WO2010090830A1 - Modulation de l'expression de sirt1 - Google Patents

Modulation de l'expression de sirt1 Download PDF

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Publication number
WO2010090830A1
WO2010090830A1 PCT/US2010/021478 US2010021478W WO2010090830A1 WO 2010090830 A1 WO2010090830 A1 WO 2010090830A1 US 2010021478 W US2010021478 W US 2010021478W WO 2010090830 A1 WO2010090830 A1 WO 2010090830A1
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sirtl
animal
antisense
metabolic
glucose
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PCT/US2010/021478
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English (en)
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Sanjay Bhanot
Gerald Shulman
Xing-Xian Yu
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Isis Pharmaceuticals, Inc.
Yale University
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Publication of WO2010090830A1 publication Critical patent/WO2010090830A1/fr

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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4453—Non condensed piperidines, e.g. piperocaine only substituted in position 1, e.g. propipocaine, diperodon
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    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01098—Histone deacetylase (3.5.1.98), i.e. sirtuin deacetylase
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    • C12N2310/321—2'-O-R Modification
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Definitions

  • the present invention provides compounds and methods for decreasing Sirtuin 1 (SIRTl) expression and for the treatment of metabolic and cardiovascular diseases and other related disorders.
  • SIRTl Sirtuin 1
  • Obesity is a chronic condition that is characterized by a body mass index (BMI) over 25. Both congenital and environmental factors, such as exercise and eating habits, contribute to the disease. For instance, the hormone leptin has been shown to be involved in fat accumulation and regulating eating behavior. Several animal models of obesity result from mutations in the leptin and/or leptin receptor gene. In addition to affecting the lifestyle of an individual, obesity can lead to a number of complications and diseases, including insulin resistance, Type II diabetes, gallbladder disease, hypertension, cardiovascular disease, hyperlipidemia, sleep apnea, coronary artery disease, knee osteoarthritis, gout, infertility, breast cancer, endometrial cancer, colon cancer and lower back pain.
  • BMI body mass index
  • Diabetes affects over 18.2 million people in the United States, representing over 6% of the population. Diabetes is characterized by the inability to produce or properly use insulin. Both congenital and environmental factors, such as exercise and eating habits, contribute to the disease. The pathogenic causes of diabetes are insulin productive disorders, secretion disorders or reductions in activities and sensitivities of the secreted insulin. Diabetes is largely grouped into the following two types: insulin-dependent diabetes mellitus (also known as Type I diabetes) and non-insulin- dependent diabetes mellitus (also known as Type II diabetes). Insulin resistance in Type II diabetes prevents maintenance of blood glucose within desirable ranges, despite normal to elevated plasma levels of insulin. The incidence of Type II diabetes is remarkably increased in obese patients.
  • Diabetes and obesity are interrelated in that obesity is known to exacerbate the pathology of diabetes and greater than 60% of diabetics are obese. Most human obesity is associated with insulin resistance and leptin resistance. In fact, it has been suggested that obesity may have an even greater impact on insulin action than diabetes itself (Sindelka et al., Physiol Res., 2002, 51, 85-91). Additionally, several compounds on the market for the treatment of diabetes are known to induce weight gain, a very undesirable side effect to the treatment of this disease.
  • Cardiovascular disease is also interrelated to obesity and diabetes. Cardiovascular disease encompasses a wide variety of etiologies and has an equally wide variety of causative agents and interrelated players. Many causative agents contribute to symptoms such as elevated plasma levels of cholesterol, including non-HDL cholesterol, as well as other lipid-related disorders. Such lipid- related disorders are generally referred to as dyslipidemia; include hypercholesterolemia and hypertriglyceridemia among other indications.
  • Non-HDL cholesterol is firmly associated with atherogenesis and its sequalea, including cardiovascular diseases such as arteriosclerosis, coronary artery disease, myocardial infarction, ischemic stroke, and other forms of heart disease. These rank as the most prevalent types of illnesses in industrialized countries. Indeed, an estimated 12 million people in the United States suffer with coronary artery disease and about 36 million require treatment for elevated cholesterol levels.
  • Metabolic syndrome is a combination of medical disorders that increase one's risk for cardiovascular disease.
  • the risk factors include obesity, diabetes, hypertension, and dyslipidemia (Grundy, S.M., J. Clin. Endocrinol. Metab., 2004, 89 (6), 2595-600). In some studies, the prevalence in the USA is calculated as being up to 25% of the population.
  • Metabolic syndrome is known under various other names, such as (metabolic) syndrome X, insulin resistance syndrome, Reaven's syndrome or CHAOS.
  • Sirtuin 1 SIRTl
  • a reduction of Sirtuin 1 (SIRTl) expression may prove to be a useful method for treating a wide range of metabolic and cardiovascular conditions, including but not limited to the interrelated conditions of diabetes, obesity, metabolic syndrome, dyslipidemia and their associated etiology and sequalea as provided herein.
  • SIRTl specific inhibitors may, for example, inhibit nucleic acid encoding SIRTl or processing thereof.
  • SIRTl protein expression or activity is inhibited.
  • SIRTl specific inhibitors are nucleic acids, proteins, or small molecules.
  • inhibition can occur in a cell or tissue.
  • the cell or tissue is in an animal.
  • the animal is a human.
  • SIRTl mRNA levels are reduced.
  • SIRTl protein levels are reduced. Such reduction can occur in a time-dependent manner or in a dose- dependent manner.
  • Such metabolic and/or cardiovascular diseases include, but not limited to atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, insulin resistance, and hypercholesterolemia (including polygenic hypercholesterolemia).
  • atherosclerosis includes atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyper
  • Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common.
  • Certain risk factors and causes for the development of a metabolic and/or cardiovascular diseases include, but are not limited to, age; weight; genetic predisposition, and diet.
  • Certain other risk factors and causes for development of a metabolic and/or cardiovascular diseases include, but are not limited to, those associated with elevated glucose levels, reduced insulin sensitivity, adipose tissue accumulation, lipid dysregulation, fat dysregulation, adipocyte dysregulation, and glucose dysregulation.
  • methods of treating multiple such diseases, disorders, and conditions Such multiple such diseases, disorders, and conditions can include any of the disease and disorders provided herein.
  • the multiple diseases and disorder can have one or more risk factors, causes or outcomes in common.
  • methods of treatment include administering a SIRTl specific inhibitor to an individual in need thereof.
  • SIRTl ASO is effective and decreases basal glucose.
  • Body weight of pair fed rats (A).
  • SIRTl ASO decreased liver mRNA (B) and immunoblots of isolated SIRTl protein (C).
  • SIRTl ASO decreased WAT mRNA (D), PPAR ⁇ (E), and PPAR ⁇ 2 (F).
  • Fasting basal glucose values were decreased (G), and basal insulin (H) and glucagon (I) remained unchanged.
  • FIG. 3 Peripheral and hepatic insulin responsiveness was assed using the hyperinsulemic- euglycemic clamp. Basal endogenous glucose production was decreased with SIRTl ASO (A). Overall glucose infusion rate was increased in SIRTl ASO (B), and that can be attributed to the decrease in clamp hepatic glucose production (C). Insulin stimulated whole body glucose metabolism (D), and glucose uptake in the soleus (E) and white adipose tissue was similar between groups. * P ⁇ 0.05
  • 2'-O-methoxyethyl refers to an O-methoxy-ethyl modification of the 2' position of a furosyl ring.
  • a 2'-O-methoxyethyl modified sugar is a modified sugar.
  • 2'-O-methoxyethyl nucleotide means a nucleotide comprising a 2'-O-methoxyethyl modified sugar moiety.
  • 5-methylcytosine means a cytosine modified with a methyl group attached to the 5' position.
  • a 5-methylcytosine is a modified nucleobase.
  • “Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits.
  • Active pharmaceutical ingredient means the substance or substances in a pharmaceutical composition that provides a desired effect.
  • Active target region means a target region to which one or more active antisense compounds is targeted.
  • Active antisense compounds means antisense compounds that reduce target nucleic acid levels.
  • Adipogenesis means the development of fat cells from preadipocytes.
  • Lipogenesis means the production or formation of fat, either fatty degeneration or fatty infiltration.
  • Body fat distribution can be estimated by skin-fold measures, waist-to-hip circumference ratios, or techniques such as ultrasound, computed tomography, or magnetic resonance imaging. According to the Center for Disease Control and Prevention, individuals with a body mass index (BMI) of 30 or more are considered obese.
  • BMI body mass index
  • obesity includes, but is not limited to, the following conditions: adult-onset obesity; alimentary obesity; endogenous or metabolic obesity; endocrine obesity; familial obesity; hyperinsulinar obesity; hyperplastic-hypertrophic obesity; hypogonadal obesity; hypothyroid obesity; lifelong obesity; morbid obesity and exogenous obesity.
  • administering 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.
  • administering means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.
  • “Amelioration” refers to a lessening of at least one indicator of the severity of a condition or disease.
  • the severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.
  • “Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
  • Antisense compound means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • Antisense inhibition means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
  • Antisense oligonucleotide means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
  • Atherosclerosis means a hardening of the arteries affecting large and medium-sized arteries and is characterized by the presence of fatty deposits.
  • the fatty deposits are called “atheromas” or “plaques,” which consist mainly of cholesterol and other fats, calcium and scar tissue, and damage the lining of arteries.
  • Bicyclic sugar means a furosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar.
  • Body fat content refers to the total amount of an animal's adipose tissue.
  • Body weight refers to an animal's whole body weight, inclusive of all tissues including adipose tissue.
  • 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.
  • cardiovascular diseases include, but are not limited to, aneurysm, angina, arrhythmia, atherosclerosis, cerebrovascular disease (stroke), coronary heart disease, and hypertension.
  • Chimeric antisense compounds means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.
  • Cholesterol is a sterol molecule found in the cell membranes of all animal tissues. Cholesterol must be transported in an animal's blood plasma by lipoproteins including very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and high density lipoprotein (HDL).
  • VLDL very low density lipoprotein
  • IDL intermediate density lipoprotein
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • 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 pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses administration in parallel or sequentially.
  • “Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. “Comply” means the adherence with a recommended therapy by an individual.
  • Contiguous nucleobases means nucleobases immediately adjacent to each other.
  • Deoxyribonucleotide means a nucleotide having a hydrogen at the 2' position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.
  • 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 glucose in the urine (glycosuria), excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.
  • Type 2 diabetes (also known as “type 2 diabetes mellitus” or “diabetes mellitus, type 2”, and formerly called “diabetes mellitus type II” , “non-insulin-dependent diabetes (NIDDM)", “obesity related diabetes”, or “adult-onset diabetes”) is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia.
  • NIDDM non-insulin-dependent diabetes
  • Diabetic dyslipidemia or "Type II diabetes with dyslipidemia” means a condition characterized by Type II diabetes, reduced HDL, elevated serum triglycerides, and elevated small, dense LDL particles.
  • “Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable.
  • the diluent may be a liquid, e.g. saline solution.
  • Dose means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period.
  • a dose may be administered in two or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection.
  • two or more injections may be used to achieve the desired dose.
  • a dose may be administered in two or more injections to minimize injection site reaction in an individual.
  • the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week or month.
  • Dosage unit means a form in which a pharmaceutical agent is provided, e.g. pill, tablet, or other dosage unit known in the art.
  • a dosage unit is a vial containing lyophilized antisense oligonucleotide.
  • a dosage unit is a vial containing reconstituted antisense oligonucleotide.
  • Dyslipidemia refers to a disorder of lipid and/or lipoprotein metabolism, including lipid and/or lipoprotein overproduction or deficiency. Dyslipidemia may be manifested by elevation of lipids such as cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.
  • LDL low-density lipoprotein
  • “Elevated total cholesterol” means total cholesterol at a concentration in an individual at which lipid-lowering therapy is recommended, and includes, without limitation, “elevated LDL-C", “elevated VLDL-C,” “elevated IDL-C” and “elevated non-HDL-C.” In certain embodiments, total cholesterol concentrations of less than 200 mg/dL, 200-239 mg/dL, and greater than 240 mg/dL are considered desirable, borderline high, and high, respectively.
  • LDL-C concentrations of 100 mg/dL, 100-129 mg/dL, 130-159 mg/dL, 160-189 mg/dL, and greater than 190 mg/dL are considered optimal, near optimal/above optimal, borderline high, high, and very high, respectively.
  • “Elevated lipoprotein” means a concentration of lipoprotein in a subject at which lipid- lowering therapy is recommended.
  • “Elevated triglyceride” means a concentration of triglyceride in the serum or liver at which lipid-lowering therapy is recommended, and includes “elevated triglyceride” and “elevated liver triglyceride.” In certain embodiments, triglyceride concentration of 150-199 mg/dL, 200-499 mg/dL, and greater than or equal to 500 mg/dL is considered borderline high, high, and very high, respectively.
  • “Fully complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid.
  • a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
  • an antisense oligonucleotide is a first nucleic acid and a target nucleic acid is a second nucleic acid.
  • “Gapmer” means an antisense compound in which an internal position having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having one or more nucleotides that are chemically distinct from the nucleosides of the internal region.
  • a "gap segment” means the plurality of nucleotides that make up the internal region of a gapmer.
  • a “wing segment” means the external region of a gapmer.
  • Gap- widened means an antisense compound has a gap segment of 12 or more contiguous 2'-deoxyribonucleotides positioned between and immediately adjacent to 5' and 3' wing segments having from one to six nucleotides having modified sugar moieties.
  • Glucose is a monosaccharide used by cells as a source of energy and metabolic intermediate.
  • Plasma glucose refers to glucose present in the plasma or serum.
  • Gluconeogenesis refers to the synthesis of glucose in the body of all mammals from non- carbohydrate precursors, such as pyruvate, amino acids and glycerol. It takes place in the liver and serves to maintain blood glucose under conditions of starvation and intense exercise.
  • High density lipoprotein-C means cholesterol associated with high density lipoprotein particles. Concentration of HDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L.
  • serum HDL-C and “plasma HDL-C” mean HDL-C in the serum and plasma, respectively.
  • Hybridization means the annealing of complementary nucleic acid molecules.
  • complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target.
  • complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.
  • “Hypercholesterolemia” means a condition characterized by elevated cholesterol or circulating(plasma) cholesterol, LDL-cholesterol and VLDL-cholesterol, as per the guidelines of the Expert Panel Report of the National Cholesterol Educational Program (NCEP) of Detection, Evaluation of Treatment of high cholesterol in adults (see, Arch. Int. Med. (1988) 148, 36-39).
  • NCEP National Cholesterol Educational Program
  • “Hyperglycemia” means a condition characterized by elevated serum glucose levels and/or circulating (plasma) glucose levels.
  • “Hypercholesterolemia” means a condition characterized by elevated serum cholesterol levels and/or circulating (plasma) cholesterol levels.
  • “Hyperlipidemia” or “hyperlipemia” is a condition characterized by elevated serum lipids or circulating (plasma) lipids. This condition manifests an abnormally high concentration of fats.
  • the lipid fractions in the circulating blood are cholesterol, low density lipoproteins, very low density lipoproteins and triglycerides.
  • “Hypertriglyceridemia” means a condition characterized by elevated triglyceride levels. "Identifying an animal having a metabolic or cardiovascular disease” means identifying an animal having been diagnosed with a metabolic disease, a cardiovascular disease, or a metabolic syndrome; and/or, identifying an animal having any symptom of a metabolic disease, cardiovascular disease, or metabolic syndrome including, but not limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension increased insulin resistance, decreased insulin sensitivity, above normal body weight, and/or above normal body fat content.
  • Such identification may be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring serum or circulating (plasma) cholesterol, measuring serum or circulating (plasma) blood-glucose, measuring serum or circulating (plasma) triglycerides, measuring blood-pressure, calculating BMI, measuring body fat content, measuring body weight, determining body fat distribution, and/or measuring waist circumference and the like.
  • "Identifying a diabetic animal” means identifying an animal having been identified as diabetic or identifying an animal having any symptom of diabetes (type 1 or type 2) such as, but not limited to, having a fasting glucose of at least 110 mg/dL, glycosuria, polyuria, polydipsia, increased insulin resistance, and/or decreased insulin sensitivity.
  • Such identification may be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring fasting serum or circulating (plasma) blood-glucose, and the like.
  • Identifying an obese animal means identifying an animal having been diagnosed as obese or identifying an animal with a BMI over 30 and/or a waist circumference of greater than 102 cm in men or greater than 88 cm in women. Such identification may be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as calculating BMI, measuring body fat content, measuring body weight, determining body fat distribution, and/or measuring waist circumference and the like.
  • Improved cardiovascular outcome means a reduction in the occurrence of adverse cardiovascular events, or the risk thereof.
  • adverse cardiovascular events include, without limitation, death, reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiac arrest, and atrial dysrhythmia.
  • “Individual” means a human or non-human animal selected for treatment or therapy.
  • “Individual compliance” means adherence to a recommended or prescribed therapy by an individual.
  • SIRTl inhibiting the expression or activity of SIRTl refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of the SIRTl expression or activity.
  • injection site reaction means inflammation or abnormal redness of skin at a site of injection in an individual.
  • Insulin resistance means the condition in which normal amounts of insulin are in adequate to produce a normal insulin response from fat, muscle, and liver cells. Insulin resistance in fat cells results in hydrolysis of stored triglycerides, which elevates free fatty acids in the blood plasma. Insulin resistance in muscle reduced glucose update whereas insulin resistance in liver reduced 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 insulin sensitivity effectively processes glucose whereas an individual with low insulin sensitivity does not effectively process glucose.
  • Internucleoside linkage refers to the chemical 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 serum lipids in a subject over time.
  • Lipid-lowering agent means an agent; for example, a CREB inhibitor; provided to a subject to achieve a lowering of lipids in the subject.
  • a lipid- lowering agent is provided to a subject to reduce one or more of ApoB, LDL-C, cholesterol, and triglycerides.
  • Lipid-lowering therapy means a therapeutic regimen provided to a subject to reduce one or more lipids in a subject.
  • a lipid-lowering therapy is provided to reduce one or more of ApoB, cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C, triglycerides, small dense LDL particles, and Lp(a) in a subject.
  • Lpoprotein such as VLDL, LDL and HDL, refers to a group of proteins found in the serum, plasma and lymph and are important for lipid transport. The chemical composition of each lipoprotein differs in that the HDL has a higher proportion of protein versus lipid, whereas the VLDL has a lower proportion of protein versus lipid.
  • LDL-C Low density lipoprotein-cholesterol
  • Low HDL-C means a concentration of HDL-C in a subject at which lipid-lowering therapy is recommended. In certain embodiments, lipid-lowering therapy is recommended when low HDL-C is accompanied by elevations in non-HDL-C and/or elevations in triglyceride. In certain embodiments, HDL-C concentrations of less than 40 mg/dL are considered low. In certain embodiments, HDL-C concentrations of less than 50 mg/dL are considered low.
  • Metabolic disease refers to a condition characterized by an alteration or disturbance in metabolic function.
  • Metabolic and metabolic disorders are terms well known in the art and generally include the whole range of biochemical processes that occur within a living organism. Metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (Type I and Type II), obesity, insulin resistance, and metabolic syndrome.
  • Metabolic syndrome means a condition characterized by a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin.
  • 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 less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).
  • mismatch or non-complementary nucleobase means a nucleobase of first nucleic acid that is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
  • Mated dyslipidemia means a condition characterized by elevated serum cholesterol and elevated serum triglycerides.
  • Intravenous administration means administration into a vein.
  • Modified internucleoside linkage refers to a substitution and/or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
  • Modified nucleobase means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil.
  • An "unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleotide means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.
  • a “modified nucleoside” means a nucleotide having, independently, a modified sugar moiety or modified nucleobase.
  • Modified oligonucleotide means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
  • Modified sugar refers to a substitution and/or any change from a natural sugar. "Motif means the pattern of unmodified and modified nucleosides in an antisense compound.
  • Naturally occurring internucleoside linkage means a 3' to 5' phosphodiester linkage.
  • Natural sugar means a sugar found in DNA (2'-H) or RNA (2'-OH).
  • Non-high density lipoprotein-cholesterol (Non-HDL-C) means cholesterol associated with lipoproteins other than high density lipoproteins, and includes, without limitation, LDL-C, VLDL-C, and IDL-C.
  • Nucleic acid refers to molecules composed of monomelic nucleotides.
  • a nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single- stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
  • RNA ribonucleic acids
  • DNA deoxyribonucleic acids
  • siRNA small interfering ribonucleic acids
  • miRNA microRNAs
  • Nucleobase means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
  • Nucleobase sequence means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.
  • Nucleoside means a nucleobase linked to a sugar.
  • Nucleotide means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
  • nucleoside mimetic is intended to include those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino , cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units.
  • Oleity means an excessively high amount of body fat or adipose tissue in relation to lean body mass. The amount of body fat (or adiposity) includes concern for both the distribution of fat throughout the body and the size of the adipose tissue deposits.
  • Body fat distribution can be estimated by skin-fold measures, waist-to-hip circumference ratios, or techniques such as ultrasound, computed tomography, or magnetic resonance imaging. According to the Center for Disease Control and Prevention, individuals with a body mass index (BMI) of 30 or more are considered obese.
  • BMI body mass index
  • “Over-weight” refers to a condition in which a subject has a body mass index of greater or equal to 25.0. The body mass index and other definitions are according to the "NIH Clinical Guidelines on the Identification and Evaluation, and Treatment of Overweight and Obesity in Adults” (1998).
  • “Oligomeric compound” means a polymer of linked monomelic subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
  • Oligonucleoside means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.
  • Oligonucleotide means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
  • Parenteral administration means administration through injection or infusion.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
  • Peptide means a molecule formed by linking at least two amino acids by amide bonds.
  • peptide refers to polypeptides and proteins.
  • “Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual.
  • an antisense oligonucleotide targeted to SIRTl is pharmaceutical agent.
  • “Pharmaceutically acceptable carrier” means a medium or diluent that does not interfere with the structure of the oligonucleotide. Certain, of such carries enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject.
  • “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
  • “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual.
  • a pharmaceutical composition may comprise one or more antisense oligonucleotides and a sterile aqueous solution.
  • Phosphorothioate internucleoside linkage or "phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non- bridging oxygen atoms with a sulfur atom.
  • a phosphorothioate linkage is a modified internucleoside linkage.
  • “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.
  • “Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • Side effects means physiological responses attributable to a treatment other than desired effects.
  • side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies.
  • increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality.
  • increased bilirubin may indicate liver toxicity or liver function abnormality.
  • Single-stranded oligonucleotide means an oligonucleotide which is not hybridized to a complementary strand.
  • SIRTl nucleic acid means any nucleic acid encoding SIRTl.
  • a SIRTl nucleic acid includes, without limitation, a DNA sequence encoding SIRTl, an RNA sequence transcribed from DNA encoding SIRTl, and an mRNA sequence encoding SIRTl.
  • SIRTl mRNA means an mRNA or pre-mRNA encoding a SIRTl protein.
  • SIRTl specific inhibitor or “SIRTl inhibitor (s)” refers to any agent capable of specifically inhibiting the expression or activity of SIRTl at the molecular level.
  • SIRTl specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression or activity of SIRTl.
  • SIRTl specific inhibitors may affect other components of the phosphorylation signaling cascade including downstream components.
  • SIRTl specific inhibitors may affect other molecular processes in an animal.
  • SIRTl inhibiting sirtuin (SIRTl) protein
  • the level of protein may be reduced and/or one or more of its biological activities. The level or activities may be reduced, for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • Subcutaneous administration means administration just below the skin.
  • sugar surrogate overlaps with the slightly broader term “nucleoside mimetic” but is intended to indicate replacement of the sugar unit (furanose ring) only.
  • the tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system.
  • “Targeted” or “targeted to” means having a nucleobase sequence that will allow specific hybridization of an antisense compound to a target nucleic acid to induce a desired effect.
  • a desired effect is reduction of a target nucleic acid.
  • a desired effect is reduction of SIRTl mRNA.
  • Targeting means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
  • Target nucleic acid means a nucleic acid capable of being targeted by antisense compounds.
  • Target region means a portion of a target nucleic acid to which one or more antisense compounds is targeted.
  • target segment refers to a smaller portion or sub-portion of a region within a target nucleic acid.
  • a target segment can be the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted.
  • “Therapeutically effective amount” or “effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
  • Effective amount in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of active ingredient to a subject in need of such inhibition, treatment or prophylaxis, either in a single dose or as part of a series, that is effective for inhibition of that effect, or for treatment or prophylaxis or improvement of that condition.
  • the effective amount will vary depending upon the health and physical condition of the subject to be treated, the taxonomic group of subjects to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.
  • Therapeutic lifestyle change means dietary and lifestyle changes intended to lower cholesterol and reduce the risk of developing heart disease, and includes recommendations for dietary intake of total daily calories, total fat, saturated fat, polyunsaturated fat, monounsaturated fat, carbohydrate, protein, cholesterol, insoluble fiber, as well as recommendations for physical activity.
  • Total cholesterol means all types of cholesterol, including, but not limited to, LDL-C, HDL-C, IDL-C and VLDL-C. Concentration of total cholesterol in serum (or plasma) is typically quantified in mg/dL or nmol/L.
  • Trophobic lipids means lipids that are the triesters of glycerol.
  • Sporum triglycerides means triglycerides present in serum.
  • Liver triglycerides mean triglycerides present in liver tissue.
  • Unmodified nucleotide means a nucleotide composed of naturally occuring nucleobases, sugar moieties and internucleoside linkages.
  • an unmodified nucleotide is an RNA nucleotide (i.e., ⁇ -D-ribonucleosides) or a DNA nucleotide (i.e., ⁇ -D-deoxyribonucleoside).
  • VLDL-C Very low density lipoprotein-cholesterol
  • Concentration of VLDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L.
  • Serum VLDL-C and “plasma VLDL-C” mean VLDL-C in the serum or plasma, respectively.
  • White Adipose Tissue or WAT refers to one of the two types of adipose tissue found in mammals. In humans, white adipose tissue composes as much as 20% of the body weight in men and 25% of the body weight in women. The white adipose tissue is used as a store of energy and also acts as a thermal insulator, helping to maintain body temperature.
  • SIRTl has been developed as an antisense target.
  • the present invention relates generally to treatment of diseases associated with metabolic and cardiovascular disorders.
  • treatment with SIRTl inhibitors reduces adipocytes, plasma glucose, cholesterol and triglycerides in vivo. This finding is bolstered by a concomitant reduction in fatty acid synthesis, increased fatty acid oxidation and changes in gene expression related to cholesterol metabolism.
  • white adipose tissue (WAT) weight was reduced by treatment with a SIRTl inhibitor. It is therefore an objective herein to treat dyslipidemia and obesity.
  • lipid dysregulation and obesity are significant factors associated with metabolic and/or cardiovascular disease.
  • SIRTl inhibitor Activation of SIRTl has been shown previously to promote fat mobilization in white adipocytes .
  • Sirtl protein bind to and represses genes controlled by the fat regulator PPAR-gamma (peroxisome proliferator-activated receptor-gamma), including genes mediating fat storage.
  • PPAR-gamma peroxisome proliferator-activated receptor-gamma
  • SIRTl Repression of PPAR-gamma by SIRTl is also evident in 3T3-L1 adipocytes, where overexpression of SIRTl attenuates adipogenesis, and RNA interference of SIRTl enhances it.
  • upregulation of SIRTl triggers lipolysis and loss of fat.
  • provided herein are methods of inhibiting SIRTl resulting in the reduction of adipogenesis or fat and further reducing lipolysis and improving insulin sensitivity. Therefore provided herein are methods for the treatment of metabolic and cardiovascular diseases, for example, but not limited to diabetes, obesity, and dyslipidemia.
  • treatment with a SIRTl inhibitor also reduces plasma glucose levels and increases insulin sensitivity. This finding is confirmed by a concomitant reduction in gluconeogenesis as further indicated by expression levels of key gluconeogenic genes.
  • SIRTl inhibitor Another significant finding provided herein is improved hepatic insulin sensitivity achieved by administering a SIRTl inhibitor.
  • antisense oligonucleotide reduction of SIRTl expression significantly improves hepatic insulin responsiveness.
  • Reduction of SIRTl mRNA and/or SIRTl protein by a SIRTl inhibitor, particularly an antisense oligonucleotide results in the benefit of improved hepatic insulin sensitivity.
  • Reduction of SIRTl mRNA and/or SIRTl protein also can result in a reduction of hepatic lipids.
  • hepatic insulin resistance or disease characterized by hepatic insulin resistance and/or hepatic lipid content such as NAFLD and NASH.
  • NAFLD associated hepatic insulin resistance is a major factor contributing to hyperglycemia in Type 2 diabetes; it is a specific objective herein to treat type 2 diabetes and/or type 2 diabetes with dyslipidemia with an antisense oligonucleotide.
  • SIRTl inhibition with an antisense oligonucleotide reduced SIRTl mRNA and/or SIRTl protein levels specifically in both adipose tissue and liver tissue.
  • antisense oligonucleotide inhibitors of SIRTl are useful agents for the treatment of disorders characterized by SIRTl expression in adipose (such as adipogenesis and obesity) and liver tissues (such as hepatic steatosis, NAFLD and NASH).
  • the added benefit of using antisense oligonucleotide inhibitors of SIRTl includes the ability to target both adipose and liver tissues simultaneously, both of which play key roles in metabolic disorders like obesity and diabetes.
  • the present invention also provides herein, methods of modulating the levels of SIRTl in a cell, or tissue by contacting the cell or tissue with a SIRTl inhibitor.
  • the levels can include but are not limited to SIRTl mRNA levels and SIRTl protein levels.
  • the cell or tissue is in an animal.
  • the animal is a human.
  • SIRTl levels are reduced. Such reduction can occur in a time-dependent manner or in a dose- dependent manner or both.
  • the disease or disorder is a cardiovascular and/or metabolic disease or disorder.
  • the disease or disorder is characterized by dyslipidemia, more specifically hyperlipidemia, even more specifically hypercholesterolemia and/or hypertriglyceridemia.
  • the disease or disorder is atherosclerosis.
  • the disease or disorder is diabetes, more specifically Type 2 diabetes.
  • the disease or disorder is obesity.
  • the SIRTl inhibitor reduces lipid accumulation or lipid levels.
  • the lipid levels can be cholesterol levels or triglyceride levels or both.
  • such inhibitor is useful for treating dyslipidemia or conditions characterized by dyslipidemia such as cardiovascular diseases, such as atherosclerosis and coronary heart disease, obesity, lipoma, nonalcoholic fatty liver disease (NAFLD), hyperfattyacidemia.
  • the reduction in lipid levels can be in combination with a reduction in glucose levels and/or insulin resistance.
  • such inhibitor is useful for treating conditions characterized by both dyslipidemia and glucose dysregulation such as metabolic disorder including diabetes and metabolic syndrome.
  • the SIRTl inhibitor reduces adiposity, lipogenesis, lipogenic genes, body weight and/or body fat.
  • such inhibitor is useful for treating obesity and/or obesity related diseases and disorders. Such reduction can be in combination with a reduction in glucose levels and/or insulin resistance.
  • such inhibitor is useful for treating metabolic syndrome and other disorders associated with diabesity.
  • the SIRTl inhibitor reduces lipid levels, adipocyte levels, and glucose levels.
  • such inhibitor is useful for treating any number of cardiovascular, metabolic and obesity related diseases and disorders as further provided herein.
  • kits for identifying a subject having dyslipidemia are provided herein.
  • identifying a subject having obesity or a condition of localized increase in adipogenesis are methods of identifying a subject having obesity or a condition of localized increase in adipogenesis and administering to the subject a SIRTl inhibitor.
  • methods of identifying a subject having or at risk of having a cardiovascular disorder are methods of identifying a subject having or at risk of having a cardiovascular disorder and administering to the subject a SIRTl inhibitor.
  • identifying a subject having a metabolic disease characterized by dyslipidemia or a change in fat accumulation are methods of identifying a subject having elevated cholesterol levels and administering to the subject a SIRTl inhibitor, thereby reducing cholesterol levels.
  • methods of identifying a subject having elevated triglyceride levels and administering to the subject a SIRTl inhibitor, thereby reducing triglyceride levels are methods of identifying a subject having elevated triglyceride levels and administering to the subject a SIRTl inhibitor, thereby reducing triglyceride levels.
  • hepatic insulin sensitivity in another embodiment provided herein, are methods of identifying a subject having reduced hepatic insulin sensitivity and administering to the subject a SIRTl inhibitor, thereby improving hepatic insulin sensitivity.
  • a subject having elevated lipid levels, increased fat accumulation, reduced hepatic insulin sensitivity or a combination thereof comprising selecting a subject having elevated lipid levels, increased fat accumulation, reduced hepatic insulin sensitivity or a combination thereof; and administering to the subject a SIRTl inhibitor.
  • the invention also provides methods of preventing or delaying the onset of or reducing the risk-factors for a cardiovascular-related or metabolic-related disease or disorder in an animal comprising administering a therapeutically or prophylactically effective amount of a SIRTl inhibitor, hi one aspect, the animal is a human.
  • the metabolic and cardiovascular disorders includes, but is not limited to atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, insulin resistance, and hypercholesterolemia (including polygenic hypercholesterolemia).
  • atherosclerosis includes atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyper
  • Methods of administration of SIRTl inhibitors of the invention to a subject are intravenously, subcutaneously, or orally. Administrations can be repeated once or multiple times.
  • the SIRTl inhibitor is a SIRTl inhibitor, for example an antisense compound targeted to inhibit SIRTl expression.
  • the SIRTl compound is selected from: an oligonucleotide, a antisense oligonucleotide, a ssRNA, a dsRNA, a ribozyme, a triple helix molecule or a siRNAs.
  • a SIRTl inhibitor can be co-administered with at least one additional therapy.
  • the SIRTl inhibitor and additional therapy are administered concomitantly.
  • the SIRTl inhibitor and additional therapy are administered hi a single formulation, hi some embodiments, the SIRTl inhibitor is administered in combination with a non inhibitor of SIRTl .
  • the SIRTl inhibitor is administered in combination with a cholesterol-lowering agent and/ or glucose-lowering agent and/or a lipid-lowering agent.
  • the present invention also provides a SIRTl inhibitor, as described herein for use in treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein.
  • the invention provides a SIRTl inhibitor as described herein for use in treating or preventing atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, insulin resistance, and hypercholesterolemia (including polygenic hypercholesterolemia).
  • NAFLD non-alcoholic fatty liver disease
  • the present invention also provides the use of a SIRTl inhibitor, as described herein in the manufacture of a medicament for treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein.
  • the invention provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for treating or preventing atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, insulin resistance, and hypercholesterolemia (including polygenic hypercholesterolemia).
  • NAFLD non-alcoholic fatty liver disease
  • the invention also provides a SIRTl inhibitor, as described herein for reducing serum lipid levels, e.g. for reducing serum lipid levels in a subject having elevated serum lipid levels.
  • the present invention also provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for reducing serum lipid levels, e.g. for reducing serum lipid levels in a subject having elevated serum lipid levels.
  • the invention also provides a SIRTl inhibitor, as described herein for reducing cholesterol levels, e.g. for reducing cholesterol levels in a subject having elevated cholesterol levels.
  • the present invention also provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for reducing cholesterol levels, e.g. for reducing cholesterol levels in a subject having elevated cholesterol levels.
  • the invention also provides a SIRTl inhibitor as described herein for reducing triglyceride levels, e.g. for reducing triglyceride levels in a subject having elevated triglyceride levels.
  • the present invention also provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for reducing triglyceride levels, e.g. for reducing triglyceride levels in a subject having elevated triglyceride levels.
  • the invention also provides a SIRTl inhibitor as described herein for improving hepatic insulin sensitivity, e.g. for improving hepatic insulin sensitivity in a subject having reduced hepatic insulin sensitivity.
  • the present invention also provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for improving hepatic insulin sensitivity, e.g. for improving hepatic insulin sensitivity in a subject having reduced hepatic insulin sensitivity.
  • the invention also provides a SIRTl inhibitor as described herein for reducing adipogenesis, e.g. for reducing adipogenesis in a subject having elevated adipocyte levels.
  • the present invention also provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for reducing adipogenesis, e.g. for reducing adipogenesis in a subject having elevated adipocyte levels.
  • the invention also provides a SIRTl inhibitor, as described herein for treating diabetes, e.g. for treating diabetes in a subject having type II diabetes with dyslipidemia.
  • the present invention also provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for treating diabetes, e.g. for treating diabetes in a subject having type II diabetes with dyslipidemia.
  • the invention also provides a SIRTl inhibitor, as described herein for treating metabolic syndrome, e.g. for treating metabolic syndrome in a subject having metabolic syndrome or one or more risk factors of metabolic syndrome.
  • the present invention also provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for treating metabolic syndrome, e.g. for treating metabolic syndrome in a subject having metabolic syndrome or one or more risk factors of metabolic syndrome.
  • the invention also provides a SIRTl inhibitor, as described herein for use in treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein by combination therapy with an additional therapy as described herein.
  • the invention also provides a pharmaceutical composition comprising a SIRTl inhibitor, as described herein in combination with an additional therapy as described herein.
  • the invention also provides the use of a SIRTl inhibitor as described herein in the manufacture of a medicament for treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein by combination therapy with an additional therapy as described herein.
  • the invention also provides the use of a SIRTl inhibitor, as described herein in the manufacture of a medicament for treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein in a patient who has previously been administered an additional therapy as described herein.
  • the invention also provides the use of a SIRTl inhibitor, as described herein in the manufacture of a medicament for treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein in a patient who is subsequently to be administered an additional therapy as described herein.
  • the invention also provides a kit for treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein, said kit comprising: (i) a SIRTl inhibitor as described herein; and (ii) an additional therapy as described herein.
  • the invention also provides a kit for treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein, said kit comprising: (i) a SIRTl inhibitor as described herein; and (ii) an additional therapy as described herein.
  • kit for treating or preventing a cardiovascular and/or metabolic disease or disorder as described herein said kit comprising: (i) a SIRTl modified oligonucleotide as described herein; and (ii) an additional therapy as described herein.
  • kits of the invention may further include instructions for using the kit to treat or prevent a cardiovascular and/or metabolic disease or disorder as described herein by combination therapy as described herein.
  • the SIRTl inhibitor is an antisense compound.
  • the antisense compound is a nucleic acid.
  • the nucleic acid is a modified oligonucleotide.
  • the modified oligonucleotide may be a single-stranded or double- stranded oligonucleotide.
  • the modified oligonucleotide may be 70, 75, 80, 85, 90, 95, or 100% complementary to a human SIRTl nucleic acid.
  • the modified oligonucleotide may have at least one modified internucleoside linkage.
  • the internucleoside linkage may be a phosphorothioate intemucleoside linkage.
  • the modified oligonucleotide may have at least one modified sugar.
  • the modified sugar may be a bicyclic sugar.
  • the modified sugar may comprise a 2'-O-methoxyethyl.
  • the modified oligonucleotide may comprise at least one nucleoside having a modified nucleobase.
  • the antisense compounds targeting SIRTl may have the nucleobase sequence of any of SEQ ID NOs: 3 to 80.
  • the method comprises identifying an animal having a metabolic or cardiovascular disease and administering to the animal having a metabolic or cardiovascular disease a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to human SIRTl.
  • the metabolic or cardiovascular disease is obesity, diabetes, or dyslipidemia, or a combination thereof.
  • the disease is dyslipidemia.
  • the disease the dyslipidemia is hyperlipidemia.
  • the hyperlipidemia is hypercholesterolemia, hypertriglyceridemia, or both hypercholesterolemia and hypertriglyceridemia.
  • the method results in a reduction of triglyceride levels.
  • the method results in a reduction of triglyceride levels of at least 20, 30, 35, or 40%.
  • the method results in a reduction of cholesterol levels.
  • the method results in a reduction of cholesterol levels by at least 10, 20, 30, 35 or 40%.
  • the method results in a reduction of glucose levels.
  • the method results in a reduction of glucose levels by at least 5 or 10%.
  • the method results in a reduction of body weight.
  • the method results in a reduction of body weight by at least 10 or
  • the method results in a reduction of body fat. In one such embodiment, the method results in a reduction of body fat by at least 10, 20, 30, or 40%.
  • the method results in a reduction of triglyceride levels, cholesterol levels, glucose levels, body weight, fat content, insulin resistance, or any combination thereof, wherein levels are independently reduced by 5%, 10%, or 15%.
  • the method comprises identifying an obese animal and administering to the obese animal a therapeutically effective amount of a SIRTl inhibitor.
  • the method comprises identifying a diabetic animal and administering to the diabetic animal a therapeutically effective amount of a SIRTl inhibitor. In one such embodiment, the method results in a reduction of glucose levels. In another such embodiment, the method results in a reduction of glucose level by at least 10 or 15%.
  • the method comprises identifying an animal having a metabolic and/or cardiovascular disease and administering to the animal having a metabolic and/or cardiovascular disease a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to human SIRTl having no more than 3 mismatched nucleobases.
  • the method comprises identifying an animal having a metabolic and/or cardiovascular disease and administering to the animal having a metabolic and/or cardiovascular disease a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 nucleobases in length targeted to at least an 8 nucleobase portion of nucleotides of SEQ ID NO: 2 encoding SIRTl, wherein said compound is at least 95% complementary to SEQ ID NO: 2.
  • administration of an antisense compound targeted an SIRTl nucleic acid is parenteral administration. Parenteral administration may be intravenous or subcutaneous administration. Accordingly, in another embodiment, administration of an antisense compound targeted to an SIRTl nucleic acid is intravenous or subcutaneous administration. Administration may include multiple doses of an antisense compound targeted to an SIRTl nucleic acid. In one embodiment, administration of an antisense compound targeted an SIRTl nucleic acid is oral administration. Certain Indications
  • the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions of the present invention.
  • the individual has a metabolic disorder or a cardiovascular disorders, or both a metabolic disorder and a cardiovascular disorder.
  • the disorder is atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, insulin resistance, and hypercholesterolemia (including polygenic hypercholesterolemia).
  • NAFLD non-alcoholic fatty liver disease
  • administration of a therapeutically effective amount of an antisense compound targeted to a SIRTl nucleic acid is accompanied by monitoring plasma glucose, plasma triglycerides, and plasma cholesterol levels in the serum of an individual, to determine an individual's response to administration of the antisense compound.
  • body weight is monitored. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
  • administration of an antisense compound targeted to a SIRTl nucleic acid results in reduction of SIRTl expression by at least 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.
  • administration of an antisense compound targeted to a SIRTl nucleic acid results in a change in plasma glucose, plasma triglycerides, plasma cholesterol, and/or body weight.
  • administration of a SIRTl antisense compound decreases plasma glucose, plasma triglycerides, plasma cholesterol, and/or body weight by at least 15, 20, 25, 30, 35, 40, 45, or 50%, or a range defined by any two of these values.
  • a pharmaceutical composition comprising an antisense compound targeted to SIRTl is used for the preparation of a medicament for treating a patient suffering or susceptible to a metabolic disorder.
  • a pharmaceutical composition comprising an antisense compound targeted to SIRTl is for use in therapy.
  • the therapy is the reduction of blood glucose, body fat content, or fat tissue weight in an individual.
  • the therapy is the treatment of atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, insulin resistance, and hypercholesterolemia (including polygenic hypercholesterolemia).
  • NAFLD non-alcoholic fatty liver disease
  • composition comprising an antisense compound targeted to SIRTl is used for the preparation of a medicament for reduction of blood glucose, blood glucose, body fat content, or fat tissue weight.
  • pharmaceutical composition comprising an antisense compound targeted to SIRTl is used for the preparation of a medicament for reducing body fat content and obesity.
  • an antisense compound targeted to SIRTl is used for the preparation of a medicament for the treatment of metabolic syndrome disorders.
  • an antisense compound targeted to SIRTl is used for the preparation of a medicament of atherosclerosis, coronary heart disease, hyperlipoprotenemia, obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, insulin resistance, and hypercholesterolemia (including polygenic hypercholesterolemia).
  • diabetes including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia
  • dyslipidemia including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia
  • NAFLD non-alcoholic fatty liver disease
  • hyperfattyacidemia including hepatic steatosis and steatohepatitis
  • metabolic syndrome including hepatic steatosis and steatohepatitis
  • Conditions associated with and included in metabolic disorders encompass, but are not limited to obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, and insulin resistance.
  • diabetes including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia
  • dyslipidemia including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia
  • NAFLD non-alcoholic fatty liver disease
  • hyperfattyacidemia including hepatic steatosis and steatohepatitis
  • metabolic syndrome including hyperglycemia, and insulin resistance.
  • Blood sugar regulation is the process by which the levels of blood sugar, primarily glucose, are maintained by the body. Blood sugar levels are regulated by negative feedback in order to keep the body in homeostasis. If the blood glucose level falls glucagon is released. Glucagon is a hormone whose effects on liver cells act to increase blood glucose levels. They convert glycogen storage into glucose, through a process is called glycogenosis. The glucose is released into the bloodstream, increasing blood sugar levels. When levels of blood sugar rise, whether as a result of glycogen conversion, or from digestion of a meal, insulin is released, and causes the liver to convert more glucose into glycogen (glycogenesis), and forces about 2/3 of body cells to take up glucose from the blood, thus decreasing blood sugar levels. Insulin also provides signals to several other body systems, and is the chief regulatory metabolic control in humans.
  • Diabetes mellitus type 2 is caused by insulin resistance which, if untreated, results in hyperglycemia.
  • Insulin resistance is 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 reduces the effects of insulin and results in elevated hydrolysis of stored triglycerides in the absence of measures which either increase insulin sensitivity or which provide additional insulin. Increased mobilization of stored lipids in these cells elevates free fatty acids in the blood plasma.
  • Insulin resistance in muscle cells reduces glucose uptake and causes local storage of glucose as glycogen, whereas insulin resistance in liver cells reduces storage of glycogen, making it unavailable for release into the blood when blood insulin levels fall. High plasma levels of insulin and glucose due to insulin resistance often lead to metabolic syndrome and type 2 diabetes, and other related complications ⁇
  • Nonalcoholic fatty liver disease is strongly associated with hepatic insulin resistance in patients with poorly controlled type 2 diabetes mellitus (T2DM) (Petersen, K.F., et al., 2005, Diabetes 54:603-608; Petersen, K.F., et al. 2002, JCUn Invest 109:1345-1350; Yki-Jarvinen, H., Helve, E., et al., 1989. Am J Physiol 256:E732- 739).
  • T2DM poorly controlled type 2 diabetes mellitus
  • Metabolic syndrome is the clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. It has been closely linked to the generalized metabolic disorder known as insulin resistance.
  • NCEP National Cholesterol Education Program
  • ATPIII Adult Treatment Panel III established criteria for diagnosis of metabolic syndrome when three or more of five risk determinants are present.
  • the five risk determinants are abdominal obesity defined as waist circumference of greater than 102 cm for men or greater than 88 cm for women, triglyceride levels greater than or equal to 150 mg/dL, HDL cholesterol levels of less than 40 mg/dL for men and less than 50 mg/dL for women, blood pressure greater than or equal to 130/85 mm Hg and fasting glucose levels greater than or equal to 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).
  • the World Health Organization definition of metabolic syndrome is diabetes, impaired fasting glucose, impaired glucose tolerance, or insulin resistance (assessed by clamp studies) and at least two of the following criteria: waist-to-hip ratio greater than 0.90 in men or greater than 0.85 in women, triglycerides greater than or equal to 1.7 mmol/1 or HDL cholesterol less than 0.9 mmol in men and less then 1.0 mmol in women, blood pressure greater than or equal to 140/90 mmHg, urinary albumin excretion rate greater than 20 .mu.g/min or albumin-to-creatinine ratio greater than or equal to 30 mg/g (Diabetes Care, 2005, 28(9): 2289-2304).
  • Another embodiment is a method of treating metabolic and cardiovascular disorder risk with SIRTl modulating agents targeted to SIRTl.
  • SIRTl modulating agents targeted to SIRTl.
  • the gluconeogenic enzymes, cytosolic phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial PEPCK and the transcriptional co-activator peroxisomal proliferator activated receptor gamma coactivator-1 -alpha (PGC-l ⁇ ) mRNA levels were decreased by 43%, 55% and 54% respectively in the liver of the SIRTl antisense oligonucleotide groups .
  • a subject preferable an animal, even more preferably a human, suspected of having a metabolic disorder which can be treated by modulating the expression of SIRTl.
  • a subject is treated by administering a SIRTl inhibitor, preferably a SIRTl inhibitor, for example a SIRTl inhibitor such as an antisense compounds targeting SIRTl .
  • SIRTl inhibitors include, but are not limited to proteins, peptides, polypeptides, antibodies, antisense compounds including oligonucleotides and antisense oligonucleotides, ssRNA, dsRNA molecules, ribozymes, triple helix molecules, siRNAs, and small molecule inhibitors.
  • the antisense compounds included herein can operate by an RNaseH or RNAi mechanism or by other known mechanism such as splicing.
  • SIRTl inhibitors for example antisense compounds targeting SIRTl that reduce SIRTl mRNA and or protein.
  • SIRTl inhibitors for example antisense compounds targeting SIRTl that reduce liver SIRTl mRNA.
  • SIRTl inhibitors for example antisense compounds targeting SIRTl that reduce liver SIRTl mRNA.
  • SIRTl inhibitors for example antisense compounds targeting SIRTl that reduce liver SIRTl mRNA.
  • SIRTl that reduce white adipose tissue (WAT) SIRTl mRNA.
  • WAT white adipose tissue
  • antisense compounds targeting SIRTl and methods of their use prophylactically, for example, to prevent or delay the progression or development of metabolic disorders such as diabetes or elevated blood glucose levels.
  • a metabolic disorder in a subject, comprising administering one or more SIRTl inhibitors, hi certain embodiments, the subject has metabolic disorders or conditions including, but not limited obesity, diabetes (including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia), dyslipidemia (including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia), non-alcoholic fatty liver disease (NAFLD) (including hepatic steatosis and steatohepatitis), hyperfattyacidemia, metabolic syndrome, hyperglycemia, and insulin resistance.
  • diabetes including Type 1 diabetes, Type 2 diabetes and Type 2 diabetes with dyslipidemia
  • dyslipidemia including hyperlipidemia, hypertriglyceridemia, and mixed dyslipidemia
  • NAFLD non-alcoholic fatty liver disease
  • hyperfattyacidemia including hepatic steatosis and steatohepatitis
  • metabolic syndrome including hyperglycemia, and insulin resistance.
  • a method of decreasing blood glucose levels and/or increasing insulin sensitivity comprises selecting a subject in need of a decrease in blood glucose or increase in insulin sensitivity, and administering to the subject a therapeutically effective amount of a SIRTl inhibitor.
  • a method of reducing risk of development of type 2 diabetes and metabolic syndrome includes selecting a subject having elevated blood glucose levels or reduced insulin sensitivity and one or more additional indicators risk of development of type 2 diabetes or metabolic syndrome, and administering to the subject a therapeutically effective amount of a SIRTl inhibitor, for example a antisense compound.
  • administration of a therapeutically effective amount of a SIRTl inhibitor targeted a SIRTl nucleic acid is accompanied by monitoring of glucose levels in the serum of a subject, to determine a subject's response to administration of the SIRTl inhibitor.
  • a subject's response to administration of the SIRTl inhibitor is used by a physician to determine the amount and duration of therapeutic intervention.
  • administering a therapeutically effective amount of an antisense compound targeted a SIRTl nucleic acid is accompanied by monitoring of glucose levels in the serum or insulin sensitivity of a subject, to determine a subject's response to administration of the antisense compound.
  • a subject's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
  • antisense compounds targeting SIRTl that reduce diet induced obesity in animals.
  • antisense compounds targeting SIRTl are useful in treating, preventing or - delaying obesity.
  • antisense compounds targeting SIRTl that reduce white adipose tissue mass in Type 2 diabetic animals. Further described herein, are antisense compounds targeting SIRTl that reduce fasting plasma leptin concentrations in Type 2 diabetic animals. Further described herein, are antisense compounds targeting that reduce plasma insulin in Type 2 diabetic animals
  • antisense compounds targeting SIRTl that reduce plasma glucose in Type 2 diabetic animals. Further described herein, are antisense compounds targeting SIRTl that improve insulin sensitivity.
  • antisense compounds targeting SIRTl that reduce fasting plasma insulin concentrations.
  • antisense compounds targeting SIRTl that reduce fasting plasma glucose concentrations in Type 2 diabetic animals.
  • antisense compounds targeting SIRTl that reduce the gluconeogenic enzymes, for example, cytosolic phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial PEPCK, and the transcriptional co-activator peroxisomal proliferator activated receptor gamma coactivator-1 alpha (PGC- l ⁇ ) mRNA.
  • PEPCK cytosolic phosphoenolpyruvate carboxykinase
  • mitochondrial PEPCK mitochondrial
  • POC- l ⁇ transcriptional co-activator peroxisomal proliferator activated receptor gamma coactivator-1 alpha
  • a physician may determine the need for therapeutic intervention for subjects in cases where more or less aggressive blood glucose or triglyceride-lowering therapy is needed.
  • the practice of the methods herein may be applied to any altered guidelines provided by the NCEP, or other entities that establish guidelines for physicians used in treating any of the diseases or conditions listed herein, for determining coronary heart disease risk and diagnosing metabolic syndrome.
  • Various SIRTl inhibitors targeting SIRTl such as antisense compounds, can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier.
  • Use of the compounds and methods of the invention may also be useful prophylactically to prevent such diseases or disorders, e.g., to prevent or delay undue weight gain, diabetes, other metabolic disorders, or cardiovascular disorders.
  • Cardiovascular Disorders Conditions associated with risk of developing a cardiovascular disorders include, but are not limited to: history of myocardial infarction, unstable angina, stable angina, coronary artery procedures (angioplasty or bypass surgery), evidence of clinically significant myocardial ischemia, noncoronary forms of atherosclerotic disease (peripheral arterial disease, abdominal aortic aneurysm, carotid artery disease), diabetes, cigarette smoking, hypertension, low HDL cholesterol, family history of premature coronary heart disease, obesity, physical inactivity, elevated triglyceride (hypertriglyceridemia), hypercholesterolemia (including polygenic hypercholesterolemia), coronary heart disease (early onset coronary heart disease), elevated ApoB, or elevated cholesterol (including elevated LDL-cholesterol, elevated VLDL-cholesterol, elevated IDL-cholesterol, and elevated non- HDL cholesterol). (Jama, 2001 , 285, 2486-2497; Grundy et al., Circulation, 2004, 110,
  • Hypertriglyceridemia denotes high blood levels of triglycerides.
  • a triglyceride is glyceride in which the glycerol is esterified with three fatty acids. Elevated triglyceride levels have been associated with atherosclerosis, even in the absence of hypercholesterolemia (high cholesterol levels). It can also lead to pancreatitis in excessive concentrations.
  • a related term is "hyperglyceridemia” or “hyperglyceridaemia”, which refers to a high level of all glycerides, including monoglycerides, diglycerides and triglycerides
  • Triglycerides as major components of very low density lipoprotein (VLDL) and chylomicrons, play an important role in metabolism as energy sources and transporters of dietary fat. Fat and liver cells can synthesize and store triglycerides. When the body requires fatty acids as an energy source, the hormone glucagon signals the breakdown of the triglycerides by hormone- sensitive lipase to release free fatty acids. The glycerol component of triglycerides can be converted into glucose, via gluconeogenesis, for brain fuel when it is broken down.
  • antisense compounds targeting SIRTl that reduce plasma triglycerides in Type 2 diabetic animals.
  • the studies show a significant reduction in plasma triglyceride levels after treatment with the SIRTl antisense oligonucleotide.
  • These studies indicate that reduction of SIRTl expression can provide therapeutic benefit in subjects having metabolic disorders like obesity and Type 2 Diabetes, with the added benefit of preventing or reducing associated dyslipidemia that can also lead to the risk of cardiovascular disorders characterized by hypercholesterolemia and hypertriglyceridemia.
  • antisense inhibitors of SIRTl could be candidate therapeutic agents for the treatment of conditions characterized by hypercholesterolemia, and hypertriglyceridemia, or conditions of dyslipidemia associated with NAFLD, Type 2 diabetes, obesity and other metabolic disorders.
  • Hypercholesterolemia is the presence of high levels of cholesterol in the blood. It is not a disease but a metabolic derangement that can be secondary to many diseases and can contribute to many forms of disease, most notably cardiovascular disorder. It is closely related to "hyperlipidemia” (elevated levels of lipids) and “hyperlipoproteinemia” (elevated levels of lipoproteins).
  • Conditions with elevated concentrations of oxidized LDL particles, especially "small dense LDL” (sdLDL) particles are associated with atheroma formation in the walls of arteries, a condition known as atherosclerosis, which is the principal cause of coronary heart disease and other forms of cardiovascular disorder.
  • sdLDL small dense LDL particles
  • HDL particles especially large HDL
  • Increased concentrations of HDL correlate with lower rates of atheroma progressions and even regression.
  • Elevated levels of the lipoprotein fractions, LDL, IDL and VLDL are regarded as atherogenic (prone to cause atherosclerosis). Levels of these fractions correlate with the extent and progress of atherosclerosis. Conversely, the cholesterol can be within normal limits, yet be made up primarily of small LDL and small HDL particles, under which conditions atheroma growth rates would still be high. In contrast, however, if LDL particle number is low (mostly large particles) and a large percentage of the HDL particles are large, then atheroma growth rates are usually low, even negative, for any given cholesterol concentration.
  • SIRTl antisense oligonucleotide Further described herein is a significant reduction in plasma cholesterol levels after treatment with the SIRTl antisense oligonucleotide.
  • These studies indicate that reduction of SIRTl expression can provide therapeutic benefit in subjects having metabolic disorders like obesity and Type 2 Diabetes, with the added benefit of preventing or reducing associated dyslipidemia that can also lead to the risk of cardiovascular disorders characterized by hypercholesterolemia and hypertriglyceridemia.
  • antisense inhibitors of SIRTl could be candidate therapeutic agents for the treatment of conditions characterized by hypercholesterolemia, and hypertriglyceridemia, or conditions of dyslipidemia associated with NAFLD, Type 2 diabetes, obesity and other metabolic disorders.
  • Atherosclerosis is a disease affecting arterial blood vessels. It is a chronic inflammatory response in the walls of arteries, in large part due to the accumulation of macrophage white blood cells and promoted by low density (especially small particle) lipoproteins (plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high density lipoproteins (HDL), (see apoA-1 Milano). It is commonly referred to as a "hardening" or "furring" of the arteries. It is caused by the formation of multiple plaques within the arteries. Atherosclerosis can lead to coronary heart disease, stroke, peripheral vascular disease, or other cardiovascular- related disorders.
  • SIRTl antisense oligonucleotides could be candidate therapeutic agents for the treatment of conditions characterized the progression of atherosclerosis.
  • a subject preferable an animal, even more preferably a human, suspected of having a cardiovascular disorder which can be treated by modulating the expression of SIRTl .
  • a subject is treated by administering a SIRTl inhibitor, for example an antisense compounds targeting SIRTl.
  • a further embodiment is a method of treating cardiovascular disorders wherein, the SIRTl inhibitor is, for example a SIRTl antisense oligonucleotide.
  • SIRTl inhibitors herein include, but are not limited to proteins, peptides, polypeptides, antibodies, antisense compounds including oligonucleotides and antisense oligonucleotides, ssRNA, dsRNA molecules, ribozymes, triple helix molecules, siRNAs, and small molecule inhibitors.
  • the antisense compounds included herein can operate by an RNaseH or RNAi mechanism or by other known mechanism such as splicing. .
  • SIRTl inhibitors for example antisense compounds targeting SIRTl that reduce SIRTl mRNA and/or SIRTl protein.
  • SIRTl inhibitors for example antisense compounds targeting SIRTl that reduce liver SIRTl mRNA.
  • SIRTl inhibitors for example antisense compounds targeting SIRTl that reduce white adipose tissue (WAT) SIRTl mRNA.
  • WAT white adipose tissue
  • antisense compounds targeting SIRTl and methods of their use prophylactically, for example, to prevent or delay the progression or development of cardiovascular disorders such as elevated cholesterol and/or triglyceride levels.
  • SIRTl inhibitors include, but are not limited to proteins, peptides, polypeptides, antibodies, antisense compounds including oligonucleotides and antisense oligonucleotides, ssRNA, dsRNA molecules, ribozymes, triple helix molecules, siRNAs, and small molecule inhibitors.
  • the antisense compounds included herein can operate by an RNaseH or RNAi mechanism or by other known mechanism such as splicing.
  • SIRTl inhibitors such as antisense compounds
  • Use of the compounds and methods of the invention may also be useful prophylactically to prevent such diseases or disorders, e.g., to prevent or delay undue weight gain, diabetes, other metabolic disorders, or cardiovascular disorders.
  • a therapeutically effective amount of a SIRTl antisense compound is administered to a subject having atherosclerosis.
  • a therapeutically effective, amount of antisense compound targeted to a SIRTl nucleic acid is administered to a subject susceptible to atherosclerosis.
  • Atherosclerosis is assessed directly through routine imaging techniques such as, for example, ultrasound imaging techniques that reveal carotid intimomedial thickness. Accordingly, treatment and/or prevention of atherosclerosis further include monitoring atherosclerosis through routine imaging techniques, hi one embodiment, administration of a SIRTl antisense compound leads to a lessening of the severity of atherosclerosis, as indicated by, for example, a reduction of carotid intimomedial thickness in arteries.
  • measurements of cholesterol, lipoproteins and triglycerides are obtained using serum or plasma collected from a subject.
  • Methods of obtaining serum or plasma samples are routine, as are methods of preparation of the serum samples for analysis of cholesterol, triglycerides, and other serum markers.
  • a physician may determine the need for therapeutic intervention for subjects in cases where more or less aggressive blood glucose or triglyceride-lowering therapy is needed.
  • the practice of the methods herein may be applied to any altered guidelines provided by the NCEP, or other entities that establish guidelines for physicians used in treating any of the diseases or conditions listed herein, for determining coronary heart disease risk and diagnosing metabolic syndrome.
  • antisense compounds targeting SIRTl that reduce triglycerides levels in Type 2 diabetic animals.
  • antisense compounds targeting SIRTl are useful in treating, preventing or delaying cardiovascular disorder.
  • antisense compounds targeting SIRTl that reduce total plasma cholesterol in Type 2 diabetic animals.
  • the reduced hepatic lipid content includes, but is not limited to, a reduction in lipids such as triglycerides, diacylglycerols, and long chain CoAs.
  • antisense compounds targeting SIRTl increase rate of fatty acid oxidation in Type 2 diabetic animals.
  • antisense compounds targeting SIRTl increase rate of hepatic insulin sensitivity in Type 2 diabetic animals.
  • a SIRTl inhibitor that decreases the hepatic expression of SIRTl mRNA improves hepatic insulin sensitivity associated with fatty liver and hepatic insulin resistance.
  • one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents.
  • such one or more other pharmaceutical agents are designed to treat the same disease or condition as the one or more pharmaceutical compositions of the present invention.
  • such one or more other pharmaceutical agents are designed to treat a different disease or condition as the one or more pharmaceutical compositions of the present invention.
  • such one or more other pharmaceutical agents are designed to treat an undesired effect of one or more pharmaceutical compositions of the present invention.
  • one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent.
  • one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.
  • pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising an antisense compound targeted to a SIRTl nucleic acid include glucose-lowering agents and therapies.
  • the glucose-lowering agent is a PPAR agonist (gamma, dual, or pan), a dipeptidyl peptidase (IV) inhibitor, a GLP-I analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, a meglitinide, a thiazolidinedione, or a sulfonylurea.
  • the glucose-lowering drug includes all oral hypoglycemic agents such as sulfonylureas that increase insulin secretion (for example, tolbutamide, chlorpropamide and glibenclamide), biguanides (for example, metformin and buformin) that increase glucose uptake and utilization and .alpha.-glucosidase inhibitors (for example, acarbose and voglibose).
  • thiazolidinediones such as troglitazone, rosiglitazone and pioglitazone, are used to ameliorate insulin-resistance.
  • thiazolidinedione intake is usually associated with a weight gain.
  • Sirtuin sient mating type information regulation 2 homolog
  • SIR2L1 transcriptional coactivator 1
  • SIRTl gluconeogenic/glycolytic pathways in liver in response to fasting signals through the transcriptional coactivator, PGC-I alpha .
  • SIRTl induces gluconeogenic genes and hepatic glucose output through PGC- 1 alpha, by modulating the effects of PGC- 1 alpha repression of glycolytic genes in response to fasting and levels of pyruvate .
  • the glucose-lowering therapeutic is a GLP-I analog.
  • the GLP-I analog is exendin-4 or liraglutide.
  • the glucose-lowering therapeutic is a sulfonylurea.
  • the sulfonylurea is acetohexamide, chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, a glyburide, or a gliclazide.
  • the glucose-lowering drug is a biguanide.
  • the biguanide is metformin, and in some embodiments, blood glucose levels are decreased without increased lactic acidosis as compared to the lactic acidosis observed after treatment with metformin alone.
  • the glucose-lowering drug is a meglitinide.
  • the meglitinide is nateglinide or repaglinide.
  • the glucose-lowering drug is a thiazolidinedione.
  • the thiazolidinedione is pioglitazone, rosiglitazone, or troglitazone.
  • blood glucose levels are decreased without greater weight gain than observed with rosiglitazone treatment alone.
  • the glucose-lowering drug is an alpha-glucosidase inhibitor.
  • the alpha-glucosidase inhibitor is acarbose or miglitol.
  • a co-administered glucose-lowering agent is ISIS 113715.
  • glucose-lowering therapy is therapeutic lifestyle change.
  • the glucose-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention.
  • the glucose -lowering agent is administered following administration of a pharmaceutical composition of the present invention.
  • the glucose - lowering agent is administered at the same time as a pharmaceutical composition of the present invention.
  • the dose of a co-administered glucose -lowering agent is the same as the dose that would be administered if the glucose -lowering agent was administered alone.
  • the dose of a co-administered glucose -lowering agent is lower than the dose that would be administered if the glucose -lowering agent was administered alone. In certain such embodiments the dose of a co-administered glucose -lowering agent is greater than the dose that would be administered if the glucose -lowering agent was administered alone.
  • the pharmaceutical agent may be co-administered with a pharmaceutical composition comprising an antisense compound targeted to a SIRTl nucleic acid include anti-obesity agents.
  • anti-obesity agents include but are not limited to Orlistat, Sibutramine, or Rimonabant, and may be administered as described above as adipose or body weight lowering agents.
  • the antisense compound may be co-administered with appetite suppressants.
  • appetite suppressants include but are not limited to diethylpropion tenuate, mazindol, orlistat, phendimetrazine, phentermine, and sibutramine and may be administered as described herein.
  • pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising an antisense compound targeted to a SIRTl nucleic acid include antipsychotic agents.
  • antipsychotic agents therapeutics may be administered as described above to reduce metabolic abnormalities associated with treatment with antipsychotic agents.
  • SIRTl antisense oligonucleotides Due to the ability of SIRTl antisense oligonucleotides to increase metabolic rate and insulin sensitivity and reduce adiposity and weight gain, these compounds can be administered to reduce metabolic abnormalities associated with treatment with antipsychotic agents.
  • the SIRTl antisense oligonucleotide is delivered in a method of reducing metabolic abnormalities associated with the therapeutic use of psychotherapeutic agents.
  • weight inducing antipsychotic agents include, but are not limited to clozapine, olanzapine, aripiprazole, risperidone and ziprasidone.
  • the SIRTl antisense oligonucleotide is delivered concomitant with delivery of the psychotherapeutic agent.
  • SIRTl antisense oligonucleotide is administered prior to the treatment with antipsychotic agents.
  • the SIRTl antisense oligonucleotide is administered after treatment with an obesity inducing drug or agent is ceased.
  • administering of the SIRTl antisense compound results in increased metabolic rate or decreasing adiposity or both without affecting the CNS effects of the psychotherapeutic agent
  • SIRTl antisense oligonucleotides are administered in combination either in the same formulation or separate formulations with other anti-obesity drugs or agents.
  • the anti-obesity agents are CNS based such as, but not limited to, sibutramine or GLP-I based such as, but not limited to, liraglutide.
  • pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include lipid-lowering agents.
  • pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to atorvastatin, simvastatin, rosuvastatin, and ezetimibe.
  • the lipid-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention.
  • the lipid-lowering agent is administered following administration of a pharmaceutical composition of the present invention, hi certain such embodiments the lipid-lowering agent is administered at the same time as a pharmaceutical composition of the present invention, hi certain such embodiments the dose of a co-administered lipid-lowering agent is the same as the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is lower than the dose that would be administered if the lipid-lowering agent was administered alone. In certain such embodiments the dose of a co-administered lipid-lowering agent is greater than the dose that would be administered if the lipid-lowering agent was administered alone.
  • a co-administered lipid-lowering agent is a HMG-CoA reductase inhibitor
  • the HMG-CoA reductase inhibitor is a statin
  • the statin is selected from atorvastatin, simvastatin, pravastatin, fluvastatin, and rosuvastatin.
  • a co-administered lipid-lowering agent is a cholesterol absorption inhibitor, hi certain such embodiments, cholesterol absorption inhibitor is ezetimibe.
  • a co-administered lipid-lowering agent is a co-formulated HMG- CoA reductase inhibitor and cholesterol absorption inhibitor, hi certain such embodiments the co- formulated lipid-lowering agent is ezetimibe/simvastatin.
  • a co-administered lipid-lowering agent is a microsomal triglyceride transfer protein inhibitor (MTP inhibitor).
  • MTP inhibitor microsomal triglyceride transfer protein inhibitor
  • a co-administered lipid-lowering agent is an oligonucleotide targeted to ApoB.
  • a co-administered pharmaceutical agent is a bile acid sequestrant.
  • the bile acid sequestrant is selected from cholestyramine, colestipol, and colesevelam.
  • a co-administered pharmaceutical agent is a nicotinic acid.
  • the nicotinic acid is selected from immediate release nicotinic acid, extended release nicotinic acid, and sustained release nicotinic acid.
  • a co-administered pharmaceutical agent is a fibric acid.
  • a fibric acid is selected from gemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.
  • compositions of the present invention include, but are not limited to, corticosteroids, including but not limited to prednisone; immunoglobulins, including, but not limited to intravenous immunoglobulin (IVIg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-I inhibitors, and COX-2, inhibitors); salicylates; antibiotics; antivirals; antifungal agents; antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan,
  • corticosteroids including but not limited to prednisone
  • compositions of the present invention may be administered in conjunction with a lipid-lowering therapy.
  • a lipid-lowering therapy is therapeutic lifestyle change.
  • a lipid-lowering therapy is LDL apheresis.
  • Antisense compounds include, but are not limited to, oligomeric compounds, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense oligonucleotides, and siRNAs.
  • Antisense compounds may target a nucleic acid, meaning that the antisense compound is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • an antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
  • an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
  • an antisense compound targeted to a SIRTl nucleic acid is 12 to 30 subunits in length. In other words, antisense compounds are from 12 to 30 linked subunits. In certain embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked subunits. In certain embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15,
  • the linked subunits are linked nucleobases, nucleosides, or nucleotides.
  • the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.
  • the antisense compounds comprise at least 8 contiguous nucleobases of an antisense compound disclosed herein. In certain embodiments, the antisense compounds comprises at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of an antisense compound disclosed herein. Antisense compounds 12 to 30, 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.
  • Further antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5 '-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5 '-terminus of the antisense compound that is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases).
  • antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3 '-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3 '-terminus of the antisense compound that is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases).
  • antisense compounds may be represented by oligonucleotide sequences that comprise at least 8 consecutive nucleobases from an internal portion of the sequence of an illustrative preferred antisense compound, and may extend in either- or both directions until the oligonucleotide contains about 8 to about 80 nucleobases.
  • a shortened or truncated antisense compound targeted to a SIRTl nucleic acid has a single subunit deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation).
  • a shortened or truncated antisense compound targeted to a SIRTl nucleic acid may have two subunits deleted from the 5' end, or alternatively may have two subunits deleted from the 3' end, of the antisense compound.
  • the deleted subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit deleted from the 5' end and one subunit deleted from the 3' end.
  • the subunits are nucleobases, nucleosides, or nucleotides.
  • the additional subunit may be located at the 5' or 3' end of the antisense compound.
  • the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the antisense compound.
  • the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5' end and one subunit added to the 3' end.
  • the subunits are nucleobases, nucleosides, or nucleotides.
  • an antisense compound such as an antisense oligonucleotide
  • introduce mismatch bases without eliminating activity.
  • an antisense compound such as an antisense oligonucleotide
  • a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model.
  • Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
  • Gautschi et al demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.
  • antisense compounds targeted to a SIRTl nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
  • 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, or increased inhibitory activity.
  • a second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
  • Antisense compounds having a gapmer motif are considered chimeric antisense compounds.
  • a gapmer an internal region having a plurality of nucleosides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleosides that are chemically distinct from the nucleosides of the internal region.
  • the gap segment In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides, hi certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region.
  • each distinct region comprises uniform sugar moieties.
  • wing-gap- wing motif is frequently described as "X-Y-Z", where "X” represents the length of the 5' wing region, "Y” represents the length of the gap region, and “Z” represents the length of the 3' wing region.
  • Any of the antisense compounds described herein can have a gapmer motif.
  • X and Z are the same, in certain other embodiments, they are different, hi certain embodiments, 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, 25, 30 or more nucleotides.
  • gapmers of the present invention include, but are not limited to, for example 5-10-5, 4-8-4, 4-12-3, 5-13-3, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1 or 2-8-2.
  • the antisense compound as a "wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration.
  • wingmer configurations of the present invention include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10 or 8-2.
  • antisense compounds targeted to a SIRTl nucleic acid possess a 5- 10-5 gapmer motif.
  • an antisense compound targeted to a SIRTl nucleic acid has a gap- widened motif
  • an antisense oligonucleotide targeted to a SIRTl nucleic acid has a gap-widened motif
  • a gap-widened antisense oligonucleotide targeted to a SIRTl nucleic acid has a gap segment of fourteen 2'-deoxyribonucleotides positioned between wing segments of three chemically modified nucleosides, hi certain embodiments, the chemical modification comprises a 2 '-sugar modification. In certain embodiments, the chemical modification comprises a 2'-MOE sugar modification.
  • Nucleotide sequences that encode SIRTl include, without limitation, the following:
  • Nucleotide sequences that encode SIRTl include, without limitation, the following: GENBANK Accession No. 2984 063B, and incorporated herein as SEQ ID NO: 1 and with GENBANK Accession No: NM_012238.3, and incorporated herein as SEQ ID NO: 2.
  • antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
  • Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.
  • a target region is a structurally defined region of the nucleic acid.
  • a target region may encompass a 3' UTR, a 5' UTR, an exon, an intron, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region.
  • the structurally defined regions for SIRTl can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference.
  • 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.
  • Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs.
  • the desired effect is a reduction in mRNA target nucleic acid levels.
  • the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid, hi certain embodiments, the reduction is 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, or 100% at a concentration of 10O nM in cells.
  • 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 other emodiments, target segments within a target region are separated by no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid. In certain embodiments, target segments within a target region are separated by no more than about 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous.
  • Suitable target segments may be found within a 5' UTR, a coding region, a 3' UTR, an intron, or an exon.
  • Target segments containing a start codon or a stop codon are also suitable target segments.
  • a suitable target segment may specifcally exclude a certain structurally defined region such as the start codon or stop codon.
  • the determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids.
  • This comparison can prevent the selection of antisense compound sequences that may hybridize in a non manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).
  • reductions in SIRTl mRNA levels are indicative of reduction of SIRTl expression.
  • Reductions in levels of a SIRTl protein are also indicative of reduction of target mRNA expression.
  • phenotypic changes are indicative of reduction of SIRTl expression. For example, phenotypic changes may include reduction in adipose tissue accumulation and reduction in body weight.
  • hybridization occurs between an antisense compound disclosed herein and a SIRTl nucleic acid.
  • the most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
  • Hybridization can occur under varying conditions. Stringent conditions are sequence- dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
  • the antisense compounds provided herein are specifically hybridizable with a SIRTl nucleic acid.
  • An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense reduction of a target nucleic acid, such as a SIRTl nucleic acid).
  • Non-complementary nucleobases between an antisense compound and a SIRTl nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid.
  • an antisense compound may hybridize over one or more segments of a SIRTl nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
  • the antisense compounds provided herein are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% complementary to a SIRTl nucleic acid. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
  • an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize would represent 90 percent complementarity.
  • the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention.
  • Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. MoI. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489). In certain embodiments, the antisense compounds provided herein are fully complementary
  • antisense compound may be fully complementary to a SIRTl nucleic acid, or a target region, or a target segment or target sequence thereof.
  • "fully complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid.
  • the location of a non-complementary nucleobase may be at the 5 ' end or 3 ' end of the antisense compound.
  • the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound.
  • non-complementary nucleobases When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous, hi certain embodiments, non- complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
  • antisense compounds up to 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 a SIRTl nucleic acid.
  • antisense compounds up to 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 a SIRTl nucleic acid.
  • the antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid.
  • 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.
  • the antisense compounds are complementary to at least an 8 nucleobase portion of a target segment.
  • the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment.
  • the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment.
  • 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.
  • the antisense compounds provided herein include those comprising a portion which consists of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases of the nucleobase sequence set forth in SEQ ID NOs: 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, 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.
  • the antisense compounds are complementary to an equal-length portion of SEQ ID NOs: 1 or 2. In certain embodiments, the antisense compounds are at least 75%, 80%, 85%, 90%, 95%, or 100% (fully) complementary to SEQ ID NOs: 1 or 2.
  • the antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NOs, or compound represented by a specific Isis number.
  • an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine.
  • Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
  • the antisense compounds 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 3-80, or a portion thereof, disclosed herein.
  • a nucleoside is a base-sugar combination.
  • the nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to the 2', 3 ' or 5' hydroxyl moiety of the sugar.
  • Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide.
  • the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
  • Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
  • Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
  • RNA and DNA The naturally occuring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
  • Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom.
  • Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
  • antisense compounds targeted to a SIRTl nucleic acid comprise one or more modified internucleoside linkages.
  • the modified internucleoside linkages are phosphorothioate linkages.
  • each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
  • Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified.
  • Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity or some other beneficial biological property to the antisense compounds.
  • nucleosides comprise a chemically modified ribofuranose ring moieties.
  • Examples of chemically modified sugars include 2'-F-5 '-methyl substituted nucleoside (see PCT International Application WO 2008/101157 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 substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5 '-substitution of a BNA (see PCT International Application WO 2007/134181 Published on 11/22/07 wherein LNA is substituted with for example a 5'-methyl or a 5 '-vinyl group).
  • nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH 3 and 2'-O(CH 2 ) 2 OCH 3 substituent groups.
  • R m and R n are, independently, H or substituted or unsubstituted C 1 -C 10 alkyl.
  • bicyclic nucleic acids examples include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms.
  • antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4'-(CH 2 )-O-2' (LNA); 4'-(CH 2 )-S-2'; 4'-(CH 2 )-O-2' (LNA); 4'-(CH 2 ) 2 -O-2' (ENA); 4'-C(CH 3 ) 2 -O-2' (see PCT/US2008/068922); 4'-CH(CH 3 )-O-2' and 4'-CH(CH 2 OCH 3 )-O- T (see U.S.
  • Patent 7,399,845, issued on July 15, 2008); 4'-CH 2 -N(OCH 3 )-2' (see PCT/US2008/ - 064591); 4'-CH 2 -O-N(CH 3 )-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH 2 -N(R)-O-2' (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH 2 -C(CH 3 )-2'and 4'-CH 2 -C( CH 2 )-2' (see PCT/US2008/ 066154); and wherein R is, independently, H, Cj-C 12 alkyl, or a protecting group.
  • BNAs include various stereochemical sugar configurations including for example ⁇ -L-ribofuranose and ⁇ -D-ribofuranose (see PCT international application PCT7DK98/00393, published on March 25, 1999 as WO 99/14226).
  • nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate.
  • modification includes without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as one having one of the formula:
  • bicyclo and tricyclo sugar surrogate ring systems are also know in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Christian J.). Such ring systems can undergo various additional substitutions to enhance activity. Methods for the preparations of modified sugars are well known to those skilled in the art.
  • nucleobase moieties In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
  • antisense compounds targeted to a SIRTl nucleic acid comprise one or more nucleotides having modified sugar moieties.
  • the modified sugar moiety is 2'-MOE.
  • the 2'-MOE modified nucleotides are arranged in a gapmer motif.
  • Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5- methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 0 C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
  • Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone.
  • Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • antisense compounds targeted to a SIRTl nucleic acid comprise one or more modified nucleobases.
  • gap-widened antisense oligonucleotides targeted to a SIRTl nucleic acid comprise one or more modified nucleobases.
  • the modified nucleobase is 5-methylcytosine.
  • each cytosine is a 5-methylcytosine.
  • Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
  • Antisense compound targeted to a SIRTl nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS).
  • PBS is a diluent suitable for use in compositions to be delivered parenterally.
  • a pharmaceutical composition comprising an antisense compound targeted to a SIRTl nucleic acid and a pharmaceutically acceptable diluent.
  • the pharmaceutically acceptable diluent is PBS.
  • the antisense compound is an antisense oligonucleotide.
  • compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • a prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.
  • Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides.
  • Typical conjugate groups include cholesterol moieties and lipid moieties.
  • Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery or localization within a cell. The cap can be present at the 5'- terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5'- stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on January 16, 2003. Cell culture and antisense compounds treatment
  • the effects of antisense compounds on the level, activity or expression of SIRTl nucleic acids can be tested in -vitro in a variety of cell types.
  • Cell types used for such analyses are available from commerical 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 instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, CA).
  • Illustrative cell types include, but are not limited to, Hep3B cells and primary hepatocytes.
  • Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
  • cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
  • One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, CA).
  • Antisense oligonucleotides are mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN® 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 LIPOFECT AMINE® (Invitrogen, Carlsbad, CA).
  • Antisense oligonucleotide is mixed with LIPOFECTAMINE® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE® concentration that typically ranges 2 to 12 ⁇ g/ ⁇ L per 100 nM antisense oligonucleotide.
  • Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time mRNA levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.
  • the concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 500 nM.
  • RNA Isolation RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.
  • RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL® Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocols.
  • target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR.
  • RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM® 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA and used according to manufacturer's instructions.
  • 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 Biosystems, Foster City, CA) according 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 subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA).
  • RNA quantification by RIBOGREEN® is taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).
  • a CYTOFLUOR® 4000 instrument is used to measure RIBOGREEN® fluorescence.
  • Probes and primers are designed to hybridize to a SIRTl nucleic acid.
  • Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS® Software (Applied Biosystems, Foster City, CA).
  • Antisense inhibition of SIRTl nucleic acids can be assessed by measuring SIRTl protein levels. Protein levels of SIRTl can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, histone deacytelase activity), immunohistochemistry, immunocytochemistry or fluorescence- activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
  • Antisense compounds for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of SIRTl and produce phenotypic changes, such as reduction in amyloid fibril formation and increase in lifespan.
  • Amyloid fibril formation may be measured by light scattering and Congo red-binding assay, for example.
  • Lifespan may be measured by increased length of life of a treated animal in comparison to a non-treated animal.
  • a relevant tissue e.g., liver tissue for systemic delivery and brain tissue for CNS delivery
  • antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with 17, specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.
  • the compounds of the present invention can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.
  • expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and ViIo, FEBS Lett., 2000, 480, 17-24; Celis, et al, FEBS Lett., 2000, 480, 2- 16), SAGE (serial analysis of gene expression)(Madden, et al, Drug Discov. Today, 2000, 5, 415- 425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol, 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al, Proc. Natl. Acad. Sci. U. S.
  • the antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding SIRTl.
  • oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective SIRTl inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively.
  • primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding SIRTl and in the amplification of said nucleic acid molecules for detection or for use in further studies of SIRTl.
  • Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding SIRTl can be detected by means known in the art.
  • Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of SIRTl in a sample may also be prepared.
  • antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans.
  • Antisense oligonucleotide drugs including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.
  • an animal preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of SIRTl is treated by administering antisense compounds in accordance with this invention.
  • the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a SIRTl inhibitor.
  • the SIRTl inhibitors of the present invention effectively inhibit the activity of the SIRTl protein or inhibit the expression of the SIRTl protein. In one embodiment, the activity or expression of SIRTl in an animal is inhibited by about 10%.
  • the activity or expression of SIRTl in an animal is inhibited by about 30%. More preferably, the activity or expression of SIRTl in an animal is inhibited by 50% or more.
  • the oligomeric antisense compounds modulate expression of SIRTl mRNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%.
  • the reduction of the expression of SIRTl may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal.
  • the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding SIRTl protein and/or the SIRTl protein itself.
  • the antisense compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.
  • the compounds of the invention may also be admixed, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, or other formulations, for assisting in uptake, distribution and/or absorption.
  • Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S.: 5,108,921;
  • the antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • pharmaceutically acceptable salts include salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • oligonucleotides preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Patent 6,287,860, which is incorporated herein in its entirety.
  • Sodium salts have been shown to be suitable forms of oligonucleotide drugs.
  • the present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention.
  • the pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intracerebral administration, intrathecal administration, intraventricular administration, ventricular administration, intracerebroventricular administration, cerebral intraventricular administration or cerebral ventricular administration.
  • Oligonucleotides with at least one 2'-O-methoxyethyl modification are believed to be particularly useful for oral administration.
  • Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). hi general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations.
  • the pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
  • Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ m in diameter.
  • Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in the aqueous phase, oily phase or itself as a separate phase.
  • Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Patent 6,287,860, which is incorporated herein in its entirety.
  • Formulations of the present invention include liposomal formulations.
  • liposome means a vesicle composed of amphophilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Liposomes and their uses are further described in U.S. Patent 6,287,860, which is incorporated herein in its entirety.
  • the pharmaceutical formulations and compositions of the present invention may also include surfactants.
  • Surfactants and their uses are further described in U.S. Patent 6,287,860, which is incorporated herein in its entirety.
  • the present invention employs various penetration enhancers to affect the efficient delivery of nucleic acids, particularly oligonucleotides. Penetration enhancers and their uses are further described in U.S. Patent 6,287,860, which is incorporated herein in its entirety.
  • formulations are routinely designed according to their intended use, i.e. route of administration.
  • Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g.
  • compositions and formulations for parenteral administration including intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion, or intracranial may include sterile aqueous solutions which may 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.
  • compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis- chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
  • cancer chemotherapeutic drugs such as daunorubicin
  • chemotherapeutic agents When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5- FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).
  • chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5- FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and
  • Anti-inflammatory drugs including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
  • compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target.
  • compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.
  • Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient.
  • Optimum dosages may vary depending on the relative potency of individual oligonucleotides. In general, dosage is from 0.01 ⁇ g to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or at desired intervals.
  • oligonucleotide is administered in maintenance doses, ranging from 0.01 ⁇ g to 100 g per kg of body weight, once or more daily.
  • Example 1 Antisense inhibition of Rat Sirtuin 1 in primary rat hepatocytes
  • Antisense oligonucleotides targeted to a Sirtuin 1 (SIRTl) nucleic acid were tested for their effects on SIRTl mRNA in vitro.
  • Cultured primary rat hepatocytes at a density of 20,000 cells per well were treated with 165 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and SIRTl mRNA levels were measured by quantitative real-time PCR. SIRTl mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN ® . Results are presented as percent inhibition of SIRTl, relative to untreated control cells.
  • All antisense oligonucleotides in Table 1 are chimeric oligonucleotides ('gapmers') 20 nucleotides in length, composed of a central 'gap' region consisting often 2'-deoxynucleotides, which is flanked on both sides (5 1 and 3' directions) by five-nucleotide 'wings'.
  • the wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides.
  • 'Target start site' indicates the 5'-most nucleotide to which the antisense oligonucleotide is targeted.
  • 'Target Stop Site' indicates the 3 '-most nucleotide to which the antisense oligonucleotide is targeted.
  • Table 1 lists the antisense oligonucleotides targeted to rat SIRTl mRNA, SEQ ID NO: 1 (GENBANK Accession No. 2984_063B).
  • Table 1 Inhibition of rat SIRTl mRNA levels by chimeric oligonucleotides having 5-10-5
  • rat oligonucleotides listed in Table 2 show cross-reactivity with the human SIRTl mRNA (GENBANK Accession No. NM_012238.3) listed here as SEQ ID No. 2.
  • 'Human Target Start Site' indicates the 5'-most nucleotide in the human mRNA (GENBANK Accession No. NM 012238.3) to which the antisense oligonucleotide is targeted.
  • 'Human Target Stop Site' indicates the 3'-most nucleotide in the human mRNA (GENBANK Accession No. NM_012238.3) to which the antisense oligonucleotide is targeted.
  • 'Number of mismatches' indicates the mismatches between the rat oligonucleotide and the human mRNA sequence to which it is targeted.
  • rat oligonucleotides listed above show cross-reactivity with the human SIRTl mRNA (GENBANK Accession No. NM_012238.3) listed here as SEQ ID NO. 2.
  • the rat oligonucleotides listed above have 3 or less nucleobase mismatches with the human SIRTl mRNA.
  • the oligonucleotide sequences listed herein are therefore representative of human oligonucleotides sequences.
  • Chimeric antisense oligonucleotides having 5-10-5 wings and deoxy gap, and a phosphorothioate backbone were designed to target rat SIRTl (GENEBANK Accession No. 2984_063B), incorporated herein as SEQ ID NO: 1.
  • the oligonucleotides ('gapmers') are 20 nucleotides in length, composed of a central 'gap' region consisting often 2'-deoxynucleotides, which is flanked on both sides (5 1 and 3' directions) by five-nucleotide 'wings'.
  • the wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides.
  • the antisense oligonucleotides were evaluated for their ability to reduce rat SIRTl mRNA in rat primary hepatocytes.
  • Antisense oligonucleotides ISIS 384803 (GGAATGAAAGCCATTAGTGA, target site 580), incorporated herein as SEQ ID NO: 3; ISIS 384817 (TGGATTCCTGCAACCTGCTC, target site 1173), incorporated herein as SEQ ID NO: 17; ISIS 384819 (ACAAATCAGGCAAGATGCTG, target site 1225), incorporated herein as SEQ ID NO: 19; ISIS 384834
  • Rat SIRTl primer probe set RTS 2569 (forward sequence CACAAATACTGCCAAGATGTGAATT (SEQ ID NO: 81); reverse sequence TCCAAAATATTACACTCTCCCCAGTAG (SEQ ID NO: 82); probe sequence AATATGCAAAGCCTTTCTGACCCTAATGATGG (SEQ ID NO: 83) was used to measure mRNA levels.
  • SIRTl mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN ® . Results are presented as percent inhibition of SIRTl 5 relative to untreated control cells (UTC). As illustrated in Table 3, SIRTl mRNA levels were reduced in a dose-dependent manner.
  • Treatment Antisense oligonucleotides, ISIS 384856 and ISIS 384834 were evaluated for their ability to reduce the SIRTl mRNA in vivo.
  • Antisense oligonucleotides, ISIS 384856 and ISIS 384834 were examined in male Sprague Dawley rats and compared to a control group treated with PBS.
  • ISIS 384856 was injected subcutaneously at a dose of 50 mg/kg twice a week for 3 weeks.
  • ISIS 384834 was injected subcutaneously at a dose of 50 mg/kg twice a week for 3 weeks.
  • PBS was injected subcutaneously twice a week for 3 weeks. After the treatment period, rat livers were harvested for RNA analysis of SIRTl mRNA expression.
  • ISIS 384856 and ISIS 384834 resulted in a significant reduction of rat SIRTl mRNA, as compared to the PBS control.
  • ISIS 384856 inhibited SIRTl mRNA expression by 51 %
  • ISIS 384834 inhibited expression by 42 %.
  • both the ISIS antisense oligonucleotides were able to significantly inhibit the expression of SIRTl mRNA.
  • Example 4 Antisense inhibition of SIRTl in a rat model of type 2 diabetes mellitus (T2DM)
  • the high-fat diet-fed, Streptozotocin (STZ)-treated rat model provides a novel animal model for T2DM that simulates the human syndrome and is suitable for the testing of antidiabetic compounds .
  • This model of type 2 diabetes in a non-obese, outbred rat strain replicates the natural history and metabolic characteristics of the human syndrome and is suitable for pharmaceutical research.
  • the rats were injected intraperitoneally with 175 mg/kg dose of nicotinamide. After 15 mins, the rats were injected intraperitoneally with 65 mg/kg streptozotocin dissolved in PBS. After 3 days of recovery, afternoon blood glucose levels were determined, and the rats were randomized based on blood glucose values prior to the first antisense oligonucleotide injection. During the treatment period, the rats were pair- fed a combination of high-fructose diet (60%), and a high fat diet. As such, the T2DM rat model provides for a novel animal model for type 2 diabetes that simulates the human syndrome, and is suitable for the testing of antidiabetic compounds.
  • liver mRNA of SIRTl expression was significantly reduced in the SIRTl antisense oligonucleotide-treated rats compared to the control rats.
  • Treatment with ISIS 384856 resulted in 77 % reduction in liver SIRTl mRNA levels compared to the control.
  • WAT mRNA expression of SIRTl was also reduced in the ISIS 384856-treated rats compared to the control rats.
  • SIRTl mRNA was reduced 91 % (Table 4 and Fig. ID).
  • antisense inhibitors of SIRTl are candidate therapeutic agents for the treatment of disorders characterized by SIRTl expression in adipose (such as adipogenesis and obesity) and liver tissues (such as hepatic steatosis, NAFLD and NASH).
  • Example 5 Effect of antisense inhibition of SIRTl in a rat model of type 2 diabetes mellitus (T2DM) on metabolic parameters
  • Table 5 Metabolic profiles after control oligonucleotide or ISIS 384856 treatments in the T2DM rat model
  • ISIS 384856 significantly decreased epididymal white adipose tissue, plasma free fatty acids, leptin, IL-6, and adiponectin concentrations versus the control.
  • the decrease in epididymal white adipose tissue mass in ISIS 384856-treated rats can be attributed to a 78 % and 72 % reduction in PPAR ⁇ l and PPAR ⁇ 2 transcriptions, respectively, compared to the control (Table 5 and Fig. IE- F).
  • Example 6 Effect of antisense inhibition of SIRTl in a rat model of type 2 diabetes mellitus (T2DM) on basal glucose production SIRTl antisense oligonucleotide, ISIS 348856 was tested for its ability to affect basal glucose production in rats of the T2DM model. Rat T2DM model was used to determine basal glucose production because basal hepatic glucose production in these models is increased 19 % compared to normal rats. This increase is comparable to the percentage increase seen in patients with T2DM . In the first cohort, ISIS 384856 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks.
  • T2DM type 2 diabetes mellitus
  • control oligonucleotide ISIS 141923 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks.
  • plasma glucose values were determined using a Beckman Glucose Analyzer II (Beckman Coulter) by a glucose oxidase method.
  • Plasma insulin and glucagon levels were measured by a LINCOplex assay system (Linco). The various parameters are presented in Table 6 and Fig. 1 (G-I).
  • ISIS 384856 decreased basal glucose levels (9 %), basal insulin levels (24 %), but did not significantly reduce basal glucagon levels (7 %) compared to the control.
  • Example 7 Effect of antisense inhibition of SIRTl in a rat model of type 2 diabetes mellitus (T2DM) on hepatic insulin sensitivity
  • ISIS 384856 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks.
  • control oligonucleotide ISIS 141923 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks.
  • hepatic and peripheral insulin sensitivity was assessed using hyperinsulinemic-euglycemic clamps. Seven to nine days prior to the clamp, catheters were inserted into the right internal jugular vein extending to the right atrium and the left carotid artery extending into the aortic arch. Subsequently, the rats were fasted for 24 hours, and then infused with 99% [6, 6- 2 H] glucose (1.1 mg/kg prime, 0.1 mg/kg) infusion to assess basal glucose turnover.
  • the hyperinsulinemic-euglycemic clamp was conducted for 140 minutes with a primed/continuous infusion of insulin (400 mU/kg prime over 5 minutes, 4mU/kg per minute infusion subsequently) and a variable infusion of 20% dextrose spiked with approximately 2.5% [6,6- 2 H] glucose to maintain euglycemia.
  • the results are presented in Table 7 and Fig 3.
  • Table 7 Hepatic insulin sensitivity in ISIS-384856-treated rats compared to the control in the T2DM model
  • ISIS 384856-treated rats show that basal hepatic glucose production rates were decreased by 12.5 % (Table 7 and Fig. 3A).
  • the rate with which glucose was infused to maintain euglycemia was 20 % higher in the ISIS 384856-treated group compared to the control (Table 7 and Fig. 3B).
  • the increased glucose infusion rate may be attributed to a 47 % decrease in hepatic glucose production during the clamp (Table 7 and Fig. 3C).
  • ISIS 384856 treatment had no effect on peripheral insulin-stimulated whole body glucose metabolism (Table 7 and Fig. 3D).
  • Example 8 Effect of antisense inhibition of SIRTl in a rat model of type 2 diabetes mellitus (T2DM) on cholesterol and triglyceride content
  • SIRTl antisense oligonucleotide ISIS 384856 was tested for its ability to affect genes regulating cholesterol and triglyceride levels in the T2DM model.
  • ISIS 384856 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks.
  • control oligonucleotide, ISIS 141923 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks.
  • Table 8 Cholesterol and triglyceride profiles in control rats and SIRTl antisense oligonucleotide in the T2DM rat model
  • ISIS 384856 did not affect plasma triglyceride or liver triglyceride content (Table 8). Plasma ⁇ -hydroxybutyrate concentrations were decreased 25 % in the ISIS 384856-treated group compared to the control (Table 8). ISIS 384856 also decreased total plasma cholesterol by 29 %. (Table 8 and Fig. 2A-B). Therefore, ISIS 384856 is able to specifically regulate cholesterol levels in the T2DM model.
  • SIRTl antisense oligonucleotide shows a significant reduction in plasma cholesterol levels after treatment with the SIRTl antisense oligonucleotide. Also, ⁇ -hydroxybutyrate, which is linked to increased food intake and body weight gain , is decreased by ISIS 384856 treatment. These studies indicate that reduction of SIRTl expression can provide therapeutic benefit in subjects having metabolic disorders like obesity and Type 2 Diabetes, with the added benefit of preventing or reducing associated dyslipidemia that can also lead to the risk of cardiovascular disorders characterized by hypercholesterolemia and hypertriglyceridemia.
  • antisense inhibitors of SIRTl are candidate therapeutic agents for the treatment of conditions characterized by hypercholesterolemia, and hypertriglyceridemia, or conditions of dyslipidemia associated with NAFLD, Type 2 diabetes, obesity and other metabolic disorders.
  • Example 9 Effect of antisense inhibition of SIRTl in a rat model of type 2 diabetes mellitus (T2DM) on cholesterol transport and metabolism SIRTl antisense oligonucleotide ISIS 384856 was tested for its ability to affect genes regulating cholesterol metabolism in the T2DM model, hi the first cohort, ISIS 384856 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks. In the second cohort, control oligonucleotide, ISIS 141923 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks. After the treatment period, several key cholesterol transport and metabolism enzyme transcriptions were measured, as presented in Table 9 and Fig. 2 (C-I).
  • T2DM type 2 diabetes mellitus
  • ISIS 384856 reduced Liver-X receptor (LXR)- ⁇ transcription, an important factor in cholesterol transport and metabolism (Table 9 and Fig. 2C).
  • LXR ⁇ transcriptional target HDL scavenger receptor, class B (SR-Bl) was also down-regulated (Table 9 and Fig. 2D).
  • SR-Bl HDL scavenger receptor
  • LDLR low density lipoprotein receptor
  • the cholesterol 7-alpha hydroxylase (Cyp7Al) gene transcription was up-regulated with ISIS 384856 treatment, indicating greater bile acid synthesis (Table 9 and Fig. 21).
  • ATP-binding cassette G8 gene (ABCG8)
  • SREBP sterol-responsive element-binding protein-2 gene
  • HMG ⁇ -hydroxy ⁇ - methylglutaryl
  • antisense inhibitors of SIRTl are candidate therapeutic agents for the treatment of conditions characterized by hypercholesterolemia, and hypertriglyceridemia, or conditions of dyslipidemia associated with NAFLD, Type 2 diabetes, obesity and other metabolic disorders.
  • Example 10 Effect of antisense inhibition of SIRTl in a rat model of type 2 diabetes mellitus (T2DM) on regulatory enzymes of the glucose pathway
  • T2DM type 2 diabetes mellitus
  • ISIS 384856 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks.
  • control oligonucleotide, ISIS 141923 was injected intraperitoneally at a dose of 37.5 mg/kg twice a week for 4 weeks.
  • various gluconeogenic enzyme post-translational modifications, and activation were measured, and are presented in Table 10 and Fig. 4.
  • the acetylation and activation of forkhead transcription factor, FOXOl, which is linked to insulin signaling in pancreatic ⁇ -cells is increased by 33 % compared to the control (Table 10 and Fig. 4C).
  • STAT3 is a potent transcriptional inhibitor of gluconeogenesis , and its phosphorylation and acetylation were both increased by ISIS 384856 by 76 % and 52 % respectively, compared to the control (Table 10 and Fig. 4A-B).
  • the increased acetylation of STAT3 resulted in 20 %, 50 % and 50 % decreases in the transcription of the gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase (FBPase), and glucose-6-phosphatase (G6Pase), respectively, compared to the control (Table 10 and Fig. 4E-G).
  • PEPCK phosphoenolpyruvate carboxykinase
  • FBPase fructose 1,6-bisphosphatase
  • G6Pase glucose-6-phosphatase
  • the present invention provides for SIRTl antisense oligonucleotides that modulate or inhibit SIRTl expression, activity, or processing.
  • Such agents are candidate therapeutic agents for the treatment of both metabolic and cardiovascular disorders, such as Type 2 diabetes, metabolic syndrome, obesity, dyslipidemia, and hypercholesterolemia, or any combination thereof.
  • Example 11 Effect of antisensc inhibition of SIRTl in a Zucker Diabetic fatty (ZDF) rat model of on metabolic parameters
  • the leptin receptor deficient Zucker diabetic fatty (ZDF) rat is another useful model for the investigation of type 2 diabetes. Diabetes develops spontaneously in these male rats at ages 8-10 weeks, and is associated with hyperphagia, polyuria, polydipsia, and impaired weight gain, symptoms which parallel the clinical symptoms of diabetes (Phillips MS, et al., 1996, Nat Genet 13, 18-19).
  • hyperglycemia is initially manifested at about 7 weeks of age, and all obese male rats fed Purina 5008 are fully diabetic by 12 weeks. Between 7 and 10 weeks, blood insulin levels are high, but these subsequently drop as the pancreatic beta cells cease to respond to the glucose stimulus.
  • ZDF/GmiCrl-;/fl/ya (ZDF) male rats were purchased from Charles River Laboratories
  • ISIS 384856 inhibited Sirtl mRNA levels in both liver and WAT compared to the PBS controls.
  • Table 11 Percent inhibition of Sirtl mRNA in the WAT and Liver tissue relative to saline control
  • Glucose, cholesterol, triglycerides and free fatty acids were measured using an automated clinical chemistry analyzer (Olympus Clinical Analyzer 400E). Insulin levels were measured using an Alpco Insulin Ultrasensitive EIA Kit. The results are presented in Table 12.
  • SIRTl mRNA inhibition demonstrating an improvement in insulin sensitivity.
  • Treatment with ISIS 284856 also leads to recovery from the loss of glucose stimulated insulin secretion (GSIS), which is a characteristic of this model. Insulin levels are elevated by 120 % in the fed state in response to the increase in glucose levels, and by 67% in the fasted state.
  • GSIS 384856 also resulted in decrease of plasma cholesterol, triglyceride and free fatty acid levels, indicating that reduction of SIRTl expression could have therapeutic benefit in subjects having metabolic disorders, like obesity and metabolic syndrome as well as other related disorders.
  • antisense inhibitors of SIRTl could be candidate therapeutic agents for the treatment of conditions characterized by elevated adipose tissue such as obesity and related metabolic disorders.
  • this data presents the ability of ISIS 384856 to lower fat in vivo by specifically modulating or inhibiting SIRTl expression.

Abstract

L'invention concerne des procédés pour réduire l'expression de SIRT1 par administration d'un inhibiteur de SIRT1. Elle concerne également des procédés pour traiter des troubles cardiovasculaires et métaboliques chez un sujet ou pour retarder ou prévenir leurs facteurs de risque par la réduction de l'expression de SIRT1. La présente invention concerne également des procédés de réduction des teneurs en lipides chez un sujet, ou de prévention ou de retard du déclenchement d'une augmentation des teneurs en lipides chez un sujet, comprenant l'administration audit sujet d'un inhibiteur de SIRT1.
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