Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • 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 or pantothenic acid
  • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • 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
  • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
  • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
  • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
  • A61K38/28—Insulins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/33—Chemical structure of the base
  • C12N2310/334—Modified C
  • C12N2310/3341—5-Methylcytosine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/341—Gapmers, i.e. of the type ===---===
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/34—Spatial arrangement of the modifications
  • C12N2310/346—Spatial arrangement of the modifications having a combination of backbone and sugar modifications

Definitions

• Sequence Listing is provided as a file entitled BIOL0174WOSEQ.txt, created March 19, 2013, which is 216 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. FIELD
• inventions provide compositions and methods for modulating the expression of hypoxia inducible factor 1 alpha (HlFla).
• HlFla hypoxia inducible factor 1 alpha
• embodiments provided herein relate to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding HlFla and methods of treating metabolic disorders, such as metabolic syndrome, obesity, and diabetes with such compounds.
• Obesity is considered a long-term metabolic disease. There are several serious medical sequelae related to obesity. There are over 1 billion overweight individuals worldwide with 100 million clinically obese. The increasing health care costs of treating obesity related diseases in the US alone are estimated at over $ 100 billion annually.
• Current methods for treating obesity include behavioral modification, diet, surgery (gastroplasty), administering pharmaceutical agents that block appetite stimulating signals or absorption of nutrients (fat), and administering agents that increase thermogenesis or fat metabolism. Some of these methods have disadvantages in that they rely on patient resolve, are invasive, or have unwanted side effects. An understanding of the mechanisms by which obesity is regulated may provide important therapeutic information.
• Obesity is frequently associated with insulin resistance and together constitutes risk factors for later development of type 2 diabetes and cardiovascular diseases. Insulin resistance occurs well before development of type 2 diabetes, and insulin is overproduced to compensate for the insulin resistance and to maintain normal glucose levels. Type 2 diabetes ensues, as the pancreas can no longer produce enough insulin to maintain normal glucose levels. Early stages of type 2 diabetes are associated with elevated levels of insulin but as the disease progresses the pancreas may fail to produce insulin, resulting in increased blood glucose levels. Diabetes is a significant risk factor for both heart disease and stroke and is the leading cause of blindness and end-stage renal failure.
• Diabetes is a disorder characterized by hyperglycemia due to deficient insulin action that may result from reduced insulin production or insulin resistance or both.
• Diabetes mellitus is a polygenic disorder affecting a significant portion of the people in the world. It is divided into two types. In type I diabetes, or insulin-dependent diabetes mellitus (IDDM), patients produce little or no insulin, the hormone that regulates glucose utilization.
• IDDM insulin-dependent diabetes mellitus
• noninsulin-dependent diabetes mellitus In type 2 diabetes, or noninsulin-dependent diabetes mellitus (NIDDM), patients often have plasma insulin levels that are the same compared to nondiabetic humans; however, these patients have developed a resistance to the insulin stimulating effect of glucose and lipid metabolism in the main insulin- sensitive tissues, i.e., muscle, liver and adipose tissues, and the plasma insulin levels are insufficient to overcome the pronounced insulin resistance. Additionally, glucotoxicity, which results from long-term hyperglycemia, induces tissue-dependent insulin resistance (Nawano et al., Am. J. Physiol. Endocrinol. Metab., 278, E535-543) exacerbating the disease.
• Type 2 diabetes accounts for over 90% of all diabetes cases. It is a metabolic disorder characterized by hyperglycemia leading to secondary complications such as neuropathy, nephropathy, retinopathy, hypertriglyceridemia, obesity, and other cardiovascular diseases generally referred to as metabolic syndrome.
• Metabolic syndrome is a combination of medical disorders that increase one's risk for cardiovascular disease and diabetes.
• the symptoms including high blood pressure, high triglycerides, decreased HDL and obesity, tend to appear together in some individuals.
• Metabolic syndrome is known under various other names, such as (metabolic) syndrome X, insulin resistance syndrome or Reaven's syndrome.
• 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., 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. Therefore, a compound that has the potential to treat both diabetes and obesity would provide a significant improvement over current treatments.
• hypoxia inducible factor is a key regulator of oxygen homeostasis.
• Hypoxia induces the expression of genes participating in many cellular and physiological processes, including oxygen transport and iron metabolism, erythropoiesis, angiogenesis, glycolysis and glucose uptake, transcription, metabolism, pH regulation, growth-factor signaling, response to stress and cell adhesion. These gene products participate in either increasing oxygen delivery to hypoxic tissues or activating an alternative metabolic pathway (glycolysis) which does not require oxygen.
• Hypoxia-induced pathways in addition to being required for normal cellular processes, can also aid tumor growth by allowing or aiding angiogenesis, immortalization, genetic instability, tissue invasion and metastasis (Harris, Nat. Rev.
• HIF is a heterodimer composed of an alpha subunit complexed with a beta subunit, both of which are basic helix-loop-helix transcription factors.
• the beta subunit of HIF is a constitutive nuclear protein.
• the alpha subunit is the regulatory subunit specific to the oxygen response pathway, and can be one of three subunits, HIFla, 2 alpha or 3 alpha (HIF- ⁇ , HIF-2a and HIF-3a, respectively) (Maxwell et al., Curr. Opin. Genet. Dev., 2001, 11, 293-299; Safran and Kaelin, J. Clin. Invest, 2003, 111, 779-783).
• HIFla expression and HIF- 1 transcriptional activity are precisely regulated by cellular oxygen concentration.
• the beta subunit is a constitutive nuclear protein, while the alpha subunit is the regulatory subunit.
• HIFla mRNA is expressed at low levels in tissue culture cells, but it is markedly induced by hypoxia or ischemia in vivo (Yu et al., J. Clin. Invest., 1999, 103, 691-696).
• HIFla protein is negatively regulated in non-hypoxic cells by ubiquitination and proteasomal degradation (Huang et al., Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 7987-7992).
• HIFla protein levels increase dramatically, and the fraction that is ubiquitinated decreases. HIFla then translocates to the nucleus and dimerizes with a beta subunit (Sutter et al., Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 4748- 4753).
• HIF 1 a Although the role of HIF 1 a in cancer is well documented, its role in metabolism and related diseases such as obesity and diabetes remains unclear. Recent studies provided inconsistent results from various HIFla loss-of- function mouse models regarding the role HIFla in metabolism and related pathologies. Transgenic mice with adipose tissue-selective expression of a dominant negative version of HIF 1 a developed more severe obesity and were more susceptible to high fat diet-induced glucose intolerance and insulin resistance compared to wild type mice, suggesting HIFl should not be targeted to treat obesity or diabetes (Zhang X. et al. JBC 285: 32869-32877, 2010).
• mice bearing an adipocyte-specific knockout of the HIF 1 a gene exhibited reduced fat formation, protection from obesity, and insulin resistance in response to a high fat diet (Jiang C. et al. Diabetes 60:2484-2495, 201 1). These studies demonstrate that the role of HIFla in metabolism and related pathologies is uncertain and the effects of targeting HIFla for inhibition are unpredictable.
• Effective treatments are needed for diabetes, obesity, metabolic syndrome and other diseases and conditions thereof.
• Provided herein is data establishing a role for HIFla in both diabetes and obesity.
• the data supports targeting of HIFla for treatment of a range of metabolic conditions, including diabetes, obesity and metabolic syndrome. Therefore, among the objectives herein, it is an object to provide compounds, compositions and methods for the treatment of such diseases and conditions.
• compositions and methods for modulating HIFla expression can be used to treat diabetes, obesity, metabolic syndrome and other diseases and conditions thereof.
• Embodiments provided herein are directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding hypoxia inducible factor 1 alpha (HIF 1 a), and which modulate the expression of HIF la.
• Pharmaceutical and other compositions comprising the compounds of the invention are also provided.
• methods of modulating the expression of HIF 1 a in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention.
• methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of HIF la by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.
• Methods of reducing HIF la expression, activity and/or nucleic acid levels include administering to an individual a compound targeted to a nucleic acid encoding HIF 1 a.
• the compound is administered in a composition.
• Compounds targeted to a HIF la nucleic acid may be targeted to sequences as set forth in GENBANK® Accession Nos. described herein. All of the in GENBANK® Accession Nos. along with their associated sequence and structural data pertaining to such sequences including gene organization and structural elements that may be found in sequence databases such as the National Center for Biotechnology Information (NCBI) are incorporated herein by reference in their entirety.
• NCBI National Center for Biotechnology Information
• HIF la specific inhibitors are provided. In certain embodiments, the HIF la specific inhibitors are antisense compounds. In certain embodiments, the antisense compounds are oligomeric antisense compounds. In certain embodiments the oligomeric antisense compounds are or include modified or unmodified oligonucleotides. In certain embodiments, the antisense compounds are single stranded modified oligonucleotides.
• compositions for the treatment, prevention, slowed progression of and/or amelioration of diabetes, obesity, metabolic syndrome or related diseases or conditions thereof are provided herein.
• the methods, compounds and compositions are for improving insulin sensitivity.
• methods, compounds and compositions for the reduction of glucose levels can be blood, plasma and/or serum glucose levels.
• such glucose levels can be fed or fasting glucose levels.
• such glucose levels are fed or fasting blood glucose levels.
• such methods include administering to a subject a HIF la specific inhibitor, such as an antisense compound targeted to a nucleic acid encoding HIF la. Further provided are methods for treating, preventing and/or ameliorating diabetes, obesity or metabolic syndrome, or another disease or condition thereof in an individual.
• Such method includes selecting an individual diagnosed with diabetes, obesity or metabolic syndrome or other disease or condition, administering to the individual a therapeutically effective amount of a HIFla specific inhibitor, such as an antisense compound targeted to a HIF 1 a nucleic acid, and monitoring factors related to diabetes, obesity or metabolic syndrome or other related disease or condition.
• a HIFla specific inhibitor such as an antisense compound targeted to a HIF 1 a nucleic acid
• Such methods include administering to a subject a HIFla specific inhibitor, such as an antisense compound targeted to a nucleic acid encoding HIFla. In certain embodiments, such methods include the administration of a therapeutically effective amount of a HIF 1 a specific inhibitor, such as an antisense compound targeted to a HIFla nucleic acid. In certain embodiments, the compound is administered in a composition. In certain embodiments the subject is an animal. In certain embodiments the animal is a human.
• the subject to which the HIFla specific inhibitor, such as an antisense compound, is administered and in which metabolic rate is increased and/or weight or fat content is lowered has one or more of the diseases or disorders listed above.
• the subject to which the HIFla specific inhibitor, such as an antisense compound, is administered and in which metabolic rate is increased and/or weight or fat content is lowered has obesity, diabetes or metabolic syndrome.
• LDL low-density lipoprotein
• the methods, compounds and compositions are for the treatment, prevention and/or amelioration of diabetes, obesity and metabolic syndrome. In certain embodiments, the methods, compounds and compositions are for the treatment, prevention and/or amelioration of type 2 diabetes, and type 2 diabetes with dyslipidemia. In certain embodiments, such methods, compounds and compositions are used to treat, slow, prevent, delay or ameliorate the sequelae of diabetes including, but not limited to, retinopathy, neuropathy, cardiovascular complications and nephropathy.
• a HIFl a nucleic acid may be the sequence set forth in any of SEQ ID NOs: 1 -21.
• the antisense compound may be targeted to a HIFl a nucleic acid as set forth in any of SEQ ID NOs: 1 -21.
• administration of the antisense compound may comprise parenteral administration.
• the parenteral administration may further comprise subcutaneous or intravenous administration.
• the antisense compound may have least 80%, at least 90%, or at least 95% complementarity to any of SEQ ID NOs: 1 -21. Alternatively, the antisense compound may have 100%) complementarity to any of SEQ ID NOs: 1 -21.
• the antisense compounds provided herein and employed in any of the described methods may be 8 to 80 subunits in length, 12 to 50 subunits in length, 12 to 30 subunits in length, 15 to 30 subunits in length, 18 to 24 subunits in length, 19 to 22 subunits in length, or 20 subunits in length.
• the antisense compounds employed in any of the described methods may be antisense oligonucleotides 8 to 80 nucleotides in length, 12 to 50 nucleotides in length, 12 to 30 nucleotides in length 15 to 30 nucleotides in length, 18 to 24 nucleotides in length, 19 to 22 nucleotides in length, or 20 nucleotides in length.
• the antisense compound may be an antisense oligonucleotide.
• the antisense oligonucleotide may be chimeric.
• the chimeric antisense oligonucleotide may be a gapmer antisense oligonucleotide.
• the gapmer antisense oligonucleotide may comprise a gap segment of ten 2'-deoxynucleotides positioned between wing segments of five 2'-MOE nucleotides.
• the antisense compounds may have at least one modified internucleoside linkage. Additionally, each internucleoside linkage may be a phosphorothioate internucleoside linkage. Each cytosine may be a 5-methyl cytosine.
• a compound for treatment of obesity, diabetes, or metabolic syndrome may be an antisense compound 12 to 30 nucleobases targeted to a HIF l a nucleic acid.
• the compound may have at least 70%> to
• the antisense oligonucleotide may be a gapmer antisense oligonucleotide.
• the gapmer antisense oligonucleotide may comprise a gap segment of ten 2'- deoxynucleotides positioned between wing segments of five 2'-MOE nucleotides.
• the antisense compounds may have at least one modified internucleoside linkage. Additionally, each internucleoside linkage may be a phosphorothioate internucleoside linkage. Each cytosine may be a 5- methyl cytosine.
• NCBI National Center for Biotechnology Information
• 2'-MOE nucleoside (also 2'-0-methoxyethyl nucleoside) means a nucleoside comprising a 2'- MOE modified sugar moiety.
• 2 '-substituted nucleoside means a nucleoside comprising a substituent at the 2 '-position of the furanosyl ring other than H or OH.
• 2' substituted nucleosides include nucleosides with bicyclic sugar modifications.
• 3' target site refers to the nucleotide of a target nucleic acid which is complementary to the 3'- most nucleotide of a particular antisense compound.
• 5' target site refers to the nucleotide of a target nucleic acid which is complementary to the 5'- most nucleotide of a particular antisense compound.
• administering refers to routes of introducing an antisense compound provided herein to a subject to perform its intended function.
• routes of administration includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.
• Base complementarity refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.”
• Bicyclic sugar moiety means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure.
• the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2 '-carbon and the 4 '-carbon of the furanosyl.
• Bicyclic nucleic acid or " BNA” or “BNA nucleosides” means nucleic acid monomers having a bridge connecting two carbon atoms between the 4' and 2'position of the nucleoside sugar unit, thereby forming a bicyclic sugar.
• bicyclic sugar include, but are not limited to A) a-L-Methyleneoxy (4'- CH 2 -0-2') LNA , (B) ⁇ -D-Methyleneoxy (4'-CH 2 -0-2') LNA , (C) Ethyleneoxy (4'-(CH 2 ) 2 -0-2') LNA , (D) low.
• Examples of 4'- 2' bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: -[C(Ri)(R 2 )] n -, -[C(Ri)(R 2 )] n -0-, -C(RiR 2 )-N(R -O- or -C(RiR 2 )-O-N(R -.
• bridging groups encompassed with the definition of LNA are 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'- (CH 2 ) 3 -2', 4'-CH 2 -0-2', 4'-(CH 2 ) 2 -0-2', 4'-CH 2 -0-N(Ri)-2' and 4'-CH 2 -N(R 1 )-0-2'- bridges, wherein each R t and R 2 is, independently, H, a protecting group or C1-C12 alkyl.
• LNAs in which the 2'- hydroxyl group of the ribosyl sugar ring is connected to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH 2 -0-2') bridge to form the bicyclic sugar moiety.
• the bridge can also be a methylene (-CH 2 -) group connecting the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4'- CH 2 -0-2') LNA is used.
• ethyleneoxy (4'-CH 2 CH 2 -0-2') LNA is used, a -L- methyleneoxy (4'-CH 2 - 0-2'), an isomer of methyleneoxy (4'-CH 2 -0-2') LNA is also encompassed within the definition of LNA, as used herein.
• cEt or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4'- carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH 3 )-0-2'.
• Consstrained ethyl nucleoside (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH 3 )-0-2' bridge.
• “Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
• 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.
• Designing or “Designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.
• Downstream refers to the relative direction toward the 3' end or C-terminal end of a nucleic acid.
• “Fully complementary” or “100% 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.
• “Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions.
• the internal region may be referred to as the "gap” and the external regions may be referred to as the "wings.”
• “HIF 1 a” means hypoxia inducible factor 1 alpha.
• 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.
• “Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.
• mismatch or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
• “Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occuring or modified.
• Microtif means the pattern of unmodified and modified nucleosides in an antisense compound.
• Natural sugar moiety means a sugar moiety found in DNA (2'-H) or RNA (2'-OH).
• Naturally occurring internucleoside linkage means a 3' to 5' phosphodiester linkage.
• Non-complementary nucleobase refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.”
• 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, and double-stranded nucleic acids.
• Nucleotide means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
• Olemeric 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.
• 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
• Regular is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
• “Ribonucleotide” means a nucleotide having a hydroxy at the 2' position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.
• Sites are defined as unique nucleobase positions within a target nucleic acid.
• Specifically hybridizable refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.
• Stringent hybridization conditions or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.
• Target refers to a protein, the modulation of which is desired.
• Target gene refers to a gene encoding a target.
• 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.
• Body fat is defined as 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
• Insulin resistance is defined as the condition in which normal amounts of insulin are inadequate to produce a normal insulin response from fat, muscle and liver cells. Insulin resistance in fat cells results in hydrolysis of stored triglycerides, which elevates free fatty acids in the blood plasma. Insulin resistance in muscle reduces glucose uptake whereas insulin resistance in liver reduces glucose storage, with both effects serving to elevate blood glucose. High plasma levels of insulin and glucose due to insulin resistance often leads to metabolic syndrome and type 2 diabetes.
• Diabetes mellitus is (also called diabetes mellitus), is a metabolic disorder typically characterized by high levels of blood glucose. Diabetes mellitus can take one of two forms: type I or type 2.
• Type I diabetes also known as insulin-dependent diabetes mellitus or IDDM— strikes people under age 35, typically appearing suddenly between the ages of 10 and 16. In this form of the illness, which affects 10 percent of diabetics, a virus or autoimmune reaction probably destroys the insulin-producing cells. Insulin normally enables sugar to pass from the blood into the body's cells. Since a person with type I diabetes has completely stopped producing insulin, lifelong treatment means taking insulin several times daily.
• Type 2 diabetes also known as diabetes mellitus type 2, and formerly called diabetes mellitus type 2, non-insulin-dependent diabetes (NIDDM), obesity related diabetes, or adult-onset diabetes
• NIDDM non-insulin-dependent diabetes
• Type 2 diabetes is a metabolic disorder that is primarily characterized by insulin resistance, relative insulin deficiency, and hyperglycemia.
• Diabetic dyslipidemia or "Type 2 diabetes with dyslipidemia” means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated serum triglycerides, and elevated small, dense LDL particles.
• Metal disorder refers to a condition characterized by an alteration or disturbance in metabolic function.
• Metal and “metabolism” are terms well know 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 2), obesity, insulin resistance and metabolic syndrome.
• Metabolic syndrome means a condition characterized by a clustering of lipid and non-lipid 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-C less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 1 10 mg/dL.
• IPGTT Intraperitoneal Glucose Tolerance Testing
• Intraperitoneal injection to determine how quickly it is cleared from the blood. The test is usually used to test for diabetes, insulin resistance, and sometimes reactive hypoglycemia.
• ITT Insulin Tolerance Test
• the test is usually used to test for diabetes, insulin resistance, and sometimes reactive hypoglycemia.
• Metabolic rate means the rate of metabolism or the amount of energy expended in a given period. Metabolic rate also is the amount of energy expended while at rest in a neutrally temperate environment, in the post-absorptive state (meaning that the digestive system is inactive, which requires about twelve hours of fasting in humans). The release of energy in this state is sufficient only for the functioning of the vital organs, such as the heart, lungs, brain and the rest of the nervous system, liver, kidneys, sex organs, muscles and skin.
• Metabolic rate decreases with age and with the loss of lean body mass. Increased cardiovascular exercise and muscle mass can increase metabolic rate. Illness, previously consumed food and beverages, environmental temperature, and stress levels can affect one's overall energy expenditure, and can affect one's metabolic rate as revealed by gas analysis. It is measured when the person is at complete rest, but awake. . "Prevention" refers to delaying or forestalling the onset or development of a condition or disease for a period of time from hours to days, preferably weeks to months.
• “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.
• Treatment refers to administering a composition of the invention to effect an alteration or improvement of the disease or condition. Prevention, amelioration, and/or treatment may require administration of multiple doses at regular intervals, or prior to onset of the disease or condition to alter the course of the disease or condition. Moreover, a single agent may be used in a single individual for each prevention, amelioration, and treatment of a condition or disease sequentially, or concurrently.
• “Cures” means a method or course that restores health or a prescribed treatment for an illness.
• “Expression” refers to all the functions and steps by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.
• Modulation refers to a perturbation of function or activity when compared to the level of the function or activity prior to modulation.
• modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression.
• modulation of expression can include perturbing splice site selection of pre-mRNA processing.
• 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.
• Subject refers to an animal, including, but not limited to a human, to whom a pharmaceutical composition is administered.
• Antisense activity means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
• Antisense compound means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
• antisense compounds include, but are not limited to, single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, and shRNAs.
• Antisense inhibition means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
• 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.
• Bicyclic sugar means a furanosyl ring modified by the bridging of two atoms. A bicyclic sugar is a modified sugar.
• BNA Bicyclic nucleoside
• the bridge connects the 4 '-carbon and the 2 '-carbon of the sugar ring.
• Oligonucleotide means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
• Single-stranded modified oligonucleotide means a modified oligonucleotide which is not hybridized to a complementary strand.
• Nucleoside means a nucleobase linked to a sugar.
• Linked nucleosides means adjacent nucleosides which are bonded together.
• Linked deoxynucleoside means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.
• Nucleobase means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
• Contiguous nucleobases means nucleobases immediately adjacent to each other.
• Modified oligonucleotide means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
• Modified nucleoside means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.
• Modified nucleotide means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.
• nucleobase complementarity refers to a nucleobase that is capable of base pairing with another nucleobase.
• adenine (A) is complementary to thymine (T).
• adenine (A) is complementary to uracil (U).
• complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid.
• nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid
• the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
• Nucleobase sequence means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.
• Internucleoside linkage refers to the chemical bond between nucleosides.
• 5-methylcytosine means a cytosine modified with a methyl group attached to the 5' position.
• a 5- methylcytosine is a modified nucleobase.
• 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 refers to any nucleobase other than adenine, cytosine, guanine or thymidine.
• 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.
• 2'-0-methoxyethyl refers to an O-methoxy-ethyl modification of the 2' position of a furosyl ring.
• a 2'-0-methoxyethyl modified sugar is a modified sugar.
• Modified sugar refers to a substitution and/or any change from a natural sugar.
• Cap structure or "terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
• “Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2'- O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-0- methoxy ethyl modifications.
• Chimeric antisense compound means an antisense compound that has at least two chemically distinct regions.
• “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.
• Salts mean 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.
• “Pharmaceutically acceptable carrier” means a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal.
• the excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition.
• “Pharmaceutical agent” refers to a substance provides a therapeutic benefit when administered to a subject.
• “Therapeutically effective amount” refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.
• a therapeutically effective amount of antisense compound targeted to a HIF la nucleic acid is an amount that improves adiposity in the individual.
• prophylactically effective amount refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.
• a prophylactically effective amount of antisense compound targeted to a HIFla nucleic acid is an amount that improves adiposity in the individual.
• a “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual.
• a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution.
• administering means providing a pharmaceutical agent or composition to an individual, and includes, but is not limited to administering by a medical professional and self-administering.
• Co-administration refers to administration of two or more pharmaceutical agents to an animal.
• 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.
• administering refers to the administration of two agents at the same time 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.
• “Individual” means a human or non-human animal selected for treatment or therapy. "Individual”, “subject”, and “human” may be used interchangeably herein.
• Duration means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent are administered.
• Parenteral administration means administration through injection or infusion.
• Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
• Subcutaneous administration means administration just below the skin.
• Intravenous administration means administration into a vein.
• Intraperitoneal administration means administration through infusion or injection into the peritoneum.
• Dose means a specified quantity of a pharmaceutical agent provided in a single administration.
• 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.
• Dosage unit means a form in which a pharmaceutical agent is provided.
• a dosage unit is a vial containing lyophilized antisense oligonucleotide.
• a dosage unit is a vial containing reconstituted antisense oligonucleotide.
• Active pharmaceutical ingredient means the substance in a pharmaceutical composition that provides a desired effect.
• Major risk factors refers to factors that contribute to a high risk for a particular disease or condition
• “Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits. “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.
• injection site reaction means inflammation or abnormal redness of skin at a site of injection in an individual.
• “Individual compliance” means adherence to a recommended or prescribed therapy by an individual.
• Therapeutic lifestyle change means dietary and lifestyle changes intended to lower overall body weight and reduce the risk of developing diabetes and obesity, 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.
• Unmodified nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
• 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).
• Upstream refers to the relative direction toward the 5' end or N-terminal end of a nucleic acid.
• the antisense compounds provided herein are useful for treating a number of metabolic conditions, including diabetes, obesity and metabolic syndrome. Such treatments encompass a therapeutic regimen that results in a clinically desirable outcome.
• the clinically desired outcomes may be tied to glucose metabolism.
• the antisense compounds and methods provided herein are useful for improving blood glucose control or tolerance and for improving insulin sensitivity in a subject in need thereof.
• the antisense compounds and methods provided herein are also useful for reducing glucose levels in a subject in need thereof.
• the compounds and methods are particularly useful for reducing blood, plasma and/or serum glucose levels.
• the compounds and methods are useful for reducing both fed and fasting glucose levels.
• Such clinical outcomes are desirable in disease and disorders related to glucose metabolism and insulin resistance including, for example, diabetes, particularly type 2 diabetes, obesity and metabolic syndrome. Therefore, the antisense compounds and methods provided herein are useful for the treatment of such diseases and disorders.
• the compounds and methods are also particularly useful for increasing metabolic rate and, in turn, lowering body weight gain.
• the compounds and methods are also particularly useful for lowering epididymal and perirenal fat pad weight and whole body fat content.
• Such clinical outcomes are desirable in conditions such as metabolic syndrome, obesity, diabetes, in particular type 2 diabetes, type 2 diabetes with dyslipidemia. Therefore, the antisense compounds and methods provided herein are useful for the treatment of such diseases and disorders.
• Metabolic syndrome is a condition characterized by a clustering of lipid and non-lipid 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-C less than 40 mg/dL in men or less than 50 mg/dL in women; blood pressure of at least 130/85 mmHg; and fasting glucose of at least 1 10 mg/dL.
• waist circumference of greater than 102 cm in men or greater than 88 cm in women
• serum triglyceride of at least 150 mg/dL
• HDL-C less than 40 mg/dL in men or less than 50 mg/dL in women
• blood pressure of at least 130/85 mmHg
• fasting glucose of at least 1 10 mg/dL.
• Obesity is characterized by an excess of subcutaneous fat in proportion to lean body mass.
• adipose tissue as opposed to most tissues in the body, will continue to grow. Growth of the adipose tissue results from both the enlargement of mature adipocytes and the formation of new adipocytes from adipocyte precursor cells (preadipocytes). Thus, fat accumulation is associated with increase in the size (hypertrophy) as well as the number (hyperplasia) of adipose tissue cells.
• an antisense oligonucleotide targeted to HIF 1 a to an animal model of obesity resulted in antisense inhibition of HIF 1 a, a reduction in body weight, total body fat content, and percentage fat content of both epididymal and perirenal fat. Particularly, expression of HIF 1 a was reduced.
• antisense inhibition of HIF la results in reduced glucose levels, reduced adiposity, improved insulin sensitivity, and increased metabolic rate.
• methods of reducing glucose levels, body fat, or body weight through the administration of an antisense compound targeted to an HIF la nucleic acid. Blood glucose is considered a risk factor for development of diabetes, obesity and metabolic syndrome.
• methods for the treatment, prevention and/or amelioration of diabetes, obesity and metabolic syndrome and for the treatment, prevention and/or amelioration of associated disorders.
• the invention provides methods of treating an individual comprising administering one or more HIF la specific inhibitors, such as an antisense compound targeted to a HIF la nucleic acid, described herein.
• the individual has diabetes, obesity, metabolic syndrome and/or associated disorders including but not limited to type 2 diabetes, type 2 diabetes with dyslipidemia, dyslipidemia, hyperlipidemia, or non-alcoholic fatty liver disease.
• a HIF la specific inhibitor such as an antisense compound targeted to a HIF 1 a nucleic acid.
• a HIF la specific inhibitor such as an antisense compound targeted to a HIF 1 a nucleic acid.
• a HIF la specific inhibitor such as an antisense compound targeted to a HIF la nucleic acid.
• improved insulin sensitivity is observed when administration of an antisense compound targeted to a HIF 1 a nucleic acid results in lower blood glucose and insulin levels.
• a method of decreasing body fat content comprises selecting an individual in need of a decrease in body fat content, and administering to the individual a therapeutically effective amount of a HIF la specific inhibitor, such as an antisense compound targeted to a HIF la nucleic acid.
• a HIF la specific inhibitor such as an antisense compound targeted to a HIF la nucleic acid.
• the body fat is epidydimal fat or peri-renal fat.
• a method of decreasing body weight comprises selecting an individual in need of a decrease in body weight, and administering to the individual a therapeutically effective amount of a HIF la specific inhibitor, such as an antisense compound targeted to a HIF la nucleic acid.
• a HIF la specific inhibitor such as an antisense compound targeted to a HIF la nucleic acid.
• a method of reducing risk of development of obesity, diabetes, or metabolic syndrome in an individual includes administering to the individual a therapeutically effective amount of a HIF la specific inhibitor, such as an antisense compound targeted to a HIF la nucleic acid.
• a method comprises selecting an individual having elevated blood glucose or triglyceride levels.
• a method of increasing metabolic rate in an individual includes administering to the individual a therapeutically effective amount of a HlFla specific inhibitor, such as an antisense compound targeted to a HlFla nucleic acid.
• an increase in metabolic rate is observed when administration of an antisense compound targeted to a HIF la nucleic acid results in decreased adiposity with unchanged food intake.
• Also provided are methods for reducing serum triglyceride levels or plasma cholesterol levels in a subject which include selecting a subject having elevated serum triglyceride levels or plasma cholesterol levels, and administering to the individual a therapeutically effective amount of a HIF la specific inhibitor, such as an antisense compound targeted to a HIF la nucleic acid, and additionally monitoring serum triglyceride levels or plasma cholesterol levels.
• a HIF la specific inhibitor such as an antisense compound targeted to a HIF la nucleic acid
• serum triglyceride levels or plasma cholesterol levels In certain embodiments, the cholesterol is LDL.
• the individual is an animal. In certain embodiments the individual is a human.
• HIF la specific inhibitors suitable for use in the methods of several embodiments provided herein include, but are not limited to, antibodies, peptides, and small molecule chemical compounds that specifically inhibit HIF la.
• a HIF la specific inhibitor can include any agent capable of specifically inhibiting the expression of HIF la mRNA and/or HIF la protein at the molecular level.
• HIF la specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of HIF la mRNA and/or HIF la protein.
• Certain embodiments provide a method for treating an animal having diabetes, obesity, metabolic syndrome and/or associated disorders including but not limited to type 2 diabetes, type 2 diabetes with dyslipidemia, dyslipidemia, hyperlipidemia, or non-alcoholic fatty liver disease comprising: a) identifying said animal with such disease, disorder or condition, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NOs: 1- 21 , as measured over the entirety of said modified oligonucleotide.
• the therapeutically effective amount of the compound or composition administered to the animal treats or reduces the disease, disorder or condition in the animal.
• the modified oligonucleotide is an antisense oligonucleotide of Table 2, 3, or 4 provided herein.
• Certain embodiments provide a method for decreasing blood glucose levels in an animal comprising: a) identifying said animal with elevated blood glucose levels, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NOs: 1-21, as measured over the entirety of said modified oligonucleotide.
• the modified oligonucleotide is an antisense oligonucleotide of Table 2, 3, or 4 provided herein.
• Certain embodiments provide a method for improving insulin sensitivity in an animal comprising: a) identifying said animal with elevated blood glucose levels, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NOs: 1 -21 , as measured over the entirety of said modified oligonucleotide.
• the modified oligonucleotide is an antisense oligonucleotide of Table 2, 3, or 4 provided herein.
• improved insulin sensitivity is observed when administration of an antisense compound targeted to a HIFl a nucleic acid, such as an antisense oligonucleotide of Table 2, 3, or 4, results in lower blood glucose and insulin levels.
• Certain embodiments provide a method for decreasing body fat in an animal comprising: a) identifying said animal in need of a decrease in body fat, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90% complementary to any of SEQ ID NOs: 1 - 21 , as measured over the entirety of said modified oligonucleotide.
• the modified oligonucleotide is an antisense oligonucleotide of Table 2, 3, or 4 provided herein.
• the body fat is epidydimal fat or peri-renal fat.
• Certain embodiments provide a method for decreasing body weight in an animal comprising: a) identifying said animal in need of a decrease in body weight, and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90%> complementary to any of SEQ ID NOs: 1 -21 , as measured over the entirety of said modified oligonucleotide.
• the modified oligonucleotide is an antisense oligonucleotide of Table 2, 3, or 4 provided herein.
• Certain embodiments provide a method for reducing risk of development of obesity, diabetes, or metabolic syndrome in an animal comprising: a) identifying said animal at risk of developing obesity, diabetes, or metabolic syndrome and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90%> complementary to any of SEQ ID NOs: 1 -21 , as measured over the entirety of said modified oligonucleotide.
• the modified oligonucleotide is an antisense oligonucleotide of Table 2, 3, or 4 provided herein.
• Certain embodiments provide a method for increasing metabolic rate in an animal comprising: a) identifying said animal having or at risk of developing obesity, diabetes, or metabolic syndrome and b) administering to said animal a therapeutically effective amount of a compound or composition comprising a modified oligonucleotide consisting of 14 to 20 linked nucleosides and having a nucleobase sequence at least 90%) complementary to any of SEQ ID NOs: 1 -21 , as measured over the entirety of said modified oligonucleotide.
• the modified oligonucleotide is an antisense oligonucleotide of Table 2, 3, or 4 provided herein.
• an increase in metabolic rate is observed when administration of an antisense compound targeted to a HIFla nucleic acid results in decreased adiposity with unchanged food intake.
• administration of a therapeutically effective amount of an antisense compound targeted a HIFla nucleic acid is accompanied by monitoring of glucose levels in the serum of an individual, to determine an individual's response to administration of the antisense compound.
• An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.
• a physician may determine the need for therapeutic intervention for individuals in cases where more or less aggressive blood glucose or adiposity 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 and diagnosing metabolic syndrome.
• administration of an antisense compound targeted to a HIFla nucleic acid is via parenteral administration.
• Parenteral administration may be intravenous or subcutaneous administration.
• administration of an antisense compound targeted to a HIFla nucleic acid is intravenous or subcutaneous administration.
• Administration may include multiple doses of an antisense compound targeted to a HIF 1 a nucleic acid.
• a pharmaceutical composition comprising an antisense compound targeted to HIFla 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 obesity, metabolic syndrome, mixed dyslipidemia, type 2 diabetes, type 2 diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, or non-alcoholic fatty liver disease.
• pharmaceutical composition comprising an antisense compound targeted to HIFla 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 HIFla is used for the preparation of a medicament for reducing body fat content and obesity.
• an antisense compound targeted to HIFla is used for the preparation of a medicament for the treatment of metabolic syndrome disorders.
• an antisense compound targeted to HIFla is used for the preparation of a medicament of type 2 diabetes, type 2 diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease.
• one or more pharmaceutical compositions of the present invention are coadministered 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.
• the glucose-lowering agent is a PPAR agonist (gamma, dual, or pan), a dipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or an insulin analog, an insulin secretagogue, a SGLT2 inhibitor, a human amylin analog, a biguanide, an alpha-glucosidase inhibitor, a meglitinide, a thiazolidinedione, or a sulfonylurea.
• the glucose-lowering therapeutic is a GLP- 1 analog.
• the GLP- 1 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. In some embodiments, the meglitinide is nateglinide or repaglinide. In some embodiments, the glucose-lowering drug is a thiazolidinedione. In some embodiments, the thiazolidinedione is pioglitazone, rosiglitazone, or troglitazone. In some embodiments, 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.
• glucose-lowering therapy is therapeutic lifestyle change.
• the glucose-lowering agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain such embodiments, the glucose-lowering agent is administered following administration of a pharmaceutical composition of the present invention. In certain such embodiments the glucose-lowering agent is administered at the same time as a pharmaceutical composition of the present invention. In certain such embodiments 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. In certain such embodiments 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.
• anti-obesity agents include but are not limited to Orlistat and may be administered as described above as adipose or body weight lowering agents.
• pharmaceutical agents that may be co-administered with a pharmaceutical composition comprising an antisense compound targeted to a HIFla nucleic acid include antipsychotic agents.
• antipsychotic agents therapeutics may be administered as described above to reduce metabolic abnormalities associated with treatment with antipsychotic agents.
• HIFla antisense oligonucleotides Due to the ability of HIFla 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 HIFla 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 HIF 1 a antisense oligonucleotide is delivered concomitant with delivery of the psychotherapeutic agent. Alternatively, delivery can be in the same formulation or can be administered separately. In certain embodiments, HIF 1 a antisense oligonucleotide is administered prior to the treatment with antipsychotic agents. In a certain embodiment, the HIFla antisense oligonucleotide is administered after treatment with an obesity inducing drug or agent is ceased. In certain embodiments, HIF 1 a antisense oligonucleotides are administered in combination either in the same formulation or separate formulations with other anti-obesity drugs or agents. In certain embodiment, the anti-obesity agents are GLP- 1 based such as, but not limited to, liraglutide.
• an antisense compound targeted to a HIF la nucleic acid via injection and further including administering a topical steroid at the injection site.
• 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 (IVlg); analgesics (e.g., acetaminophen); anti-inflammatory agents, including, but not limited to non-steroidal anti-inflammatory drugs (e.g., ibuprofen, COX-1 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, and
• the pharmaceutical 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 provided herein refer to oligomeric compounds capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
• Examples of antisense compounds include single-stranded and double-stranded compounds, such as but not limited to, antisense oligonucleotides, siRNAs and shRNAs.
• 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 HIF la nucleic acid is 12 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or 20 linked subunits.
• the antisense compounds are 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values.
• the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleosides.
• an antisense compound targeted to a HIF 1 a nucleic acid can have antisense portions of 10 to 50 nucleobases in length.
• antisense compounds having antisense portions of 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
• an antisense compound targeted to a HIF la nucleic acid can have antisense portions of 12 to 30 nucleobases in length.
• antisense compounds having antisense portions of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
• an antisense compound targeted to a HIF 1 a nucleic acid can have antisense portions of 12 or 13 to 24 nucleobases in length.
• antisense portions 12 or 13 to 24 nucleobases in length.
• an antisense compound targeted to a HIF 1 a nucleic acid can have antisense portions of 19 to 23 nucleobases in length.
• antisense portions 19 to 23 nucleobases in length.
• antisense compounds targeted to a HIF 1 a nucleic acid may be shortened or truncated.
• a single subunit may be deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation).
• a shortened or truncated antisense compound targeted to a HIF la 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 nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3 ' end.
• 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.
• an antisense compound such as an antisense oligonucleotide
• an antisense oligonucleotide it is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity.
• 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 1 1 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 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, and/or increased inhibitory activity.
• a second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
• Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect.
• the amount and/or activity of the target nucleic acid is modulated.
• the amount and/or activity of the target nucleic acid is reduced.
• hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation.
• hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation.
• the presence of the antisense compound hybridized with the target nucleic acid results in a modulation of antisense activity.
• antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms.
• antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.
• Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.
• antisense activity results at least in part from degradation of target RNA by RNase H.
• RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid.
• antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides.
• the modified nucleosides do not support RNase H activity.
• such antisense compounds are gapmers, as described herein.
• the gap of the gapmer comprises DNA nucleosides.
• the gap of the gapmer comprises DNA-like nucleosides.
• the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.
• Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds.
• an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides 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.
• the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region.
• sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include ⁇ -D-ribonucleosides, ⁇ -D-deoxyribonucleosides, 2'- modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-0-CH 3 , among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl).
• nucleosides in the wings may include several modified sugar moieties, including, for example 2'-MOE and bicyclic sugar moieties such as constrained ethyl or LNA.
• wings may include several modified and unmodified sugar moieties.
• wings may include various combinations of 2'-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2'-deoxynucleosides.
• Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties.
• wing-gap-wing motif is frequently described as "X-Y-Z", where "X” represents the length of the 5'- wing, “Y” represents the length of the gap, and “Z” represents the length of the 3 '-wing.
• "X” and “Z” may comprise uniform, variant, or alternating sugar moieties.
• "X” and “Y” may include one or more 2'-deoxynucleosides.”
• Y may comprise 2'-deoxynucleosides.
• a gapmer described as "X-Y-Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5 '-wing and the 3' wing.
• any of the antisense compounds described herein can have a gapmer motif.
• "X” and “Z” are the same; in other embodiments they are different.
• "Y” is between 8 and 15 nucleosides.
• X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.
• the antisense compound targeted to a HIFla nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, or 16 linked nucleosides.
• the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J) m -(B) n -(J) p -(B) r -(A) t -(D) g -(A) v -(B) w -(J) x -(B) y -(J) z
• each A is independently a 2 '-substituted nucleoside
• each B is independently a bicyclic nucleoside
• each J is independently either a 2 '-substituted nucleoside or a 2'-deoxynucleoside;
• each D is a 2'-deoxynucleoside
• At least one of m, n, and r is other than 0;
• At least one of w and y is other than 0;
• antisense compounds are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short- interfering RNA or siRNA) and single- stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.
• RNAi interfering RNA compounds
• siRNA double-stranded RNA compounds
• ssRNAi compounds single- stranded RNAi compounds
• antisense compounds including those particularly suited for use as single- stranded RNAi compounds (ssRNA) comprise a modified 5 '-terminal end.
• the 5 '-terminal end comprises a modified phosphate moiety.
• such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5'-phosphate).
• such 5 '-terminal nucleosides stabilize the 5 '-phosphorous moiety.
• Certain modified 5'- terminal nucleosides may be found in the art, for example in WO/201 1/139702.
• ce e 5 '-nucleoside of an ssRNA compound has Formula lie:
• Ti is an optionally protected phosphorus moiety
• T 2 is an internucleoside linking group linking the compound of Formula lie to the oligomeric compound
• A has one of the formulas:
• Qi and Q 2 are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-C6 alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(R 3 )(R4);
• Q 3 is O, S, N(R 5 ) or C(R6)( 7 );
• each R 3 , R4 R 5 , R ⁇ 5 and R 7 is, independently, H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl or Ci-C 6 alkoxy;
• Ri4 is H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, Ci-C 6 alkoxy, substituted Ci-C 6 alkoxy, C 2 -C 6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
• Ri5, Ri6, Ri7 and Rig are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
• Bxi is a heterocyclic base moiety
• Bx 2 is a heterocyclic base moiety and Bxi is H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2- C 6 alkynyl or substituted C 2 -C 6 alkynyl;
• J4, J 5 , Je and J 7 are each, independently, H, halogen, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, Ci-C 6 alkoxy, substituted Ci-C 6 alkoxy, C 2 -C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl or substituted C 2 -C 6 alkynyl;
• each Rig, R 2 o and R21 is, independently, H, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
• each R 8 and R9 is, independently, H, halogen, Ci-Ce alkyl or substituted Ci-Ce alkyl;
• Xi is O, S or N(Ei);
• Z is H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E 2 )(E 3 );
• Ei, E 2 and E 3 are each, independently, H, Ci-Ce alkyl or substituted Ci-Ce alkyl;
• n is from 1 to about 6;
• n 0 or 1 ;
• j is 0 or 1 ;
• each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ b N(Ji)(J 2 ),
• X 2 is O, S or NJ 3 ;
• each Ji, J 2 and J 3 is, independently, H or Ci-Ce alkyl
• said oligomeric compound comprises from 8 to 40 monomelic subunits and is hybridizable to at least a portion of a target nucleic acid.
• Qi and Q 2 are each, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, Ci-Ce alkoxy or substituted Ci-Ce alkoxy. In certain embodiments, Qi and Q 2 are each H. In certain embodiments, Qi and Q 2 are each, independently, H or halogen. In certain embodiments, Qi and Q 2 is H and the other of Qi and Q 2 is F, CH 3 or OCH 3 .
• Ti has the formula: wherein:
• R a and R c are each, independently, protected hydroxyl, protected thiol, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, Ci-Ce alkoxy, substituted Ci-Ce alkoxy, protected amino or substituted amino; and
• R b is O or S. In certain embodiments, R b is O and R a and R c are each, independently, OCH 3 ,
• G is F, OCH 3 or 0(CH 2 ) 2 -OCH 3 .
• G is 0(CH 2 ) 2 -OCH 3 .
• antisense compounds including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif.
• Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
• the oligonucleotides comprise or consist of a region having uniform sugar modifications.
• each nucleoside of the region comprises the same RNA-like sugar modification.
• each nucleoside of the region is a 2'-F nucleoside.
• each nucleoside of the region is a 2'-OMe nucleoside.
• each nucleoside of the region is a 2'-MOE nucleoside.
• each nucleoside of the region is a cEt nucleoside.
• each nucleoside of the region is an LNA nucleoside.
• the uniform region constitutes all or essentially all of the oligonucleotide.
• the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.
• oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type.
• nucleosides of both types are RNA-like nucleosides.
• the alternating nucleosides are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA, and cEt.
• the alternating modificatios are 2'-F and 2'- OMe. Such regions may be contiguous or may be interupted by differently modified nucleosides or conjugated nucleosides.
• the alternating region of alternating modifications each consist of a single nucleoside (i.e., the patern is (AB) x A y wheren A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1).
• one or more alternating regions in an alternating motif includes more than a single nucleoside of a type.
• oligonucleotides may include one or more regions of any of the following nucleoside motifs:
• a and B are each selected from 2'-F, 2'-OMe, BNA, and MOE.
• oligonucleotides having such an alternating motif also comprise a modified 5' terminal nucleoside, such as those of formula lie or He.
• oligonucleotides comprise a region having a 2-2-3 motif. Such regions comprises the following motif:
• A is a first type of modifed nucleosde
• B and C are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;
• x and y are from 1 to 15.
• A is a 2'-OMe modified nucleoside.
• B and C are both 2'-F modified nucleosides.
• A is a 2'-OMe modified nucleoside and B and C are both 2'-F modified nucleosides.
• oligonucleosides have the following sugar motif:
• Q is a nucleoside comprising a stabilized phosphate moiety.
• Q is a nucleoside having Formula lie or He;
• A is a first type of modifed nucleoside
• B is a second type of modified nucleoside
• D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1 , then D must be differently modified than A. In certain embodiments, D differs from both A and B.
• X is 5-15;
• Y is O or 1 ;
• Z is 0-4.
• oligonucleosides have the following sugar motif:
• Q is a nucleoside comprising a stabilized phosphate moiety.
• Q is a nucleoside having Formula lie or He;
• A is a first type of modifed nucleoside
• D is a modified nucleoside comprising a modification different from A.
• A, B, C, and D in the above motifs are selected from: 2'-OMe, 2'-F, V- MOE, LNA, and cEt.
• D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance).
• the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.
• antisense compounds comprising those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif.
• oligonucleotides comprise a region having an alternating internucleoside linkage motif.
• oligonucleotides comprise a region of uniformly modified internucleoside linkages.
• the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages.
• the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages.
• each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate.
• each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
• the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages.
• the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3 ' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3 ' end of the oligonucleotide.
• Oligonucleotides having any of the various sugar motifs described herein may have any linkage motif.
• the oligonucleotides including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:
• antisense compounds are double-stranded RNAi compounds (siRNA).
• siRNA double-stranded RNAi compounds
• one or both strands may comprise any modification motif described above for ssRNA.
• ssRNA compounds may be unmodified RNA.
• siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.
• compositions comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.
• compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function.
• the target nucleic acid is HIFla.
• the degradation of the targeted HIFla is facilitated by an activated RISC complex that is formed with compositions of the invention.
• compositions of the present invention are directed to double-stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex.
• the compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes.
• the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.
• compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif.
• Each strand of the compositions of the present invention can be modified to fulfil a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role.
• the antisense strand can be modified at the 5'-end to enhance its role in one region of the RISC while the 3 '-end can be modified differentially to enhance its role in a different region of the RISC.
• the double-stranded oligonucleotide molecules can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
• the double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e.
• each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof).
• the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self- complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non- nucleic acid-based linker(s).
• the double-stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
• the double-stranded oligonucleotide can be a circular single- stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
• the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions.
• the double-stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene.
• the double-stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
• double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides.
• the short interfering nucleic acid molecules lack 2'-hydroxy (2'-OH) containing nucleotides.
• short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
• double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups.
• double-stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
• siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others.
• siRNA short interfering RNA
• dsRNA double-stranded RNA
• miRNA micro-RNA
• shRNA short hairpin RNA
• siRNA short interfering oligonucleotide
• short interfering nucleic acid short interfering modified oligonucleotide
• ptgsRNA post-transcriptional gene silencing RNA
• RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
• sequence specific RNA interference such as post transcriptional gene silencing, translational inhibition, or epigenetics.
• double-stranded oligonucleotides can be used to epigenetically silence genes at both the post-transcriptional level and the pre -transcriptional level.
• epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).
• compositions of several embodiments provided herein can target HIFla by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., "hairpin" or stem- loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA.
• a dsRNA-mediated gene silencing or RNAi mechanism including, e.g., "hairpin" or stem- loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA.
• the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.
• the dsRNA or dsRNA effector molecule may be a single molecule with a region of self- complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule.
• a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides.
• the dsRNA may include two different strands that have a region of complementarity to each other.
• both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides.
• the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence.
• the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA.
• the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin.
• the dsRNA has one or more single stranded regions or overhangs.
• RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.
• an antisense strand or region e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid
• RNA strand or region that is a sense strand or region e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid
• the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.
• a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell.
• the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.)
• Exemplary circular nucleic acids include lariat structures in which the free 5' phosphoryl group of a nucleotide becomes linked to the 2' hydroxyl group of another nucleotide in a loop back fashion.
• the dsRNA includes one or more modified nucleotides in which the 2' position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2' position contains a hydrogen or an hydroxyl group.
• the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally- occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages.
• the dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661.
• the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.
• the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S.
• dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.
• antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway.
• antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid.
• the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.
• Nucleotide sequences that encode human HIFla include, without limitation, the following: GENBANK® Accession No. NM 001530.3, incorporated herein as SEQ ID NO: 1 GENBANK® Accession No. AF050127.1, incorporated herein as SEQ ID NO: 2; GENBANK® Accession No. AU123241.1, incorporated herein as SEQ ID NO: 3; GENBANK® Accession No. CD1 10339.1, incorporated herein as SEQ ID NO: 4; GENBANK® Accession No. BG772697.1, incorporated herein as SEQ ID NO: 5; GENBANK® Accession No.
• Nucleotide sequences that encode Mus musculus HIFla include, without limitation, the following: GENBANK® Accession No. NM 010431.2, incorporated herein as SEQ ID NO: 16; an alternative mRNA composed of exons la, exons 2-10, exon l ib, exons 12-14, and partial exon 15, incorporated herein as SEQ ID NO: 17; GENBANK® Accession No. CV558871.1, incorporated herein as SEQ ID NO: 18; GENBANK® Accession No. AK034087.1, incorporated herein as SEQ ID NO: 19; and GENBANK® Accession No. NT 039551.7 truncated from nucleotides 32326001 to 32378000, incorporated herein as SEQ ID NO: 20.
• Nucleotide sequences that encode human HIFla include, without limitation, the following:
• SEQ ID NO: 3 GENBANK® Accession No. CD1 10339.1, incorporated herein as SEQ ID NO: 4;
• SEQ ID NO: 8 GENBANK® Accession No. AK299551.1, incorporated herein as SEQ ID NO: 9;
• an antisense oligonucleotide targets any one of SEQ ID NOs: 1-15 and 21.
• an antisense oligonucleotide that is targeted to any of SEQ ID NOs: 1-15 and 21 is at least 90% complementary to the corresponding SEQ ID NOs: 1-15 and 21. In certain such embodiments, an antisense oligonucleotide that is targeted to any of SEQ ID NOs: 1-15 and 21 is at least 95% complementary to the corresponding SEQ ID NOs: 1-15 and 21. In certain such embodiments, an antisense oligonucleotide that is targeted to any of SEQ ID NOs: 1-15 and 21 is 100% complementary to the corresponding SEQ ID NOs: 1-15 and 21. In certain embodiments, an antisense oligonucleotide targeted to any of SEQ ID NOs: 1- 15 and 21 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Tables 2 and 3.
• gapmer antisense compounds are targeted to a HIFla nucleic acid. In certain such embodiments, gapmer antisense compounds are targeted to any one of SEQ ID NOs: 1-15 and 21. In certain such embodiments, the nucleotide sequences illustrated in Table 2 have a 5- 10-5 gapmer motif. Table 2 illustrates gapmer antisense compounds targeted to GENBANK® Accession No. NM 001530.3, incorporated herein as SEQ ID NO: 1, or GENBANK® Accession No. NM l 81054.2, incorporated herein as SEQ ID NO: 7.
• the gapmer antisense compounds in Table 2 have a 5- 10-5 motif, where the gap segment comprises 2'-deoxynucleotides and each wing segment comprises nucleotides comprising a 2'-0- methoxyethyl sugar modification.
• Internucleoside linkages are phosphorothioate, and cytosines are 5- methylcytosines.
• junction 1 723 acttacttacctcacaacgt 88 intron: exon
• junction 1 1548 aatctgtgtcctttaaaaca 89
• gapmer antisense compounds are targeted to a HIFla nucleic acid.
• the nucleotide sequences illustrated in Table 3 have a 5-10-5 gapmer motif.
• Table 3 illustrates gapmer antisense compounds targeted to GENBANK® Accession No. NM 001530.3, incorporated herein as SEQ ID NO: 1.
• “Target site” indicates the first (5 '-most) nucleotide number on the particular target sequence to which the compound binds.
• All compounds in Table 3 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap” region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings".
• the wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides.
• Nucleotide sequences that encode Mus musculus HIF la include, without limitation, the following: GENBANK® Accession No. NM 010431.2, incorporated herein as SEQ ID NO: 16; an alternative mRNA composed of exons la, exons 2-10, exon l ib, exons 12-14, and partial exon 15, incorporated herein as SEQ ID NO: 17; GENBANK® Accession No. CV558871.1, incorporated herein as SEQ ID NO: 18; GENBANK® Accession No. AK034087.1, incorporated herein as SEQ ID NO: 19; and GENBANK® Accession No. NT 039551.7 truncated from nucleotides 32326001 to 32378000, incorporated herein as SEQ ID NO: 20.
• an antisense oligonucleotide targets any one of SEQ ID NOs: 16-20.
• an antisense oligonucleotide that is targeted to any of SEQ ID NOs: 16-20 is at least 90% complementary to the corresponding SEQ ID NOs: 16-20. In certain such embodiments, an antisense oligonucleotide that is targeted to any of SEQ ID NOs: 16-20 is at least 95%> complementary to the corresponding SEQ ID NOs: 16-20. In certain such embodiments, an antisense oligonucleotide that is targeted to any of SEQ ID NOs: 16-20 is 100% complementary to the corresponding SEQ ID NOs: 16-20. In certain embodiments, an antisense oligonucleotide targeted to any of SEQ ID NOs: 16-20 comprises a nucleotide sequence selected from the nucleotide sequences set forth in Table 4.
• gapmer antisense compounds are targeted to SEQ ID NOs: 16-20.
• the nucleotide sequences illustrated in Table 4 have a 5-10-5 gapmer motif.
• Table 4 illustrates gapmer antisense compounds targeted to GENBANK® Accession No. NM_010431.2, incorporated herein as SEQ ID NO: 16.
• “Target site” indicates the first (5'-most) nucleotide number on the particular target sequence to which the compound binds.
• All compounds in Table 4 are chimeric oligonucleotides ("gapmers") 20 nucleotides in length, composed of a central "gap” region consisting of ten 2'-deoxynucleotides, which is flanked on both sides (5' and 3' directions) by five-nucleotide "wings".
• the wings are composed of 2'-methoxyethyl (2'-MOE) nucleotides.
• a target region is a structurally defined region of the target nucleic acid.
• a target region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region.
• the structurally defined regions for a target 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.
• a target segment is a smaller, sub-portion of a target region within a nucleic acid.
• a target segment can be the sequence of nucleotides of a target nucleic acid to which one or more antisense compound is targeted.
• 5' target site refers to the 5 '-most nucleotide of a target segment.
• 3' target site refers to the 3 '-most nucleotide of a target segment.
• 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.
• a target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain emodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values.
• target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5' target sites or 3' target sites listed herein.
• Suitable target segments may be found within a 5' UTR, a coding region, a 3' UTR, an intron, an exon, or an exon/intron junction.
• Target segments containing a start codon or a stop codon are also suitable target segments.
• a suitable target segment may 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.
• the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).
• HIFla mRNA levels are indicative of inhibition of HIF 1 a expression.
• Reductions in levels of a HIF 1 a protein are also indicative of inhibition of HIFla expression.
• phenotypic changes are indicative of inhibition of HIFla expression.
• human HIFla target segments include, but are not limited to, nucleobases 191-210, 250-269, 292-31 1, 388-407, 436-455, 490-509, 554-573, 705-724, 723-742, 759-778, 789-808, 846-865, 994- 1013, 1 141- 1 160, 1 176-1 195, 1231-1250, 1265-1284, 1279-1298, 1364- 1383, 1423-1442, 1520-1539, 1548- 1567, 1582- 1601, 1814-1833, 1839-1858, 1905-1924, 2123-2142, 2135-2154, 2179-2198, 2225-2244, 2266- 2285, 2362-2381, 2460-2479, 2461-2480, 2471-2490, 2544-2563, 2636-2655, 2875-2894, 2968-2987, 3070- 3089, 3166-3185, 3233-3252, 3240-3259, 3332-3351, 3455-34
• NM 001530.3 incorporated herein as SEQ ID NO: 1
• hybridization may occur between an antisense compound disclosed herein and a HIFla 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 HIF 1 a 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 inhibition of a target nucleic acid, such as a HIF l a nucleic acid).
• Non-complementary nucleobases between an antisense compound and a HIF 1 a 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 HIF 1 a 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, or a specified portion thereof are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementary to a HIF l a nucleic acid, a target region, target segment, or specified portion thereof.
• Percent complementarity of an antisense compound with a HIF l a acid can be determined using routine methods. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity.
• 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. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981 , 2, 482 489).
• the antisense compounds provided herein, or specified portions thereof are fully complementary (i.e. 100% complementary) to a HIF l a nucleic acid, or specified portion thereof.
• antisense compound may be fully complementary to a HIF 1 a 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.
• a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound.
• Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid.
• a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long.
• the 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound.
• the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
• 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.
• two or more non-complementary nucleobases may be contiguous (i.e. linked) or non-contiguous.
• a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
• antisense compounds that are, or are up to, 12, 13, 14, 15, 16, 17, 18, 19, or
• 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 HIFla nucleic acid, or specified portion thereof.
• antisense compounds that are, or are up to, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid or specified portion thereof.
• 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.
• nucleobase portion of a target segment 9, 10, 1 1, 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 may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, 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.
• an 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%, at least 75%, at least 80%, at least 85%), at least 90%, at least 95% or 100% identical to one or more of the antisense compounds disclosed herein. Modifications
• a nucleoside is a base-sugar combination.
• the nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety.
• Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.
• Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
• 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 an Androgen Receptor 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 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 chemically modified ribofuranose ring moieties.
• Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(Ri)(R 2 ) (R, Ri and R 2 are each independently H, C 1 -C 12 alkyl or a protecting group) and combinations thereof.
• substitutent groups including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA)
• BNA bicyclic nucleic acids
• R, Ri and R 2 are each independently H, C 1 -C 12 alkyl or a protecting group
• Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleoside (see PCT International Application WO 2008/101 157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16,
• nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH 3 , 2'-OCH 2 CH 3 , 2'-OCH 2 CH 2 F and - 0(CH 2 ) 2 OCH 3 substituent groups.
• tricyclic nucleosides refer to modified nucleosides comprising a bicyclic sugar moiety.
• bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms.
• antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4' to 2' bridge.
• 4' to 2' bridged bicyclic nucleosides include but are not limited to one of the formulae: 4'-(CH 2 )-0-2' (LNA); 4'-(CH 2 )-S-2'; 4'-(CH 2 ) 2 -0-2' (ENA); 4'-CH(CH 3 )-0-2' (also referred to as constrained ethyl or cEt) and 4'-CH(CH 2 OCH 3 )- 0-2' (and analogs thereof see U.S.
• Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and ⁇ -D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
• the bridge of a bicyclic sugar moiety is -[C(R a )(R b )] n -, -[C(R a )(R b )] n -0-, -C(R a R b )-N(R)-0- or -C(R a R b )-0-N(R)-.
• the bridge is 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'- (CH 2 ) 3 -2', 4'-CH 2 -0-2', 4'-(CH 2 ) 2 -0-2', 4'-CH 2 -0-N(R)-2' and 4'-CH 2 -N(R)-0-2'- wherein each R is, independently, H, a protecting group or C 1 -C 12 alkyl.
• bicyclic nucleosides are further defined by isomeric configuration.
• a nucleoside comprising a 4'-2' methylene-oxy bridge may be in the a-L configuration or in the ⁇ - D configuration.
• a-L-methyleneoxy (4'-CH 2 -0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al, Nucleic Acids Research, 2003, 21, 6365- 6372).
• bicyclic nucleosides include, but are not limited to, (A) a-L-methyleneoxy
• Bx is the base moiety and R is independently H, a protecting group, C 1 -C 12 alkyl or C 1 -C 12 alkoxy.
• bicyclic nucleosides are provided having Formula I:
• Bx is a heterocyclic base moiety
• R c is C 1 -C 12 alkyl or an amino protecting group
• T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
• bicyclic nucleosides are provided having Formula II:
• Bx is a heterocyclic base moiety
• T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
• Z a is Ci-Ce alkyl, C 2 -C6 alkenyl, C 2 -C6 alkynyl, substituted Ci-Ce alkyl, substituted C 2 -C6 alkenyl, substituted C 2 -C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
• bicyclic nucleosides are provided having Formula III:
• Bx is a heterocyclic base moiety
• T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
• bicyclic nucleosides are provided having Formula IV:
• Bx is a heterocyclic base moiety
• T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
• Rd is Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
• each q a , q b , q c and qd is, independently, H, halogen, Ci-Ce alkyl, substituted Ci-Ce alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, Ci-Ce alkoxyl, substituted Cp Ce alkoxyl, acyl, substituted acyl, Ci-Ce aminoalkyl or substituted Ci-Ce aminoalkyl;
• bicyclic nucleosides are provided having Formula V:
• Bx is a heterocyclic base moiety
• T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
• q g and q 3 ⁇ 4 are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
• BNA methyleneoxy (4'-CH 2 -0-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
• bicyclic nucleosides are provided having Formula VI:
• Bx is a heterocyclic base moiety
• T a and T b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
• 4 '-2' bicyclic nucleoside or “4' to 2' bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2' carbon atom and the 4' carbon atom of the sugar ring.
• nucleosides refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties.
• sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
• 2'-modified sugar means a furanosyl sugar modified at the 2' position.
• modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl.
• Other - substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, F, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties.
• modifed nucleosides comprise a 2'-MOE side chain (Baker et al, J. Biol. Chem., 1997, 272, 1 1944-12000).
• 2'-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'- O- methyl, O-propyl, and O-aminopropyl.
• Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv.
• a "modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran "sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate).
• Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841 -854) or fluoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below:
• HNA hexitol nucleic acid
• ANA anitol nucleic acid
• MNA manitol nucleic acid
• F-HNA fluoro HNA having a tetrahydropyran ring system as illustrated below:
• sugar surrogates are selected having Formula VII:
• Bx is a heterocyclic base moiety
• T a and T b are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T a and T b is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T a and T b is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;
• ⁇ Qi, 3 ⁇ 43, 3 ⁇ 44, qs, 3 ⁇ 46 and q 7 are each independently, H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 -C 6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and each of Ri and R2 is selected from hydrogen, hydroxyl, halogen, subsitituted or unsubstituted alkoxy, NJiJ 2 , SJi, N 3 , and CN, wherein X is O, S or NJi and each J J 2 and J 3 is, independently, H or Ci-Cg alkyl.
• the modified THP nucleosides of Formula VII are provided wherein qi, q 2 , q 3 , q4, q 5 , q6 and q 7 are each H. In certain embodiments, at least one of qi, q2, q 3 , q4, qs, q6 and q 7 is other than H. In certain embodiments, at least one of qi, q2, q 3 , q4, qs, q6 and q 7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R2 is fluoro.
• Ri is fluoro and R2 IS H; Ri is methoxy and R2 is H, and Ri is methoxyethoxy and R2 is H.
• sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom.
• nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al, Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5, 185,444; and 5,034,506).
• morpholino means a sugar surrogate having the following formula:
• morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure.
• sugar surrogates are referred to herein as "modifed morpholinos.”
• Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid see PCT International Application WO 2007/134181, published on 1 1/22/07 wherein a 4'-CH 2 -0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group.
• PCT International Application WO 2007/134181 published on 1 1/22/07 wherein a 4'-CH 2 -0-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group.
• carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described ⁇ see, e.g., Srivastava et al, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
• antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides.
• Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on April 10, 2010, Robeyns et al, J. Am. Chem. Soc, 2008, 130(6), 1979-1984; Horvath et al, Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al, J.
• Bx is a heterocyclic base moiety
• T 3 and T 4 are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T 3 and T 4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T 3 and T 4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5'-or 3'-terminal group; and
• ⁇ 3 ⁇ 4, 3 ⁇ 43, 3 ⁇ 44, 3 ⁇ 45, 3 ⁇ 46, 3 ⁇ 47, 3 ⁇ 48 and q 9 are each, independently, H, Ci-C 6 alkyl, substituted Ci-C 6 alkyl, C 2 - C 6 alkenyl, substituted C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl or other sugar substituent group.
• 2 '-modified or “2 '-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH.
• 2 '-modified nucleosides include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2'substituents, such as allyl, amino, azido, thio, O-allyl, O-C Ci 0 alkyl, -OCF 3 , 0-(CH 2 ) 2 -0-CH 3 , 2'-0(CH 2 ) 2 SCH 3 , 0-(CH 2 ) 2 -0- N(R m )(R n ), or where each R m and R n is, independently, H or substituted or unsubstituted Ci-C
• 2'-F refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position of the sugar ring.
• 2'-OMe or “2'-OCH 3 " or “2'-0-methyl” each refers to a nucleoside comprising a sugar comprising an -OCH 3 group at the 2' position of the sugar ring.
• oligonucleotide refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
• RNA ribonucleosides
• DNA deoxyribonucleosides
• bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.
• 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 comprise one or more nucleosides having modified sugar moieties.
• the modified sugar moiety is 2'-MOE.
• the 2'-MOE modified nucleosides are arranged in a gapmer motif.
• the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH 3 )-0-2') bridging group.
• the (4'- CH(CH 3 )-0-2') modified nucleosides are arranged throughout the wings of 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 can 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°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
• Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C ⁇ C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted urac
• Heterocyclic base moieties can 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 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
• antisense compounds targeted to a HIFla nucleic acid comprise one or more modified nucleobases.
• gapmer antisense oligonucleotides targeted to a HIFla 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 and/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.
• An antisense compound targeted to a HIFla 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.
• employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a HIFla 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.
• compositions of the present invention comprise one or more oligonucleotides and one or more excipients.
• excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulosem and polyvinylpyrrolidone.
• a pharmaceutical composition of the present invention is prepared using known techniques, including, but not limited to mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tabletting processes.
• a pharmaceutical composition of the present invention is a liquid (e.g., a suspension, elixir and/or solution).
• a liquid pharmaceutical composition is prepared using ingredients known in the art, including, but not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.
• a pharmaceutical composition of the present invention is a solid (e.g., a powder, tablet, and/or capsule).
• a solid pharmaceutical composition comprising one or more oligonucleotides is prepared using ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents.
• a pharmaceutical composition of the present invention is formulated as a depot preparation. Certain such depot preparations are typically longer acting than non-depot preparations. In certain embodiments, such preparations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, depot preparations are prepared using suitable polymeric or hydrophobic materials (for example an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• a pharmaceutical composition of the present invention comprises a delivery system.
• delivery systems include, but are not limited to, liposomes and emulsions.
• Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds.
• certain organic solvents such as dimethylsulfoxide are used.
• a pharmaceutical composition of the present invention comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types.
• pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
• a pharmaceutical composition of the present invention comprises a co- solvent system.
• co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
• co-solvent systems are used for hydrophobic compounds.
• VPD co-solvent system is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80.TM., and 65% w/v polyethylene glycol 300.
• the proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
• identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80. TM.; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
• a pharmaceutical composition of the present invention comprises a sustained-release system.
• a sustained-release system is a semi-permeable matrix of solid hydrophobic polymers.
• sustained-release systems may, depending on their chemical nature, release pharmaceutical agents over a period of hours, days, weeks or months.
• a pharmaceutical composition of the present invention is prepared for oral administration.
• a pharmaceutical composition is formulated by combining one or more oligonucleotides with one or more pharmaceutically acceptable carriers.
• Certain of such carriers enable pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject.
• pharmaceutical compositions for oral use are obtained by mixing oligonucleotide and one or more solid excipient.
• Suitable excipients include, but are not limited to, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
• PVP polyvinylpyrrolidone
• such a mixture is optionally ground and auxiliaries are optionally added.
• pharmaceutical compositions are formed to obtain tablets or dragee cores.
• disintegrating agents e.g., cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate are added.
• dragee cores are provided with coatings.
• concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to tablets or dragee coatings.
• compositions for oral administration are push-fit capsules made of gelatin.
• Certain of such push- fit capsules comprise one or more pharmaceutical agents of the present invention in admixture with one or more filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• pharmaceutical compositions for oral administration are soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• one or more pharmaceutical agents of the present invention are be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added.
• compositions are prepared for buccal administration. Certain of such pharmaceutical compositions are tablets or lozenges formulated in conventional manner.
• a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.).
• a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
• other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives).
• injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like.
• compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
• Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
• such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
• a pharmaceutical composition is prepared for transmucosal administration.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• a pharmaceutical composition is prepared for administration by inhalation.
• Certain of such pharmaceutical compositions for inhalation are prepared in the form of an aerosol spray in a pressurized pack or a nebulizer.
• Certain of such pharmaceutical compositions comprise a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• the dosage unit may be determined with a valve that delivers a metered amount.
• capsules and cartridges for use in an inhaler or insufflator may be formulated.
• Certain of such formulations comprise a powder mixture of a pharmaceutical agent of the invention and a suitable powder base such as lactose or starch.
• a pharmaceutical composition is prepared for rectal administration, such as a suppositories or retention enema.
• Certain of such pharmaceutical compositions comprise known ingredients, such as cocoa butter and/or other glycerides.
• a pharmaceutical composition is prepared for topical administration.
• Certain of such pharmaceutical compositions comprise bland moisturizing bases, such as ointments or creams.
• ointment bases include, but are not limited to, petrolatum, petrolatum plus volatile silicones, lanolin and water in oil emulsions such as Eucerin.TM., available from Beiersdorf (Cincinnati, Ohio).
• suitable cream bases include, but are not limited to, Nivea.TM.
• Cream available from Beiersdorf (Cincinnati, Ohio), cold cream (USP), Purpose Cream.TM., available from Johnson & Johnson (New Brunswick, N.J.), hydrophilic ointment (USP) and Lubriderm.TM., available from Pfizer (Morris Plains, N.J.).
• a pharmaceutical composition of the present invention comprises an oligonucleotide in a therapeutically effective amount.
• the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
• one or more oligonucleotides of the present invention is formulated as a prodrug.
• a prodrug upon in vivo administration, is chemically converted to the biologically, pharmaceutically or therapeutically more active form of the oligonucleotide.
• prodrugs are useful because they are easier to administer than the corresponding active form.
• a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form.
• a prodrug may have improved solubility compared to the corresponding active form.
• prodrugs are less water soluble than the corresponding active form.
• a prodrug is an ester.
• the ester is metabolically hydrolyzed to carboxylic acid upon administration.
• the carboxylic acid containing compound is the corresponding active form.
• a prodrug comprises a short peptide (polyaminoacid) bound to an acid group.
• the peptide is cleaved upon administration to form the corresponding active form.
• a prodrug is produced by modifying a pharmaceutically active compound such that the active compound will be regenerated upon in vivo administration.
• the prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug.
• a pharmaceutical composition comprising one or more pharmaceutical agents of the present invention is useful for treating a conditions or disorders in a mammalian, and particularly in a human, subject.
• Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, intraperitoneal, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous).
• pharmaceuticals are administered to achieve local rather than systemic exposures.
• pharmaceutical compositions may be injected directly in the area of desired effect (e.g., in the renal or cardiac area).
• a pharmaceutical composition of the present invention is administered in the form of a dosage unit (e.g., tablet, capsule, bolus, etc.).
• a dosage unit e.g., tablet, capsule, bolus, etc.
• such pharmaceutical compositions comprise an oligonucleotide in a dose of 1- 1000 mg.
• the dose is administered at intervals ranging from once per week to once per month, for as long as needed to sustain the desired effect.
• a pharmaceutical agent is sterile lyophilized oligonucleotide that is reconstituted with a suitable diluent, e.g., sterile water for injection.
• a suitable diluent e.g., sterile water for injection.
• the reconstituted product is administered as a subcutaneous injection or as an intravenous infusion after dilution into saline.
• the lyophilized drug product consists of the oligonucleotide which has been prepared in water for injection, adjusted to pH 7.0-9.0 with acid or base during preparation, and then lyophilized.
• the lyophilized oligonucleotide may be 1 - 1000 mg of the oligonucleotide.
• the lyophilized drug product may be packaged in a 2 mL Type I, clear glass vial (ammonium sulfate -treated), stoppered with a bromobutyl rubber closure and sealed with an aluminum FLIP- OFF® overseal.
• compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
• the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
• the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.
• auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.
• 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 and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3'- cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5 '-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on January 16, 2003.
• antisense compounds are modified by attachment of one or more conjugate groups.
• conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance.
• Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligonucleotide.
• Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.
• Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.
• HIFla nucleic acids The effects of antisense compounds on the level, activity or expression of HIFla 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 A549 cells. In vitro testing of antisense oligonucleotides
• 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 they 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
• LIPOFECTAMINE® (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 ug/mL per 100 nM antisense oligonucleotide.
• Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-
• RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein.
• 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 300 nM.
• RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of 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. Analysis of inhibition of target levels or expression
• 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.
• RNA 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.
• RT reverse transcriptase
• cDNA complementary DNA
• 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). RT, real-time-PCR reactions are carried out by methods well known to those skilled in the art.
• Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as GAPDH, or by quantifying total RNA using RIBOGREEN® (Invetrogen, Inc. Carlsbad, CA). GAPDH expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN® RNA quantification reagent (Invetrogen, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREEN® are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A
• CYTOFLUOR® 4000 instrument PE Applied Biosystems is used to measure RIBOGREEN® fluorescence.
• Antisense inhibition of HIFla nucleic acids can be assessed by measuring HIFla protein levels. Protein levels of HIFla can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence- activated cell sorting (FACS).
• 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. Antibodies useful for the detection of human and mouse HIFla are commercially available.
• Antisense compounds for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of HIFla and produce phenotypic changes, such as decreases in body weight. Testing may be performed in normal animals, or in experimental disease models.
• antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight.
• RNA is isolated from various tissues and changes in HIFla nucleic acid expression are measured. Changes in HIFla protein levels may also be measured.
• Antisense oligonucleotides targeted to a murine HIF- 1 a nucleic acid were tested for their effects on HIF- ⁇ mRNA in vitro.
• Cultured A549 cells were transfected with 150 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and mouse HIF- 1 a mRNA levels were measured by quantitative real-time PCR and with primer probe set HTS3931 (forward sequence
• HIF- la mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN.
• ISIS 298745 (GTGCAGTATTGTAGCCAGGC; SEQ ID NO: 148), which was one of the antisense oligonucleotides tested in the assay, was designed as a 5-10-5 MOE gapmer, and is 20 nucleosides in length, wherein the central gap segment is comprised of ten 2'-deoxynucleosides and is flanked on both sides (in the 5' and 3' directions) by wings comprising 5 nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2' -MOE modification.
• ISIS 298745 is targeted to nucleobases 3075 to 3094 of mouse HIF- ⁇ (GENBANK Accession No. NM 010431.2), incorporated herein as SEQ ID NO: 16. ISIS 298745 is also cross-reactive with nucleobases 2975 to 2944 of human HIF- ⁇ ⁇ mRNA (GENBANK Accession No. NM 001530.3, incorporated herein as SEQ ID NO: 1). ISIS 298745 reduced murine HIF- ⁇ mRNA expression by 78%.
• Example 2 Effect of in vivo antisense inhibition of murine HIF- ⁇ in high-fat diet fed mice
• ISIS 298745 which demonstrated significant activity in vitro (see Example 1), was selected for further evaluation in vivo.
• mice Male 8-week old C57BL/6 mice were fed a 58% high-fat diet for 4 weeks prior to the start of the dosing regimen. The mice were then randomly divided into groups. One group of mice was treated with 25 mg/kg of ISIS 298745, administered twice a week for 9 weeks. One group of mice was treated with 25 mg/kg of a control oligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC (SEQ ID NO: 166), 5-10-5 MOE gapmer with no known murine target), administered twice a week for 9 weeks. A third group of mice was treated with PBS, administered twice a week for 9 weeks. High-fat diet feeding was continued throughout the study. A set of C57BL/6 mice fed normal mouse chow was also included in the study as a control.
• mice from all groups were measured each week. The results are presented in Table 6 and indicate that treatment with ISIS 298745 did not cause any effect on food intake. Treatment with the control oligonucleotide also did not affect food intake significantly, as expected.
• mice from all groups were measured each week. The results are presented in Table 7.
• PBS-treated mice and the control oligonucleotide-treated mice had increases in body weight in a time-dependent manner.
• ISIS 298745 prevented further increase in body weight after 2 weeks of treatment.
• the body weight in ISIS 298745-treated mice was lower than that of either PBS-treated or the control oligonucleotide-treated mice.
• mice from all groups were measured at different time points with an Echo- MRI body composition analyzer. The results are presented in Tables 8 and 9.
• Body fat content in both PBS- treated mice and the control oligonucleotide-treated mice increased in a time-dependent manner.
• treatment with ISIS 298745 prevented the increase in body fat content.
• body fat content in ISIS 298745-treated mice was lower than that in either PBS-treated mice or the control oligonucleotide-treated mice
• mice The fat depot weights of mice from all groups were harvested and weighed at sacrifice of the mice. The results are presented in Table 10. Mice treated with ISIS 298745 had 53% lower epididymal fat and 73% lower peri-renal fat compared to the PBS treated mice.
• Body fat content (% of body weight) of high- fat- fed mice
• LPL lipoprotein lipase
• ACC acetyl-CoA carboxylase
• FES fatty acid synthase
• SCD steroyl-CoA desaturase
• SCD2 SCD2
• DGAT diacylglycerol acyltransferase
• mice treated with ISIS 298745 had decreased expression of lipogenic genes without any change in the levels of lipolytic genes, compared to the PBS treated mice and the control oligonucleotide -treated mice. This suggests that treatment with ISIS 298745 resulted in decreased lipogenesis with unchanged lipolysis.
• the expression levels of the lipogenic genes ACC1, ACC2, FAS, SCD1, SCD2, DGAT2, and sterol regulatory element -binding protein (SREBP) lc in the liver were measured. The results are presented in Table 13, expressed in percent inhibition compared to the levels in the PBS control. The data indicates that treatment with ISIS 298745 caused down-regulation in lipogenic genes, suggesting decreased lipogenesis in the liver.
• SREBP sterol regulatory element -binding protein
• Plasma glucose levels were determined by using a clinical analysis Olympus AU400e Beckman Coulter and were measured before dosing and on weeks 3 and 8. Plasma insulin concentrations were determined by an ELISA kit (ALPCO Diagnostics, Inc) and were measured on week 8. The results are presented in Tables 14 and 15. The data demonstrates that both glucose and insulin were reduced after treatment with ISIS 298745, indicating improved insulin sensitivity in the mice. Mice treated with ISIS 298745 had glucose levels equivalent to those of normal chow-fed mice.
• ALT alanine transaminase
• AST aspartate transaminase
• Example 3 In vivo metabolic effects of antisense inhibition of murine HIF-lalpha in diet-induced obesity (DIO) mice
• mice Male C57BL/6 mice purchased at 6 weeks of age from the Jackson Laboratory were fed a 58% high- fat diet (Research Diet 12330) for 4.5 months to induce obesity. The mice were then divided into different groups based on body weight and body fat content. Groups of mice were treated with control oligonucleotide, ISIS 141923, or ISIS 298745 (SEQ ID NO: 148) targeting HIF-lalpha at doses of 25 mg/kg twice a week for 1 1 weeks. A control group of mice was treated with PBS twice a week for 1 1 weeks. During the treatment, the mice continued to be fed the high-fat diet. A group of mice was fed normal chow and was used as a reference group.
• control oligonucleotide ISIS 141923, or ISIS 298745 (SEQ ID NO: 148) targeting HIF-lalpha at doses of 25 mg/kg twice a week for 1 1 weeks.
• a control group of mice was treated with PBS twice a week for 1 1 weeks. During the
• Body weights, liver and spleen weights were measured at the end of the study, and are presented in Tables 17 and 18. Treatment with ISIS 298745 resulted in decreased body weight. There was negligible change in organ weights in the treatment groups.
• Body compositions were monitored with an Echo MRI Body Composition Analyzer. Epidermal and perirenal white adipose tissue weights were measured at the end of the study, and are presented in Table 19. The total body fat content was also measured and is presented in Table 20. Treatment with ISIS 298745 resulted in decreased fat depot weight, as well as decreased total fat content. The lean mass of ISIS 298745- treated mice did not change compared to the controls.
• the metabolic rate was assessed by measuring the oxygen consumption and respiratory quotient of the mice. Both parameters were measured with an indirect calorimetry system (Oxymax system, Columbus Instruments). Metabolic rate was assessed both in darkness, when the mice are typically more active, and in light. The results are presented in Tables 21 and 22. The results indicate that treatment with ISIS 298745 increased whole body oxygen consumption rate without affecting the respiratory quotient of the mice. Hence, antisense inhibition of HIF- ⁇ expression in obese subjects with a high calorie diet would be beneficial as it would increase the metabolic rate in the subject.
• Plasma glucose values were determined at week 7 by using a glucose oxidase method (Beckman Glucose Analyzer II; Beckman Coulter). Plasma insulin concentrations were determined also at week 7 by an RIA Assay system (Linco). The results are presented in Table 23. The data demonstrates that insulin levels were significantly reduced while plasma glucose levels remained relatively unchanged after treatment with ISIS 298745. This data indicates that reduction of HIF-1 alpha caused an improvement in insulin sensitivity.
• Glucose tolerance was measured via the oral glucose tolerance test (OGTT). The mice were fasted overnight and then an oral administration of glucose at 0.75 mg/kg was given. Blood glucose levels were measured before the glucose challenge and at different time points after challenge up to 180 min.
• OGTT oral glucose tolerance test
• mice were fasted for 4 hours, injected with insulin at 1.0 U/kg and tested for blood glucose levels over a period of 2 hours.
• the blood glucose levels (mg/dL) are shown below in Table 25.
• the glucose levels in ISIS 298745-treated mice were significantly lower than controls during ITT. This finding confirms that HIF- 1 alpha inhibition by antisense treatment demonstrated an improvement in insulin sensitivity.
• mice were euthanized and blood was collected via cardiac puncture. Lipid levels were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Results are presented in Table 26-29, expressed as mg/dL. The data indicates that ISIS 298745 significantly lowered total cholesterol, HDL and LDL levels but had no effect on plasma triglyceride levels after 1 1 weeks of treatment.
• ISIS oligonucleotides To evaluate the effect of ISIS oligonucleotides on hepatic function, levels of various plasma chemistry markers were measured on week 1 1 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 30, expressed in IU/L. Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also presented in Table 30. The results indicate that treatment with ISIS 298745 was tolerable in this model.
• Example 4 In vivo metabolic effects of antisense inhibition of murine HIF-lalpha in ob/ob mice
• Leptin is a hormone produced by fat that regulates appetite. Deficiency of this hormone in both humans and in non-human animals, leads to obesity, ob/ob mice have a mutation in the leptin gene which results in hyperglycemia and obesity. As such, these mice are a useful model for the investigation of obesity and diabetes and related conditions provided herein.
• mice Male ob/ob (C57Bl/6J-Lep ob /Lep ob ) mice purchased at 6 weeks of age from the Jackson laboratory were fed a regular rodent chow and divided into different groups based on body weight and body fat content. The mice were treated with PBS, or with control oligonucleotide, ISIS141923, or with ISIS 298745 targeting HIF- 1 alpha at doses of 25 mg/kg twice a week for 13 weeks. During the treatment, body weights and food intake were measured weekly. Body composition, and plasma insulin, glucose and lipid levels were measured at different time points. At the end of the treatment, the mice were sacrificed and different fat depots were dissected and weighed. Tissue HIF- 1 alpha expression was measured.
• the primer probe set mHIFla forward sequence GCCTGATGCTCTCACTCTGCT, designated herein SEQ ID NO: 170; reverse sequence TGTGTCATCGCTGCCAAAAT, designated herein SEQ ID NO: 171 ; probe sequence CTGCCGGCGACACCATCATCTCTC, designated herein as SEQ ID NO: 172
• Body weights were measured at the end of the study, and are presented in Table 32. Treatment with ISIS 298745 resulted in decreased body weight compared to the control groups.
• Body compositions were monitored with an Echo MRI Body Composition Analyzer. Total body fat content was measured and is presented in Table 33. Total body lean mass is presented in Table 34. Treatment with ISIS 298745 resulted in decreased total fat content. The lean mass of ISIS 298745-treated mice did not change compared to the controls.
• Plasma glucose values were determined by using a glucose oxidase method (Beckman Glucose Analyzer II; Beckman Coulter). Plasma insulin concentrations were determined by a RIA Assay system (Linco). The results are presented in Tables 35 and 36. The data demonstrates that glucose and insulin levels were reduced in both the fed and fasted states after treatment with ISIS 298745.
• Plasma insulin levels (ng/mL) in ob/ob
• Plasma glucose levels (mg/dL) in ob/ob
• mice were euthanized and blood was collected via cardiac puncture. Lipid levels were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Results are presented in Table 37-40, expressed as mg/dL. The data indicates that ISIS 298745 significantly lowered total cholesterol, HDL levels, LDL levels and plasma triglyceride levels in both the fed and fasted states.
• Plasma levels of non-esterified fatty acids which are the end-products of fatty acid oxidation, were measured using an automated clinical chemistry analyzer (Olympus Clinical Analyzer). The results are presented in Table 41. Treatment with ISIS 298745 resulted in an increase in free fatty acid levels in the plasma over the PBS control.
• ISIS oligonucleotides To evaluate the effect of ISIS oligonucleotides on hepatic function, levels of various plasma chemistry markers were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Tables 42 and 43, expressed in IU/L. The results indicate that treatment with ISIS 298745 was tolerable in this model.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Epidemiology (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biomedical Technology (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Wood Science & Technology (AREA)
• Organic Chemistry (AREA)
• General Engineering & Computer Science (AREA)
• Endocrinology (AREA)
• Gastroenterology & Hepatology (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Plant Pathology (AREA)
• Diabetes (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=49223304

Family Applications (1)

Country Status (1)

Citations (3)

• 2013
  • 2013-03-19 WO PCT/US2013/032993 patent/WO2013142509A1/fr not_active Ceased

Patent Citations (3)

Non-Patent Citations (3)

Similar Documents

Legal Events