WO2012082765A2 - Méthodes pour réduire le poids corporel et traiter le diabète - Google Patents

Méthodes pour réduire le poids corporel et traiter le diabète Download PDF

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WO2012082765A2
WO2012082765A2 PCT/US2011/064690 US2011064690W WO2012082765A2 WO 2012082765 A2 WO2012082765 A2 WO 2012082765A2 US 2011064690 W US2011064690 W US 2011064690W WO 2012082765 A2 WO2012082765 A2 WO 2012082765A2
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hifla
subject
mice
decreasing
inhibitor
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WO2012082765A3 (fr
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Frank J. Gonzalez
Changtao Jiang
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US Department of Health and Human Services
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    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • This disclosure relates to methods for treating diabetes and reducing body weight, particularly using inhibitors of hypoxia- inducible factor la.
  • Obesity has been identified as a major risk factor for chronic diseases such as type 2 diabetes, cardiovascular disease, hepatosteatosis, and even cancer (Bluher, Exp. Clin. Endocrinol. Diabetes 117:241-250, 2009).
  • Insulin resistance defined as dysregulation of the insulin- signaling cascade in insulin target tissues, such as adipose tissue, skeletal muscle, and liver, is a hallmark of type 2 diabetes (Kahn and Flier, J. Clin. Invest. 106:473-481, 2000).
  • Increasing evidence shows that obesity is strongly associated with insulin resistance (Kim, Cell Metab. 4:417-419, 2006).
  • hypoxia a marker of hypoxia
  • lactate production a marker of lactate
  • hypoperfusion in both obese human and animal models.
  • hypoxia is observed in white adipose tissue of obese individuals (Hosogai et al., Diabetes 56:901-911, 2007).
  • hypoxia is observed in white adipose tissue of obese individuals (Hosogai et al., Diabetes 56:901-911, 2007).
  • hypoxia is observed in white adipose tissue of obese individuals (Hosogai et al., Diabetes 56:901-911, 2007).
  • hypoxia is observed in white adipose tissue of obese individuals (Hosogai et al., Diabetes 56:901-911, 2007).
  • hypoxia is observed in white adipose tissue of obese individuals (Hosogai et al., Diabetes 56:901-911, 2007).
  • hypoxia is observed in white adipose tissue of obese individuals (Hosogai et al
  • HIF hypoxia inducible factor
  • HIFl , HIF2a, HIF3 , and ARNT are all expressed in adipose tissue (Hatanaka et ah, Biol. Pharm. Bull. 32: 1166-1172, 2009; Shimba et al, J. Biol. Chem. 279:40946-40953, 2004; Ye, Int. J. Obes. (Lond.) 33:54-66, 2009).
  • the methods include
  • a composition including an inhibitor of hypoxia-inducible factor l (HIFl ) to a subject with diabetes or a subject in need of reduction of body weight.
  • HIFl hypoxia-inducible factor l
  • administration of a therapeutically effective amount of a HIFla inhibitor to a subject decreases body weight by decreasing total body weight, decreasing body mass index (BMI), decreasing waist circumference, decreasing total body fat, or a combination of two or more thereof, as compared to a control.
  • administration of a therapeutically effective amount of a HIFla inhibitor to a subject treats diabetes in the subject by increasing glucose tolerance, decreasing insulin resistance, decreasing triglyceride levels, decreasing free fatty acid levels, decreasing hemoglobin Ale (HbAlc) levels, or a combination of two or more thereof, as compared to a control.
  • the inhibitor of HIFla decreases expression or half-life of
  • HIFla mRNA or protein such as decreasing transcription and/or translation of HIFla or increasing degradation of HIFla mRNA and/or protein
  • activity of HIFla protein or a combination thereof relative to a control.
  • the inhibitor is a small molecule (for example, acriflavine), a nucleic acid molecule, or an antibody.
  • the HIFla inhibitor is targeted to adipose tissue (such as white adipose tissue), for example using a peptide or antibody that specifically localizes to adipose tissue.
  • the composition includes a HIFlcc inhibitor and at least one pharmaceutically acceptable carrier.
  • the inhibitor of HIFlcc is administered orally.
  • FIGS. 1A and IB are bar graphs of qPCR analysis of Arnt mRNA expression in the indicated tissues or adipocytes from Ami F/F and Arnt A dipo mice (FIG. 1A) or
  • FIGS. 1C and ID are bar graphs showing qPCR analysis of CYP1 Al mRNA induction by 2,3,7, 8-tetrachlorodibenzo-/?-dioxin (TCDD) treatment in WAT (FIG. 1C) and BAT (FIG. ID) of ArntTM or Arnt AAdipo mice.
  • TCDD 2,3,7, 8-tetrachlorodibenzo-/?-dioxin
  • FIG. 2E is a pair of bar graphs showing cumulative food intake adjusted to body weight and metabolic efficiency for 2 weeks in HiflaTM and Hifla AAdipo mice after 4-6 weeks of HFD.
  • FIG. 2F is a pair of bar graphs showing resting and total 0 2 consumption adjusted to body weight.
  • FIG. 2G is a pair of bar graphs showing resting and total respiratory exchange ratio (RER; vC0 2 /v02).
  • FIG. 2H is a pair of bar graphs showing ambulatory and total activity levels. Data shown in FIGS. 2E-H are from the same set of HiflaTM and Hifla AAdipo mice. Indirect calorimetry (FIGS.
  • FIG. 3A-C is a series of bar graphs showing body weight (FIG. 3A), cumulative food intake (FIG. 3B), and metabolic efficiency (FIG. 3C) for 2 weeks in 6 to 8 week old male mice maintained on a chow diet.
  • FIG. 3E is a bar graph showing cumulative food intake for 2 weeks in mice fed HFD from 4-6 weeks.
  • FIG. 3F is a pair of bar graphs showing resting and total 0 2 consumption in mice after 7 weeks of high fat diet (same mice as data shown in FIG. 3E). Data are mean + SEM.
  • FIG. 5C is a graph showing time courses of blood glucose during the hyperinsulinemic-euglycemic clamp.
  • FIG. 5D is a bar graph showing plasma glucose in the basal state and during the clamp.
  • FIG. 5E is a bar graph showing plasma insulin levels in the basal state and during the clamp.
  • FIG. 5B is a graph showing ITT in Hifla F/F and Hifla A dip
  • FIG. 5F is a graph showing glucose infusion rate during the clamp.
  • FIG. 5G is a bar graph showing basal and clamp endogenous glucose production (EGP), glucose infusion rate (GIR), whole body glucose disposal (Rd), and glucose uptake in skeletal muscle, WAT, and BAT.
  • FIGS. 6A and 6B are a series of graphs showing body mass, GTT, and ITT of HiflaTM and Hifla AAdipo mice fed HFD for 4 weeks (FIG. 6A) or 8 weeks (FIG. 6B).
  • FIG. 6C is a series of graphs showing body mass, fasted glucose, fasted serum insulin levels, and HOMA index of Hifl aF and Hifla AAdipo mice after 6 weeks on a HFD.
  • FIG. 7A is a graph showing gene expression in WAT in wild-type C57BL/6 mice fed chow or HFD for 12 weeks. *P ⁇ 0.05, **P ⁇ 0.01 compared to chow-fed mice.
  • FIG. 8A and B are a pair of bar graphs showing qPCR analysis of gene expression in WAT of Arnt vlv and Arai AAdipo (FIG. 8 A) or Hifla ' v and Hifla Adipo mice (FIG. 8B).
  • FIG. 8C is a series of bar graphs showing total adiponectin, HMW adiponectin, and HMW/total adiponectin ratio in Arn lv and Arnt A dipo mice (top) and HiflaTM and Hifla AAdipo mice (bottom) after 12 weeks of HFD.
  • FIG. 8D is a pair of bar graphs showing qPCR analysis of fibrosis-related gene expression in WAT of Arnt vlv and Arai AAdipo mice (top) and HiflaTM and Hifla Adipo mice
  • FIG. 8E is a pair of bar graphs showing Socs3 expression determined by qPCR in WAT of ArntTM and Arai AAdipo mice (top) and HiflaTM and Hifla Adipo mice (bottom).
  • FIG. 8F is a pair of bar graphs showing quantitation of SOCS3 expression and STAT3 phosphorylation. Relative protein levels were normalized to levels for
  • Hifla F/F littermates For qPCR analysis, expression was normalized to p-actin. Data are mean + S.D. *P ⁇ 0.05, **P ⁇ 0.01 compared to wild-type floxed littermates.
  • FIG. 9A is a bar graph showing qPCR analysis of F4/80 and CD68 mRNA in adipose tissue in ArntTM and Arai AAdipo mice after 12 weeks of HFD.
  • FIG. 9B a bar graph showing qPCR analysis of F4/80 and CD68 mRNA in adipose tissue in HiflaTM and HIFla A dipo mice after 12 weeks of HFD.
  • the expression was normalized to ⁇ -actin and each bar represents the mean value + S.D. **P ⁇ 0.01 compared to wild- type floxed littermates.
  • FIG. 10A is a bar graph showing qPCR analysis of Arnt mRNA expression in peritoneal macrophages ( ⁇ - ⁇ ) from Arai F/F and Arai AAdipo mice.
  • FIGS. 10B and IOC are a pair of bar graphs showing qPCR analysis of HIFla, SOCS3, and adiponectin mRNA expression in adipocytes (FIG. 10B) and SVF- ⁇ (FIG. IOC) of Hifla lv and HIFla A dipo mice after 12 weeks on HFD.
  • the expression was normalized to ⁇ -actin and each bar represents the mean value + S.D. **P ⁇ 0.01 compared to wild- type floxed littermates.
  • FIG. 11 A is a pair of bar graphs showing SOCS3 mRNA in 3T3-L1 adipocytes after hypoxia (1% 0 2 ) and hypoxia mimicking reagent 100 ⁇ CoCl 2 treatment.
  • FIG. 1 IB is a series of bar graphs showing ARNT, SOCS3, and HIFla expression.
  • 3T3-L1 adipocytes were transfected with scrambled siRNA, ARNT siRNA, or HIFla siRNA. After 24 hours, cells were incubated with 100 ⁇ CoCl 2 . For qPCR analysis, the expression was normalized to ⁇ -actin and each bar represents the mean value + S.D.
  • FIG. 11C is a bar graph showing the of 3T3-L1 adipocytes transiently transfected with the Socs3 promoter luciferase construct and co- transfected with empty vector, HIFla or HIF2a expression plasmids. **P ⁇ 0.01 compared to control.
  • FIG. 1 ID is a pair of digital images of ChIP assays of the Socs3 promoter in 3T3-L1 cells treated with hypoxia for 8 hours using primers flanking HRE1 (left panel) or HRE2 and 3 (right panel).
  • FIG. 12A is a pair of bar graphs showing the effect of SOCS3 siRNA on expression of Socs3 (left) and adiponectin (right) in 3T3-L1 adipocytes.
  • FIG. 12B is a bar graph of adiponectin concentrations in medium of 3T3-L1 adipocytes transfected with scrambled siRNA or Socs3 siRNA. After 24 hours, cells were incubated for another 24 hours with 100 ⁇ CoCl 2 .
  • FIG. 12C is a digital image of a Western blot showing STAT3 phosphorylation in 3T3-L1 adipocytes with the indicated treatment.
  • FIG. 12A is a pair of bar graphs showing the effect of SOCS3 siRNA on expression of Socs3 (left) and adiponectin (right) in 3T3-L1 adipocytes.
  • FIG. 12B is a bar graph of adiponectin concentrations in medium of 3T3
  • 12D is a bar graph showing expression of adiponectin mRNA in 3T3-L1 cells treated with the indicated siRNA and the STAT3 inhibitor NSC 74859 (100 ⁇ ) in hypoxia conditions by 100 ⁇ CoCl 2 .
  • the expression was normalized to ⁇ -actin and each bar represents the mean value + S.D. **P ⁇ 0.01 compared to control (scramble siRNA), #P ⁇ 0.05 compared to scrambled siRNA + CoCl 2 (100 ⁇ ) or Hifla siRNA treatment.
  • FIGS. 13A and B are a series of graphs showing body mass (FIG. 13 A) and total fat mass and fat mass ratio (FIG. 13B) in mice treated with 12 weeks HFD and vehicle or NSC 74859 treatment for 6 weeks.
  • FIGS. 13C and D show GTT (FIG. 13C), and ITT (FIG. 13D) in mice treated with 11 weeks HFD and vehicle or NSC 74859 treatment for 5 weeks.
  • FIG. 13E is a series of graphs showing fasted glucose, fasted serum insulin levels, and HOMA index.
  • FIG. 13F is a series of graphs showing serum total adiponectin, HMW adiponectin, and HMW/total adiponectin ratio.
  • FIG. 13G is a digital image of Western blot analysis of STAT3
  • FIG. 14A is a graph showing growth curves of vehicle and ACF treated mice on HFD.
  • FIG. 14B is a pair of bar graphs showing fat mass and fat mass ratio of vehicle and ACF treated mice on HFD for 12 weeks.
  • FIG. 14C is a pair of graphs showing GTT (left) of vehicle and ACF treated mice on HFD for 11 weeks and ITT (right) of vehicle and ACF treated mice on HFD for 12 weeks. *P ⁇ 0.05 compared to ACF-treated mice.
  • FIG. 14D is a series of bar graphs showing fasted glucose, fasted serum insulin levels, and HOMA index of vehicle and ACF treated mice on HFD for 12 weeks.
  • FIG. 14E is a series of graphs showing serum total adiponectin, HMW adiponectin, and HMW/total adiponectin ratio of vehicle and ACF treated mice on HFD for 12 weeks.
  • FIG. 15A is a bar graph showing the effect of ACF (5 ⁇ ), CoCl 2 , or ACF plus CoCl 2 on expression of SOCS3 mRNA in 3T3-L1 adipocytes.
  • FIG. 15B is a pair of graphs showing GTT after 4 and 8 weeks of HFD.
  • FIG. 15C is a pair of graphs showing ITT after 5 and 9 weeks of HFD.
  • FIG. 15D is a series of graphs showing fasted glucose, fasted serum insulin, and HOMA index in vehicle and ACF treated mice after 6 weeks of HFD.
  • FIG. 15A is a bar graph showing the effect of ACF (5 ⁇ ), CoCl 2 , or ACF plus CoCl 2 on expression of SOCS3 mRNA in 3T3-L1 adipocytes.
  • FIG. 15B is a pair of graphs showing GTT after 4 and 8 weeks of HFD.
  • FIG. 15C is a pair of graphs showing ITT after 5 and 9 weeks of H
  • FIG. 16A is a graph showing typical growth curves of ACF-treated Hifla F/F (left panel) and Hifl a AAdipo (right panel) mice maintained on high-fat diet (HFD).
  • FIG. 17A is a graph showing blood glucose levels in 2-hour glucose tolerance test (GTT, top) and the area under the curve (AUC; bottom) 11 weeks after ACF treatment.
  • FIGS. 18A-B are a series of graphs showing fasted serum triglyceride, cholesterol, and FFA levels of ACF-treated HiflaTM and Hifla AAdipo mice, respectively, after 16 weeks of ACF treatment. *P ⁇ 0.05, **P ⁇ 0.01 compared to vehicle-treated mice.
  • FIGS. 19A-B are a series of graphs showing total adiponectin levels, HMW adiponectin, and the ratio of HMW to total adiponectin of ACF-treated Hifla F/F and Hifla A dipo mice, respectively, after 16 weeks of ACF treatment. *P ⁇ 0.05 compared to vehicle-treated mice.
  • Sequence_Listing.txt which was created on December 1, 2011, and is 16,823 bytes, which is incorporated by reference herein.
  • SEQ ID NOs: 1-4 are primers for chromatin immunoprecipitation assays.
  • SEQ ID NOs: 5-7 are primers for SOCS3 luciferase assays.
  • SEQ ID NOs: 8-13 are RT-PCR primers.
  • SEQ ID NOs: 14-67 are qPCR primers.
  • SEQ ID NOs: 68-73 are siRNAs.
  • SEQ ID NOs: 74-76 are exemplary white adipose tissue targeting peptides.
  • Adiponectin Also known as adiponectin, C1Q and collagen domain containing; 30 kDa adipocyte complement-related protein; adipose specific collagen-like factor; gelatin-binding protein 28 ⁇ e.g., GenBank Gene ID No. 9370). Adiponectin is expressed in adipose tissue. Receptors for adiponectin are found primarily in skeletal muscle and liver and adiponectin improves insulin sensitivity by increasing energy expenditure, fatty acid oxidation, and expression of PPARcc target genes (Rasouli and Kern, J. Clin. Endocrinol. Metab. 93:S64-S73, 2008).
  • Adiponectin nucleic acid and protein sequences are publicly available.
  • GenBank Accession Nos. NM_004797 and NM_001177800 disclose exemplary human adiponectin nucleic acid sequences
  • GenBank Accession Nos. NP_004788 and NP_00117127 disclose exemplary human adiponectin protein sequences, each of which is incorporated herein by reference as present in GenBank on December 16, 2010.
  • Body mass index A mathematical formula for measuring body mass in humans, also sometimes called Quetelet's Index. BMI is calculated by dividing weight (in kg) by height 2 (in meters 2 ). The current standards for both men and women accepted as "normal” are a BMI of 20-24.9 kg/m". In one embodiment, a BMI of greater than 25 kg/m can be used to identify an obese subject. Grade I obesity (also called “overweight”) corresponds to a BMI of 25-29.9 kg/m . Grade II obesity corresponds to a BMI of 30-40 kg/m ; and Grade III obesity corresponds to a BMI greater than 40 kg/m 2 (Jequier, Am. J Clin. Nutr., 45: 1035-47, 1987). Ideal body weight will vary among individuals based on height, body build, bone structure, and sex.
  • control refers to a sample or standard used for comparison with an experimental sample.
  • the control is a sample obtained from a healthy patient.
  • the control is a historical control or standard reference value or range of values (such as a previously tested sample, subject, or group of samples or subjects).
  • Diabetes mellitus A disease caused by a relative or absolute lack of insulin leading to uncontrolled carbohydrate metabolism, commonly simplified to
  • diabetes though diabetes mellitus should not be confused with diabetes insipidus. As used herein, “diabetes” refers to diabetes mellitus, unless otherwise indicated.
  • a “diabetic condition” includes pre-diabetes and diabetes.
  • Type 1 diabetes (sometimes referred to as “insulin-dependent diabetes” or “juvenile-onset diabetes”) is an auto- immune disease characterized by destruction of the pancreatic ⁇ cells that leads to a total or near total lack of insulin.
  • T2DM type 2 diabetes
  • non-insulin-dependent diabetes or “adult-onset diabetes”
  • Symptoms of diabetes include: excessive thirst (polydipsia); frequent urination (polyuria); extreme hunger or constant eating (polyphagia); unexplained weight loss; presence of glucose in the urine (glycosuria); tiredness or fatigue; changes in vision; numbness or tingling in the extremities (hands, feet); slow- healing wounds or sores; and abnormally high frequency of infection.
  • Diabetes may be clinically diagnosed by a fasting plasma glucose (FPG) concentration of greater than or equal to 7.0 mmol/L (126 mg/dL), or a plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two hours after an oral glucose tolerance test (OGTT) with a 75 g load.
  • FPG fasting plasma glucose
  • OGTT oral glucose tolerance test
  • Hypoxia-inducible factor 1 A transcription factor found in mammalian cells cultured under reduced oxygen tension (hypoxia) that plays a role in cellular and systemic response to hypoxia.
  • HIF1 is a heterodimer composed of an alpha subunit and a beta subunit.
  • the beta subunit is the aryl hydrocarbon receptor nuclear translocator (Arnt; also known as HIFip).
  • Arnt is constitutively present in the cell nucleus.
  • HIFlcc HIF2cc (also known an EPAS1)
  • HIF3CC Hypoxia-inducible factor 1
  • HIF alpha subunits are hydroxylated by a prolyl hydroxylase and targeted for proteasome dependent degradation.
  • the prolyl hydroxylase is inhibited in hypoxia, leading to accumulation of HIF alpha subunits.
  • HIF alpha subunits dimerize with Arnt to form a functional transcription factor capable of binding DNA at hypoxia response elements (HRE) and transcriptional activation.
  • HRE hypoxia response elements
  • HIF1 nucleic acid and protein sequences are publicly available.
  • GenBank Accession Nos. NM_001530 and NM_181054 disclose exemplary human HIFlcc nucleic acid sequences and GenBank Accession Nos. NP_001521 and NP_851397 disclose exemplary human HIFlcc protein sequences.
  • GenBank Accession No. NM_001430 discloses an exemplary human HIF2cc nucleic acid sequence and GenBank Accession No. NP_001421 discloses an exemplary human HIF2cc protein sequence.
  • GenBank Accession Nos. NM_152795, NM_022462, and NM_152794 disclose exemplary human HIF3cc nucleic acid sequences and GenBank Accession Nos.
  • NP_690008, NP_071907, and NP_690007 disclose exemplary human HIF3 protein sequences.
  • GenBank Accession Nos. NM_001668 and NM_178427 disclose exemplary human Arnt nucleic acid sequences
  • GenBank Accession Nos. NP_001659 and NP_848514 disclose exemplary human Arnt protein sequences. Each of these sequences is incorporated by reference herein, as present in GenBank on December 16, 2010.
  • Inhibitor Any chemical compound, nucleic acid molecule, or peptide (such as an antibody), specific for a gene product that can reduce activity of a gene product or directly interfere with expression of a gene, such as genes that encode HIF1, such as HIFlcc.
  • An inhibitor of the disclosure can inhibit the activity of a HIF la protein either directly or indirectly. Direct inhibition can be accomplished, for example, by binding to a HIF la protein and thereby preventing the protein from binding an intended target, such as a dimerization partner (e.g., Arnt) or a DNA sequence (such as a HRE).
  • a dimerization partner e.g., Arnt
  • DNA sequence such as a HRE
  • Indirect inhibition can be accomplished, for example, by binding to a HIF la protein intended target, such as a receptor or binding partner, thereby blocking or reducing activity of HIF la.
  • an inhibitor of the disclosure can inhibit a HIF la gene by reducing or inhibiting expression of the gene, inter alia by interfering with gene expression (transcription, processing, translation, post-translational modification), for example, by interfering with the mRNA and blocking translation of the HIF la gene product or by altering post-translational modification of a HIFla gene product (such as prolyl
  • Insulin resistance A state in which the cells of a subject do not respond appropriately to insulin, and increased amounts of insulin are required for glucose to be taken up by the cells.
  • insulin resistance is defined as a state where 200 units of insulin per day or more are required to attain glycemic control and prevent ketosis.
  • Subjects with insulin resistance often have increased plasma glucose levels, increased plasma insulin levels, or both, as compared with a subject without insulin resistance or standard normal ranges.
  • insulin resistance is determined by measuring blood glucose (such as fasting plasma glucose) and/or blood insulin (such as fasting plasma insulin) levels.
  • insulin resistance is determined by oral glucose tolerance test, glucose clamp (such as hyperinsulinemic euglycemic clamp), modified insulin suppression test, homeostatic model assessment, or quantitative insulin sensitivity check index (QUICKI).
  • Obesity A condition in which excess body fat may put a person at health risk (see Barlow and Dietz, Pediatrics 102: E29, 1998; National Institutes of Health, Obes. Res. 6 (suppl. 2):51S-209S, 1998). Excess body fat is a result of an imbalance of energy intake and energy expenditure.
  • the Body Mass Index (BMI) is used to assess obesity.
  • waist circumference is used to assess obesity.
  • a waist circumference of 102 cm or more is considered obese, while in women a waist circumference of 89 cm or more is considered obese.
  • obesity affects both the morbidity and mortality of individuals.
  • an obese individual is at increased risk for heart disease, non-insulin dependent (type 2) diabetes, hypertension, stroke, cancer ⁇ e.g. endometrial, breast, prostate, and colon cancer), dyslipidemia, gall bladder disease, sleep apnea, reduced fertility, and osteoarthritis, amongst others (see Lyznicki et ah, Am. Fam. Phys. 63:2185, 2001).
  • compositions useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor,
  • compositions and formulations suitable for pharmaceutical delivery of compounds such as an inhibitor of HIFlcc (for example, acriflavine).
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified preparation of a HIFlcc inhibitor is one in which the HIFlcc inhibitor is more enriched than in its
  • a preparation is purified such that the HIFlcc inhibitor represents at least 50% of the total content of the preparation, for example, at least 50% by weight.
  • the HIFlcc inhibitor is at least 50%, for example at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more free of proteins, lipids, carbohydrates or other materials with which it is originally associated.
  • RNA interference refers to a cellular process that inhibits expression of genes, including cellular and viral genes. RNAi is a form of antisense- mediated gene silencing involving the introduction of double stranded RNA-like oligonucleotides leading to the sequence- specific reduction of RNA transcripts. Double-stranded RNA molecules that inhibit gene expression through the RNAi pathway include siRNAs, miRNAs, and shRNAs.
  • siRNA Short (or Small) Interfering Nucleotide Sequence: A nucleotide sequence capable of interfering with gene expression, for instance by inducing gene- specific inhibition of expression. Typically, the sequence of a siRNA is
  • RNA substantially identical to a portion of a transcript of a target gene (mRNA) for which interference or inhibition of expression is desired.
  • mRNA target gene
  • small, double stranded RNAs of about 15 to about 40 nucleotides in length (the length of each of the individual strands of the dsRNA), such as about 15 to about 25 nucleotides in length (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides), that interfere with, or inhibit, expression of a target sequence.
  • the RNA backbone and/or component nucleotides can be unmodified or modified.
  • the dsRNA can contain one or more deoxynucleic acids. Synthetic small dsRNAs may be used to induce gene-specific inhibition of expression.
  • the dsRNAs can be formed from complementary single stranded RNAs ("ssRNAs") or from a ssRNA that forms a hairpin or from expression from a DNA vector.
  • ssRNAs complementary single stranded RNAs
  • these small interfering nucleotide sequences have 3' and/or 5' overhangs on each strand of the duplex. These overhangs can be 0 nucleotides (that is, blunt ends) to 5 nucleotides in length.
  • siRNA molecules can be used as reverse genetic and therapeutic tools in mammalian cells, including human cells, both in vitro and in vivo.
  • These small interfering nucleotide sequences are suitable for interference or inhibition of expression of a target gene wherein the sequence of the small interfering nucleotide sequence is substantially identical to a portion of an mRNA or transcript of the target gene for which interference or inhibition of expression is desired.
  • nucleotides suitable for inhibiting or interfering with the expression of a target sequence include nucleotide derivatives and analogs.
  • a non-natural linkage between nucleotide residues can be used, such as a phosphorothioate linkage.
  • the nucleotide strand can be derivatized with a reactive functional group or a reporter group, such as a fluorophore.
  • the 2'-hydroxyl at the 3' terminus can be readily and selectively derivatized with a variety of groups.
  • nucleotide derivatives incorporate nucleotides having modified carbohydrate moieties, such as 2'-0-alkylated residues or 2'-deoxy- 2'-halogenated derivatives.
  • modified carbohydrate moieties include 2'-0-methyl ribosyl derivatives and 2'-0-fluoro ribosyl derivatives.
  • the nucleotide bases can be modified. Any modified base useful for inhibiting or interfering with the expression of a target sequence can be used. For example, halogenated bases, such as 5-bromouracil and 5-iodouracil can be incorporated.
  • the bases may also be alkylated, for example, 7-methylguanosine may be incorporated in place of a guanosine residue.
  • Non-natural bases that yield successful inhibition can also be incorporated.
  • Subject A living multi-cellular vertebrate organism, a category that includes both human and non-human mammals.
  • Therapeutically effective amount An amount or dose sufficient to prevent advancement, or to cause regression of a disease or syndrome or is capable of relieving symptoms of a disease or syndrome (such as diabetes, obesity, or insulin resistance).
  • Treatment Refers to both prophylactic inhibition of initial disease or syndrome, and therapeutic interventions to alter the natural course of a disease process or syndrome, such as diabetes, obesity, or insulin resistance.
  • the methods include administering a therapeutically effective amount of a composition including a HIFla inhibitor (such as acriflavine) to a subject, thereby decreasing body weight of the subject.
  • the methods include administering a therapeutically effective amount of a composition including a HIFla inhibitor (such as acriflavine) to a subject having diabetes, thereby treating diabetes in the subject.
  • administering an inhibitor of HIFla to a subject increases levels of total adiponectin, high molecular weight (HMW) adiponectin, and/or the ratio of HMW to total adiponectin, for example in a serum sample from the subject.
  • Adiponectin is a bioactive peptide secreted from adipocytes that is considered an adipokine.
  • Receptors for adiponectin are found primarily in skeletal muscle and liver and adiponectin improves insulin sensitivity by increasing energy expenditure, fatty acid oxidation, and expression of PPARa target genes (Rasouli and Kern, J. Clin. Endocrinol. Metab. 93:S64-S73, 2008).
  • administration of a HIFla inhibitor reduces body weight and/or treats diabetes by increasing circulating levels of total adiponectin, HMW adiponectin, HMW:total adiponectin ratio, or a combination of two or more thereof.
  • one or more of total adiponectin, HMW adiponectin, or HMW:total adiponectin ratio is increased by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,
  • blood adiponectin levels are increased to more than about 8 ⁇ g/mL (such as about 8-13.9 ⁇ g/mL).
  • the disclosed methods include measuring adiponectin (such as total adiponectin, HMW adiponectin, and/or HMW:total adiponectin ratio) in a sample from a subject.
  • the disclosure includes decreasing body weight of a subject by administering a therapeutically effective amount of a composition including an inhibitor of HIFlcc to a subject.
  • the method includes selecting a subject in need of decreasing body weight (such as an overweight or obese subject).
  • a subject may be considered overweight or obese if their BMI is greater than 25 kg/m2, their waist circumference is greater than 35 inches (female) or 40 inches (male) or body fat percentage is greater than 25% (male) or 32% (female).
  • decreasing body weight includes one or more of decreasing total body weight, decreasing BMI, decreasing waist
  • the disclosed methods include measuring total body weight, BMI, waist circumference, and/or body fat amount in a subject.
  • decreasing body weight of a subject includes reducing total body weight of the subject by at least about 1% (such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more).
  • reduction in total body weight is determined relative to the starting total body weight of the subject (for example, prior to treatment with a HIFlcc inhibitor).
  • decreasing body weight of a subject includes decreasing BMI of the subject by at least about 1% (such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more).
  • BMI is calculated by dividing weight (in kg) by height 2 (in meters 2 ).
  • the current standards for both men and women accepted as "normal” are a BMI of 20-24.9 kg/m".
  • Grade I obesity (also called “overweight”) corresponds to a BMI of 25-29.9 kg/m .
  • Grade II obesity corresponds to a BMI of 30-40 kg/m ; and
  • Grade III obesity corresponds to a
  • BMI greater than 40 kg/m reduction in BMI is determined relative to the starting BMI of the subject (for example, prior to treatment with a HIFl inhibitor).
  • decreasing BMI of a subject includes reduction of BMI from a starting point (for example BMI greater than 30 kg/m ) to a target level (for example, BMI less than 30 kg/m 2 , 29 kg/m 2 , 28 kg/m 2 , 27 kg/m 2 , 26 kg/m 2 , or 25 kg/m 2 ).
  • decreasing body weight of a subject includes decreasing waist circumference by at least 1% (such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more).
  • reduction in waist circumference is determined relative to the starting waist circumference of the subject (for example, prior to treatment with a HIFla inhibitor).
  • decreasing waist circumference of a subject includes reduction of waist
  • a starting point for example greater than 40 inches for men or greater than 35 inches for women
  • a target level for example, waist
  • decreasing body weight of a subject includes decreasing body fat (such as total body fat, subcutaneous body fat, or visceral body fat) of the subject by at least 1% (such as at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, or more).
  • body fat such as total body fat, subcutaneous body fat, or visceral body fat
  • Methods of determining body fat are known to one of skill in the art. Such methods include near-infrared interactance, dual energy X-ray absorptiometry, body average density measurement, bioelectrical impedance analysis, skinfold tests (for example, Durnin- Womersley skinfold method or Jackson-Pollock skinfold method), and U.S. Navy circumference method.
  • reduction in body fat is determined relative to the starting body fat of the subject (for example, prior to treatment with a HIFla inhibitor).
  • decreasing body fat of a subject includes reduction of body fat from a starting point (for example greater than about 25% body fat for men or greater than about 32% body fat for women) to a target level (for example, body fat of less than about 25% for men or less than about 32% for women).
  • a target body fat level may be about 14-24% body fat for men or about 21-31% body fat for women.
  • the disclosure includes treating diabetes or prediabetes in a subject by administering a therapeutically effective amount of a composition including an inhibitor of HIFlcc to the subject.
  • the method includes selecting a subject with diabetes or at risk for diabetes (such as a subject with pre-diabetes).
  • a subject with diabetes may be clinically diagnosed by a fasting plasma glucose (FPG) concentration of greater than or equal to 7.0 mmol/L (126 mg/dL), or a plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL) at about two hours after an oral glucose tolerance test (OGTT) with a 75 g load, or in a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma glucose concentration of greater than or equal to 11.1 mmol/L (200 mg/dL), or HbAlc levels of greater than or equal to 6.5%.
  • FPG fasting plasma glucose
  • OGTT oral glucose tolerance test
  • a subject with pre-diabetes may be diagnosed by impaired glucose tolerance (IGT).
  • An OGTT two-hour plasma glucose of greater than or equal to 140 mg/dL and less than 200 mg/dL (7.8-11.0 mM), or a fasting plasma glucose (FPG) concentration of greater than or equal to 100 mg/dL and less than 125 mg/dL (5.6-6.9 mmol/L), or HbAlc levels of greater than or equal to 5.7% and less than 6.4% (5.7-6.4%) is considered to be IGT, and indicates that a subject has pre-diabetes. A more detailed description of diabetes may be found in
  • treating diabetes includes one or more of increasing glucose tolerance, decreasing insulin resistance (for example, decreasing plasma glucose levels, decreasing plasma insulin levels, or a combination thereof), decreasing serum triglycerides, decreasing free fatty acid levels, and decreasing HbAlc levels in the subject.
  • the disclosed methods include measuring glucose tolerance, insulin resistance, plasma glucose levels, plasma insulin levels, serum triglycerides, free fatty acids, and/or HbAlc levels in a subject.
  • administration of a HIFlcc inhibitor treats diabetes by increasing glucose tolerance, for example, by decreasing blood glucose levels (such as two-hour plasma glucose in an OGTT or FPG) in a subject.
  • the method includes decreasing blood glucose by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control.
  • a decrease in blood glucose level is determined relative to the starting blood glucose level of the subject (for example, prior to treatment with a HIFlcc inhibitor).
  • decreasing blood glucose levels of a subject includes reduction of blood glucose from a starting point (for example greater than about 126 mg/dL FPG or greater than about 200 mg/dL OGTT two-hour plasma glucose) to a target level (for example, FPG of less than 126 mg/dL or OGTT two-hour plasma glucose of less than 200 mg/dL).
  • a target FPG may be less than 100 mg/dL.
  • a target OGTT two-hour plasma glucose may be less than 140 mg/dL.
  • the disclosed methods include treating a subject with diabetes by decreasing insulin resistance in the subject.
  • a subject with insulin resistance is a subject with diabetes, while in other examples, a subject with insulin resistance does not have diabetes, but may, for example, be pre-diabetic.
  • Insulin resistance is a decreased sensitivity or responsiveness to the metabolic actions of insulin. In some examples, insulin resistance results in increased blood glucose and/or increased blood insulin levels (such as fasting blood glucose or fasting blood insulin levels).
  • insulin resistance is determined by hyperinsulinemic euglycemic clamp (glucose clamp), which measures the amount of glucose necessary to compensate for increased insulin levels without causing hypoglycemia (see, e.g., DeFronzo et ah, Am. J. Physiol. 237:E214-E223, 1979).
  • the glucose clamp method includes infusing insulin in a subject at 10-120
  • glucose such as ⁇ 4 mg/min
  • ITT impaired glucose tolerance
  • administering decreases insulin resistance by increasing the amount of glucose required to maintain blood glucose levels in a glucose clamp in a subject, for example, by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control.
  • the method includes increasing the amount of glucose required to maintain blood glucose levels in a glucose clamp to >4 mg/min glucose. In other examples, the method includes increasing the amount of glucose required to maintain blood glucose levels in a glucose clamp to >7.5 mg/min glucose.
  • insulin resistance is determined by the frequently sampled intravenous glucose tolerance test (FSIVGTT; Bergman, Diabetes 38: 1512- 1527, 1989).
  • FSIVGTT is performed by administering intravenous glucose with frequent blood sampling to determine glucose and insulin levels. Insulin is injected 20 minutes after the start of glucose administration.
  • the insulin sensitivity index (SI) reflecting increase in fractional glucose disappearance per unit of insulin increase, is calculated. In some examples, an SI value of ⁇ 2 ⁇ /min/mL indicates insulin resistance.
  • administration of a FHFlcc inhibitor decreases insulin resistance by increasing the insulin sensitivity index of a subject, for example, by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIFlcc inhibitor).
  • the method includes increasing the insulin sensitivity index to >2 ⁇ /min/mL.
  • insulin resistance is determined by QUICKI (Katz et ah, J. Clin. Endocrinol. Metab. 85:2402-2410, 2000).
  • administration of an inhibitor of HIFlcc decreases insulin resistance by increasing the QUICKI value in a subject by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIFla inhibitor).
  • the method includes increasing the subject's QUICKI to >0.350.
  • insulin resistance is determined by the homeostasis model assessment (HOMA-IR; Matthews et al, Diabetologia 28:412-429, 1985).
  • HOMA-IR is calculated from fasting glucose and fasting insulin levels:
  • HOMA-IR [fasting plasma insulin x fasting plasma glucose]/22.5 wherein fasting plasma insulin is expressed as ⁇ /mL and fasting plasma glucose is expressed as mM.
  • administration of an inhibitor of HIFl decreases insulin resistance by decreasing the HOMA-IR value in a subject by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIFla inhibitor).
  • the method includes decreasing HOMA-IR to ⁇ 4.
  • administering decreases insulin resistance by decreasing plasma insulin levels (such as fasting plasma insulin or 2-hour insulin levels following OGTT) in a subject, for example, decreasing plasma insulin levels by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to
  • the method includes decreasing fasting plasma insulin levels to ⁇ 15 ⁇ /mL.
  • Methods to measure plasma insulin in a subject for example, in a blood sample from a subject, such as immunoassays, are routine.
  • insulin sensitive subjects include the top 25 th percentile of insulin sensitive subjects in a given cohort where insulin levels are measured in the same central reference laboratory.
  • insulin resistant subjects include the bottom 25 th percentile of insulin sensitive subjects in a given cohort where insulin levels are measured in the same central reference laboratory.
  • impaired glucose tolerance can be defined according the results of an oral glucose tolerance test using guidelines that are published by the American Diabetes Association. See, e.g., Diabetes Care 33:S62-S69, 2010.
  • the disclosed methods include treating a subject with diabetes by decreasing triglyceride or free fatty acid levels in the subject.
  • administration of an inhibitor of HIFlcc treats diabetes by decreasing blood triglyceride levels in a subject, for example decreasing triglyceride levels by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIFlcc inhibitor).
  • the method includes decreasing triglyceride levels in a subject to ⁇ 150 mg/dL. Methods of determining triglyceride levels in a subject (for example in a blood sample from a subject) are routine.
  • administration of a HIF1 a inhibitor treats diabetes by decreasing blood free fatty acid levels in a subject, for example, decreasing free fatty acid levels by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the FHFlcc inhibitor).
  • the method includes decreasing free fatty acid levels below 0.6 mmol/L (such as about 0.1-0.6 mmol/L). Methods of determining free fatty acid levels in a subject (for example, in a blood sample from a subject) are routine.
  • the disclosed methods include treating a subject with diabetes by decreasing HbAlc levels in the subject.
  • HbAlc glycated hemoglobin
  • HbAlc level is a marker for average blood glucose levels over time (such as weeks or months) and is an indicator of glycemic control in a subject.
  • administering treats diabetes by decreasing HbAlc levels in a subject, for example decreasing HbAlc levels by at least 5% (such as at least 10%, 15%, 20%, 25%, 30%, 35%, or more) as compared with a control (such as the subject prior to administration of the HIFlcc inhibitor).
  • the method includes decreasing HbAlc levels in a subject to less 7% (such as 6.5%, 6%, 5.5%, 5%, or less).
  • Methods of determining HbAlc levels in a subject are routine, for example, by HPLC or immunoassay.
  • the disclosed methods include comparing one or more indicators of body weight or obesity (such as body weight, body mass index, waist circumference, or body fat) to a control, wherein a decrease in the particular indicator relative to the control indicates effective reduction of body weight or treatment of obesity.
  • body weight or obesity such as body weight, body mass index, waist circumference, or body fat
  • the control can be any suitable control against which to compare the indicator of body weight or obesity in a subject.
  • the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of subjects which are overweight or obese, or group of samples from subjects which are not overweight or obese).
  • the control is a reference value, such as a standard value obtained from a population of individuals that is used by those of skill in the art. Similar to a control population, the value of the sample from the subject can be compared to the mean reference value or to a range of reference values (such as the high and low values in the reference group or the 95% confidence interval).
  • control is the subject (or group of subjects) treated with placebo compared to the same subject (or group of subjects) treated with the therapeutic compound in a cross-over study. In further examples, the control is the subject (or group of subjects) prior to treatment with the HIFlcc inhibitor.
  • the disclosed methods include comparing one or more indicator of diabetes (such as glucose tolerance, insulin resistance, triglyceride levels, free fatty acid levels, or HbAlc levels) to a control, wherein an increase or decrease in the particular indicator relative to the control (as discussed above) indicates effective treatment of diabetes.
  • the disclosed methods include comparing one or more indicator of insulin resistance (such as blood glucose levels, blood insulin levels, insulin sensitivity index, HOMA-IR, or QUICKI) to a control, wherein a decrease in the particular indicator relative to the control (as discussed above) indicates effective treatment of insulin resistance.
  • the disclosed methods include comparing adiponectin levels to a control, wherein an increase in adiponectin levels (such as total adiponectin, HMW adiponectin, and/or the ratio of HMW to total adiponectin) indicates an effective treatment of diabetes or obesity.
  • adiponectin levels such as total adiponectin, HMW adiponectin, and/or the ratio of HMW to total adiponectin
  • the control can be any suitable control against which to compare the indicator of diabetes in a subject.
  • the control is a sample obtained from a healthy subject (such as a subject without diabetes).
  • the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of subjects with diabetes, or group of samples from subjects that do not have diabetes).
  • the control is a reference value, such as a standard value obtained from a population of normal individuals that is used by those of skill in the art. Similar to a control population, the value of the sample from the subject can be compared to the mean reference value or to a range of reference values (such as the high and low values in the reference group or the 95% confidence interval).
  • control is the subject (or group of subjects) treated with placebo compared to the same subject (or group of subjects) treated with the therapeutic compound in a cross-over study. In further examples, the control is the subject (or group of subjects) prior to treatment with the HIFlcc inhibitor. IV. HIFlcc Inhibitors
  • HIFlcc inhibitors include compounds that decrease the expression, longevity (e.g., half-life) or activity of HIFl cc (directly or indirectly), for example, relative to a control. Direct inhibition can be
  • HIF la protein accomplished, for example, by binding to a HIF la protein and thereby preventing the protein from binding an intended target, such as a dimerization partner (e.g., Arnt) or a DNA sequence (such as a HRE).
  • a dimerization partner e.g., Arnt
  • a DNA sequence such as a HRE
  • Indirect inhibition can be accomplished, for example, by binding to a HIF la protein intended target, such as a receptor or binding partner, thereby blocking or reducing activity of the protein.
  • an inhibitor of the disclosure can inhibit a HIF la gene by reducing or inhibiting expression of the gene, inter alia by interfering with gene expression (transcription, processing, translation, post-translational modification, or stability), for example, by interfering with the mRNA and blocking translation of the HIFl gene product or by altering post-translational modification of a HIFla gene product (such as prolyl hydroxylation), or by causing changes in intracellular localization.
  • HIFla inhibitors can include small organic molecules. Some small molecule inhibitors may inhibit multiple HIF alpha subunits (such as HIFla, HIF2a, and/or HIF3a), while others may be specific for HIFla. In some examples, a small molecule inhibitor inhibits more than one HIF alpha subunit, including HIFla. In particular examples, a small molecule inhibitor specifically inhibits HIFla expression or activity (such as dimerization, DNA binding, and/or transcriptional activity).
  • the small molecule inhibitor of HIFla is a previously identified HIFla inhibitor.
  • the inhibitor is acriflavine (ACF; see, e.g., Lee et al, Proc. Natl. Acad. Sci. USA 106:17910-17915, 2009).
  • ACF is a mixture of 3,6-diamino-lO-methylacridinium chloride (trypaflavin) and 3,6-diaminoacridine (proflavine), such as about a 1:1 mixture.
  • a HIFla small molecule inhibitor is a cardiac glycoside, such as digoxin, ouabain, proscillaridin A, digitoxin, acetydigitoxin, convallatoxin, peruvoside, strophanthin K, nerifolin, cymarin, or periplocymarin (see e.g., hang et al., Proc. Natl. Acad. Sci.
  • rapamycin or an analog thereof such as rapamycin, everolimus, temsirolimus, or tacrolimus
  • an anthracycline or analog thereof such as doxorubicin or daunorubicin
  • a proteasome inhibitor such as bortezomib (PS-341)
  • camptothecin or an analog thereof such as CRLX-101, SN-38, EZN-2208, irinotecan, or topotecan.
  • a HIFla small molecule inhibitor includes echinomycin (see, e.g., Kong et al., Cancer Res.
  • a small molecule inhibitor of HIFla is aminoflavone (e.g., Terzuoli et al, Cancer Res. 20:6837-6848, 2010). It is to be understood that HIFla inhibitors for use in the present disclosure also include novel HIFl small molecule inhibitors developed in the future.
  • HIFla inhibitors also include nucleic acid molecules, including, but not limited to antisense molecules. Some HIFla nucleic acid inhibitors may inhibit multiple HIF alpha subunits (such as HIFla, HIF2a, and/or HIF3a), while others may be specific for HIFla. In some examples, a nucleic acid inhibitor inhibits more than one HIF alpha subunit, including HIFla. In particular examples, a nucleic acid inhibitor specifically inhibits HIFla expression or activity (such as dimerization, DNA binding, and/or transcriptional activity).
  • the nucleic acid inhibitor of HIFla decreases expression of HIFla, such as an antisense molecule or an RNAi molecule (such as siRNA, miRNA, or shRNA).
  • a HIFla siRNA includes SEQ ID NOs: 70 or 71.
  • the inhibitor is RX-0047 or RX-0149 (see, e.g., U.S. Pat. No. 7,205,283), double- stranded HIF decoy oligonucleotides (e.g., WO 2005/056795), ACU-HHY-011 (Opko Health), or EZN-2968 (see, e.g., U.S. Pat. Nos. 7,589,190 and 7,737,264).
  • the nucleic acid inhibitor of HIFla decreases activity of HIFla, such as a G-rich
  • HIFla inhibitors for use in the present disclosure also include novel HIFla nucleic acid molecule inhibitors developed in the future.
  • inhibitors of HIFl can include HIFl - specific binding agents, such as polyclonal or monoclonal antibodies.
  • HIF la-specific binding agents include HIFla-specific antibodies or functional fragments thereof, for instance monoclonal antibodies or fragments of monoclonal antibodies, including humanized monoclonal antibodies. Methods for producing antibodies (including monoclonal antibodies) using standard procedures are described in a number of texts, including Harlow and Lane (Antibodies, A).
  • Monoclonal or polyclonal antibodies may be produced to either the normal HIFla protein or mutant forms of this protein, for instance particular portions that contain a mutation and therefore may provide a distinguishing epitope.
  • antibodies raised against these proteins or peptides specifically detect the protein or peptide with which the antibodies are generated. That is, an antibody generated to the HIFla protein or a fragment thereof would recognize and bind the HIFla protein and would not substantially recognize or bind to other proteins found in mammalian cells (for example, human cells).
  • Antibodies for HIFla can also be obtained from commercially available sources, including from Santa Cruz Biotechnology (Santa Cruz, CA), Abeam (Cambridge, MA), and Millipore (Billerica, MA). It is to be understood that HIFla antibodies for use in the present disclosure also include novel anti-HIFla antibodies developed in the future.
  • the HIFl inhibitor is targeted to adipose tissue (such as white adipose tissue).
  • WAT targeting motifs include peptides that preferentially bind to WAT cells or WAT vasculature.
  • the targeting peptide includes CKGGRAKDC (SEQ ID NO: 74), CMLAGWIPC (SEQ ID NO: 75), and
  • an adipose tissue targeting molecule includes an antibody that preferentially binds to adipose tissue (such as WAT), for example an antibody that preferentially binds to resistin.
  • a HIFl inhibitor is coupled to an adipose tissue targeting molecule (such as a peptide or antibody).
  • compositions that include an inhibitor of HIFl can be formulated with an appropriate pharmaceutically acceptable carrier, depending upon the particular mode of administration chosen.
  • the pharmaceutical composition includes a HIFl inhibitor and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition consists essentially of an inhibitor of HIFla and a pharmaceutically acceptable carrier.
  • parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • injectable fluids such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like.
  • compositions ⁇ e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non- toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, pH buffering agents, or the like, for example sodium acetate or sorbitan monolaurate.
  • Excipients that can be included are, for instance, other proteins, such as human serum albumin or plasma preparations.
  • the HIFlcc inhibitor is included in a controlled release formulation, for example, a microencapsulated formulation.
  • a controlled release formulation for example, a microencapsulated formulation.
  • biodegradable and biocompatible polymers methods can be used, and methods of encapsulating a variety of synthetic compounds, proteins and nucleic acids, have been well described in the art (see, for example, U.S. Pat. Publication Nos.
  • the HIFlcc inhibitor is included in a nanodispersion system.
  • Nanodispersion systems and methods for producing such nanodispersions are well known to one of skill in the art. See, e.g., U.S. Pat. No. 6,780,324; U.S. Pat. Publication No. 2009/0175953.
  • a nanodispersion system includes a biologically active agent and a dispersing agent (such as a polymer, copolymer, or low molecular weight surfactant).
  • Exemplary polymers or copolymers include polyvinylpyrrolidone (PVP), poly(D,L-lactic acid) (PLA), poly(D,L-lactic-co-glycolic acid (PLGA), poly(ethylene glycol).
  • Exemplary low molecular weight surfactants include sodium dodecyl sulfate, hexadecyl pyridinium chloride, polysorbates, sorbitans, poly(oxyethylene) alkyl ethers, poly(oxyethylene) alkyl esters, and combinations thereof.
  • the nanodispersion system includes PVP and a HIFlcc inhibitor (such as 80/20 w/w).
  • the nanodispersion is prepared using the solvent evaporation method. See, e.g., Kanaze et ah, Drug Dev. Indus. Pharm. 36:292-301, 2010; Kanaze et al, J. Appl. Polymer Sci. 102:460-471, 2006.
  • the HIFlcc inhibitor includes pharmaceutically acceptable salts of such compounds.
  • “Pharmaceutically acceptable salts” of the presently disclosed compounds include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, ⁇ , ⁇ '- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, diethylamine, piperazine,
  • salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a pharmaceutically acceptable salt thereof.
  • “Pharmaceutically acceptable salts” are also inclusive of the free acid, base, and zwitterionic forms. Description of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002).
  • the pharmaceutical compositions disclosed herein comprise a HIFlcc inhibitor and at least one pharmaceutically acceptable carrier.
  • the composition consists essentially of a HIFlcc inhibitor and at least one pharmaceutically acceptable carrier.
  • additional active compounds for example additional inhibitors of HIFlcc
  • other inert agents such as fillers, wetting agents, or the like
  • the dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. For instance, in addition to injectable fluids, topical, inhalation, oral and suppository formulations can be employed. Topical preparations can include eye drops, ointments, sprays, patches and the like.
  • Inhalation preparations can be liquid (e.g., solutions or suspensions) and include mists, sprays and the like.
  • Oral formulations can be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules).
  • Suppository preparations can also be solid, gel, or in a suspension form.
  • conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, cellulose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
  • compositions that include an inhibitor of HIFlcc can be formulated in unit dosage form, suitable for individual administration of precise dosages.
  • a unit dosage contains from about 1 mg to about 1 g of an HIFlcc inhibitor (such as about 10 mg to about 100 mg, about 50 mg to about 500 mg, about 100 mg to about 900 mg, about 250 mg to about 750 mg, or about 400 mg to about 600 mg HIFlcc inhibitor).
  • the amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated.
  • compositions of this disclosure including an inhibitor of HIFlcc can be administered to humans or other animals on whose tissues they are effective in various manners such as orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally, intrathecally, subcutaneously, via inhalation or via suppository.
  • the composition is administered orally.
  • site-specific administration of the composition can be used, for example by administering a HIFlcc inhibitor to adipose tissue (for example by injection in adipose tissue, such as visceral adipose tissue).
  • the particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g.
  • Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.
  • treatment involves once daily dose of a HIFlcc inhibitor (such as acriflavine).
  • a therapeutically effective amount of a HIFlcc inhibitor is about 0.01 mg/kg to about 50 mg/kg (for example, about 0.5 mg/kg to about 25 mg/kg or about 1 mg/kg to about 10 mg/kg). In a specific example, a therapeutically effective amount of a HIFlcc inhibitor is about 1 mg/kg to about 5 mg/kg, for example about 2 mg/kg. In a particular example, a therapeutically effective amount of a HIFlcc inhibitor includes about 1 mg/kg to about 10 mg/kg acriflavine, such as about 2 mg/kg acriflavine.
  • a therapeutically effective amount of a HIFlcc inhibitor can be the amount of a HIFlcc inhibitor necessary to treat diabetes or reduce body weight in a subject.
  • a therapeutically effective amount of an inhibitor of HIFlcc can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the therapeutically effective amount will be dependent on the subject being treated, the severity and type of the affliction, and the manner of
  • the present disclosure also includes combinations of a HIFlcc inhibitor with one or more other agents useful in the treatment of diabetes or insulin resistance or in the reduction of body weight.
  • the compounds of this disclosure can be administered in combination with effective doses of anti-diabetic agents (such as biguanides, thiazolidinediones, or incretins) and/or lipid lowering compounds (such as statins or fibrates).
  • anti-diabetic agents such as biguanides, thiazolidinediones, or incretins
  • lipid lowering compounds such as statins or fibrates.
  • administration in combination or “coadministration” refers to both concurrent and sequential administration of the active agents.
  • Administration of a HIFlcc inhibitor may also be in combination with lifestyle modifications, such as increased physical activity, low fat diet, and smoking cessation.
  • a subject that has diabetes or a subject with insulin resistance is a candidate for treatment using the therapeutic methods disclosed herein.
  • a subject in need of a reduction in body weight for example, a subject with overweight or obesity (for example, a subject with a body mass index of 25 kg/m or more) is a candidate for treatment using the therapeutic methods herein.
  • mice were on the C57BL/6 background. Only male mice were used for experiments.
  • the adipocyte-specific knockout mice of the Arnt and Hifl a genes were designated Arnt Adipo and Hifl a Adipo mice, respectively.
  • Mice were housed in temperature- and light-controlled rooms and were supplied with water and pelleted NIH-31 standard chow diet consisting of 10 kcal fat ad libitum.
  • HFD high-fat diet
  • 6-week-old male mice were given HFD consisting of 60 kcal fat (BioServ, Frenchtown, NJ) for 12 weeks. All animal studies were performed in accordance with Institute of Laboratory Animal Resources guidelines and approved by the National Cancer Institute Animal Care and Use Committee.
  • mice were fasted for 16 hours, blood was drawn, and mice were injected intraperitoneally (i.p.) with lg/kg glucose.
  • ITT insulin tolerance tests
  • mice were fasted for 4 hours, blood was drawn, and then mice were injected i.p. with insulin (Humulin® R, Eli Lilly, Indianapolis, IN), lU/kg body weight. Blood samples were taken from the tail vein at 15, 30, 60, 90, and 120 minutes after injection and glucose was measured using a Glucometer (Elite, Bayer Health Care, Tarrytown, NY).
  • Hyperinsulinemic- euglycemic clamps were performed in awake mice fasted for 12 hours as previously described (Toyoshima et al., Endocrinology 146:4024-4035, 2005) with modifications. Primed-continuous infusion of [3- H] glucose was used; 2.5 ⁇ bolus, 0.05 ⁇ / ⁇ during the basal state, and 0.1 ⁇ / ⁇ during the clamp period. Insulin (Humulin® R, Eli Lilly, Indianapolis, IN) was infused as a bolus of 18 mU/kg over a period of 3 minutes, followed by continuous insulin infusion at the rate of 3.5 mU/kg lean mass/minute (in Hifla F/F mice) and 9.4 m/kg lean
  • mice were fasted overnight and injected i.p. with 5 U/kg body weight of insulin (Humulin®, Eli Lilly). Mice were killed 5 minutes after insulin stimulation. Liver, white adipose tissue and quadriceps were removed, snap-frozen in liquid nitrogen, and stored at - 80°C until use.
  • Body composition was measured in non- anesthetized mice using an Echo 3-in-l NMR analyzer (Echo Medical Systems, Houston, TX). Daily food intake was measured in individually caged mice using glass metabolic chambers for 24 hours (Jencons, Leighton
  • adiponectin ELISA kit (Crystal Chem Inc., Downers Grove, IL). Fasted serum cholesterol, free fatty acids, and triglycerides were measured using reagents from Wako (Richmond, VA). Adiponectin serum levels were measured with a mouse adiponectin ELISA kit (ALPCO, Salem, NH). The amounts of adiponectin in culture medium for 3T3-L1 adipocytes were measured using Quantikine® Mouse Adiponectin/ Acrp30
  • RNA Analysis Total RNA was extracted from adipose tissue, liver, and skeletal muscle using TRIzol reagents (Invitrogen, Carlsbad, CA). Quantitative real-time PCR (qPCR) was performed using cDNA generated from 1 ⁇ g of total RNA with the Superscript® II Reverse Transcriptase kit (Invitrogen). qPCR reactions were carried out by use of SYBR® green PCR master mix (Applied Biosystems, Carlsbad, CA) in an ABI Prism 7900HT sequence detection system (Applied Biosystems). Values were quantified by comparative CT methods and normalized to ⁇ -actin. Primer sequences are listed in Table 1.
  • Dye-coupled cDNAs were purified with a MiniElute PCR purification kit (Qiagen) and hybridized to an Agilent 44 K mouse 60-mer oligo microarray (Agilent Technologies, Santa Clara, CA). The procedures were repeated for replicate experiments with independent hybridization and processing, and the data processed and analyzed by Genespring GX software (Agilent
  • 3T3-L1 fibroblasts were grown in Dulbecco' s modified Eagle's medium supplemented with 10% Calf Serum and differentiated into adipocytes as described previously (Jiang et ah, J. Clin. Invest. 106:473-481, 2003).
  • 3T3-L1 adipocytes were transfected with siRNA duplexes by electroporation using Amaxa® Cell Line Nucleofector® Kit L (Amaxa Biosystems, Cologne, Germany). Sequences corresponding to the siRNA of ARNT, HIFl , and SOCS3 are listed in Table 1.
  • a scrambled Stealth RNAi duplex (catalog nos. 12935200 and 12935300, Invitrogen) served as a negative control.
  • Adipocytes and SVF- ⁇ were isolated from epididymal WAT as previously described (Sugii et ah, Proc. Natl. Acad. Sci. USA 106:22504-22509, 2009).
  • 3T3-L1 adipocytes were incubated for the indicated time in a water-jacketed C0 2 incubator containing 1% 0 2 , 5% C0 2 , and 94% nitrogen gas.
  • Luciferase Assay The mouse Socs 3 luciferase reporter plasmids were constructed by cloning the upstream regions using the primers listed in Table 1. The PCR fragments were cloned into the Mlul and Xhol restriction sites in the pGL3- basic vector (Promega, Madison WI). Oxygen stable mouse HIFla or HIF2a were generated by mutating the prolines in the degradation domain to alanines by site- directed mutagenesis (Stratagene, La Jolla, CA).
  • luciferase reporters were co- transfected with the mammalian expression plasmid for an oxygen stable HIFl or oxygen stable HIF2a into differentiated 3T3-L1 adipocytes by using Amaxa® Cell Line Nucleofector® Kit L (Amaxa Biosystems, Cologne, Germany). Cells were incubated for 24 hours. Standard dual luciferase assay was used and normalized to a co-transfected control reporter (Promega).
  • ChIP assays Chromatin immunoprecipitation (ChIP) assays were performed using the SimpleChIPTM Enzymatic Chromatin IP Kit (Cell Signaling Technology, Danvers, MA). Briefly, 1x10 differentiated 3T3L1 cells were exposed to normoxia or hypoxia (1% oxygen) for 8 hours, crosslinked in 1% formaldehyde and lysed. Chromatin was fragmented by partial digestion with micrococcal nuclease, sheared in a chilled Bioruptor (Diagenode, vide, Belgium) and the nuclear lysate was cleared by centrifugation at 16,000 g for 5 minutes.
  • Soluble chromatin was immunoprecipitated with primary antibody for HIFl (Santa Cruz Biotechnology, Santa Cruz, CA). The de-crosslinked samples were incubated with RNase A and proteinase K. DNA was purified using DNA purification spin columns, and 2 of samples was amplified by PCR by using primers listed in Table 1.
  • ACF HIFlcc Inhibitor Acriflavine Treatment Mice: ACF was purchased from Sigma- Aldrich (St. Louis, MO). Male C57BL/6 mice were administrated vehicle (saline) or ACF (2 mg/kg) daily via i.p. injection and fed a HFD for 12 weeks from 6 weeks of age.
  • Results are expressed as mean ⁇ SD. Differences between groups were examined for statistical significance with Student's t test. A P value of less than 0.05 was considered statistically significant.
  • HIF-2a FWD 5 '-TG AGTTGGCTC ATG AGTTGC-3 ' 66
  • HIF-2a REV 5 '-T ATGTGTCCGA AGG AAGCTG-3 ' 67 siRNA
  • ARNT antisense 5 '-UU AG AUCGG AAUCGG A AC AUGACGG-3 ' 69 SEQ ID NO: 1
  • SOCS3 sense 5'-GCCACUUCUUCACGUUGAGCGUCAA-3' 72
  • This example describes generation of transgenic mice with adipocyte- specific knockout of Arnt or HIFla genes and their initial characterization.
  • a PCR amplicon for the Arnt null allele amplified as a 340 base pair product, and was detected in genomic DNA isolated from adipocytes or adipose tissue of Arnt A dipo mice, and not in adipose DNA isolated from Arnt F/F mice.
  • the floxed allele was the only band detected in adipose tissue from Arnt F/F mice, and from all non-adipose tissues in Arnt A dipo mice.
  • the Hifla null amplicon amplified as a 355 base pair product, and was detected in genomic DNA isolated from adipose tissue of
  • Hifla A dipo mice and was not detected in adipose tissue DNA isolated from HiflaTM mice. The null allele was only detected in adipose tissue and not in liver, skeletal muscle, spleen, and kidney from Hifla A dipo mice.
  • the expression of Arnt mRNA was specifically decreased by 50% in white adipose tissue (WAT) and brown adipose tissue (BAT) in the Arnt A dipo mice compared to Arnt 7iF mice; no decrease was evident from liver or skeletal muscle.
  • WAT white adipose tissue
  • BAT brown adipose tissue
  • qPCR showed nearly absent expression of ARNT mRNA in the adipocytes of Arnt A dipo mice (FIG. 1A). Similar results were obtained from tissues of Hifla A dipo mice and an approximately 88% decrease in Hifla mRNA from adipocytes was observed (FIG. IB).
  • This example describes characterization of Arnt A dipo or Hifla A dipo mice fed a high fat diet (HFD).
  • HFD high fat diet
  • HIF1 in fat metabolism and glucose homeostasis
  • mice were fed either a chow diet or HFD.
  • Arnt A dipo and Hifla A dipo mice grew at a rate similar to that of Arnt 7iF and Hifla lv mice, respectively.
  • 12 weeks of HFD led to weight gain in Arnt F/F and Hifla F/F mice while Arnt A dipo and Hifla A dipo mice were resistant to the HFD-induced weight gain (FIGS. 2A and 2B).
  • Arnt Adipo and Hifla Adipo mice maintained on a HFD for 12 weeks exhibited a significant reduction in body mass compared to littermate controls.
  • Hifla A dipo mice however, the metabolic efficiency was decreased in the Hifl AAdipo mice (FIG. 2E), suggesting an increase in the metabolic rate.
  • the indirect calorimetry performed on week 7 of HFD showed that resting and total 0 2 consumption adjusted to body weight were significantly increased in Hifla A dipo mice at both 24°C and 30°C (FIG. 2F).
  • Hifla AAdipo mice had reduced resting and total RER at 30°C, suggesting increased fatty acid oxidation (FIG. 2G) and a tendency towards increased activity (FIG. 2H). All of these changes in Hifla A dipo mice could contribute to the decrease in weight gain on a HFD as compared to Hifla lv mice.
  • GTT and ITT glucose and insulin tolerance tests
  • Insulin sensitivity was further determined by performing ITT. Sixty minutes after insulin challenge, glucose levels in Arnt A dipo mice decreased to approximately 50% of baseline with only a 15% decline noted in Arn 17 mice; similar results were obtained from the Hifla A dipo mice, suggesting a significant improvement in insulin sensitivity by adipose HIFl disruption (FIGS. 4B and 5B). Moreover, fed glucose, fasted glucose, and fasted serum insulin levels were significantly lower in Arnt A dipo and Hifla A dipo mice compared to Arn 17 and Hifla F/F mice, respectively, after 12 weeks of HFD. The calculated homeostasis model assessment (HOMA) measure of insulin resistance was significantly decreased in Ami AAdipo and Hifla A dipo mice (Table 2). Arnt A dipo and Hifla A dipo mice also had reduced fasted serum triglycerides and free fatty acid levels consistent with improved glucose tolerance and insulin sensitivity in these mice (Table 2).
  • HOMA homeostasis model assessment
  • Insulin sensitivity at earlier time points on the HFD prior to the change in body weight between Arnt A dipo and Hifla A dipo mice was investigated to determine whether improved glucose metabolism was due to the reduced adiposity in these mice.
  • body mass was similar between Hifla lv and Hifla A dipo mice after 4 weeks, 6 weeks and 8 weeks of HFD, GTT and ITT revealed that glucose tolerance and insulin sensitivity began to be improved in Hifla A dipo mice from 4 weeks on the HFD (FIGS. 6A and 6B).
  • fasted glucose was similar while fasted insulin levels and HOMA index in Hifla A dipo mice were significantly decreased (FIG. 6C).
  • Hifla 17 and Hifla AAdipo mice were significantly reduced plasma glucose and insulin levels; basal endogenous glucose production (EGP) was similar between genotypes (FIG. 5C-E and G).
  • EDP basal endogenous glucose production
  • GIR glucose infusion rate
  • Hifla A dipo mice FIGS. 5F and G
  • the GIR was significantly increased in Hifla A dipo mice (FIGS. 5F and G), which confirmed improved whole-body insulin sensitivity in Hifla A dipo mice.
  • insulin induced a more marked suppression of EGP in Hifla A dipo mice, suggesting increased insulin sensitivity in the liver.
  • Whole body glucose disposal (Rd) and glucose uptake into skeletal muscle and adipose tissue were significantly increased in Hifla A dipo mice (FIG. 5G).
  • HIF1 deficiency in adipocytes improved the HFD-impaired insulin signaling pathway in WAT, liver and skeletal muscle, and targeted disruption of Hifla in adipocytes completely mimicked the protective effects of adipose Ami-deficiency on HFD-induced insulin resistance.
  • This example describes the effect of HIF1 disruption on expression of lipid and glucose metabolism-related genes.
  • Hifla expression was significantly induced, resulting in activation of HIF1 target genes such as Glutl and Vegf (FlG. 7 A).
  • HIF1 target genes such as Glutl and Vegf (FlG. 7 A).
  • these target genes and adiponectin expression were similar between Hifl ' 7 and Hifla A dipo mice (FIG. 7B).
  • Hif la protein expression was no significant in WAT from chow-fed mice, while it was induced in HFD-fed mice.
  • Hifl a mice on a chow diet while different on a HFD Hifl a mice on a chow diet while different on a HFD.
  • Atgl and Hsl and the mitochondrial biogenesis related genes Pgcla and Pgclfi, were increased, and expression of inflammation related genes Tnfa and Pai-1, were downregulated in Arnt Adipo and Hifla Adipo mice (FIGS. 8A and 8B).
  • expression of adiponectin was upregulated in Arnt A dipo and Hifla A dipo mice.
  • Total serum adiponectin, the HMW form of adiponectin, and the ratio of HMW to total adiponectin were all increased in Arnt AAdipo and Hifla AAdipo mice on a 12 week HFD (FIG. 8C).
  • HMW adiponectin and ratio of HMW to total adiponectin began to be increased on a 6 week HFD before the decrease in body weight (FIG. 6D).
  • Macrophage marker F4/80 staining of adipose tissue sections revealed that macrophage infiltration to WAT decreased significantly in Arnt A dipo and Hifla A dipo mice.
  • Expression of mRNAs encoding the macrophage markers F4/80 and CD68 were also decreased in Arnt AAdipo mice and Hifla AAdipo mice (FIGS. 9A and 9B). Since it was reported that aP2 is expressed in macrophage (Makowski et al, Nat.
  • HIF1 Hifl a gene disruption in peritoneal macrophages ( ⁇ - ⁇ ) from Arnt A dipo mice.
  • Socs3 expression was significantly upregulated in parallel with increased Hifla expression in WAT from HFD wild-type mice (FIG. 7A). Further, haploinsufficiency of Socs3 significantly protected mice against the development of diet-induced obesity and associated metabolic complications (Howard et ah, Nat. Med. 10:734-738, 2004). Consistent with these findings, SOCS3 mRNA was also found to be downregulated in Arnt A dipo and Hifla A dipo mice as revealed by microarray analysis. qPCR confirmed that expression of SOCS3 mRNA in
  • Arnt Adipo and Hifla Adipo mice was significantly lower than that in Arnt vlv and
  • Hifla F/F mice were prepared from WAT of Hifl AAdipo and Hifla 17 mice after 12 weeks of HFD, and the macrophage enriched in SVF (SVF- ⁇ ). In adipocytes, no significant expression of HIFl mRNA in Hifl AAdipo mice was found. The expression of Socs3 in Hifla AAdipo mice was about 80% decreased as compared to that in HiflaTM mice. Adiponectin was upregulated in Hifla A dipo mice (FIG. 10B).
  • Hifla Adipo and HiflaTM mice (FIG. IOC).
  • HIFla protein expression was not significantly induced in BAT after HFD treatment.
  • qPCR analysis revealed that HIFla and expression of its target genes (Glutl, Vegf and Socs3) were not significantly different in BAT between chow diet and HFD treatment, while Adiponectin, Ucp-1 and Pgcla expression were decreased after HFD treatment.
  • Ucp-1 expression was upregulated in Hifla A dipo mice compared to Hifla ' F mice after HFD.
  • This example describes analysis of regulation of Socs3 expression by HIFl in adipocytes.
  • SOCS3 mRNA and protein levels were decreased in WAT of Arnt A dipo mice and Hifla A dipo mice after HFD.
  • Socs3 was a direct target gene of HIFl, experiments were conducted in vitro with 3T3-L1 adipocytes.
  • HRE HIF response elements
  • Hifla A dipo mice were fed a HFD for 6 weeks and then administered the STAT3 inhibitor NSC 74859 (5 mg/kg) while the mice were maintained on the HFD.
  • the body weight, fat mass, and fat mass ratio were increased significantly after 6 weeks of NSC 74859 treatment (FIGS. 13A-B).
  • Mice fed the inhibitor also exhibited exacerbated insulin resistance (FIGS. 13C-E).
  • the activation of STAT3 in WAT was significantly reduced, coincident with the decrease of total adiponectin levels, HMW adiponectin, and the ratio of HMW to total adiponectin (FIGS. 13F and G).
  • This example describes the effect of treating wild-type mice fed a HFD with a HIFl inhibitor.
  • HFD-fed mice were administered acriflavine (ACF), a specific inhibitor of HIFla.
  • ACF was identified as a drug that binds directly to HIFl and inhibits HIFl dimerization and transcriptional activity (Lee et al, Proc. Natl. Acad. Sci. USA 106: 17910-17915, 2009).
  • GTT and ITT revealed that insulin sensitivity began to be improved in ACF-treated mice as early as 4 weeks on HFD, and the improvement became more significant on 8- and 12-weeks HFD (FIGS. 14C and 15B-C).
  • This example describes the effect of treating wild- type and Hifl o ⁇ dipo mice with a specific inhibitor of HIFlcc.
  • HiflaTM and Hifla Adipo mice were fed a HFD for 7 weeks, and then administered 2 mg/kg ACF i.p. while the mice were maintained on a HFD.
  • Hifla F/F mice at 15, 30, 60, 90, and 120 minutes after glucose loading, while in Hifla A dipo mice blood glucose was lower at 90, and 120 minutes after ACF treatment (FIG. 17A).
  • Insulin sensitivity was further determined by performing ITT. In Hifl a F/F mice, ACF treatment decreased blood glucose significantly at 30, 60 and 120 minutes, however, in Hifla A dipo mice blood glucose was decreased at 60, and 90 minutes after 16 weeks of ACF treatment ( Figure 17B). Fasted glucose, fasted serum insulin levels and HOMA were significantly lower in
  • This example describes methods that can be used to assess the effect of treating an animal model of obesity and/or diabetes with a HIFl inhibitor.
  • Animal models of obesity and/or diabetes are used to assess the in vivo effects of HIFl inhibitors.
  • the animal model is the ob/ob mouse (Zhang et al., Nature 372:425-432, 1994), or db/db mouse (Chen et al., Cell 84:491-495, 1996) which carry mutations in leptin and the leptin receptor, respectively.
  • the animal model is the KK-A y mouse.
  • Heterozygous KK-A y males develop hyperglycemia, hyperinsulinemia, glucose intolerance, and obesity by about eight weeks of age (e.g., Reddi and Camerini- Davalos, Adv. Exp. Med. Biol. 246:7-15, 1988).
  • the animal model is the Hifla AAdipo mouse (described herein).
  • One of skill in the art can identify additional animal models of obesity and/or diabetes, for example available from The Jackson Laboratory (jax.org/jaxmice).
  • Suitable control mice are also utilized, such as wild type mice of the same strain as the test animal (e.g., C57BL/6 of Svl29 mice) or a parental strain (e.g., Hifla F/F in the case of Hifla AAdipo mice).
  • mice are maintained on a HFD for a period of time (such as about 4-18 weeks) prior to administration of a HIFla inhibitor.
  • Mice are then administered a HIFla inhibitor (for example, orally or by i.p. injection) while continuing to be fed HFD.
  • Measurements of parameters related to obesity and diabetes such as body mass, fat mass, food intake, metabolic efficiency, activity, oxygen consumption, blood glucose (for example in a GTT or ITT), fasting plasma glucose, fasted serum insulin, HOMA index, serum triglycerides, serum free fatty acids, and serum adiponectin (such as total, HMW, and/or HMW/total adiponectin) are measured at various time points.
  • mice are administered ACF (such as about 0.1 mg/kg to about 10 mg/kg) by i.p. injection daily.
  • a Hifl ashRNA is administered to the mice, for example, under the control of an adipose-specific promoter (such as the ap2 promoter).
  • a Hifl ashRNA may be included in a viral vector (such as a lentiviral vector), which is administered i.p. at a dose of about 10 9 -1012 particles.
  • a decrease in body weight for example, decreased body mass, decreased fat mass, or a combination of two or more thereof, for example, compared with mice on HFD but not treated with the HIFl indicates the effectiveness of the treatment.
  • increased glucose tolerance e.g., decreased fasting plasma glucose or decreased fasting plasma insulin or decreased HOMA score
  • decreased triglycerides e.g., decreased free fatty acids, increased adiponectin, or a combination of two or more thereof, indicates the effectiveness of the treatment.
  • a subject who is in need of a reduction in body weight is selected.
  • a therapeutically effective dose of a composition including an inhibitor of HIFl is administered to the subject.
  • the amount of the composition administered to decrease body weight of the subject depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition.
  • a therapeutically effective amount of an agent is the amount sufficient to decrease body weight in a subject without causing a substantial cytotoxic effect in the subject.
  • a decrease in body weight for example, decreased total body weight, decreased BMI, decreased waist circumference, decreased body fat, or a
  • a subject who has been diagnosed with diabetes or prediabetes is identified. Following subject selection, a therapeutically effective dose of a composition including an inhibitor of HIFl is administered to the subject.
  • the amount of the composition administered to prevent, reduce, inhibit, and/or treat diabetes depends on the subject being treated, the severity of the disorder, and the manner of administration of the therapeutic composition.
  • a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the condition (e.g. , diabetes) in a subject without causing a substantial cytotoxic effect in the subject.
  • a reduction in the clinical symptoms associated with diabetes indicates the effectiveness of the treatment.
  • decreased insulin resistance e.g., decreased fasting plasma glucose or decreased fasting plasma insulin or decreased QUICKI score
  • decreased triglycerides e.g., decreased free fatty acids, decreased HbAlc, or a combination of two or more thereof, indicates the effectiveness of the treatment.

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Abstract

Cette invention concerne des méthodes pour traiter le diabète (par ex., augmenter la tolérance au glucose, réduire la résistance à l'insuline, et abaisser les lipides sériques) et/ou réduire le poids corporel (par ex., traiter le surpoids ou l'obésité), lesdites méthodes comprenant l'administration d'une quantité thérapeutiquement efficace d'une composition contenant un inhibiteur du facteur 1α inductible par l'hypoxie (HIF1α) à un sujet souffrant du diabète ou un sujet ayant besoin de réduire son poids corporel. L'inhibiteur HIF1α peut être administré en association avec un véhicule de qualité pharmaceutique. Dans certains exemples, l'inhibiteur HIF1α est administré par voie orale.
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Cited By (5)

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US9938525B2 (en) 2012-10-26 2018-04-10 Nlife Therapeutics, S.L. Compositions and methods for selective delivery of oligonucleotide molecules to cell types
KR20180126436A (ko) * 2015-08-17 2018-11-27 더 존스 홉킨스 유니버시티 조직 복원용 간엽 세포-결합 복합 재료
WO2020225427A1 (fr) 2019-05-09 2020-11-12 Teresa Pereira Traitement du diabète
WO2022040161A1 (fr) * 2020-08-18 2022-02-24 The Regents Of The University Of California Méthodes et compositions pour le traitement du diabète et la régénération de cellules bêta
CN116615241A (zh) * 2020-08-18 2023-08-18 加利福尼亚大学董事会 用于糖尿病治疗和β细胞再生的方法和组合物

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