CH3)-O-2’) BNA, (G) methylene-thio (4’-CH2-S-2’) BNA, (H) methylene-amino (4’-CH2-N(R)-2’) BNA, (1) methyl carbocyclic (4’-CH2-CH(CH3)-2’) BNA, and (J) propylene carbocyclic H2)3-2’) BNA as depicted below.
(A) (B) (C) E O BX H3C \o E O BX E O BX E O BX E O BX (G) (H)R (D wherein BX is the base moiety and R is independently H, a protecting group or C1-C12 alkyl.
In certain embodiments, bicyclic nucleosides are provided having Formula I: wherein: BX is a heterocyclic base moiety; -Qa-Qb-Qc- is -CH2-N(Rc)-CH2-, -C(=O)-N(Rc)-CH2-, -N(RC)-, -CH2-N(Rc)-O- or - N(Rc)'O'CH2; RC is C1-C12 alkyl or an amino protecting group; and Ta and Tb 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.
In certain ments, bicyclic sides are provided having Formula II: 2012/035694 wherein: BX is a cyclic base moiety; Ta and Tb 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; Za is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, tuted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
In one embodiment, each of the substituted groups is, independently, mono or poly tuted with substituent groups independently selected from halogen, oxo, yl, OJC, NJch, SIC, N3, OC(=X)JC, and NJeC(=X)NJch, wherein each Jc, Id and I6 is, independently, H, C1- C6 alkyl, or substituted C1-C6 alkyl and X is O or NJC.
In certain embodiments, bicyclic nucleosides are provided having Formula III: o BX o \0 I III Tb wherein: BX is a heterocyclic base moiety; Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a orus moiety or a covalent attachment to a support medium; Zb is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 l, substituted C2-C6 alkynyl or substituted acyl (C(=O)-).
In certain embodiments, bicyclic nucleosides are provided having Formula IV: qa qb Ta_0 BX O/Tb 1?] IV wherein: BX is a heterocyclic base moiety; Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a ve phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; Rdis C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, each qa, qb, qc and qd is, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 l or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 lkyl or substituted C1-C6 aminoalkyl; In certain embodiments, bicyclic sides are provided having Formula V: Ta—O BX O—Tb wherein: BX is a heterocyclic base moiety; Ta and Tb are each, ndently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a orus moiety or a covalent attachment to a support medium; qa, qb, qe and qf are each, independently, hydrogen, halogen, C1-C12 alkyl, substituted C1- C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxy, substituted C1-C12 , OJj, SJj, SOJj, SOZJj, NJij, N3, CN, C(=O)OJ-, C(=O)NJij, C(=O)Jj, O-C(=O)NJij, N(H)C(=NH)NJij, N(H)C(=O)NJij orN(H)C(=S)NJij; or qe and qf together are =C(qg)(qh); qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
The synthesis and preparation of the methyleneoxy (4’-CH2-O-2’) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and c acid recognition properties have been described (Koshkin el al., edron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of methyleneoxy 2-O-2’) BNA and 2'—thio-BNAs, have also been prepared (Kumar el al., Bioorg. Med. Chem. Left, 1998, 8, 222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel el al., W0 99/14226 ). Furthermore, synthesis of 2'—amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh el al., J. Org. Chem., 1998, 63, 1003 5-10039). In addition, 2'-amino- and 2'—methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA s has been previously reported.
In certain embodiments, bicyclic sides are provided having Formula VI: Ta_0 BX O‘Tb (lj VI wherein: BX is a heterocyclic base moiety; Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive orus group, a phosphorus moiety or a nt attachment to a support medium; each qi, qj, qk and q1 is, independently, H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, tuted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxyl, substituted C1-C12 alkoxyl, OJj, SJj, SOJj, SOZJj, NJij, N3, CN, C(=O)OJj, C(=O)NJij, C(=O)Jj, O-C(=O)NJij, N(H)C(=NH)NJij, N(H)C(=O)NJij C(=S)NJij; and qi and qJ- or ql and qk together are =C(qg)(qh), wherein qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
One carbocyclic bicyclic nucleoside having a 2)3-2' bridge and the alkenyl analog bridge CH-CH2-2' have been described (Freier el al., Nucleic Acids Research, 1997, (22), 4429-4443 and Albaek el al., J. Org. Chem, 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic sides along with their oligomerization and biochemical studies have also been described (Srivastava el al., J. Am. Chem. Soc, 2007, 129(26), 8362- 8379) 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 se ring connects the 2’ carbon atom and the 4’ carbon atom of the sugar ring.
As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
As used herein, “2’-modified sugar” means a syl sugar modified at the 2’ position.
In certain embodiments, such modifications include substituents selected from: a halide, including, but not d to substituted and unsubstituted alkoxy, substituted and unsubstituted kyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2’ modifications are ed from substituents including, but not limited to: O[(CH2)nO]mCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nF, O(CH2)nONH2, =O)N(H)CH3, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2'— substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, F, CF3, OCF3, SOCH3, SOzCH3, ONOz, N02, N3, NHz, cycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a er group, an intercalator, a group for improving pharmacokinetic properties, or a group for ing the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modifed nucleosides comprise a 2’-MOE side chain (Baker el 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’- 0- methyl, O-propyl, and O-aminopropyl. ucleotides 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 el al., , 1996, 50, 168-176; Altmann el al., Biochem. Soc. Trams, 1996, 24, 630-637; and n el al., Nucleosides Nucleotides, 1997, 16, 917-926).
As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran ” substituted in for the pentofilranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is ed 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), fiuoro HNA (F-HNA) or those compounds having Formula VII: (11 C12 Ta_O (13 C17 C14 (16 BX Tb/ R1 R2 (15 wherein independently for each of said at least one ydropyran nucleoside analog of Formula VII: BX is a heterocyclic base moiety; Ta and Tb are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of Ta and Tb is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the nse compound and the other of Ta and Tb is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group; q1, q2, q3, q4, q5, q6 and q7 are each independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and each of R1 and R2 is selected from en, hydroxyl, halogen, ituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, wherein X is O, S or NJ1 and each J1, J2 and J3 is, independently, H or C1-C6 alkyl.
In n embodiments, the d THP nucleosides of Formula VII are provided wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In n embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R1 and R2 is fluoro. In certain embodiments, R1 is fluoro and R2 is H; R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.
As used , “2’-modif1ed” or “2’-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2’ position other than H or OH. 2’-modif1ed nucleosides, include, but are not d to, bicyclic nucleosides n the bridge connecting two carbon atoms of the sugar ring connects the 2’ carbon and another carbon of the sugar ring; and sides with non-bridging 2’substituents, such as allyl, amino, azido, thio, O-allyl, O-Cl-Clo alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-O-N(Rm)(Rn), or O-CH2-C(=O)- N(Rm)(Rn), where each RIn and RH is, independently, H or substituted or unsubstituted C1-C10 alkyl. 2’-modifed nucleosides may r comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.
As used , “2’-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2’ position.
As used herein, “2’-OMe” or “2’-OCH3” or “2’-O-methyl” each refers to a nucleoside comprising a sugar comprising an -OCH3 group at the 2’ position of the sugar ring.
As used herein, “MOE” or “2’-MOE” or “2’-OCH2CH2OCH3” or “2’-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a -OCH2CH2OCH3 group at the 2’ position of the sugar ring.
As used herein, "oligonucleotide" refers to a nd comprising a plurality of linked 2O 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).
Many other o and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example reView article: Leumann, Bioorg. Med. Chem, 2002, 10, 841-854).
Such ring systems can o s additional substitutions to enhance actiVity.
Methods for the preparations of modified sugars are well known to those skilled in the art.
In nucleotides haVing modified sugar moieties, the nucleobase moieties (natural, modified or a combination f) are maintained for hybridization with an appropriate c acid target.
In certain embodiments, nse compounds comprise one or more sides having modified sugar moieties. In certain embodiments, the d sugar moiety is 2’-MOE. In certain embodiments, the 2’-MOE modified nucleosides are arranged in a gapmer motif. In n embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4’-CH(CH3)- 0-2’) bridging group. In certain embodiments, the (4’-CH(CH3)-O-2’) modified nucleosides are arranged throughout the wings of a gapmer motif. In certain embodiments, the modified sugar moiety is a cEt. In certain embodiments, the cEt modified tides are arranged throughout the wings of a gapmer motif.
Modified Nucleobases Nucleobase (or base) modifications or substitutions are structurally guishable from, yet fiinctionally interchangeable with, naturally occurring or synthetic unmodified nucleobases.
Both l and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart se stability, binding affinity or some other beneficial biological property to antisense nds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are ularly useful for increasing the binding affinity of an antisense compound for a target c acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid dupleX stability by 0.6-1 .2°C (Sanghvi, Y.S., Crooke, ST. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278). onal 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 e and guanine, uracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-CEC-CH3) 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 es and guanines, 5-halo particularly 5-bromo, 5-trifiuoromethyl and other S- tuted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino- adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine.
Heterocyclic base moieties may include those in which the purine or pyrimidine base is replaced with other heterocycles, for e 7-deaza-adenine, 7-deazaguanosine, 2- yridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of nse compounds include 5-substituted pyrimidines, 6-azapyrimidines and N—2, N- 6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
In certain embodiments, antisense compounds ed to an ApoCIII nucleic acid comprise one or more modified bases. In certain ments, gap-widened antisense oligonucleotides targeted to an ApoCIII nucleic acid comprise one or more modified nucleobases.
In certain embodiments, the modified base is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
Compositions andMethodsfor Formulating Pharmaceutical Compositions Antisense oligonucleotides may be d 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 e, or dose to be administered.
Antisense compound targeted to an ApoCIII c acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or r.
In certain embodiments, the “pharmaceutical carrier” or “excipient” is a pharmaceutically able solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The ent 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, nylpyrrolidone 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); ants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn , polyethylene glycols, sodium benzoate, WO 49495 sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).
Pharmaceutically acceptable organic or inorganic excipients, which do not deleteriously react with nucleic acids, suitable for eral or non-parenteral stration can also be used to formulate the itions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, n, lactose, e, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in itions 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. ceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such , or an ucleotide 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 able salts of antisense compounds, prodrugs, pharmaceutically able salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can e 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.
ConjugatedAntisense Compounds nse 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, , phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
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 ures. These terminal modifications protect the antisense compound from exonuclease ation, 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 i. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5'- izing 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 January 16, 2003.
Cell Culture andAntisense nds Treatment 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 ble from commercial vendors (e. g. American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and cells are cultured according to the vendor’s ctions using cially available reagents (e. g. Invitrogen Life logies, Carlsbad, CA). 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 ofAntl'sense ucleotides Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluence in culture.
One t commonly used to introduce antisense oligonucleotides into cultured cells es the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, CA).
Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® l (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of antisense oligonucleotide and a CTIN® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into ed cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® in EM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of nse ucleotide and a CTAMINE® concentration that lly ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes Cytofectin® (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a Cytofectin® concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes OligofectamineTM (Invitrogen Life Technologies, Carlsbad, CA). Antisense oligonucleotide is mixed with OligofectamineTM in Opti-MEMTM-l reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to achieve the desired concentration of oligonucleotide with an OligofectamineTM to oligonucleotide ratio of approximately 0.2 to 0.8 uL per 100 nM.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes FuGENE 6 (Roche stics Corp., Indianapolis, IN). 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 l to 4 uL ofFuGENE 6 per 2O 100 nM.
Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation (Sambrooke and Russell in Molecular Cloning. A Laboratory Manual.
Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring , New York. 2001).
Cells are treated with antisense ucleotides 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 bed herein (Sambrooke and l in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001). In l, when ents are performed in multiple replicates, the data are presented as the average of the ate treatments.
The concentration of antisense oligonucleotide used varies from cell line to cell line.
Methods to determine the optimal nse oligonucleotide concentration for a particular cell line are well known in the art (Sambrooke and Russell in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001). Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE2000® (Invitrogen, ad, CA), ctin® (Invitrogen, ad, CA) or CytofectinTM (Genlantis, San Diego, CA). Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.
RNA ion RNA analysis can be performed on total cellular RNA or )+ mRNA. Methods of RNA isolation are well known in the art (Sambrooke and Russell in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001). RNA is prepared using methods well known in the art, for example, using the ® Reagent (Invitrogen, Carlsbad, CA) according to the cturer’s recommended protocols. is ofInhibition of Target Levels or Expression Inhibition of levels or expression of an ApoCIII nucleic acid can be assayed in a variety ofways known in the art (Sambrooke and Russell in Molecular Cloning. A Laboratory Manual.
Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001). For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain on (PCR), or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods ofRNA ion are well known in the art.
Northern blot analysis is also routine in the art. tative real-time PCR can be conveniently accomplished using the cially available ABI PRISM® 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to manufacturer’ s instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE-Applied 2012/035694 Biosystems, Foster City, CA) ing to manufacturer’s instructions. Methods of quantitative real-time PCR are well known in the art.
Prior to real-time PCR, the isolated RNA is ted 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 med sequentially in the same sample well. RT and real-time PCR reagents are obtained from ogen (Carlsbad, CA). RT and real-time-PCR reactions are d out by methods well known to those skilled in the art.
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® (Invitrogen, Inc. Carlsbad, CA). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is fied using RIBOGREEN® RNA quantification reagent (Invitrogen, Inc. Carlsbad, CA). Methods ofRNA quantification by RIBOGREEN® are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000 instrument (PE Applied Biosystems, Foster City, CA) is used to measure RIBOGREEN® fluorescence.
Probes and primers are designed to hybridize to an ApoCIII nucleic acid. Methods for designing ime PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS® Software (Applied Biosystems, Foster City, CA).
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 RiboGreenTM (Molecular Probes, Inc. , OR). GAPDH or Cyclophilin A expression can be quantified by RT, real-time PCR, by being run simultaneously with the target, lexing, or separately. Total RNA was quantified using RiboGreenTM RNA quantification t (Molecular Probes, Inc. , OR).
Analysis ofProtein Levels 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 oblotting), enzyme- linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS) (Sambrooke and Russell in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 2001). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of dies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Antibodies useful for the ion of human and mouse ApoCIII are commercially available.
In vivo testing ofantisense compounds Antisense compounds, for e, antisense oligonucleotides, are tested in animals to assess their y to inhibit expression of ApoCIII and produce phenotypic changes. Testing can be performed in normal animals, or in experimental disease . For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as ate-buffered saline. stration 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 ed from tissue and changes in I nucleic acid expression are measured. Changes in ApoCIII protein levels are also ed.
Certain Indications In certain embodiments, provided herein are methods of treating an dual comprising stering one or more pharmaceutical compositions as described herein. In certain embodiments, the individual has a cardiovascular disease or a metabolic disorder.
In certain embodiments, the cardiovascular disease is aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary heart disease, hypertension, dyslipidemia, ipidemia, hypertriglyceridemia, holesterolemia, stroke and the like. In certain embodiments, the dyslipidemia is chylomicronemia.
As shown in the es below, compounds targeted to ApoCIII as described herein have been shown to modulate physiological markers or phenotypes of a cardiovascular e.
In certain of the experiments, the compounds increased HDL levels, and decreased LDL and triglyceride levels compared to untreated animals. In certain embodiments, the increase in HDL levels and decrease in LDL and triglyceride levels was associated with an inhibition of ApoCIII by the compounds.
In certain embodiments, physiological markers of a cardiovascular disease may be quantifiable. For example, HDL levels may be ed and quantified by, for example, standard lipid tests. For such markers, in certain embodiments, the marker may be increased by about 5, , 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.
Also, provided herein are methods for preventing, treating or ameliorating a symptom associated with a cardiovascular disease in a subject in need thereof. In certain embodiments, ed is a method for reducing the rate of onset of a symptom associated with a cardiovascular disease. In certain embodiments, provided is a method for reducing the severity of a symptom associated with a cardiovascular disease. In such embodiments, the methods comprise administering to an individual in need thereof a therapeutically effective amount of a compound targeted to an I nucleic acid.
Cardiovascular diseases are characterized by numerous physical symptoms. Any symptom known to one of skill in the art to be associated with a vascular disease can be prevented, treated, ameliorated or otherwise modulated as set forth above in the s described above. In certain ments, the symptom may be any of, but not limited to, angina, chest pain, shortness of breath, palpitations, weakness, dizziness, nausea, sweating, tachycardia, ardia, hmia, atrial fibrillation, swelling in the lower extremities, cyanosis, fatigue, fainting, numbness of the face, numbness of the limbs, claudication or cramping of s, bloating of the abdomen or fever.
In certain ments, the m is . In certain embodiments, the symptom is chest pain. In certain embodiments, the symptom is shortness of breath. In certain embodiments, the symptom is palpitations. In certain embodiments, the m is weakness. In certain embodiments, the symptom is dizziness. In certain embodiments, the symptom is nausea. In certain embodiments, the symptom is sweating. In certain embodiments, the symptom is tachycardia. In certain embodiments, the symptom is bradycardia. In certain embodiments, the symptom is arrhythmia. In certain embodiments, the symptom is atrial fibrillation. In certain embodiments, the symptom is swelling in the lower extremities. In certain embodiments, the symptom is is. In certain ments, the symptom is fatigue. In certain embodiments, the symptom is fainting. In n embodiments, the m is numbness of the face. In certain 2012/035694 embodiments, the symptom is numbness of the limbs. In certain embodiments, the symptom is claudication or cramping of muscles. In certain embodiments, the symptom is bloating of the abdomen. In certain embodiments, the symptom is impaired short-term fever.
In certain embodiments, the metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia.
In n embodiments, nds targeted to ApoCIII as described herein modulate physiological markers or ypes of a metabolic er. In certain embodiments, physiological markers of a lic disorder may be quantifiable. For example, glucose levels or insulin resistance can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker may be decreased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two ofthese values. In another e, insulin ivity or HDL levels can be measured and quantified by standard tests known in the art. For such markers, in certain embodiments, the marker may be increase by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.
Also, provided herein are methods for preventing, treating or ameliorating a symptom associated with a metabolic disorder in a subject in need thereof. In certain embodiments, provided is a method for reducing the rate of onset of a symptom associated with a metabolic disorder. In certain embodiments, ed is a method for ng the severity of a symptom associated with a metabolic disorder. In such embodiments, the methods comprise administering to an individual in need thereof a therapeutically effective amount of a compound targeted to an ApoCIII nucleic acid. lic 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, d, ameliorated or otherwise modulated as set forth above in the methods described above. In certain embodiments, the m may be any of, but not limited to, excessive urine production (polyuria), excessive thirst and increased fluid intake (polydipsia), blurred vision, unexplained weight loss and lethargy.
In n embodiments, provided are methods of treating an individual comprising administering a eutically effective amount of one or more pharmaceutical compositions as described . In certain embodiments, the individual has a cardiovascular disease. In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an ApoCIII nucleic acid is accompanied by monitoring of ApoCIII levels or markers of cardiovascular disease, diabetes or other e process associated with the expression of ApoCIII, to determine an individual’s response to administration of the antisense compound. An individual’s response to stration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
In certain embodiments, stration of an antisense compound ed to an ApoCIII nucleic acid results in reduction of ApoCIII expression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two ofthese values. In certain embodiments, ApoCIII expression is reduced to S 50 mg/L, 3 60 mg/L, 3 70 mg/L, 3 80 mg/L, S 90 mg/L, S 100 mg/L, S 110 mg/L, S 120 mg/L, S 130 mg/L, S 140 mg/L, S 150 mg/L, S 160 mg/L, S 170 mg/L, S 180 mg/L, 3 190 mg/L or S 200 mg/L.
In certain embodiments, administration of an antisense compound targeted to an ApoCIII nucleic acid results in increase in HDL levels by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 %, or a range defined by any two ofthese .
In certain ments, stration of an antisense compound targeted to an ApoCIII nucleic acid results in reduction of TG (postprandial or fasting) levels by at least about 15%,, %,, 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 ments, TG (postprandial or fasting) is reduced to S 100 mg/dL, S 110 mg/dL, S 120 mg/dL, S 130 mg/dL, S 140 mg/dL, S 150 mg/dL, S 160 mg/dL, S 170 mg/dL, S 180 mg/dL, S 190 mg/dL, S 200 mg/dL, s 210 mg/dL, s 220 mg/dL, s 230 mg/dL, s 240 mg/dL, s 250 mg/dL, s 260 mg/dL, s 270 mg/dL, S 280 mg/dL, S 290 mg/dL, S 300 mg/dL, S 350 mg/dL, S 400 mg/dL, S 450 mg/dL, S 500 mg/dL, S 550 mg/dL, S 600 mg/dL, S 650 mg/dL, S 700 mg/dL, S 750 mg/dL, S 800 mg/dL, s 850 mg/dL, s 900 mg/dL, s 950 mg/dL, s 1000 mg/dL, s 1100 mg/dL, s 1200 mg/dL, s 1300 mg/dL, : 1400 mg/dL, s 1500 mg/dL, s 1600 mg/dL, s 1700 mg/dL, s 1800 mg/dL or s 1900 mg/dL.
In certain embodiments, ceutical compositions comprising an antisense compound targeted to ApoCIII are used for the preparation of a medicament for treating a patient suffering or susceptible to a cardiovascular disease.
Administration 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.
In certain embodiments, the compounds and compositions as bed herein are administered parenterally. Parenteral administration includes intravenous, intra-arterial, aneous, intraperitoneal or intramuscular injection or infusion.
In n embodiments, parenteral administration is by infusion. on can be chronic or continuous or short or intermittent. In n embodiments, d pharmaceutical agents are delivered with a pump. In n embodiments, the infusion is intravenous.
In certain embodiments, parenteral stration is by injection. The injection can be delivered with a syringe or a pump. In certain embodiments, the ion is a bolus injection. In certain embodiments, the injection is administered directly to a tissue or organ. In certain embodiments, parenteral administration is subcutaneous.
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.
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 ons in water or non-aqueous media, es, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, ts, f1ers, dispersing aids or binders can be desirable. In certain embodiments, oral ations are those in which compounds of the invention are administered in conjunction with one or more penetration enhancers, surfactants and chelators.
Dosing In n embodiments, pharmaceutical compositions are administered according to a dosing regimen (e.g., dose, dose frequency, and duration) wherein the dosing regimen can be selected to achieve a desired effect. The desired effect can be, for example, reduction of ApoCIII or the prevention, reduction, amelioration or slowing the progression of a disease or condition associated with ApoCIII.
In certain embodiments, the variables of the dosing regimen are ed 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 ceutical composition at an amount sufficient to achieve a desired effect.
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 e state is achieved. Dosing is also dependent on drug potency and metabolism. In certain embodiments, dosage is from 0.01 ug to 100mg per kg of body weight, or within a range of 0.001mg — lOOOmg 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 o maintenance therapy to prevent the recurrence of the disease state, n the oligonucleotide is administered in maintenance doses, ranging from 0.01ug to 100mg per kg of body weight, once or more daily, to once every 20 years or ranging from 0.00lmg to lOOOmg dosing.
Certain Combination Therapies In certain ments, a first agent comprising the compound described hereinis co- administered with one or more secondary agents. In certain embodiments, such second agents are ed to treat the same disease, disorder, or condition as the first agent described herein. In n embodiments, such second agents are designed to treat a ent 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 n embodiments, such second agents are designed to treat an undesired side effect of one or more pharmaceutical compositions as described . In certain ments, second agents are co- administered with the first agent to produce a ational 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 lactic effect if the agents were administered as independent therapy.
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 ion and one or more other pharmaceutical agents are stered at ent times. In certain embodiments, one or more itions 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.
In certain embodiments, second agents include, but are not limited to, ApoCIII lowering agent, cholesterol lowering agent, non-HDL lipid lowering (e.g., LDL) agent, HDL raising agent, fish oil, niacin, fibrate, statin, DCCR (salt of diazoxide), glucose-lowering agent and/or anti- diabetic agents. In certain embodiments, the first agent is stered in ation 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. es of ApoCIII ng agents include an ApoCIII antisense oligonucleotide different from the first agent, niacin or an Apo B antisense oligonucleotide.
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-l analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, an glucosidase inhibitor, metformin, sulfonylurea, itazone, meglitinide, thiazolidinedione, alpha-glucosidase tor and the like. The sulfonylurea can be acetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide, or a zide. The meglitinide can be nateglinide or repaglinide. The thiazolidinedione can be pioglitazone or rosiglitazone. The alpha-glucosidase can be acarbose or miglitol.
The terol or lipid lowering therapy can e, but is not limited to, a therapeutic lifestyle change, statins, bile acids sequestrants, nicotinic acid and fibrates. The statins can be atorvastatin, fiuvastatin, loyastatin, praVastatin, rosuVastatin and simVastatin and the like. The bile acid sequestrants can be coleseVelam, cholestyramine, colestipol and the like. The fibrates can be gemfibrozil, rate, clofibrate and the like.
HDL increasing agents include cholesteryl ester transfer protein (CETP) inhibiting drugs (such as Torcetrapib), peroxisome proliferation ted receptor agonists, Apo-Al, Pioglitazone and the like. n Treatment Populations Certain subjects with high TG levels are at a significant risk of cardiovascular and metabolic disease. In many subjects with high TG (e.g., hypertriglyceridemia), current treatments cannot reduce their TG levels to safe levels. ApoCIII plays an important role in TG metabolism and is an independent risk factor for cardiovascular disease. ApoCIII inhibition, as shown , significantly decreases TG levels which can ameliorate cardiovascular or metabolic disease, or the risk thereof.
Borderline high TG levels (150—199 mg/dL) are commonly found in the general population and are a common component of the metabolic syndrome/insulin resistance states.
High plasma TG level of2 200 mg/dL is a common al trait associated with an increased risk of cardiovascular disease (Hegele et al., Hum Mol Genet 2009, 18:4189-4194; Hegele and PolleX, Mol Cell Biochem, 2009, 326:35-43). Very high TG levels (2500 and S2000 mg/dL) are most often associated with elevated chylomicron levels as well, and are accompanied by sing risk for acute pancreatitis.
In certain embodiments, the compounds, compositions and methods disclosed herein are used to treat subjects with a TG level between 100-200 mg/dL, 100-300 mg/dL, 100-400 mg/dL, lOO-5OO mg/dL, 200-500 mg/dL, 300-500 mg/dL, 400-500 mg/dL, 500-1000 mg/dL, 600-1000 mg/dL, 700-1000 mg/dL, 800-1000 mg/dL, 00 mg/dL, 500-1500 mg/dL, 1000-1500 mg/dL, lOO-2000 mg/dL, l50-2000 mg/dL, 200-2000 mg/dL, 300-2000 mg/dL, 400-2000 mg/dL, 500-2000 mg/dL, 600-2000 mg/dL, 700-2000 mg/dL, 800-2000 mg/dL, 900-2000 mg/dL, 1000- 2000 mg/dL, 1100-2000 mg/dL, 000 mg/dL, 1300-2000 mg/dL, 1400-2000 mg/dL, or 1500-2000 mg/dL. In certain embodiments, treatment with the compounds disclosed herein is ted for a subject with a TG level of 2 100 mg/dL, 2 110 mg/dL, 2 120 mg/dL, 2 130 mg/dL, Z 140 mg/dL, Z 150 mg/dL, Z 160 mg/dL, Z 170 mg/dL, Z 180 mg/dL, Z 190 mg/dL, Z 200 mg/dL, Z 300 mg/dL, Z 400 mg/dL, Z 500 mg/dL, Z 600 mg/dL, Z 700 mg/dL, Z 800 mg/dL, 2 900 mg/dL, 2 1000 mg/dL, 2 1100 mg/dL, 2 1200 mg/dL, 2 1300 mg/dL, 2 1400 mg/dL, 2 1500 mg/dL, 2 1600 mg/dL, 2 1700 mg/dL, 2 1800 mg/dL, 2 1900 mg/dL, 2 2000 mg/dL, 2 2100 mg/dL, Z 2200 mg/dL, Z 2300 mg/dL, Z 2400 mg/dL or Z 2500 mg/dL.
Some types of hypertriglyceridemia can be terized by the Fredrickson classification system or by the classification system described by ay (Tremblay et al., J Clin Lipidol, 2011, 5:37-44). In certain ments, the compounds, compositions and s described herein are useful in treating subjects with, or at risk for, ckson Type II, IV or V hypertriglyceridemia.
Fredrickson Type IIb (also known as familial combined hyperlipoproteinemia) is a mixed hyperlipidemia (high terol 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 se in B-100 sis. They may also be caused by the decreased clearance of LDL.
Prevalence in the population is about 10%.
Fredrickson Type IV (also known as familial hypertriglyceridemia) is an autosomal dominant condition occurring in approximately 1% of the population. TG levels are elevated as a result of excess hepatic production of VLDL or heterozygous LPL deficiency, but are almost always less than 1000 mg/dL. Serum cholesterol levels are usually within normal limits. The disorder is heterogeneous and the phenotype strongly influenced by environmental factors, particularly carbohydrate and ethanol consumption. In certain ments, the compounds, compositions and methods described herein are useful in treating ts with a TG level 2 200 mg/dL and heterozygous LPL deficiency or VLDL oduction. In certain ments, the compounds, compositions and s described herein are useful in treating subjects with a TG level 2 500 mg/dL and heterozygous LPL deficiency or VLDL overproduction.
Fredrickson Type V has high VLDL and chylomicrons. It is characterized by carriers of loss-of-fiinction LPL gene variants associated with LPL activity of at least 20% (i.e. partial LPL ncy). These subjects present with lactescent plasma and severe hypertriglyceridemia because of chylomicrons and VLDL. TG levels are invariably greater than 1000 mg/dL and total cholesterol levels are always elevated. The LDL-C level is usually low. It is also associated with increased risk for acute pancreatitis, glucose intolerance and hyperuricemia. Symptoms lly present in adulthood (> 35years) and, although the prevalence is relatively rare, it is much more common than homozygous or compound heterozygous LPL deficient subjects. In certain embodiments, the compounds, compositions and methods bed herein are useful in treating subjects with Z 1000 mg/dL TG. In certain embodiments, the nds, compositions and methods described herein are useful in treating subjects with, or at risk for, pancreatitis associated with high TG levels in a t.In certain embodiments, the compounds, compositions and methods described herein are useful in treating subjects with, or at risk for, vascular or metabolic disease associated with high TG levels in a subject. In certain embodiments, the cardiovascular disease is aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary heart e, hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, stroke and the like. In certain embodiments, the dyslipidemia is chylomicronemia. In certain embodiments, the metabolic es or disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type I and type II), obesity, insulin resistance, metabolic syndrome and diabetic dyslipidemia.
In certain embodiments, treatment with the compounds disclosed herein is indicated for a human animal with a genetic defect that increases I levels and/or triglyceride levels. In certain embodiments, the genetic defect is an allelic variant or polymorphism that increases ApoCIII expression. In certain embodiments, the polymorphism are T (at on 74) to A, C (at position -641) to A, G (at position -630) to A, T (at position -625) to deletion, C (at position - 482) to T, T (at position -455) to C, C (at position 1100) to T, C (at position 3175) to G, T (at position 3206) to G, C (at position 323 8) to G, and the like. In certain embodiments, the c defect is a heterozygous LPL deficiency.
In certain embodiments, treatment with the compounds disclosed herein is indicated for a human animal with ed ApoCIII levels. In certain embodiments, the elevated ApoCIII level is Z 50 mg/L, 2 60 mg/L, 2 70 mg/L, 2 80 mg/L, 2 90 mg/L, 2 100 mg/L, 2 110 mg/L, 2 120 mg/L, 2 130 mg/L, 2 140 mg/L, 2 150 mg/L, 2 160 mg/L, 2 170 mg/L, 2 180 mg/L, 2 190 mg/L, 2 200 mg/L, 2 300 mg/L, 2 400 mg/L or Z 500 mg/L.
EXAMPLES Non-limiting disclosure and incorporation by reference While certain compounds, compositions and methods described herein have been bed with specificity in ance 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 ty.
Example 1: Effect of in vivo antisense tion of human ApoCIII in huApoCIII transgenic mice Transgenic mice with the human ApoCIII ene utilized in the study were the progeny of huApoCIII transgenic F1 hybrids on Laboratories, CA) and C57BL/6 mice.
ISIS 304801 (previously disclosed in US Patent 7,598,227) with a start site of 508 on SEQ ID NO: 1 (GENBANK Accession No. NM_000040. 1) and a start site of 3 139 on SEQ ID NO: 2 (GENBANK Accession NT_O33899.8 truncated from nucleotides 20263040 to 20266203), with the sequence 5’- TTGTCCAGCTTTAT-3’ (SEQ ID NO: 3) and a 55 MOE gapmer motifwas utilized in this assay. Another ISIS nse oligonucleotide, ‘Compound X’, with a 55 MOE gapmer motif, targeting another region of SEQ ID NO: 1 or SEQ ID NO: 2, was also ed in this assay. Another ISIS nse oligonucleotide, ‘Compound Y’, with a 55 MOE gapmer motif, targeting a rodent ApoCIII sequence (GenBank Accession No.
NM_023 1 14.3; SEQ ID NO: 5) was also ed in this assay.
Treatment Human I transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were ved in 0.9% PBS for ion.
Male and female mice were assayed separately. The male mice were divided into three treatment groups consisting of 5 mice each. Two such groups received subcutaneous injections of ISIS 304801 or Compound X at a dose of 37.5 mg/kg twice a week for 2 weeks. One group of mice received subcutaneous injections ofPBS twice a week for 2 weeks. The female mice were divided into four treatment groups consisting of 4-5 mice each. Three such groups received subcutaneous injections of ISIS 304801, Compounds X or Y at a dose of 37.5 mg/kg twice a week for 2 weeks. One group of mice received subcutaneous injections ofPBS twice a week for 2 weeks. Prior to the treatment as well as after the last dose, blood was withdrawn from each mouse and plasma samples analyzed. Two days following the final dose, the mice were euthanized, organs harvested and analyses done.
Cholesterol and ceride Levels Plasma triglycerides and cholesterol were extracted by the method of Bligh and Dyer (Bligh, E.G. and Dyer, WJ. Can. J. Biochem. Physiol. 37: 911-917, 1959) and measured with a commercially ble triglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd.).
The results of the triglyceride analyses in males and females are presented in Tables 1 and 2, and are expressed in mg/dL. As ed, triglyceride levels in all the treatment groups were significantly lowered compared to that in the control groups.
For measuring the ent fractions of cholesterol (HDL, LDL and VLDL), the plasma samples from the female groups were analyzed by HPLC and are presented in Table 3. As observed, nse inhibition of ApoCIII cantly decreased VLDL and also significantly increased levels of HDL. An increase in HDL and a decrease in VLDL levels is a cardiovascular beneficial effect of antisense inhibition of ApoCIII and can be beneficial to animals with, or at risk or, idemic diseases.
Table 1 Effect of antisense ucleotide treatment on triglyceride levels (mg/dL) in female transgenic mice Week 0 Week 2 change PBS 2144 253 3 +21 Compound X 2385 677 -72 Compound Y 2632 1644 -37 ISIS 304801 2390 542 -75 Table 2 Effect of antisense oligonucleotide treatment on triglyceride levels (mg/dL) in male transgenic mice Week 0 Week 2 Change PBS 6191 7073 +14 ISIS 304801 6588 780 -88 Compound X 5464 861 -84 Table 3 Effect of antisense oligonucleotide treatment on plasma cholesterol fractions (% total cholesterol) in female transgenic mice VLDL (%) HDL (%) PBS 77 :: 2.6 4:: 1.0 19:: 1.9 ISIS 304801 41 :: 0.6 7 :: 0.3 52 :: 0.5 CompoundX 46 :: 5.1 7:: 1.0 48 :: 5.8 Example 2: Dose-dependent nse inhibition of human ApoCIII in huApoCIII transgenic mice ISIS 304801 and nd X were further studied in a dose-dependent study using human ApoCIII transgenic mice.
Treatment Human ApoCIII transgenic mice were maintained on a r light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the ch facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and ized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
Female mice were d into nine treatment groups consisting of 3 mice each. Eight such groups received subcutaneous injections of ISIS 304801 or compound X at a dose of 1.5 mg/kg/week, 5 week, 15 mg/kg/week, or 50 mg/kg/week for 2 weeks. One group of mice received subcutaneous ions ofPBS for 2 weeks. Prior to the treatment as well as after the last dose, blood was withdrawn from each mouse and plasma samples analyzed. Two days ing the final dose, the mice were euthanized, organs harvested and analyses done.
Cholesterol and Triglyceride Levels Plasma triglycerides and cholesterol were ted by the method of Bligh and Dyer (Bligh, E.G. and Dyer, WJ. Can. J. Biochem. Physiol. 37: 911-917, 1959) and measured with a commercially available triglyceride kit (DCL Triglyceride Reagent, Diagnostic Chemicals Ltd).
The results of the cholesterol and triglyceride analyses in the mice are presented in Tables 4 and 5, and are expressed in mg/dL. As observed, HDL levels in mice treated with higher doses of ISIS 304801 were significantly elevated, indicating the beneficial effect of inhibition of ApoCIII by the oligonucleotides. LDL and triglyceride levels in the high dose treatment groups were lowered compared to that in the control groups. An increase in HDL and a decrease in LDL and TG levels is a cardiovascular beneficial effect of antisense inhibition of ApoCIII and can be beneficial to animals with, or at risk of, dyslipidemic diseases.
WO 49495 Table 4 Effect of antisense oligonucleotide treatment on cholesterol and triglyceride levels (mg/dL) in transgenic mice Dose Total TrlleCerldes. . (mg/kg/wk) Cholesterol PBS ' 124 1017 50.0 105 417 ISIS 15.0 116 593 304801 5.0 101 871 1.5 125 1092 50.0 90 496 Compound 15.0 127 1168 X 5.0 166 1506 1.5 168 1518 Table 5 Effect of nse oligonucleotide treatment on HDL and LDL cholesterol levels (mg/dL) in transgenic mice Dose HDL LDL (mg/kg/Wk) PBS - 40 :: 8 42 :: 8 50.0 62 :: 19 28 :: 7 ISIS 15.0 60 :: 9 34 :: 7 304801 5.0 44 :3 30:: 13 1.5 39 :: 2 40 :: 2 50.0 46 :: 10 25 :: 3 Compound 15.0 37 :: 40 :: 2 X 5.0 40 :: 10 47 :5 1.5 45 :: 7 44 :: 6 2012/035694 e 3: Effect of antisense tion of ApoCIII in CETP Transgenic LDL receptor null mice Compound Y was further studied in a human CETP transgenic LDLr'/' mouse model to examine the effects of a mouse ApoCIII antisense inhibitor on plasma lipids and lipoprotein metabolism in hyperlipidemic mice.
Human CETP transgenic LDLr'/' transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum a western diet (42% calories from fat, 0.2% cholesterol).
Animals were acclimated to this diet for 10 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
Eight week old male mice were divided into three treatment groups. One such group of 6 mice received subcutaneous ions of compound Y at a dose of 12.5 mg/kg/week for 4 weeks. One group of 4 mice received subcutaneous injections of the l oligonucleotide ISIS 141923 (SEQ ID NO: 4) at a dose of 12.5 mg/kg/week for 4 weeks. One group of 5 mice received subcutaneous injections ofPBS for 4 weeks. Plasma s were taken at prior to the start of dosing, and at 2 and 4 weeks of treatment.
Cholesterol and Triglyceride Levels Plasma triglycerides and cholesterol were extracted by the method of Bligh and Dyer (Bligh, E.G. and Dyer, WJ. Can. J. Biochem. Physiol. 37: 911-917, 1959) and measured with a commercially available triglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd).
The results of the cholesterol and triglyceride es in the mice are ted in Tables 6 to 7, and are expressed in mg/dL. Cholesterol and triglyceride levels in the treatment group were significantly lowered compared to those in the control group. A decrease in terol and TG levels is a cardiovascular beneficial effect of antisense inhibition of ApoCIII and can be beneficial to animals with, or at risk of, dyslipidemic diseases.
Table 6 Effect of antisense oligonucleotide treatment on cholesterol levels (mg/dL) in transgenic mice Compound Week PBS 0 185 l 1747 2 2035 878 4 2359 686 Table 7 Effect of antisense oligonucleotide treatment on triglyceride levels ) in transgenic mice Compound Week PBS 0 297 45 l 2 420 150 4 496 86 tion ofCETP protein levels and activity Plasma CETP protein levels were measured using a commercial ELISA kit (ALPCO, Cat# 47-CETHU-E01). CETP protein activity was measured using a etric assay kit (Roar Biomedical, Inc. Cat# RB-CETP). As presented in Table 8, treatment with antisense oligonucleotide reduced CETP protein expression and activity. CETP (cholesteryl ester transfer protein) tates the exchange of triglycerides and cholesterol esters n high density lipoproteins (HDL) and apoB-containing lipoproteins, such as very low density lipoproteins (VLDL), LDL and chylomicrons. A decrease in CETP is associated with increased HDL levels and decreased LDL levels (Barter P.J. et al. Artherioscler. . Vasc. Biol. 23: 160-167, 2003). Therefore, inhibition of CETP protein levels and activity is a cardiovascular beneficial effect of antisense inhibition of ApoCIII and can be beneficial to animals with, or at risk of, dyslipidemic diseases. The control oligonucleotide did not have any significant effect on CETP, as expected.
Table 8 Percent inhibition of CETP protein in enic mice Level Activity Compound Y 24 24 ISIS 141923 0 3 Increase ofapoA] Protein levels andparaoxcmase-I (PONI) ly Plasma ApoAl n levels were measured by ELISA. PONl protein activity was measured using a EnzChek® Paroxanase etric assay kit (Invitrogen, Cat# E33702). As presented in Tables 9 and 10, treatment with antisense oligonucleotide ed ApoAl protein expression and increased PONl protein activity. ApoAl and PONl are major protein components ofHDL in plasma (Aviram, M and Rosenblat, M. Curr. Opin. Lipidol. 16: 393-399, 2005). Therefore, enhancement of protein level and activity of these two protein components is a cardiovascular beneficial effect of antisense inhibition of ApoCIII and can be beneficial to animals with, or at risk of, dyslipidemic diseases. The control oligonucleotide did not have any effect on either protein, as expected.
Table 9 Percent increase in APOA1 protein levels in transgenic mice mg/dL PBS 65 Compound Y 211 ISIS 141923 106 Table 10 Percent se in PONl protein activity in enic mice . Compound ISIS M1nutes PBS Y 141923 0.2 0.5 0.2 0.8 1.1 0.8 60 2.3 3.5 2.3 120 4.9 7.5 4.8 180 7.7 11.6 7.6 Example 4: Effect of antisense inhibition of ApoCIII on HDL cholesterol clearance in CETP Transgenic LDL receptor null mice Compound Y was further studied in a human CETP transgenic LDLr'/' mouse model to examine the effects of an ApoCIII antisense inhibitor on HDL cholesterol clearance and lism in hyperlipidemic mice.
Treatment Human CETP transgenic LDLr'/' mice were maintained on a 12-hour light/dark cycle and fed ad libitum a western diet (42% calories from fat, 0.2% cholesterol). Animals were acclimated to this diet for 10 days in the research facility before initiation of the ment.
Antisense ucleotides (ASOs) were prepared in PBS and ized by ng through a 0.2 micron filter. ucleotides were dissolved in 0.9% PBS for injection.
Eight week old male mice were divided into three treatment groups. One group of 6 mice received subcutaneous injections of nd Y at a dose of 15 mg/kg/week for 6 weeks. One group of 4 mice received subcutaneous injections of the control oligonucleotide ISIS 141923 at a dose of 15 mg/kg/week for 6 weeks. One group of 5 mice received subcutaneous injections of PBS for 6 weeks. 2O Cholesterol and Triglyceride Levels Plasma triglycerides and cholesterol were extracted by the method of Bligh and Dyer (Bligh, E.G. and Dyer, WJ. Can. J. Biochem. Physiol. 37: 911-917, 1959) and measured with a commercially available triglyceride kit (DCL Triglyceride Reagent; Diagnostic Chemicals Ltd).
The results of the terol and triglyceride analyses in the mice are presented in Table 11, and are expressed in mg/dL. Cholesterol and triglyceride levels in the treatment group was significantly lowered compared to that in the l group. A decrease in cholesterol and TG levels is a cardiovascular beneficial effect of antisense tion of ApoCIII and can be beneficial to animals with, or at risk of, dyslipidemic diseases.
Table 11 Effect of antisense oligonucleotide treatment on terol and triglyceide levels (mg/dL) in transgenic mice Total T ' - r1g ycerides1 cholesterol PBS 2188 641 Compound Y 1402 170 HDL clearance Mice from all groups were injected via tail vein with 1 X 106 dpm of 3H-cholesteryl ether th)-labeled HDL. The radiolabeled cholesteryl ether is urally similar to cholesterol but it will be trapped in tissues that take it up. Therefore, the clearance of the radiolabeled cholesteryl ether from plasma and it’s accumulation in the liver can be used to evaluate effects on reverse cholesterol transport. Plasma samples were collected at 5 min, 1.5 hrs, 3 hrs, 6 hrs and 24 hrs post-injection and the radioactivity was d using a liquid scintillation counter. At 24 hours, the mice were sacrificed and liver were harvested. The liver samples were extracted in 2:1 form/Methanol and the t was blown down under nitrogen gas, solubilized in scintillation cocktail and counted using the same liquid scintillation counter.
The decrease in radiolabel, as presented in Table 12 is associated with the nce of HDL-Ceth from the plasma. The results indicate that treatment with Compound Y lead to enhanced rate ofHDL cholesterol clearance from the plasma. This was associated with the greater accumulation of radiolabeled cholesteryl etherin the liver of Compound Y-treated mice, as presented in Table 13. Therefore, the data indicates that inhibition of ApoCIII in these 2O transgenic mice improves reverse cholesterol transport and, therefore, would have a cial effect on patients with cardiovascular e such as patients with a dyslipidemic disease.
Table 12 Effect of antisense oligonucleotide treatment on plasma HDL cholesterol (% of count at 0 hrs) in trans enic mice 1.5 hr 3hr 6hr 24 hr PBS 87 79 64 38 ISIS 141923 84 82 69 39 Compound Y 78 71 57 25 Table 13 Effect of antisense oligonucleotide treatment on hepatic uptake of radiolabeled CEth in transgenic mice % se (dpm/g liver ISIS 141923 0 nd Y 14 Example 5: Comparison of the effect of antisense inhibition of human I in C57BL/6 mice with an ApoCIII ut mouse model ApoCIII knockout mice were obtained from Jackson Laboratories (stock number 002057) and were compared to ApoCIII antisense oligonucleotide treated C57BL/6 mice. Compound Z, with a 55 MOE gapmer motif and targeting a rodent ApoCIII sequence (GenBank Accession No. NM_023114.3; SEQ ID NO: 5) was used in this study.
Antisense oligonucleotide treatment C57BL/6 mice were maintained on a 12-hour dark cycle and fed a high fat diet nd Teklad lab chow #88137) for one week. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection. The mice were randomized based on total plasma cholesterol and triglyceride levels into groups of 6-8 mice each. Three groups of C57BL/6 mice received weekly intraperitoneal injections of Compound Z at doses of 3.1 mg/kg, 6.3 mg/kg, or 12.5 mg/kg for a period of 6 weeks. A group of C5 7BL/6 mice received weekly intraperitoneal ions ofPBS for a period of 6 weeks. The PBS group served as a control to which the oligonucleotide-treated groups and the ApoCIII knockout mice were compared.
Two days after the final dose, the mice were sacrificed and organs harvested. Similar groups of mice fed a normal murine chow were also tested.
Liver triglycerides Liver triglycerides were with an Olympus clinical analyzer (Hitachi Olympus AU400e, Melville, NY). The data is presented in Table 14 and demonstrates that mice treated with an ApoCIII antisense oligonucleotide have a different phenotype than ApoCIII knockout mice. The high dose ApoCIII antisense oligonucleotide treated mice had liver triglyceride levels similar to that of the PBS control. Liver triglyceride levels in the ApoCIII knockout mice were significantly higher than in C57BL/6 mice treated with an ApoCIII antisense oligonucleotide or the PBS control. Therefore, antisense inhibition of ApoCIII had the ial effect of lowering the risk of liver sis compared to the ApoCIII knockout mouse model.
Table 14 Liver triglyceride levels (mg/ liver tissue) Hi h- ams) mist PBS - 33 3.1 44 Compound Z 6.3 47 12.5 33 I KO - 60 Example 6: Effect of in vivo antisense inhibition of ApoCIII in C57BL/6 mice The effect of antisense inhibition of ApoCIII on plasma lipid levels and fat clearance was evaluated.
Treatment Male C57/BL6 mice were maintained on a 12-hour light/dark cycle and fed ad libilum a western diet (Harland Tekland 88137). Animals were acclimated for at least 7 days in the research ty before initiation of the experiment. Antisense ucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection. 2O Groups of 7-8 mice each received intraperitoneal injections of Compound Z at a dose of 12.5 mg/kg/wk for 6 weeks. Another group of mice ed intraperitoneal injections of l oligonucleotide ISIS 141923 at a dose of 12.5 mg/kg/wk for 6 weeks. A third group of mice received intraperitoneal injections ofPBS for 6 weeks. Two days after the final dose, the mice were fasted for 4 hours, ced and plasma and tissues were collected.
Inhibition oproClll mRNA Total RNA was extracted from the liver and small intestine and ApoCIII mRNA was quantitated by RT-PCR using an ApoCIII primer probe set and normalized to cyclophilin. The results are presented in Table 15, expressed as percent inhibition of ApoCIII mRNA compared to the PBS control. ISIS 141923 did not cause any reduction in ApoCIII mRNA levels, as expected.
The data demonstrated the significant tion of ApoCIII mRNA in the liver and small intestine by Compound Z compared to the PBS l.
Inhibition of intestinal ApoCIII expression could be important in the prevention of chylomicronemia (Chait et al., 1992, Adv Intern Med. 1992, -73), a dyslipidemic state caused by improper clearance of chylomicron triglyceride. Severe forms of chylomicronemia can lead to atitis, a life-threatening condition. By inhibiting intestinal ApoCIII, inhibition of lipoprotein lipase would be reduced, and icron ceride clearance would be enhanced, thereby ting pancreatitis. In addition, inhibition of intestinal ApoCIII would enhance clearance of postprandial triglyceride, thereby lowering post prandial TG a known risk factor for ry heart disease.
Table 15 Percent inhibition of ApoCIII mR\IA relative to the PBS control inhibition Liver 74 Small Intestine 13 Cholesterol and triglyceride levels 2O Plasma cholesterol were extracted by the method of Bligh and Dyer , E.G. and Dyer, WJ. Can. J. Biochem. Physiol. 37: 911-917, 1959) and measured with an Olympus clinical analyzer (Hitachi Olympus AU400e, Melville, NY). HDL and non-HDL cholesterol were dually measured by HPLC. Triglyceride levels were measured with the use of a commercially available triglyceride kit (DCL ceride Reagent; Diagnostic als Ltd., Charlottetown, Canada). The s are presented in Table 16 and are expressed in mg/dL.
Treatment with Compound Z resulted in significant reduction of total cholesterol, non-HDL cholesterol and plasma triglyceride levels compared to the PBS control. A decrease in total cholesterol, non-HDL cholesterol and TG levels is a cardiovascular beneficial effect of antisense inhibition of ApoCIII and can be beneficial to animals with, or at risk of, dyslipidemic diseases.
Table 16 Plasma cholesterol and triglyceride levels (mg/dL) in 6 mice Dose Total HDL LDL VLDL Trea ment t Tri 1 cerides (mg/kg/wk) terol cholesterol cholesterol cholesterol g y PBS - 93 7O 20 2.9 84 ISIS 141923 12.5 97 78 19 2.1 82 Compound Z 12.5 95 75 17 3.1 70 Fat clearance Plasma s were collected at 30 min, 1 hr, 2 hrs, 3 hrs, and 4 hrs post-injection and plasma total lipid content was measured with an Olympus clinical analyzer (Hitachi Olympus AU400e, Melville, NY). The lipid level in the plasma, as presented in Table 17 was an inverse indicator of lipid clearance from the . The results indicate that treatment with Compound Z lead to enhanced rate of fat nce from the plasma.
Therefore, the data indicates that inhibition of ApoCIII in these transgenic mice improves reverse cholesterol transport and, would have a beneficial effect on patients with cardiovascular disease.
Table 17 Effect of nse oligonucleotide ent on plasma lipid (mg/dL) in C57BL/6 mice Area under the 0 hr 0.5 hr 1 hr 2 hr 3 hr 4 hr curve PBS mm- -m— ISIS 141923 115 142 124 236 225 150 43677 Compoundz 15-“ 156 101 28371 Example 7: Effect of in vivo antisense inhibition of ApoCIII in C57BL/6 mice The effect of antisense inhibition of ApoCIII on ApoCIII sion levels and fat clearance was evaluated.
Treatment Male C57/BL6 mice were maintained on a 12-hour light/dark cycle and fed ad libilum a western diet (Harland Tekland 88137). Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in PBS and sterilized by filtering through a 0.2 micron filter. ucleotides were dissolved in 0.9% PBS for injection.
Groups of 5 mice each received intraperitoneal injections of an I targeting antisense oligonucleotide, Compound Z, at a dose of 12.5 mg/kg/wk for 6 weeks. Another group of mice received intraperitoneal injections of control oligonucleotide ISIS 141923 at a dose of 12.5 mg/kg/wk for 6 weeks. Two days after the final dose, the mice were fasted overnight, and a bolus of 200 uL of olive oil was administered by oral gavage. Following the bolus, plasma triglyceride levels were measured at regular als for 4 hours. The mice were sacrificed and plasma and tissues were collected.
Inhibition oproCIH mRNA Total RNA was extracted from the liver and small intestine and ApoCIII mRNA was quantitated by RT-PCR using an ApoCIII primer probe set and normalized to cyclophilin. The results are presented in Table 18, sed as percent inhibition of I mRNA compared to the oligonucleotide control. The data demonstrated the significant tion of ApoCIII mRNA in the liver and small intestine by Compound Z compared to the olionucleotide control.
As noted elsewhere herein, inhibition of intestinal ApoCIII expression could be important in the tion of chylomicronemia (Chait et al., 1992, Adv Intern Med. 1992, 37:249-73), a idemic state caused by improper clearance of chylomicron triglyceride. Severe forms of 2O icronemia can lead to pancreatitis, a life-threatening condition. By inhibiting intestinal ApoCIII, inhibition of lipoprotein lipase would be reduced, and chylomicron triglyceride clearance would be enhanced, thereby ting pancreatitis. In addition, inhibition of intestinal ApoCIII would enhance clearance of postprandial triglyceride, thereby lowering post prandial TG a known risk factor for coronary heart disease.
Table 18 Percent inhibition of ApoCIII mRNA relative to the control oligonucleotide treated C57/BL/6 mice Strain of mice inhibition Liver 74 C57BL 6/ Small Intestine 6O Fat clearance Plasma samples were ted at 0 min, 30 min, 60 min, 120 min, 180 min, and 240 min post-injection and plasma triglyceride concentrations was measured with an Olympus clinical analyzer (Hitachi Olympus AU400e, Melville, NY). The results te that treatment with Compound Z lead to enhanced rate of triglyceride clearance from the plasma.
This study can be compared to fat bolus clinical studies in which patients sing high apo-CIII levels showed increased postprandial TG trations sen KF. et al., N Engl J Med 2010; 362: 1082-1089).
Table 19 Effect of antisense oligonucleotide treatment on postprandial plasma TG (mg/dL) in C57BL/6 mice —---120 18-0 210 mm mm mm 1923 115 142 124 236 225 150 C57BL/6 Compoundz n-n 156 101 Example 8: Effect of in vivo antisense tion of ApoCIII in C57BL/6 mice The effect of antisense inhibition of ApoCIII on fat clearance was evaluated.
Treatment Male C57/BL6 mice were maintained on a 12-hour light/dark cycle and fed ad libilum a western diet (Harland Tekland 88137). Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were 2O prepared in PBS and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
Groups of 6 mice each received intraperitoneal injections of an I targeting antisense oligonucleotide, Compound Y or nd Z, at a dose of 12.5 wk, 6.3 mg/kg/week or 3.1 mg/kg/week for 6 weeks. Another group of mice received intraperitoneal injections of control oligonucleotide ISIS 141923 at a dose of 12.5 mg/kg/wk for 6 weeks. r group of mice received intraperitoneal injections ofPBS for 6 weeks. Two days after the final dose, the mice were fasted overnight, and a bolus of 200 11L of olive oil was administered by oral gavage. Following the bolus, plasma triglyceride levels were measured at regular intervals for 4 hours.
Fat clearance Plasma samples were collected at 0 min, 30 min, 60 min, 120 min, 180 min, and 240 min post-injection and plasma triglyceride concentrations were measured with an Olympus clinical analyzer (Hitachi Olympus , Melville, NY). The results te that treatment with Compound Y and Compound Z lead to enhanced rate of fat clearance from the plasma. N.d. indicates that the data set was not calculated.
This study can be compared to fat bolus clinical studies in which patients sing high apo-CIII levels showed increased postprandial TG concentrations (Petersen KF. et al., N Engl J Med 2010; 362: 1082-1089) Table 20 Effect of antisense oligonucleotide treatment on postprandial plasma TG (mg/dL) in C57BL/6 m1ce Dose 240 0 mm. 60 120 180 (mg/kg/wk) min min min min min PBS - 79 104 118 126 113 116 ISIS 141923 12.5 75 100 116 150 138 133 12.5 79 74 103 117 120 96 Compound Y 6.3 64 70 81 94 120 112 3.1 91 85 106 139 164 133 12.5 73 65 84 118 94 76 Compound Z 6.3 70 73 89 117 120 89 3.1 86 98 143 137 152 128 Example 9: Effect of ISIS antisense ucleotides targeting human I in monkey model of hypertriglyceridemia Rhesus monkeys maintained on a high fructose diet were treated with ISIS 304801.
Antisense oligonucleotide cy and tolerability, as well as the cological effect were evaluated.
Treatment The monkeys were 2-4 years old and weighed between 2 and 5 kg. The monkeys were assigned to six groups of five randomly assigned male rhesus monkeys each. About 60g of diet fied Primate Diet #5048, PMI Nutrition International, Inc.) was provided to each monkey in Groups 1-4 twice daily. An appropriate fructose supplement (i.e. approximately 15% Kool Aid® mixture) was supplied in the morning for 16 weeks prior to antisense oligonucleotide dosing. To confirm sufficient ceride level elevations, blood samples for serum try were collected from all animals 1-2 weeks prior to dosing.
The groups of monkeys were injected subcutaneously with ISIS ucleotide or PBS using a stainless steel dosing needle and syringe of appropriate size into one of 4 sites on the back of the monkeys; each site being used per dose in a clock-wise manner. Some of the groups were dosed three times a week for the first week (Days 1, 3, and 5) as loading doses, and subsequently twice a week for weeks 2-12, with 5 mg/kg, 10 mg/kg, or 20 mg/kg of ISIS 304801. Two control groups of 5 rhesus monkeys each were injected with PBS subcutaneously three times a week for the first week (Days 1, 3, and 5), and subsequently twice a week for weeks 2-12. The dosing chart is shown in Table 21. Monkeys of Groups 1-4 were sacrificed on Day 86.
An additional high fat challenge was administered to monkeys of Groups 5 and 6 in the form of a whipping cream milk shake. The milk shake was standardized to consist of 782 calories per m2 of body surface with 77.6% of calories from fat, 19.2% from carbohydrate, and 3.1% from protein. Monkeys of Groups 5 and 6 were fasted ght and the milk shake was administered once on day 84 via a gastric tube. Blood was drawn just prior to (time = 0 hours) and 1, 2, 3, 4, and 6 hours after ingestion of the fat load to assess the triglyceride excursion. The s were rested and remained otherwise g during the 6 hours post-fat challenge. Monkeys of this group were sacrificed on Day 87.
Table 21 Groups of rhesus monkeys on a high fructose diet Weekly Dose N0. of Group Test Article (mg/kg/week Animals Toxicology groups 1 —-_ 2 Male High Fat Challenge Tesl groups —-_ 5 Hepatic Target Reduction RNA analysis Approximately 150 mg of liver was collected from Groups 1-4 for ApoCIII mRNA analysis at sacrifice. The liver was divided into 2 pieces and soaked in two tubes containing RLT buffer with 1% beta-mercaptoethanol. The tissues were homogenized and ApoCIII expression was fied by RT-PCR analysis. As shown in Table 22, treatment with ISIS 304801 resulted in significant reduction of ApoCIII mRNA in comparison to the PBS control. The Table 22 Percent Inhibition of ApoCIII mRNA in the rhesus monkey liver relative to the PBS control Groups Dose % inhibition (mg/kg/week) 2 10 68 3 2O 78 4 40 83 Protein analysis Approximately 1.5 mL of blood was collected from all study animals in Groups 1-4 and placed in tubes containing Kz-EDTA and then centrifuged for plasma separation. I protein levels were fied on a clinical er using a commercially ble turbidometric assay (Kamiya Biomedical Co., Seattle, WA). As shown in Table 23, treatment with ISIS 304801 resulted in significant reduction of ApoCIII protein levels in comparison to the PBS control. The kinetics of ApoCIII protein level reduction was also analyzed and is presented in Table 24..
Table 23 Percent Inhibition of I plasma protein levels in the rhesus monkey relative to the PBS control Groups Dose % inhibition (mg/kg/week) 2 10 74 3 2O 72 4 4O 89 Table 24 ApoCIII plasma protein levels (mg/dL) on different days in the rhesus monkey relative to the PBS control Dose Da Grows Day '7 Da30y Da86y (mg/kg/wk) 16y 1 — 4.0 5.5 3.8 5.7 2 10 4.8 3.2 0.2 1.5 3 20 4.5 3.8 0.9 1.6 4 40 5.2 2.9 0.0 0.6 Lipoprotein particle analysis To establish the cs of plasma ApoCIII suppression, plasma samples were collected 7 days before the commencement of dosing, as well as on days 16, 30 and 86 of the dosing period.
The samples were subjected to N1\/[R1ipoprotein particle analysis (Liposcience, h, NC).
Since there were no significant differences in ApoCIII lowering between the ent groups (Groups 2-4), the analyses is ted only of Group 2 (treatment group receiving 10 mg/kg/week). The data is presented in Tables 25 and 26. tically significant mean changes from the baseline were observed in total plasma triglycerides (TG) and in VLDL and chylomicron TG of the treatment group at day 30. At the same time, the control monkeys demonstrated mean increases in the same parameters. Sustained treatment with ISIS 304801 in these fructose-fed monkeys led to time-dependent increases in HDL terol particle numbers by approximately 8 umol/L (Tables 27) and did not produce elevations in LDL cholesterol in these studies (Table 28). There were no significant changes in LDL cholesterol particle quantity over the 12 week treatment period, ve to the PBS l group. 2O At the time of sacrifice, livers were extracted using the Bligh and Dyer extraction method (Bligh EG and Dyer WI. Can J Biochem Physio1 1959; 37: 91 1-917) and quantified using a Wako co1orimentric TG assay. Antisense inhibition of I did not increase hepatic TG accumulation in any of the treatment groups relative to the PBS control group (Table 29).
Table 25 Change from baseline plasma TGmg/dL) on different days in the rhesus monkey Dose Day Day Day Day '7 (mg/kg/wk) 16 30 86 PBS - O 18 23 27 ISIS 10 0 —22 —32 —27 3 04801 Table 26 Change from baseline VLDL and icron TG (mg/dL) on ent days in the rhesus monkey Dose Day Day Day Day '7 (mg/kg/wk) 16 30 86 PBS - 0 17 22 28 ISIS 0 -22 -31 -26 304801 Table 27 Change from ne HDL cholesterol particles (umol/L) on different days in the rhesus monkey Dose Day Day Day Day '7 (mg/kg/wk) 16 30 86 PBS - 0.3 -5.6 -3.5 -8.1 ISIS 0.0 0.5 9.7 8.0 304801 Table 28 Total LDL cholesterol particles (nmol/L) on different days in the rhesus monkey Dose Day Day Day Day '7 (mg/kg/wk) 16 30 86 PBS - 982 928 1005 1184 ISIS 1007 938 781 910 304801 Table 29 Hepatic triglyceride content in PBS control and ISIS 304801 cohorts after 12 weeks in HTG rhesus monkeys Liver TG (ug/mg) Average PBS 9 10mg/kg/wk 16 g/wk 18 40mg/kg/wk 6 Post-prandialplasma TG clearance At 10 weeks, the post-prandial plasma TG levels in monkeys from the 10 mg/kg/week group (Group 2) were measured at 0 hr, 1 hr, 2 hr, 3 hr, and 4 hr after providing a meal to the monkeys. As shown in Tables 30 and 31, post-prandial plasma TG clearance was significantly WO 49495 increased, as shown by the 38% decrease in post-prandial TG area under the curve (AUC) in monkeys of the 10 mg/kg/week group. randial TG clearance was also assessed in Groups 5 and 6 (the PBS control and 40 mg/kg/week group after a fat challenge) at 12 weeks. The data is presented in Table 32, and also tes a significant decrease in post-prandial TG area under the curve (AUC) in monkeys of that group ed to the control.
Table 30 Plasma TG (mg/dL) in the rhesus monkey Dose 0 hr 1 hr 2 hr 3 hr 4 hr (mg/kg/wk) PBS - 167 169 146 147 131 ISIS 105 87 95 102 80 304801 Table 31 Post-prandial TG area under the curve (AUC) in the rhesus monkey PBS 610 ISIS 304801 376 Table 32 Post-prandial TG area under the curve (AUC) in the rhesus monkey after fat nge PBS 613 ISIS 304801 405 Monkeys in the 10 mg/kg/wk group had lower fasting plasma TG levels than the PBS group at 10 weeks. Results in non-human primates demonstrate that antisense inhibition of ApoCIII represent an attractive therapeutic strategy for reducing plasma TG and VLDL in dyslipidemic individuals, and treatment can concurrently raise HDL-C levels with no adverse effects on LDL-C.
Example 10: ISIS 304801 Phase I Clinical Trial In a double-blind, single and multiple ascending-dose (SAD and MAD) Phase 1 study, healthy subjects, aged 18 to 55 years, were randomly assigned in a 3 :1 ratio to e ISIS 304801 or placebo (normal saline).
SAD subjects were administered a single subcutaneous (SC) injection of 50, 100, 200, or 400 mg (n=4/cohort) at the Study Center. The subjects returned to the Study Center for an outpatient visit on Days 4 and 8 (:24 hour ) for blood sampling and for clinical evaluation. The subjects were followed until Day 15 when they were evaluated by a telephone interview.
MAD subjects were administered multiple SC injections at 50, 100, 200, and 400 mg at the Study Center. The subjects received a loading regimen of 3 doses the first week (Days 1, 3 and 5) followed by once weekly dosing for 3 weeks (Days 8, 15 and 22). The ts were followed for 8 weeks after their last dose of study drug. The subjects returned to the Study Center for an outpatient visit on Days 29, 36 and 50 (:24 hour window) for safety and clinical tory evaluations and for blood sampling for PK analysis. The subjects were followed until Day 78 (:7 day window) when they were evaluated by a telephone interview.
The MAD subjects stayed at the Study Center from days -1 to 6 and days 22 to 23, where they were provided the diet shown in Table 33. The subjects fasted for at least 12 hours before blood s were taken for evaluation at Days 5, 8, 15, 22, 23, 29, 36, 43 and 50 (:24 hour window).
Table 33 Study Center Patient Diet Serving Study Day Food Description Portion 525 m1. 0 Thai noodles with Beef & mixed vegetable, li, bean sprout, green & red peppers 375 m1. 0 Fresh fruit salad Day '1 300 m1. 0 Orange juice [Admit] 1 0 Horse shoe cake 250 ml. 0 2% Milk 1 0 Butter croissant 2 0 Eggs, led 250 m1. 0 Sliced peaches with syrup 300 m1. 0 Orange juice Day 1 75 g. o Grilled Chicken Breast [Sandwich] on Kaiser bun 1.5 cup 0 Cream of Mushroom Soup 1-pkg. o Crackers Small 0 Salad 011 side 0 Condiments 355 ml. 0 Ginger Ale WO 49495 75 g. 0 Roast Beef 250 ml. 0 Mashed Potatoes 375 ml. 0 Mixed Vegetables On side 0 Gravy 011 side 0 Garlic bread 300 “IL 0 Apple juice 1 0 Carrot muffin 250 ml. o 2% Milk 3 o Pancakes On side 0 Syrup 1 0 Banana 300 ml. 0 Orange juice 9” o Mesquite Chicken, Chicken, bacon, cheddar, tomato, red omon, & lettuce on whole wheat bread On side 0 Ranch dressing Cup 0 Broccoli & cheese soup Day 2 l-pkg. o Crackers 355 ml. 0 Ginger ale 375 g. 0 Beef Stir-fry & mixed vegetables, broccoli, carrots, celery, & onions 150 g. o Steamed rice l-pkg. o Raisin 300 ml. 0 Apple juice 250 ml. 0 Fresh fruit salad 300 ml. 0 Cranberry juice Med. 0 Cheese & vegetable omelet on whole wheat bagel toasted 300 ml. 0 Grape juice 9” 0 Black Angus Steak, mozzarella, cheddar, d onion & mushrooms on Cheese bread On side 0 Honey Bourbon Mustard, Zesty Grille Sauce Cup 0 Chicken noodle soup l-pkg. o Crackers 355 ml. 0 Ginger ale Day 3 400 g. 0 BBQ Chicken Breast, Vegetables, cooked mixed, cauliflower, s, red & green peppers, green beans 1.5 cups 0 Flavored rice l-pkg. o Grapes 300 ml. 0 Apple juice 1 o Raisin Oatmeal cookie 250 ml. 0 2% milk 1 0 Cheese Croissant 250 ml. 0 Sliced Peaches in syrup 300 ml. 0 Orange juice Day 4 2 o Flatbread Sammie, with Chicken, bacon, r, tomato & romaine lettuce On side 0 Buttermilk ranch dressing Cup 0 li & Cheese soup l-pkg. o Crackers 355 ml. 0 Ginger ale 425 g. 0 Beef Stew, tender Beef cubes, with carrots, omons, & potatoes 325 g. 0 Thai salad, with romaine lettuce, pasta, & dressings 1 0 Italian bread 250 m1. 0 Sliced Pears in syrup 300 1111. 0 Apple juice 1 o Blueberry muffin 250 ml. o 2% Milk 375 ml. 0 Corn Flakes Med. 0 Banana 250 ml. o 2% Milk 300 1111. 0 Apple juice 6 pcs. 0 n wings 250 ml. 0 Fried rice 250 ml. . Mixed cooked veggies, green beans, carrots, & red peppers with ed tomato sauce Day 5 355 ml. . 7.1110 6” Pizza 0 Pepperoni lovers Pizza a double helping of deli style pepperoni & 100% Pizza Mozzarella Small 0 Salad 355 ml. 0 Ginger Ale 1 o Almond Croissant 250 ml. o 2% Milk Day 6 45 g. o Bagel with Cream Cheese [Discharge] 300 ml. . Orange juice 450 g. 0 ast Omelets Spinach & Feta cheese 2 slices 0 Brown toast 300 ml. 0 Orange juice 9” o Zesty Grille Steak Prime Rib Steak, mozzarella, r, mushroom, sauteed onion, on Cheese bread On side 0 Honey Bourbon Mustard, Zesty Grille Sauce Cup 0 Broccoli & Cheese soup Day 22 l-pkg. o Crackers 355 ml. 0 Ginger Ale 400 g. 0 Herb Grilled n Breast with Vegetables 1.5 cups 0 Flavored rice Small 0 Fresh fmit salad 300 1111. 0 Apple juice 1 o Raisin Oatmeal cookie 250 ml. o 2% Milk Day 23 45 g. o Butter croissant [Discharge] 300 ml. . Orange juice Results Overall, ISIS 304801 demonstrated a good safety profile and was well tolerated in all subjects with no clinically meaningful elevations of transaminase enzymes and no significant adverse events. 2012/035694 The baseline characteristics ofMAD cohorts are shown in Table 34. MAD subjects showed dose-dependent sustained reductions in total apoC-III and TG levels expressed as a percentage change from baseline in Tables 35-36.
Table 34 Baseline Characteristics ofMAD Cohorts Placebo 50 mg 100 mg 200 mg 400 mg (n=4) (n=3) (n=3) (n=3) (n=3) Gender (MzF) 3:1 3:0 3:0 3:0 3:0 Age (yrs) 43.0 40.0 40.0 43.0 40.0 BMI (kg/m2) 27.7 24.0 27.3 28.0 27.5 Lipids & Lipoproteins, mg/dL Apo CIII 6.3 10.4 9.5 11.6 8.7 cerides 97 124 94 195 89 Total Cholesterol 195 157 196 185 181 HDL-C 45 42 46 43 62 Non-HDL-C 136 118 150 149 126 LDL-C 112 93 131 95 102 Per-protocol population. Values presented are the median.
Table 35 Dose-Dependent Prolonged Reduction in Serum ApoCIII: Median % change in I from Baseline Study Day Placebo 50 mg 100 mg 200 mg 400 mg Day5 38.6 33.4 -4.5 -10.8 -42.6 Day8 31.9 17.8 -5.2 -36.0 -78.6 Day 15 0.0 -22.1 -24.0 -54.1 -79.8 Day 22 11.7 -20.9 -21.0 -61.7 -86.4 Day 23 15.8 -15.6 -11.9 -58.7 -79.0 Day 29 -11.0 -l9.7 -17.3 -70.5 -77.5 Day 36 1.9 -26.6 -32.1 -57.3 -69.5 Day 50 16.4 21.9 -3.9 -63.0 -78.6 WO 49495 Table 36 Dose-Dependent Reductions in Triglycerides: Median % Change in Triglycerides from Baseline Study Day Placebo 50 mg 100 mg 200 mg 400 mg Day 5 78.5 50.0 15.9 -15.6 -7.7 Day 8 34.9 17.7 -24.5 -31.8 -38.5 Day 15 21.2 -27.4 -12.8 -50.8 -46.2 Day 22 12.2 -18.3 -9.8 -25.1 -53.8 Day 23 51.8 -4.0 1.1 -41.0 -44.2 Day 29 28.5 -19.5 -25.0 -43.1 -43.8 Day 36 15.4 -33.1 -34.8 -17.9 -36.5 Day 50 33.1 48.8 -23.9 -48.2 -48.1 Median percent change from baseline values in the 50, 100, 200 and 400 mg multiple- dose groups showed reductions of total apoC-III of 20, 17, 71, and 78% and of TG of 20, 25, 43, and 44%, respectively, one week (Day 29) after the last dose. ions were sustained for at least four weeks after the last dose in the higher dose groups.
TG levels spiked at Day 5 and 23 for the placebo group, coinciding with the subjects’ ght stays at the Study Center. It is thought that the diet provided by the Study Center led to the surge in TG levels in the subjects staying overnight at the Study Center. ISIS 304801 decreased the TG spike in a dose-dependent manner. In a manner, the results shown herein, indicate a andial effect on TG (although TG levels were assessed after a 12 hour fast) by ISIS 304801 as a diet induced surge in TG was decreased in a dose-dependent manner by ISIS 304801.
LDL-C values did not change (data not shown) while HDL-C values tended to increase in a treatment-dependent manner as shown in Table 37.
Table 37 No Deleterious Effects on HDL-C Study Day Placebo 50 mg 100 mg 200 mg 400 mg Day 5 -5.9 7.7 -3.1 -3.2 -16.1 Day 8 2.1 2.4 0.0 -11.1 -2.9 Day 15 -2.8 4.8 10.9 4.7 -9.7 Day 22 -0.6 4.8 8.7 11.6 -2.0 Day 23 0.7 7.1 12.5 19.4 -1.6 Day 29 2.1 19.0 0.0 13.9 8.0 Day 36 0.0 23.8 8.8 13.9 1.6 Day 50 -4.8 16.7 5.9 25.0 14.5 Example 11: ISIS 304801 Phase II Clinical Trial A ized, -blind, placebo-controlled, dose response study is planned to evaluate the dose/response pharmacodynamic effects of ISIS 304801 vs. placebo on fasting apoC-III associated with VLDL levels. Additional endpoints to evaluate include: the pharmacodynamic effects of ISIS 304801 vs. o on fasting total apoC-III, TG, apoC-II (total and associated with VLDL), apolipoprotein B-lOO (apoB-IOO), 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, G, non-high-density otein- cholesterol (non-HDL-C), non-HDL-TG, HDL-C, HDL-TG, chylomicron-C (CM-C), CM-TG, free fatty acids (FFA), and ol levels; the randial lipid, apolipoprotein and lipoprotein characteristics and kinetics, and glucose levels in a subset of the patients in the study and assess more extensive PK in another subset of the patients (will not be the same patients as those undergoing the post-prandial assessment); and, the , tolerability and PK of ISIS 304801.
For each patient, the participation period consists of a 35-week screening period, (which includes a 4-week tight diet control run-in qualification period), a 1-week study qualification/baseline assessment period, a 13-week treatment period, and a post-treatment evaluation period of 13 weeks, for a total of 32 weeks of study participation. Concomitant tions and adverse events (AEs) will be recorded throughout all periods of the study.
Patients will be at least 18 years of age and have fasting TG ZSOOmg/dL at screening and fasting TG Z300mg/dL and £2000mg/dL after 4-weeks of tight diet control run-in.
Seventy two (72) ts are planned for this study. There will be 24 patients planned per dose cohort (100, 200, 300 mg) with 18 ISIS 304801 e) and 6 placebo patients per cohort.
Eligible patients will be enrolled equally (1:1) into a non-extensive -prandial group (Group 1) or an extensive PIonst-prandial group (Group 2). Patients in Group 2 will be randomized equally (1:1) to an extensive PK group (Group 2a) or a post-prandial assessment group (Group 2b). Group 2a patients will be randomized equally (1 : 1 : 1) to 1 of the 3 dose cohorts (100, 200, 300 mg) and, within each dose cohort, 5:1 to receive active or placebo. Group 2b ts will be ized equally (1:1) to 1 of 2 dose cohorts (200, 300 mg) and, within each dose cohort, 2:1 to receive active or placebo. Group 1 patients will be randomized to dose cohort and treatment in a manner that achieves an overall study randomization of 1 : 1 :1 to dose cohort (100, 200, 300 mg) and 3:1 to treatment (active, placebo).
Patients will be placed on a tightly controlled diet (after screening procedures are performed) for the duration of study participation. After 28 days on the controlled diet, patients will have baseline measurements and be ed for qualification of enrollment into the treatment phase of the study. Patients who meet the enrollment criteria following diet run-in will be ed equally (1:1) into a non-extensive PIonst-prandial group (Group 1) or into an extensive PIonst-prandial group (Group 2) and ized within their Group assignment.
Study Drug and Treatment A solution of ISIS 304801 (200 mg/mL, 1.0 mL) contained in 2-mL stoppered glass vials will be provided.
The o for this study will be 0.9% sterile saline.
ISIS 304801 solution and placebo will be prepared by an unblinded pharmacist (or qualified delegate). Vials are for single-use only. A trained professional, d to the identity of the drug, will administer the Study Drug. The Study Drug will be administered as a SC injection in the abdomen, thigh, or outer area of the upper arm on each dosing day. Doses of 100 and 200 mg will be administered as a single SC injection. Doses of 300 mg will be administered as two equal volume noncontiguous SC injections.
Patients will e 13 doses of Study Drug stered 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 will complete the treatment visits on Day 1 :: 0 days and on Day 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 within :: 1 day. Patients in the extensive PK group will also visit the clinic on Day 2 and 86 :: 0 days relative to Day 1 and 85, respectively, for the 24 hour blood draw. Patients will complete the follow-up visits on Day 92 and 99 within :: 1 day, Day 127 within :: 3 days, and Day 176 within :: 5 days of the scheduled visit date. Patients in the post- prandial assessment group will also visit the clinic on Day 103 within :: 2 days and on the day following the Day 103 visit for the 24 hour blood draw.
Preceding each visit that includes a blood draw for pharmacodynamic measurements (Days 8, 15, 29, 43, 57, 71, and 85), patients will be provided a rdized pre-cooked meal for the dinner on the evening prior to their visit (to ensure equal moderation of fat intake, per patient and per time point) after which they will remain fasted. l consumption will not be allowed for 48 hrs preceding these clinic visits.
Blood will be collected for measurement of VLDL apoC-III and other codynamic markers on Days 8, 15, 29, 43, 57, 71, and 85 (prior to Study Drug stration).
Patients in the post-prandial assessment group will e 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 randial evaluation days, following the 2O blood draws, patients will consume a standardized liquid meal, which represents about a third of the daily caloric requirements, with a stable radioisotope tracer, followed by serial blood sampling. Patients will receive a standardized oked meal 9 hrs after consuming the liquid meal, after which they will fast until the 24 hour blood draw the following day.
In addition to trough sample collection, patients in the extensive PK assessment group will undergo serial blood sampling for 24 hrs after their first (Day 1-2) and last (Day 85-86) dose of Study Drug.
Post-treatment Evaluation Period Patients will be followed until Study Day 176. During this time, patients will return to the study center for outpatient clinic visits on Study Days 92, 99, 127, and 176 (and Day 103 for patients in the post-prandial assessment group) for safety and clinical laboratory evaluations (blood draws), diet counseling and monitoring, concomitant medication usage recording, and AE event collection.
Blood samples for PK and PD analysis will be collected periodically hout the post- treatment evaluation period. Laboratory measurements of serum chemistry, urinalysis, coagulation, complement, hematology, immune filnction, thyroid function, and filll lipid panel will be performed at the various times hout the study.
Post-prandial assessments will be done in a subset of ts as described below.
Post-prandial Meal, Sampling Schedule, andAssessment Post-prandial assessment for lipoproteins metabolism will be performed using a radiolabelled meal supplemented with a labeled tracer, 3H-palmitate (3 OOuCi, Perkin Elmer Inc., Woodbridge, ON, Canada),sonicated into the liquid meal. Palmitate is a fatty acid that is a common tuent 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: ).
A liquid meal (similar to a milkshake) containing a small amount (300uCi) of radiolabelled fatty acids lmitate) 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 (0.01umol/kg/min in 100 ml 25% human serum albumin; Cambridge Isotopes Laboratories Inc., Andover, MA) and a primed (1.6 g) continuous (0.05 g/min) infusion of [1,1,2,3,3-2H]—glycerol idge Isotopes Laboratories Inc.) will be administered as previously described (Normand-Lauziere et al. 2010, PLoS. One, 5: ). Plasma palmitate and ol appearance rates will be calculated using Steele’s non-steady state equation assuming a volume of distribution of 90ml/kg and 23 0ml/kg, tively (Gastaldelli et al. 1999, J Appl. Physiol, 87: 1813-1822).
Blood samples will be drawn at intervals before and after the ingestion of the radiolabelled meal on days prior to and after the Treatment phase as noted in the table below.
A standardized meal will be given to the participants after the 9 hr blood draw. Blood will be collected in tubes containing Na2 EDTA and Orlistat (30 ug/ml, Roche, Mississauga, Canada) to prevent in vitro triacylglycerol lipolysis and te samples will be collected in NaF tubes for plasma glucose determination.
The following will be ed at each time-point: 0 Plasma and CM fraction levels for 3H-tracer 0 Plasma [U-l3C]-K palmitate and [1, l, 2, 3, 3-2H]—glycerol appearance rates 0 Plasma and CM fraction levels for TG, TC, and apoB 0 Plasma and VLDL fraction levels for apo CIII, apo C11, and apo E 0 Plasma levels for e 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.

Claims (38)

1. Use of a compound targeting ApoCIII in the manufacture of a medicament for a) treating, preventing, delaying or ameliorating pancreatitis; b) increasing HDL levels and/or improving the ratio of TG to HDL in an animal y preventing, delaying or ameliorating a cardiovascular disease, disorder, condition or symptom thereof or onset of a cardiovascular disease, disorder, condition or symptom thereof in an animal; and/or c) increasing HDL levels and/or improving the ratio of TG to HDL in an animal thereby reducing the risk of a cardiovascular disease, disorder, condition or symptom in an animal; and/or d) treating, preventing, delaying or ameliorating icronemia, wherein the compound is capable of inhibiting apolipoprotein C-III.
2. The use of claim 1, wherein the animal has, or is at risk for, riglyceridemia.
3. The use of claim 2, wherein the animal has a triglyceride level between 100-200 mg/dL, 100-300 mg/dL, 100-400 mg/dL, 100-500 mg/dL, 200-500 mg/dL, 300-500 mg/dL, 400-500 mg/dL, 500-1000 mg/dL, 600-1000 mg/dL, 00 mg/dL, 800-1000 mg/dL, 900- 1000 mg/dL, 500-1500 mg/dL, 1000-1500 mg/dL, 100-2000 mg/dL, 150-2000 mg/dL, 200- 2000 mg/dL, 300-2000 mg/dL, 00 mg/dL, 500-2000 mg/dL, 600-2000 mg/dL, 700- 2000 mg/dL, 800-2000 mg/dL, 900-2000 mg/dL, 1000-2000 mg/dL, 1100-2000 mg/dL, 000 mg/dL, 1300-2000 mg/dL, 1400-2000 mg/dL, or 1500-2000 mg/dL.
4. The use of claim 2, wherein the hypertriglyceridemia is ckson Type II, IV or V.
5. The use of any preceding claim, wherein the animal has a genetic defect g to hypertriglyceridemia, wherein the genetic defect is a heterozygous LPL deficiency or an I polymorphism.
6. The use of any preceding claim, wherein the animal has a triglyceride level ≥ 500 mg/dL and/or a heterozygous LPL deficiency.
7. The use of any preceding claim, wherein ApoCIII has a nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.
8. The use of any preceding claim, wherein the compound ing ApoCIII comprises a modified oligonucleotide.
9. The use of claim 8, wherein the nucleobase sequence of the modified oligonucleotide is 80%, 90% or 100% complementary to a base sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
10. The use of claim 8 or claim 9, wherein the modified oligonucleotide consists of a single-stranded modified oligonucleotide.
11. The use of any one of claims 8-10, wherein the modified oligonucleotide consists of 12 to 30 linked nucleosides.
12. The use of claim 11, wherein the modified oligonucleotide ts of 20 linked nucleosides.
13. The use of any one of claims 8-12, wherein at least one internucleoside linkage of the modified ucleotide is a modified internucleoside linkage, wherein at least one nucleoside of the modified oligonucleotide comprises a modified sugar and/or wherein at least one nucleoside of the modified oligonucleotide comprises a modified nucleobase.
14. The use of claim 13, n each modified internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.
15. The use of claim 13, wherein at least one ed sugar is a bicyclic sugar.
16. The use of claim 13, n at least one modified sugar comprises a 2’-O- methyoxyethyl.
17. The use of claim 13, wherein the modified nucleobase is a 5-methylcytosine.
18. The use of claim 8, n the modified oligonucleotide comprises: (a) a gap segment ting of linked deoxynucleosides; (b) a 5’ wing segment consisting of linked nucleosides; (c) a 3’ wing segment ting 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.
19. The use of claim 8, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of 8-12 linked deoxynucleosides; (b) a 5’ wing segment consisting of 1-5 linked nucleosides; (c) a 3’ wing segment consisting 1-5 linked nucleosides; wherein the gap segment is positioned immediately adjacent to and n 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.
20. The use of claim 8, wherein the modified oligonucleotide comprises: (a) a gap segment consisting of 10 linked deoxynucleosides; (b) a 5’ wing segment consisting of 5 linked nucleosides; (c) a 3’ wing segment consisting 5 linked nucleosides; wherein the gap segment is positioned immediately adjacent to and between the 5’ wing segment and the 3’ wing segment and n each nucleoside of each wing t comprises a ethyoxyethyl sugar, wherein each cytosine is a hylcytosine, and wherein each internucleoside linkage is a phosphorothioate linkage.
21. The use of any preceding claim, wherein the animal has ms that may be any one of angina; chest pain; shortness of ; 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; ng of the abdomen; or fever.
22. Use of a modified oligonucleotide targeting ApoCIII, wherein the modified ucleotide comprises: (a) a gap segment consisting of 10 linked deoxynucleosides; (b) a 5’ wing segment consisting of 5 linked nucleosides; (c) a 3’ wing segment consisting 5 linked sides; 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; and n the modified oligonucleotide is capable of inhibiting the expression of apolipoprotein C-III; in the manufacture of a medicament for: (1) treating, preventing, delaying or ameliorating atitis (2) increasing HDL levels and/or improving the ratio of TG to HDL in an animal y preventing, delaying or ameliorating a cardiovascular disease, er, condition or symptom thereof or onset of a cardiovascular disease, disorder, ion or symptom thereof in an animal; (3) increasing HDL levels and/or improving the ratio of TG to HDL in an animal y reducing the risk of a cardiovascular disease, disorder, condition or symptom in an animal; and/or (4) treating, preventing, delaying or ameliorating chylomicronemia in an animal.
23. The use of any preceding claim, n the animal is human.
24. The use of any ing claim, wherein the cardiovascular disease is aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease, coronary heart e, hypertension, dyslipidemia, hyperlipidemia, hypertriglyceridemia or hypercholesterolemia.
25. The use of claim 24, wherein the dyslipidemia is chylomicronemia.
26. The use of any preceding claim, wherein the animal is at risk for pancreatitis.
27. The use of any preceding claim, wherein reducing ApoCIII levels a) prevents, treats or ameliorates pancreatitis; b) enhances clearance of postprandial cerides; and/or c) lowers andial triglycerides.
28. The use of any preceding claim, wherein the medicament is formulated for parenteral administration.
29. The use of claim 28, wherein the parenteral administration is subcutaneous administration.
30. The use of any preceding claim, wherein the medicament is formulated for use in combination with a second agent.
31. The use of claim 30, wherein the second agent is selected from an ApoCIII lowering agent, cholesterol lowering agent, non-HDL lipid lowering agent, LDL lowering agent, TG ng agent, cholesterol lowering agent, HDL g agent, fish oil, niacin, fibrate, statin, DCCR (salt of diazoxide), glucose-lowering agent or anti-diabetic agents.
32. The use of claim 30, wherein the animal fails to respond to a maximally tolerated dose of the second agent.
33. The use of claim 30, wherein the second agent is formulated for stration concomitantly or sequentially with the compound.
34. The use of any preceding claim, wherein the compound is a salt form.
35. The use of any preceding claim, wherein the medicament is formulated with a pharmaceutically acceptable carrier or diluent.
36. The use of any ing claim, wherein the compound is a conjugated compound.
37. The compound of claim 36, wherein the compound is conjugated with a carbohydrate conjugate group.
38. Use of a compound or use of a modified oligonucleotide according to any one of the preceding claims, substantially as herein sed with reference to any one or more of the examples, excluding comparative examples.
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