WO2018160772A1 - Procédé de traitement de l'obésité, de la résistance à l'insuline, d'une stéatose hépatique non alcoolique comprenant une stéatohépatite non alcoolique - Google Patents

Procédé de traitement de l'obésité, de la résistance à l'insuline, d'une stéatose hépatique non alcoolique comprenant une stéatohépatite non alcoolique Download PDF

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WO2018160772A1
WO2018160772A1 PCT/US2018/020333 US2018020333W WO2018160772A1 WO 2018160772 A1 WO2018160772 A1 WO 2018160772A1 US 2018020333 W US2018020333 W US 2018020333W WO 2018160772 A1 WO2018160772 A1 WO 2018160772A1
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hif
subject
hepatic
mice
expression
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PCT/US2018/020333
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Frank J. Gonzalez
Cen XIE
Changtao Jiang
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The United State Of America, As Represented By The Secretary, Department Of Health & Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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

Definitions

  • the present invention relates to methods of treating and preventing obesity, insulin resistance and fatty liver disease.
  • Present disclosure shows that intestine HIF-2a and neu3 would be viable target for NAFLD therapy, NASH therapy, obesity and insulin resistance.
  • the disclosure presents methods and compositions of matter relating to the targeted inhibition and/or otherwise inactivation of HIF-2a for treating and/or preventing obesity, insulin resistance, NAFLD and NASH.
  • NAFLD non-alcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • hypoxia- inducible factor (HIF)-la and HIF-2a are essential in maintaining intestinal homeostasis.
  • HIF- ⁇ and HIF-2a are also highly expressed in a wide variety of solid tumors, including those of the colon, breast, lung, and pancreas.
  • RCC renal cell carcinoma
  • HIF-la has an antitumor role and decreases tumor growth by increasing expression of proapoptotic genes.
  • HIF-2a is essential for RCC tumor growth and promotes tumor cell proliferation through augmented c-Myc activity. However, in lung cancer, HIF-2a exerts a tumor suppressive effect. These studies demonstrate the careful need to evaluate the tumor specific roles for HIF- ⁇ and HIF-2a for therapeutic targeting.
  • HIF-2a is essential in maintaining proper micronutrient balance, inflammatory response, and the regenerative and proliferative capacity of the intestine following an acute injury.
  • HIF-2a hypoxia- inducible factor
  • HIF-la-in white adipose tissue activation has already been disclosed as a potential target for treating diabetes, obesity, insulin resistance and reducing body weight.
  • HIF- 2a activation on the other hand has shown negative and opposing effects (i.e., increased obesity and insulin resistance).
  • HIF-2a inhibitors including PT2385, and its analogs, PT2567, PT2399 and PT2977. HIF-2 a inhibitors have been studied for the treatment of cancer but not for treating obesity, NAFLD, NASH or insulin resistance.
  • the disclosure presents methods and compositions of matter relating to the targeted inhibition and/or otherwise degradation of HIF-2a for treating and/or preventing obesity, insulin resistance, NAFLD and NASH.
  • Non-alcoholic fatty liver disease is becoming the most common chronic liver disease in western countries with limited therapeutic options. NAFLD can lead to NASH, fibrosis, liver cancer and even liver failure and death.
  • NAFLD intestinal hypoxia-inducible factor
  • Human intestine biopsies from patients with or without obesity revealed a relationship between activated HIF-2a but not HIF- la in increased body mass index and hepatic lipid toxicity.
  • mice with an intestine-specific HIF-2a-disruption in which high- fat diet-induced hepatic steatosis and obesity were substantially decreased.
  • PT2385 an HIF-2a- specific inhibitor, had preventive and therapeutic effects on metabolic disorders dependent on intestine HIF-2a.
  • Intestine HIF-2a inhibition markedly reduced intestine and serum ceramide levels.
  • intestine HIF-2a regulates ceramide metabolism mainly from the salvage pathway, which was revealed by the identification of the novel HIF-2a target gene encoding neuraminidase 3 (Neu3).
  • One aspect discloses a method for treating or preventing obesity, insulin resistance, and non-alcoholic fatty liver disease in a subject, comprising selectively decreasing the expression or inhibiting the activity of intestine specific HIF-2a, thereby decreasing obesity, insulin resistance, non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in the subject.
  • Another aspect discloses a method of decreasing obesity, insulin resistance, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in a subject, comprising administering a therapeutically effective amount of PT2385, thereby decreasing obesity, insulin resistance, nonalcoholic fatty acid disease and non-alcoholic steatohepatitis in the subject.
  • Certain embodiments disclose a method of decreasing obesity, insulin resistance, and non-alcoholic fatty acid disease comprises decreasing ceramide synthesis, decreased fatty acid transport and lipogenesis, and inhibition of salvage pathway or a combination of two or more thereof in the subject as compared to a control.
  • the method discloses that intestine specific HIF-2a and neu3 inhibition attenuates hepatic steatosis.
  • the subject has non-alcoholic fatty liver disease (NAFLD).
  • the method discloses that use of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof to decrease HIF-2a expression or activity relative to a control.
  • the method depicts that decreasing obesity, insulin resistance, and non-alcoholic fatty acid disease comprises inhibition of NEU3 activity.
  • NEU3 activity is inhibited by 2,3-didehydro-N-acetyl- neuraminic acid (DANA) or naringin.
  • DANA 2,3-didehydro-N-acetyl- neuraminic acid
  • naringin 2,3-didehydro-N-acetyl- neuraminic acid
  • NEU3 expression is decreased by intestine-specific HIF-2a inhibitor for treating or preventing hepatic steatosis.
  • the method of decreasing obesity, insulin resistance, non-alcoholic fatty acid disease and non-alcoholic steatohepatitis comprises decreased or inhibited ceramide synthesis.
  • the method discloses that decreasing obesity, insulin resistance, and non-alcoholic fatty acid and non-alcoholic steatohepatitis disease comprises decreased fatty acid transport and lipogenesis.
  • the method of decreasing obesity, insulin resistance, non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in some embodiments comprises inhibition of salvage pathway.
  • the method discloses that intestine specific HIF-2a inhibitor is administered orally.
  • the intestine specific HIF-2a inhibitor is PT2385.
  • the subject is a mammal.
  • the subject is a human.
  • the method uses a composition comprising 1 mg/kg- 100 mg/kg dose of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof. In some embodiments, the method uses a composition comprising 20 mg/kg. In still other embodiments, the method uses composition comprising about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, or about 100 mg/kg or more.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • a method of decreasing obesity, insulin resistance, and nonalcoholic fatty acid disease in a subject comprising administering a therapeutically effective amount of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof, thereby decreasing obesity, insulin resistance, and non-alcoholic fatty acid disease in the subject.
  • the disclosure provides a method of decreasing obesity in a subject, comprising administering a therapeutically effective amount of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof, DANA or naringin, thereby decreasing obesity in the subject.
  • the disclosure provides a method of decreasing insulin resistance in a subject, comprising administering a therapeutically effective amount of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof, DANA or naringin, thereby decreasing insulin resistance in the subject.
  • the disclosure provides a method of decreasing non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in a subject, comprising administering a therapeutically effective amount of at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof, thereby decreasing non-alcoholic fatty acid disease including non-alcoholic steatohepatitis in the subject.
  • Another aspect discloses a method of decreasing obesity, insulin resistance, non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in a subject, comprising selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine, thereby decreasing obesity, insulin resistance, non-alcoholic fatty acid disease including non-alcoholic
  • the disclosure provides a method of decreasing obesity in a subject, comprising selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine, thereby decreasing obesity in the subject.
  • the disclosure provides a method of decreasing insulin resistance in a subject, comprising selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine, thereby decreasing insulin resistance in the subject.
  • the disclosure provides a method of decreasing non-alcoholic fatty acid disease inclduing non-alcoholic steatohepatitis in a subject, comprising selectively decreasing the expression or inhibiting the activity of HIF-2a and Neu3 in the intestine, thereby decreasing non-alcoholic fatty acid disease and non-alcoholic steatohepatitis in the subject.
  • the method of decreasing obesity, insulin resistance, and nonalcoholic fatty acid disease results in decreased ceramide levels, decreased fatty acid transport and lipogenesis, and inhibition of salvage pathway or a combination of two or more thereof in the subject as compared to a control.
  • HIF-2a and neu3 inhibition attenuates hepatic steatosis.
  • the subject treatable by the methods provided herein have nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) or are at risk for NAFLD and NASH.
  • NAFLD nonalcoholic fatty liver disease
  • NASH non-alcoholic steatohepatitis
  • the subject treatable by the methods provided herein suffers from obesity or is at risk of becoming obese.
  • the subject treatable by the methods provided herein is resistant to insulin.
  • the subject treatable by the methods provided herein has type II diabetes or is at risk for developing type II diabetes.
  • the step of selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine comprises administering a therapeutically effective amount of an HIF-2a inhibitor.
  • the HIF-2a inhibitor may be administered in a selective manner that results in selective delivery to intestine epithelia cells but miminizes delivery to other cell types.
  • the HIF-2a inhibitor may be delivered by way of a pharmaceutical composition or vehicle which comprises one or more intestinal- specific targeting moieties.
  • Moieties can include, for example, intestinal-specific antibodies or ligands.
  • the HIF-2a inhibitor is PT2385 or a functional derivative thereof having the same or equivalent effect as PT2385.
  • the step of selectively decreasing the expression or inhibiting the activity of HIF-2a in the intestine comprises administering a therapeutically effective amount of a pharmaceutical composition comprising a HIF-2a inhibitor and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising a HIF-2a inhibitor and a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions described herein may further comprise a hepatic- specific targeting moiety which can include a hepatic-specific antibody that recognizes and binds to a hepatic target, or a hepatic- specific ligand.
  • FIG. 1 Increased HIF-2a signaling in human ileum biopsies is correlated with obesity
  • (a) Representative immunohistochemical staining for the expression of HIF-2a and HIF- ⁇ in human ileum biopsies from cohort 1 (n 6 subjects/group, 3 images/subject)
  • n 6/group for blot quantification
  • FIG. 2 Intestine-specific HIF-2a disruption ameliorates the development of hepatic steatosis,
  • (a) Representative H&E staining (left two panels) and Oil Red O staining (right two panels) of liver sections (n 5 mice/group, 3 images/mouse). Scale bars, 100 ⁇ .
  • the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.
  • FIG. 3 Intestinal HIF-2a deficiency reduces ceramide synthesis in the small intestine,
  • FIG. 4 The ceramide-synthesis-related gene Neu3 is a novel HIF-2a target gene in the small intestine,
  • (b-f) Correlative analysis of ileum NEU3 mRNA levels with BMI (b), ALT (c), AST (d), DMT1 mRNA (e), and DCYTB mRNA (f). n 35.
  • the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.
  • FIG. 6 PT2385 reverses HFD-induced hepatic steatosis,
  • Lipids stain positive (red color) with Oil Red O. Scale bars, 100 ⁇ . P 0.06 for steatosis score,
  • FIG. 7 Increased HIF2a signaling in human ileum biopsies is correlated with obesity
  • (a) Correlative analysis of ileum DMT1, DCYTB, and PDK1 mRNA levels with BMI, ALT, AST, triglycerides, cholesterol, HDL, LDL, and glucose, n 35. Correlations were assessed by nonparametric Spearman's test
  • the midline represents the median; box represents the interquartile range (IQR) between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.
  • IQR interquartile range
  • (f) Growth curves of chow-fed Hif2a and Hif2a mice, (g) Representative H&E staining of liver sections of chow-fed ⁇ and Hif2a Am mice (n 15, 3 images/mouse). Scale bars, 100 ⁇ .
  • FIG. 9 Loss of HIF2a in the intestine affects ceramide metabolism in HFD-fed ⁇ and Hif2a ⁇ mice,
  • e Expression of intestinal Hif2a mRNA and HIF2a target gene mRNAs.
  • mice Hif2a ⁇ mice, (a) body weight, (b) Glucose tolerance test, (c) Insulin tolerance test, (d)
  • the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.
  • FIG. 11 Intestinal HIF2a deficiency reduces ceramide synthesis in the small intestine independent of body weight changes in HFD-fed ⁇ and Hif2a Am mice,
  • (b-d) Intestinal expression of mRNAs encoded by ceramide synthesis-related genes, including the de-novo pathway (b), the
  • sphingomyelinase pathway c
  • salvage pathway d
  • e-g Ceramide levels in the small intestine (e), systematic serum (f), and portal serum (g).
  • h-j Thermogenic gene expression in scWAT (h), BAT (i), and eWAT (j).
  • the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.
  • (1) Representative UCP1 immunohistochemistry staining of scWAT sections (n 3 images/mice). Scale, 100 ⁇ .
  • FIG. 12 HIF2a regulates the ceramide synthesis in the small intestine, (a) Intestinal expression of Vhl, Hifla, Hif2a, Dmtl and Dcytb mRNAs. (b-d) Intestinal expression of mRNAs encoded by ceramide synthesis-related genes, including the de-novo pathway (b), the
  • FIG. 13 NEU3 inhibitor DANA and naringin treatments protect mice from HFD-induced obesity and hepatic steatosis,
  • a-c Neuraminidase activities in intestine (a), liver (b), and white adipose tissue (c).
  • d,e Ceramide levels in the small intestine (d) and serum (e).
  • f Growth curves,
  • FIG. 14 Administration of ceramide reverses the protective effects of intestinal HIF2a inhibition on the HFD-induced obesity and insulin resistance in HiflaTM and Hifla Am .
  • (f) Insulin tolerance test, n 5/group. Data are presented as the mean + sem. For box plots, the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.
  • i,j Liver (i) and serum (j) cholesterol content, (k) Serum ALT levels.
  • FIG. 16 PT2385 inhibits ceramide synthesis in the small intestine and alters fatty acid synthesis, metabolism, and inflammation in the liver dependent on intestinal HIF2a in ⁇ and Hif2a ⁇ mice,
  • (a) Expression of Hif2a mRNA and its target gene mRNAs in the intestine
  • e Hepatic expression of mRNAs encoding fatty acid transport and lipogenesis.
  • FIG. 17 PT2385 reverses metabolic dysfunctions in HFD-induced obese mice, (a) Growth curves, (b) glucose tolerance test, (c) Insulin tolerance test, (d) Expression levels of HIF2a target gene mRNAs in the intestine, (e) Quantitation of ceramide concentrations in serum. (f,g) Intestinal expression of mRNAs encoded by ceramide synthesis-related genes, including the de novo pathway (f) and the sphingomyelinase pathway (g). (h) Hepatic expression of mRNA encoding fatty acid transport and lipogenesis-related enzymes, (i) Hepatic expression of mRNAs encoding inflammatory cytokines and chemokines.
  • Data are presented as the mean + sem.
  • the midline represents the median; box represents the IQR between the first and third quartiles, and whiskers represent the lowest or highest values within 1.5 times IQR from the first or third quartiles.
  • FIG. 18 Full western blot gel panels, (a-d) HIF2a, HIFla, NEU3 and ⁇ -ACTIN from which the data in FIG lb (a), FIG. If (b), FIG. 10b (c), FIG. 4g (b) and FIG. 8e (d) were derived, (e, f) NUE3 and ⁇ -ACTIN from which the data in FIG. 4h (e) and FIG. 6k (f) were derived, (g) UCP1 and EIF5 from which the data in FIG. lOv were derived.
  • FIG. 19 Demographic characteristics of the herein subjects.
  • FIG. 20 Clinical biochemistry of the herein subjects.
  • FIG. 21 provides the sequences of oligonucleotide primers used herein.
  • FIG. 22 Intestine- specific HIF-2oc disruption ameliorates high-fat-diet induced obesity, insulin resistance and activate the beiging process of adipose tissues, (a) body mass (b) body fat and (c) body fat rate of intestine- specific HIF-2oc knockout mice after 8 -week high-fat-diet feeding, (d) Glucose Tolerance Test (GTT) of intestine- specific HIF-2oc knockout mice and (e) glucose area under the curve (AUC). (f) insulin tolerance test ( ⁇ ) of intestine-specific HIF-2oc knockout mice, (g) subcutaneous adipose tissues expression of mRNAs relative to beiging.
  • GTT Glucose Tolerance Test
  • AUC glucose area under the curve
  • FIG. 23 (a) Anal temperature of intestine-specific HIF-2oc knockout mice during cold stimulation (12°C). (b) representative UCP1 immunohistochemical staining of scWAT sections, (c) expression of mRNAs relative to beiging in subcutaneous adipose tissues and (d) visceral adipose tissues, (e) expression of mRNAs in liver involved in bile acid synthesis and transport, from intestine-specific HIF-2oc knockout mice after a short term 2- week high-fat-diet treatment, (f) expression of HIF-2oc target gene mRNAs in intestine. [0071] FIG.
  • Intestine-specific HIF-2oc disruption alters bile acid compositions and its relative expression bile acid transport and metabolism genes in mice, (a) expression of mRNAs in intestine involved in bile acid transport, (b) liver expression of mRNAs in liver involved in bile acid synthesis and transport, bile acid profiles in serum (c) ileum (d) and feces (e) after an 8- week high-fat-diet treatment.
  • FIG. 25 Transfer of intestine-specific HIF-2 D disrupted mice fecal bacterium
  • FIG. 26 Fecal bacterium transplanted mice have altered bile acid and its relative gene expression, (a) expression of mRNAs in liver involved in bile acid synthesis and transport, bile acid profiles in serum (b) ileum (c) and feces (d) after fecal microbiota transfer.
  • FIG. 27 Depletion of the microbiota by antibiotic treatment reverses the therapeutic effects of intestine-specific HIF-2oc disruption, (a) expression of mRNAs in liver involved in bile acid synthesis and transport, (b) expression of mRNAs in intestine involved in bile acid transport, (c) expression of mRNAs subcutaneous adipose involved in beiging. (d) expression of mRNAs visceral adipose tissues involved in beiging. (e) body mass (f) Glucose Tolerance Test (GTT) (g) glucose area under the curve (AUC) and (h) insulin tolerance test (ITT) of antibiotic treatment mice.
  • GTT Glucose Tolerance Test
  • AUC glucose area under the curve
  • ITT insulin tolerance test
  • FIG. 28 Intestine-specific HIF-2oc disruption and fecal microbiota transplantation upregulates TGR5 signaling in both subcutaneous adipose tissues and visceral adipose tissues, (a) subcutaneous adipose tissues and (b) visceral adipose tissues expression of Tgr5 and Dio2 mRNAs in intestine-specific HIF-2oc knockout mice, (c) subcutaneous adipose tissues and (d) visceral adipose tissues expression of Tgr5 and Dio2 mRNAs in microbiota-transplanted mice.
  • the liver plays a central role in maintaining overall organism energy balance by controlling carbohydrate and lipid metabolism.
  • Liver disease is a growing global health problem, as deaths from end-stage liver cirrhosis and cancer are rising across the world.
  • pharmacologic approaches to effectively treat or prevent liver disease are extremely limited.
  • NAFLD is a complex disease, with considerable variation in severity amongst individuals. NAFLD can lead to NASH.
  • NAFLD therapies typically target four main pathways.
  • the dominant approach is targeting hepatic fat accumulation and the resultant metabolic stress.
  • Medications in this group include peroxisome proliferator-activator receptor agonists (eg, pioglitazone, elafibranor, saroglitazar), medications targeting the bile acid-farnesoid X receptor axis (obeticholic acid), inhibitors of de novo lipogenesis (aramchol, NDI-010976), incretins (liraglutide) and fibroblast growth factor (FGF)-21 or FGF-19 analogues.
  • peroxisome proliferator-activator receptor agonists eg, pioglitazone, elafibranor, saroglitazar
  • medications targeting the bile acid-farnesoid X receptor axis obeticholic acid
  • inhibitors of de novo lipogenesis aramchol, NDI-010976
  • a second approach is targeting the oxidative stress, inflammation and injury that follow the metabolic stress.
  • Medications from this group include antioxidants (vitamin E), medications with a target in the tumour necrosis factor a pathway (emricasan, pentoxifylline) and immune modulators (amlexanox, cenicriviroc).
  • a third group has a target in the gut, including antiobesity agents such as orlistat or gut microbiome modulators (IMM-124e, faecal microbial transplant, solithromycin).
  • antiobesity agents such as orlistat or gut microbiome modulators (IMM-124e, faecal microbial transplant, solithromycin).
  • Hypoxia-inducible factor is a transcription factor that regulates diverse signaling pathways enabling adaptive cellular responses to perturbations of the tissue microenvironment.
  • HIF is a heterodimeric complex consisting of a constitutively expressed ⁇ -subunit and an oxygen-sensitive a-subunit.
  • HIF- la and HIF-2a are the best characterized.
  • HIF- la and HIF-2a are characteristic features of all solid tumors.
  • HIF signaling serves as a major adaptive mechanism in tumor growth in a hypoxic microenvironment.
  • HIFs represent a critical signaling node in the switch to protumorigenic inflammatory responses through recruitment of protumor immune cells and altered immune cell effector functions to suppress antitumor immune responses and promote tumor growth through direct growth-promoting cytokine production, angiogenesis, and ROS production.
  • Role of HIF- 2a in Renal Cell Carcinoma(RCC) using HIF-2a inhibitors, including PT2385, is known in the art.
  • HIF activation through hypoxia-dependent and hypoxia-independent signals have been reported in liver disease of diverse etiologies. Increased expression of HIF- la and HIF-2a has been reported in many liver diseases, including nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), IR-induced liver injury, and hepatocellular carcinoma. A common feature of these liver diseases is tissue hypoxia due to an imbalance of metabolic demand and supply.
  • HIF-1 regulates the expression of glucose transporters as well as glycolytic enzymes and is thought to contribute to the glycolytic phenotype of hepatocellular carcinomas.
  • HIF-1 a, and HIF-2a have suggested a role for HIF-1 a, and HIF-2a, in the regulation of hepatic lipid metabolism.
  • HIF-1 a is shown to inhibit adipose tissue resulting in reduction of obesity and insulin resistance whereas inhibiting adipose HIF-2a has been observed to lead to increased body weight, glucose intolerance, and insulin resistance. Indeed, the state of the art suggests that HIF-1 a and HIF-2a have opposing effects in certain contexts such as macrophage polarization. HIF-1 a inhibition leads to steatohepatitis associated with impaired fatty acid ⁇ -oxidation, decreased lipogenic gene expression, and increased lipid storage capacity.
  • the present invention discloses that inactivation of HIF-2a significantly suppresses the development of hepatic steatosis, by decreasing ceramide synthesis, decreasing fatty acid transport and lipogenesis, and inhibition of salvage pathway or a combination of two or more, resulting in regulation of hepatic lipid metabolism in vivo, reduced obsesity, and decreased insulin resistance (or increase insulin sensitivity).
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
  • “combination therapy” refer to both concurrent administration (administration of two or more agents at the same time) and time varied administration (administration of one or more agents at a time different from that of the administration of an additional agent or agents), as long as the agents are present in the area to be treated to some extent, preferably at effective amounts, at the same time.
  • one or more of the present agents described herein are co-administered in combination with at least one additional bioactive agent, especially including an antifungal, antibacterial, and/or biocide.
  • the coadministration of agents are co-administered in combination with at least one additional bioactive agent, especially including an antifungal, antibacterial, and/or biocide.
  • therapeutically effective amount means the amount required to achieve a therapeutic effect.
  • the therapeutic effect could be any therapeutic effect ranging from
  • prevention, symptom amelioration, symptom treatment, to disease termination or cure e.g., the reduction, amelioration, or otherwise decrease the level of obesity, insulin resistance, and NAFLD.
  • administering is meant to refer to a means of providing the composition to the subject in a manner that results in the composition being inside the subject's body.
  • Such an administration can be by any route including, without limitation, subcutaneous, intradermal, intravenous, intra-arterial, intraperitoneal, and intramuscular.
  • patient or “subject” is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided.
  • patient refers to that specific animal, including a domesticated animal such as a dog or cat or a farm animal such as a horse, cow, sheep, etc.
  • patient refers to a human patient unless otherwise stated or implied from the context of the use of the term.
  • compositions and methods include the recited elements, but do not exclude other elements.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the
  • compositions of this invention Embodiments defined by each of these transition terms are within the scope of this invention.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.
  • the term "functionalities” or “moieties” or “functional moieties” refer to substances or agents which are capable of being contained in, or attached, to another compound or composition of the disclosure, e.g., a HIF-2a and neu3 inhibitor or to a
  • the moiety can be a "targeting moiety," which includes any ligand, antibody, or otherwise agent which can be attached to a HIF-2a and neu3 inhibitor or to a composition or vehicle comprising said HIF- 2a inhibitor and neu3 that allows the selective binding and attachment of the HIF-2a and neu3 inhibitor or otherwise other "cargo" to a target cell or tissue, but not to other non-desired cells or tissues.
  • kits are understood to contain at least the non-standard laboratory reagents of the invention and one or more non-standard laboratory reagents for use in the methods of the invention.
  • the term “treated,” “treating” or “treatment” includes the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated.
  • a subject that has been treated can exhibit a partial or total alleviation of symptoms (for example, NAFLD or obesity), or symptoms can remain static following treatment according to the invention.
  • the term “treatment” is intended to encompass prophylaxis, therapy and cure.
  • the term “control” refers to a sample or standard used for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy patient. In other embodiments, 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 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);
  • 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
  • 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.
  • HIF- 1 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 HIF- ⁇ ).
  • Arnt is constitutively present in the cell nucleus.
  • HIF alpha subunits HIF- la
  • HIF-2a also known an EPAS 1
  • HIF-3a HIF-3a
  • 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
  • HIF-1 nucleic acid and protein sequences are publicly available. For example,
  • GenBank Accession Nos. NM_001530 and NM_181054 disclose exemplary human HIF-la nucleic acid sequences and GenBank Accession Nos. NP_001521 and NP_851397 disclose exemplary human HIF- ⁇ protein sequences.
  • GenBank Accession No. NM_001430 discloses an exemplary human HIF-2a nucleic acid sequence and GenBank Accession No. NP_001421 discloses an exemplary human HIF-2a protein sequence.
  • GenBank Accession Nos. NM_152795, NM_022462, and NM_152794 disclose exemplary human HIF3a nucleic acid sequences and GenBank Accession Nos.
  • NP_690008, NP_071907, and NP_690007 disclose exemplary human HIF3a. protein sequences. GenBank Accession Nos. NM_001668 and NM_178427 disclose exemplary human Arnt nucleic acid sequences and 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.
  • the term "inhibitor" as in an HIF-2a inhibitor refers to 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 HIF, such as HIF-2a.
  • An inhibitor of the disclosure can inhibit the activity of a HIF-2a protein either directly or indirectly. Direct inhibition can be accomplished, for example, by binding to a HIF-2a 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).
  • Indirect inhibition can be accomplished, for example, by binding to a HIF-2a protein intended target, such as a receptor or binding partner, thereby blocking or reducing activity of HIF-2a.
  • an inhibitor of the disclosure can inhibit a HIF-2a 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-2a gene product or by altering post-translational modification of a HIF-2a gene product (such as prolyl hydroxylation), or by causing changes in intracellular localization.
  • insulin resistance refers to 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).
  • BMI Body Mass Index
  • a BMI of 25.0 kg/m 2 to 29.9 kg/m 2 is overweight (also called grade I obesity), while a BMI of 30 kg/m or more is truly obese (also called grade II 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.
  • Strong evidence shows that 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
  • non-alcoholic fatty liver disease is the term for a range of conditions caused by a build-up of fat in the liver.
  • NAFLD includes simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis.
  • non-alcoholic steatohepatitis is a condition that causes inflammation and accumulation of fat and fibrous (scar) tissue in the liver. Liver enzyme levels in the blood may be more elevated than the mild elevations seen with nonalcoholic fatty liver disease (NAFLD). NAFLD can lead to NASH.
  • NASH nonalcoholic fatty liver disease
  • neuraminidase 3 or “NEU3” (the protein) or (the gene) or aliase terms SIAL3, neuraminidase 3 (membrane sialidase), neuraminidase 3, membrane sialidase, and the like, refer to a glycohydrolytic enzyme which remove sialic acid residues from glycoproteins and glycolipids. It is localized in the plasma membrane, and its activity is specific for gangliosides. It may play a role in modulating the ganglioside content of the lipid bilayer. Mechanistically, intestine HIF-2a regulates ceramide metabolism mainly from the salvage pathway, which was revealed by the identification of the novel HIF-2a target gene encoding neuraminidase 3 (Neu3).
  • the term "pharmaceutically acceptable carrier” refers to conventional convention such carriers for any type of active agent, as described for example in Remington: The Science and Practice of Pharmacy, The University of the Sciences in
  • compositions and formulations suitable for pharmaceutical delivery of compounds such as an inhibitor of HIF-2a (for example, PT2385).
  • an inhibitor of HIF-2a for example, PT2385
  • 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.
  • 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.
  • a purified preparation of a HIF-2a inhibitor is one in which the HIF-2a inhibitor is more enriched than in its environment within a cell or other preparation, such as the environment in which it is synthesized.
  • a preparation is purified such that the HIF-2a inhibitor represents at least 50% of the total content of the preparation, for example, at least 50% by weight.
  • the HIF-2a 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
  • Double- stranded RNA molecules that inhibit gene expression through the RNAi pathway include siRNAs, miRNAs, and shRNAs.
  • the sequence of a siRNA is 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.
  • Other useful nucleotide derivatives incorporate nucleotides having modified carbohydrate moieties, such as 2'-0- alkylated residues or 2'-deoxy-2'-halogenated derivatives. Particular examples of such
  • 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.
  • the present disclosure contemplates any suitable known HIF-2a inhibitor for use in the methods of the present disclosure.
  • the present disclosure also contemplates any functional derivatives or variants of known HIF-2a inhibitors for use in the methods of the present disclosure.
  • HIF-2a refers to a monomelic protein that contains several conserved structured domains: basic helix-loop-helix (bHLH), and two Per-ARNT-Sim (PAS) domains designated PAS -A and PAS-B, in addition to C-terminal regulatory regions.
  • bHLH basic helix-loop-helix
  • PAS Per-ARNT-Sim
  • PAS Per-ARNT-Sim domains
  • HIF-2a is also alternatively known by several other names in the scientific literature, including Endothelial PAS Domain Protein 1 (EPAS 1), HIF2A, PASD2, HIF-2-Alpha, HIF2- Alpha, HLF, Hypoxia- Inducible Factor 2- Alpha, HIF-1 alpha- Like Factor, and MOP2.
  • EAPNT also known as HIF- ⁇
  • HIF-2a activity has its ordinary meaning in the art.
  • HIF-2a activity for example, includes activation of gene transcription mediated by HIF-2a.
  • the HIF-2a inhibitor is not limited to any type of molecule, including small molecular weight compounds, peptides, polypeptides, antibodies or functional fragments thereof which directly interact and/or bind to HIF-2a thereby blocking, inhibiting, or otherwise interfering with its activity.
  • Inhibitors of HIF-2a may also include nucleic acid based inhibitors, e.g., siRNA based inhibitors which block, inhibit, or otherwise modulate the expression of HIF- 2a inhibitor.
  • Blocking the expression of HIF-2a may include strategies for blocking the transcription of the HIF-2a gene, RNA-based inhibitors that block translation or otherwise lead to transcript degradation (e.g., siRNA processes), or protein-degradation-based strategies (e.g., ubiquitination-based proteosome-mediated degradation of the encoded HIF-2a.
  • RNA-based inhibitors that block translation or otherwise lead to transcript degradation
  • protein-degradation-based strategies e.g., ubiquitination-based proteosome-mediated degradation of the encoded HIF-2a.
  • the present disclosure contemplates the use of any HIF-2a described in WO2015/035223 (Aryl Ethers and Uses Thereof), which is incorporated herein by reference in its entirety. This includes the disclosure of PT2385.
  • the HIF-2a inhibitor can have the following structure of formula I:
  • Ri is aryl or heteroaryl
  • R 2 is nitro, carboxaldehyde, carboxylic acid, ester, amido, cyano, halo, sulfonyl, alkyl or heteroalkyl;
  • R 3 is hydrogen, halo, cyano, alkyl, heteroalkyl, alkenyl, alkynyl, alkylamino, carboxaldehyde, carboxylic acid, oxime, ester, amido or acyl, or R 2 /R 3 and atoms they are attached to form a 5- or 6- membered carbocycle with at least one sp hybridized carbon;
  • R4 is nitro, halo, cyano, alkyl, sulfinyl, sulfonamide, sulfonyl or sulfoximinyl;
  • R5 is hydrogen, halo or alkyl. Said formula I embodies PT2385.
  • the HIF-2a inhibitor is at least one of PT2385, PT2567,
  • HIF-2a inhibitors described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of compounds having the structure of formulae described herein, as well as active metabolites of these compounds having the same type of activity.
  • the compounds described herein can exist in unsolvated as well as solvated forms with
  • the compounds described herein may exhibit their natural isotopic abundance, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature.
  • the present invention is meant to include all suitable isotopic variations of the compounds described herein.
  • hydrogen has three naturally occurring isotopes, denoted ⁇ (protium), 2 H (deuterium), and 3 H (tritium).
  • Protium is the most abundant isotope in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increased in vivo half-life and/or exposure, or may provide a compound useful for investigating in vivo routes of drug elimination and metabolism.
  • Isotopically-enriched compounds may be prepared by conventional techniques well known to those skilled in the art or by processes analogous to those described in the
  • chemical entities described herein may include, but are not limited to, when possible, their optical isomers, such as enantiomers and diastereomers, mixtures of enantiomers, including racemates, mixtures of diastereomers, and other mixtures thereof, to the extent they can be made by one of ordinary skill in the art by routine
  • the single enantiomers or diastereomers i.e., optically active forms
  • Resolution of the racemates or mixtures of diastereomers, if needed, can be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral high-pressure liquid chromatography (HPLC) column.
  • HPLC high-pressure liquid chromatography
  • chemical entities having carbon-carbon double bonds or carbon-nitrogen double bonds may exist in Z- or inform (or cis- or trans- form).
  • some chemical entities may exist in various tautomeric forms. Unless otherwise specified, chemical entities described herein are intended to include all Z-, E- and tautomeric forms as well.
  • HIF-2a inhibitors can include small organic molecules. Some small molecule inhibitors may inhibit multiple HIF alpha subunits (such as HIF- ⁇ , HIF-2a, and/or HIF-3a.), while others may be specific for HIF-2a. In some examples, a small molecule inhibitor inhibits more than one HIF alpha subunit, including HIF-2a. In particular examples, a small molecule inhibitor specifically inhibits HIF-2a expression or activity (such as dimerization, DNA binding, and/or transcriptional activity).
  • the small molecule inhibitor of HIF-2a is a previously identified HIF-2a inhibitor.
  • the inhibitor is at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof.
  • a HIF-2a 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., Zhang 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 HIF-2a small molecule inhibitor includes echinomycin (see, e.g., Kong et al., Cancer Res. 65:9047-9055, 2005), 17-allylamino- 17-demethoxygeldanamycin (see, e.g., Liu et al., Mol.
  • 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 HIF-2a inhibitors for use in the present disclosure also include novel HIF-2a small molecule inhibitors developed in the future.
  • HIF-2a inhibitors also include nucleic acid molecules, including, but not limited to antisense molecules. Some HIF-2a nucleic acid inhibitors may inhibit multiple HIF alpha subunits (such as HIF- la, HIF-2a, and/or HIF-3a.), while others may be specific for HIF-2a. In some examples, a nucleic acid inhibitor inhibits more than one HIF alpha subunit, including HIF- 2a. In particular examples, a nucleic acid inhibitor specifically inhibits HIF-2a expression or activity (such as dimerization, DNA binding, and/or transcriptional activity). [00148] In some examples, the nucleic acid inhibitor of HIF-2a decreases expression of
  • HIF-2a such as an antisense molecule or an RNAi molecule (such as siRNA, miRNA, or shRNA).
  • a HIF-2a 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 HIF-2a decreases activity of HIF-2a, such as a G-rich
  • HIF-2a inhibitors for use in the present disclosure also include novel HIF-2a nucleic acid molecule inhibitors developed in the future.
  • inhibitors of HIF-2a can include HIF-2a-specific binding agents, such as polyclonal or monoclonal antibodies.
  • HIF-2a-specific binding agents include HIF-2a-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 Laboratory Manual, CSHL, New York, 1988).
  • Monoclonal or polyclonal antibodies may be produced to either the normal HIF-2a 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 HIF-2a protein or a fragment thereof would recognize and bind the HIF-2a protein and would not substantially recognize or bind to other proteins found in mammalian cells (for example, human cells).
  • Antibodies for HIF-2a 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 HIF-2a antibodies for use in the present disclosure also include novel anti-HIF-2a antibodies developed in the future.
  • the HIF-2a 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, CMLAGWIPC, and CWLGEWLGC, See, e.g., Kolonin et al., Nat. Med. 10:625-632, 2004 and Nie et al., Stem Cells 26:2735-2745, 2008; U.S. Pat. No. 7,951,362; each of which is
  • 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 HIF-2a inhibitor is coupled to an adipose tissue targeting molecule (such as a peptide or antibody).
  • Methods of coupling molecules are well known to one of skill in the art. This includes, but is not limited to, covalently bonding one molecule to another molecule (for example, directly or via a linker molecule), noncovalently bonding one molecule to another (e.g. electrostatically bonding) (see, for example, U.S. Patent No. 6,921,496, which discloses methods for electrostatic conjugation), noncovalently bonding one molecule to another molecule by hydrogen bonding, non-covalently bonding one molecule to another molecule by van der Waals forces, and any and all
  • the HIF-2a inhibitor is targeted to hepatic tissue.
  • Hepatic- specific ligands or antibodies that bind to one or more specific hepatic cell target molecules e.g., cell surface protein unique to hepatic cells
  • Such “targeting moieties” can be coupled directly to the HIF-2a inhibitor or to the pharmaceutical composition or vehicle used to deliver the HIF-2a inhibitor to the body.
  • targeting strategies specifically for hepatic cells can be found described in the state of the art, for example, in Mishra et al., "Efficient hepatic delivery of drugs: novel strategies and their significance," BioMed Research International, Vol. 2013 (2013), Article ID 382184, the entire contents of which are incorporated by reference.
  • compositions that include an inhibitor of HIF-2a can be formulated with an appropriate pharmaceutically acceptable carrier, depending upon the particular mode of administration chosen.
  • the pharmaceutical composition includes a HIF-2a inhibitor and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition consists essentially of an inhibitor of HIF-2a 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 HIF-2a 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. 2007/0148074; 2007/0092575; and 2006/0246139; U.S. Patent Nos. 4,522, 811 ; 5,753,234; and 7,081,489; PCT Publication No. WO/2006/052285; Benita, Microencapsulation: Methods and Industrial Applications, 2 nd ed., CRC Press, 2006).
  • the HIF-2a inhibitor is included in a nanodispersion system.
  • a nanodispersion system includes a biologically active agent and a dispersing agent (such as a polymer, copolymer, or low molecular weight surfactant).
  • 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 HIF- ⁇ inhibitor (such as 80/20 w/w).
  • the nanodispersion is prepared using the solvent evaporation method. See, e.g., Kanaze et al., Drug Dev. Indus. Pharm. 36:292-301, 2010; Kanaze et al., J. Appl. Polymer Sci. 102:460-471, 2006.
  • the HIF-2a 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).
  • compositions disclosed herein comprise a
  • the composition consists essentially of a HIF-2a inhibitor and at least one pharmaceutically acceptable carrier.
  • the composition consists essentially of a HIF-2a inhibitor and at least one pharmaceutically acceptable carrier.
  • additional active compounds for example additional inhibitors of HIF- ⁇
  • 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
  • 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 HIF-2a 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 HIF- 2a 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 HIF-2a 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 HIF-2a 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 HIF-2a inhibitor to adipose tissue (for example by injection in adipose tissue, such as visceral adipose tissue).
  • 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 HIF-2a inhibitor (such as acriflavine).
  • a therapeutically effective amount of a HIF-2a 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 HIF-2a 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 HIF-2a 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 HIF-2a inhibitor can be the amount of a
  • HIF-2a inhibitor necessary to treat diabetes or reduce body weight in a subject or treat NAFLD.
  • a therapeutically effective amount of an inhibitor of HIF-2a 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 administration of the therapeutic(s).
  • the present disclosure also includes combinations of a HIF-2a inhibitor with one or more other agents useful in the treatment of diabetes or insulin resistance or in the reduction of body weight or NAFLD.
  • the compounds of this disclosure can be administered in combination with effective doses of anti-diabetic agents (such as biguanides,
  • thiazolidinediones or incretins
  • lipid lowering compounds such as statins or fibrates.
  • administration in combination or “co-administration” refers to both concurrent and sequential administration of the active agents.
  • Administration of a HIF-2a 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.
  • the present disclosure provides a method of inhibiting the activities of HIF-2a in a cell, comprising contacting the cell with an effective amount of a compound described herein, e.g., at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof.
  • a compound described herein e.g., at least one of PT2385, PT2567, PT2399, PT2977 or a combination thereof.
  • the inhibition of HIF-2a in the intestine results in reduced obesity, insulin resistance, and NAFLD.
  • the disclosed methods include administering a therapeutically effective amount of an inhibitor of HIF-2a to a subject.
  • HIF-2a inhibitors include compounds that decrease the expression, longevity (e.g., half-life) or activity of HIF-2a (directly or indirectly), for example, relative to a control. Direct inhibition can be accomplished, for example, by binding to a HIF-2a protein and thereby preventing the protein from binding an intended target, such as a
  • an inhibitor of the disclosure can inhibit a HIF— 2a 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 HIF-2a gene product or by altering post-translational modification of a HIF-2a gene product (such as prolyl hydroxylation), or by causing changes in intracellular localization.
  • a subject for example an overweight or obese subject
  • methods for treating insulin resistance and/or diabetes in a subject for example, type 2 diabetes
  • the methods include administering a therapeutically effective amount of a composition including a HIF-2a inhibitor to a subject, thereby decreasing body weight of the subject.
  • the methods include administering a therapeutically effective amount of a composition including a HIF-2a inhibitor to a subject having diabetes or insulin resistance, thereby treating diabetes or insulin resistance in the subject.
  • the administration of the HIF-2a inhibitor is targeted to the intestinal tissue, e.g., vis-a-vis a target moiety specific for a intestinal cell marker and wherein the targeting moiety is complexed or coupled directly to the HIF-2a inhibitor or to the composition or vehicle that is employed to deliver the HIF-2a inhibitor.
  • the targeting moiety is complexed or coupled directly to the HIF-2a inhibitor or to the composition or vehicle that is employed to deliver the HIF-2a inhibitor.
  • the disclosure includes decreasing body weight of a subject or reducing obesity by administering a therapeutically effective amount of a composition including an inhibitor of HIF-2a 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 circumference, and decreasing body fat (such as total body fat, subcutaneous body fat, or visceral body fat).
  • 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 HIF-2a 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".
  • 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
  • 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 HIF-2a 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 , 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 HIF-2a inhibitor).
  • decreasing waist circumference of a subject includes reduction of waist circumference from a starting point (for example greater than 40 inches for men or greater than 35 inches for women) to a target level (for example, waist circumference less than 40 inches for men or less than 35 inches for women).
  • 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 HIF-2a 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 reducing insulin resistance in a subject by administering a therapeutically effective amount of a composition including an inhibitor of HIF-2a to the subject.
  • the method includes selecting a subject with diabetes or at risk for diabetes (such as a subject with prediabetes or shows insulin resistance).
  • 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.
  • FPG fasting plasma glucose
  • 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 HIF-2a 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 HIF-2a 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.
  • 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 al., Am. J. Physiol. 237:E214-E223, 1979).
  • the glucose clamp method includes infusing insulin in a subject at 10-120 mU/m /min and infusing 20% glucose to maintain blood glucose levels between about 90-100 mg/dL. If low levels of glucose (such as ⁇ 4 mg/min) are required to maintain blood glucose levels, then the subject is considered insulin resistant. High levels of glucose (such as>7.5 mg/min) indicate that the subject is insulin sensitive, while between 4-7.5 mg/min of glucose is considered to indicate impaired glucose tolerance (IGT), which is an early sign of insulin resistance.
  • 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 frequently sampled intravenous glucose tolerance test
  • 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.
  • SI value of ⁇ 2 ⁇ /min/mL indicates insulin resistance.
  • administration of a HIF-2a 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 HIF-2a inhibitor).
  • the method includes increasing the insulin sensitivity index to >2 ⁇ /min/mL.
  • insulin resistance is determined by QUICKI (Katz et al., J.
  • administration of an inhibitor of HIF- ⁇ 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).
  • administration of an inhibitor of HIF-2a 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 HIF-2a 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 administration of the HIF-2a inhibitor).
  • the method includes decreasing fasting plasma insulin levels to ⁇ 15 ⁇ /mL.
  • 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 HIF-2a 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 HIF-2a 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 HIF-2a 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 a 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 comparing one or more indicators of body weight or obesity (such as body weight, body mass index, waist),
  • 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 HIF-2a inhibitor.
  • the disclosed methods include comparing one or more indicator of diabetes 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 HIF-2a inhibitor.
  • kits comprising a
  • composition comprising a compound described herein and a pharmaceutically acceptable carrier or excipient and an instruction for using the composition to treat a subject to reduce obesity, insulin resistance, NAFLD or NASH.
  • Example 1 Treatment of non-alcoholic fatty liver disease
  • Non-alcoholic fatty liver disease is becoming the most common chronic liver disease in western countries with limited therapeutic options.
  • related metabolic conditions that include obesity, insulin resistance, type 2 diabetes, and non-alcoholic steatohepatitis are also major health concerns throughout the world.
  • This Example demonstrates a new role for intestinal hypoxia-inducible factor (HIF) not only in NAFLD, but also in obesity, insulin resistance, type 2 diabetes, and non-alcoholic steatohepatitis.
  • the Example shows in particular that human intestine biopsies from patients with or without obesity revealed a relationship between activated HIF2a but not HIFla in increased body mass index and hepatic toxicity.
  • the causality of this correlation was verified in mice with an intestine-specific H 2 ⁇ 2-disruption, in which high-fat diet-induced hepatic steatosis and obesity were substantially decreased.
  • PT2385 a HIF2a-specific inhibitor, had preventive and therapeutic effects on metabolic disorders dependent on intestine HIF2a.
  • Intestine HIF2a inhibition markedly reduced intestine and serum ceramide levels.
  • intestine HIF2a regulates ceramide metabolism mainly from the salvage pathway, which was revealed by the identification of the novel HIF2a target gene encoding neuraminidase 3. These results suggest that intestine HIF2a would be viable target for NAFLD therapy. These results also suggest intestine HIF2a inhibition may be a viable treatment target for related conditions including obesity, insulin resistance, type 2 diabetes, and non-alcoholic steatohepatitis.
  • Non-alcoholic fatty liver disease characterized by the accumulation of ectopic triglycerides in the liver without excess alcohol consumption, is becoming the most common chronic liver disease in industrialized countries 1 .
  • Persistent NAFLD triggers the increased risk of non-alcoholic steatohepatitis (NASH) and end stage liver diseases such as cirrhosis and hepatocellular carcinoma .
  • Obesity is a well-recognized risk factor for NAFLD.
  • Pharmacologic therapy that targets NAFLD remains extremely limited .
  • HIFs hypoxia- inducible factors
  • bHLH-PAS basic helix -hoop-helix Per-Arnt-Sim
  • HIF is a heterodimer of an oxygen-sensitive a subunit and a constitutively expressed ⁇ subunit (HIF-1 ⁇ or ARNT) 5 ' 6 .
  • HIF- ⁇ HIF- la and HIF-2a
  • PHD prolyl hydroxylase domain enzymes
  • VHL von Hippel-Lindau
  • Hepatic HIF-2a but not HIF- la was further identified as a major regulator of hepatic lipid metabolism through the up-regulation of genes involved in fatty acid synthesis (Srebplc and Fasn) and fatty acid uptake (Cd36) and the down-regulation of genes involved in regulating fatty acid ⁇ -oxidation (Ppara and Acoxl) 13.
  • liver HIF-2a activation was recently observed to ameliorate hyperglycemia through the insulin-dependent pathway with increased insulin receptor substrate- 2 (Irs2), or the insulin-independent pathway with the repression of glucagon action 14"16 .
  • Irs2 insulin receptor substrate- 2
  • these studies implied that pharmacological inhibition of liver HIF-2a would not be suitable to exploit for NAFLD therapy, due to the increased risk of increased hepatic glucose production and type 2 diabetes.
  • Several novel targets in the intestine were recently implicated in the development of NAFLD 17"19 . While both HIF- la and HIF-2a are expressed in the intestinal epithelial cells, the role of the intestine HIFa on the pathogenesis of NAFLD and other metabolic diseases is poorly understood.
  • HIF-2a-NEU3 -ceramide axis has a role in NAFLD development.
  • a specific HIF-2a inhibitor PT2385 which is in clinical trials for the treatment of renal cancer, was found to prevent and reverse metabolic disorders through the inhibition of intestinal HIF-2a.
  • intestine HIF-2a is a novel target for the treatment of NAFLD, obesity and insulin resistance, as well as related conditions such as possibly type 2 diabetes and nonalcoholic steatohepatitis.
  • Intestinal HIF-2a signaling is activated in humans with obesity.
  • HIF-2a expression in distal ileum biopsies from nonobese individuals and individuals with obesity by immunohistochemical staining (FIG. la) and western blot analysis (FIG. lb), revealing notably higher HIF-2a expression in humans with obesity relative to nonobese controls.
  • Hif2a ⁇ mice exhibited less body-weight gain with HFD feeding (FIG. 8a).
  • a glucose-tolerance test (GTT) and insulin-tolerance test (ITT) revealed that abrogation of intestinal HIF-2a substantially improved insulin sensitivity (FIG. 8b,c).
  • GTT glucose-tolerance test
  • ITT insulin-tolerance test
  • H&E and Oil Red O staining showed a reduction in hepatic lipid droplets in Hif2a Am mice (FIG. 2a).
  • Hif2a Am mice displayed significantly lower liver weights and liver weight-to-body weight ratios relative to controls (FIG. 2b,c).
  • Hepatic triglyceride levels, hepatic and serum cholesterol levels, and serum ALT levels reflecting hepatic lipotoxicity were markedly lower in Hif2a ⁇ mice, with no significant difference in serum triglyceride levels as compared to the ⁇ mice (FIG. 2d-h).
  • ceramides such as C16:0 (Ml, m/z 582.5103); C18: l (M2, m/z 608.5250); C18:0 (M3, m/z 610.5403); C20:0 (M4, mJz 638.5717); C22:0 (M5, m/z 666.6019); and C24: l (M6, m/z 692.6186) (FIG. 3b).
  • the levels of ceramides especially the most abundant, C16:0 ceramide, were substantially lower in the small intestines of Hif2a ⁇ mice than in those of the controls (FIG. 3c). Similar to what was observed in the small intestine, serum ceramides were also lower in Hif2a mice than in Hif2a mice (FIG. 3d and FIG. 9a,b).
  • Ceramides are synthesized through three different pathways (FIG. 3e): a de novo pathway from palmitoyl-CoA and serine, a sphingomyelinase (SMase) pathway generated by the hydrolysis of sphingomyelin and the salvage pathway generated from the catabolism of complex sphingolipids, such as from ganglioside monosialo 3 (GM3) hydrolysis 17, 18. Consequently, sphingomyelins and glucosylceramides, the two sources for ceramide synthesis from SMase and salvage pathways, were also evaluated.
  • SMase sphingomyelinase
  • GM3 ganglioside monosialo 3
  • Gba2 in the salvage pathway were significantly downregulated in Hif2a ⁇ mice as compared to in Hif2a wa mice (FIG. 9f,g and FIG. 3h).
  • the mRNAs encoded by genes involved in ceramide catabolism were at similar levels in Hif2a ⁇ mice and in controls (FIG. 9h).
  • HIF-2a disruption, ⁇ , ⁇ and Hif2a ⁇ mice were treated with a HFD for a short duration of 1 week that did not lead to a notable alteration of body weight (FIG. 10a).
  • a GTT and an ⁇ revealed an improvement in glucose intolerance and insulin resistance in HFD-fed Hif2a ⁇ mice as compared to those of Hif2a wa mice (FIG. 10b,c).
  • the energy expenditure was substantially enhanced in Hif2a ⁇ mice, without significant changes in cumulative food intake and ambulatory activity (FIG. lOd-h).
  • Hif2a mice exhibited lower hepatic triglyceride levels and trended toward a reduction in serum ALT levels without significant changes in liver weights and liver weight-to-body weight ratios, hepatic and serum cholesterol levels, and serum triglyceride levels, as compared to ⁇ mice (FIG. lOi-o).
  • HIF-2a modulates ceramide synthesis through targeting Neu3
  • NEU3 mRNA was notably greater in the ileum biopsies of humans with obesity relative to individuals without obesity and was positively correlated with BMI, ALT, AST, DMT1 mRNA, and DCYTB mRNA expression (FIG. 4a-f).
  • An upregulation of NEU3 expression was also observed in the small intestines of HFD-treated mice as compared to chowfed mice (FIG. 4g). Lactosylceramide is the product of NEU3 and a substrate for GLB 1 in the salvage pathway.
  • HRE HIF-response elements
  • FIG. 4i which was analyzed by transient transfection using a Neu3 promoter luciferase reporter construct.
  • the hypoxia mimic CoC12 or co-transfection with a constitutively active HIF-2a triple mutant (TM) expression plasmid markedly induced the luciferase activity (FIG. 4j).
  • the HIF-2a TM induction of luciferase expression was further potentiated in cells incubated with CoC12.
  • HRE1 (AHREl) or HRE2 (AHRE2) single-deletion constructs did not change luciferase activity, whereas the activity in both HRE (AHRE) deletion constructs was markedly suppressed.
  • Intestine-derived ceramides control hepatic steatosis
  • ceramide administered by the injection of C16:0 ceramide to Hif2a ⁇ mice fed a HFD for 6 weeks resulted in increased ceramide levels in small intestine and serum that were comparable to those in vehicle-treated HFD-fed Hif2a wa mice (FIG. 14a,b).
  • the administration of ceramide substantially reversed the improvement in body weight and insulin resistance in HFD-fed Hif2a Am mice as compared to that of Hif2a wa mice (FIG. 14c-f).
  • PT2385 improves steatosis by inhibiting intestinal HIF-2a
  • Hif2a ⁇ mice (FIG. 15a-c). Histology analysis showed that PT2385 eliminated hepatic lipid accumulation in HFD-fed H ⁇ mice, but had no further inhibition on Hif2a Am mice (FIG.
  • Hif2a ⁇ mice exhibited lower liver weights, ratios of liver weight to body weight, hepatic triglycerides, hepatic and serum cholesterol content, and ALT versus ⁇ mice, and were unresponsive to the inhibition of PT2385 treatment (FIG. 15e-k). Further, small intestine and serum ceramide levels were markedly reduced by PT2385 in the Hif2a wa mice, but not in the
  • Hif2a ⁇ mice (FIG. 151,m). Accordingly, the mRNA levels of Degs2, Smpdl, Smpd3, Smpd4, Enpp7, Neu3, Glbl, and Gba2 were substantially inhibited in the HFD-fed ⁇ mice by
  • PT2385 treatment but remained similar in the HFD-fed Hif2a ⁇ mice (FIG. 16a-d).
  • PT2385 downregulated the mRNA expression of Srebplc, Cidea, Cd36, Fabp4, Fasn, Scdl, Elovl6, Plin2, Tnfa, Pail, Ccl2, and Ccl3 in the ⁇ mice, whereas no change was found in Hif2a m mice treated with PT2385 (FIG. 16e,f).
  • PT2385 treatment substantially inhibited HIF-2a signaling as indicated by the decreased target gene Dmtl and Dcytb mRNAs in the small intestine (FIG. 17d).
  • PT2385-treated mice exhibited lower ceramide levels in both the small intestine and serum relative to vehicle-treated mice, owing to suppressed HIF-2a signaling (FIG. 6i and FIG. 17e).
  • HFD treatment 27 HIF-2a and AhR as heterodimeric transcription factors share the same heterodimer, partner HIF- ⁇ . Thus, there is the potential for cross-talk between the HIF-2a and AhR signaling pathways that might influence HIF-2a signaling.
  • Intestinal HIF-2a depletion results in less susceptibility to HFD induced hepatic fatty liver and obesity, accompanied by a downregulation of intestine and serum ceramide levels.
  • the underlying mechanism revealed that intestine HIF-2a but not HIF-la inhibition markedly suppressed intestinal-derived ceramides by directly targeting ceramide biosynthesis by the key enzyme in the ceramide salvage pathway, NEU3.
  • inhibition of intestinal HIF-2a signaling by PT2385 had both preventive and therapeutic effects on NAFLD and obesity (FIG. 61).
  • HIF-2a signaling in human intestine biopsies and obesity There is a positive correlation between HIF-2a signaling in human intestine biopsies and obesity.
  • HIF-la exerts a potent protective function at the epithelial barrier by regulating adherensj unction and tight- junction genes, including claudin 1 (Cldnl), mucin 3 (Muc3), trefoil factor 1 (Tjfl), and 5- ectonucleotidase (Cd73) 32—35. It was reported that HIF-2a has a dual role in barrier function. Acute activation of HIF-2a results in the maintenance of tight-junctionassemblies and barrier integrity through the upregulation of creatine kinase 36 , whereas chronic activation of HIF-2a disrupts the tight junctions through an increase of caveolin 1 (ref. 37).
  • intestinal HIF-2a activation in intestinal epithelial cells triggers a spontaneous inflammatory response, whereas intestinal HIF-2a deficiency substantially reduces inflammation through the direct regulation of inflammatory mediators, including tumor necrosis factor-a, microsomal prostaglandin synthase 1, and cyclooxygenase 2 in colitis models 20 ' 39. It cannot be excluded that improvement of the intestinal epithelial permeability and inflammation might contribute to the metabolic benefits of intestinal HIF-2a inhibition in the mouse model of obesity.
  • Ceramide synthase enzymes 6 leads to hepatic steatohepatitis and insulin resistance, whereas liver- or adipose- specific Cers6 disruption improves fatty liver and obesity by boosting fatty acid ⁇ -oxidation 46 ' 47 .
  • Mice lacking dihydroceramide desaturase 1 are also resistant to HFD-induced obesity and insulin resistance, owing to lower levels of ceramides 48.
  • Ceramides substantially upregulate fatty acid uptake and synthesis through direct or indirect modulation of CD36 and SREBP1C signaling, respectively 15 ' 46 .
  • mice lacking intestine HIF-2a are resistant to HFD-induced hepatic steatosis and obesity, which is correlated with lower intestine and serum ceramides, with suppressed fatty acid synthesis and uptake, and with higher 'beiging' and thermogenic capacity.
  • expression of the fatty acid ⁇ -oxidation-related genes are not changed in the livers of Hif2a wa and Hif2a ⁇ mice fed a HFD for 1 week, whereas most genes encoding fatty acid-synthesis- related enzymes are substantially downregulated in the livers of Hif2a ⁇ mice, which suggests that the HIF-2a -ceramide axis mainly regulates hepatic fatty acid synthesis.
  • intestine HIF-2a but not HIF-la was defined as a novel regulator of the ceramide salvage pathway, as revealed by measuring Neu3, Glbl, and Gba2 mRNA expression.
  • NEU3 catalyzes the hydrolysis of GM3 into lactosylceramides, which can be converted into glucosylceramides by GLB 1
  • GBA2 catalyzes the generation of ceramides from glucosylceramides 53.
  • Neu3 is a direct target gene of HIF-2a.
  • NEU3 overexpression in liver was observed to increase hepatic lipid accumulation and liver weight 54 .
  • HIF-2a a bHLH-PAS domain protein
  • PT2385 is an orally bioavailable HIF-2a antagonist that specially inhibits HIF-2a transcriptional activity by allosterically blocking the heterodimerization between HIF-2a and HIF- ⁇ , while having no effect on HIF- ⁇ .
  • PT2385 is well tolerated without toxicities in a phase 1 clinical trial to treat renal cell carcinoma.
  • Distal ileum mucosa biopsies were taken from 39 individuals who underwent colonoscopy. The subjects were between 18 to 65 years, and all had a BMI between 18.0 and
  • BMI 37.6 kg/m 2.
  • the genders and ages were at similar levels at baseline between the groups as disclosed in FIG. 19.
  • the clinical biochemistry viariables are listed in FIG. 20.
  • Hifta m , Vhf ⁇ , VhUHifla , and Vhl/Hifta ⁇ mice were previously described.
  • Vhl/Hifla ]E , Vhl Hifta m , and Vhl/Hif2a m were in a mixed Svl29 and C57BL/6 background.
  • the ⁇ and Hif2a Am were on a C57BL/6N background, after backcrossing with C57BL/6N mice for over five generations.
  • HFD (60% kcal from fat) was purchased from Bioserv. Inc (Flemington, NJ).
  • Eight- to 10- weeks-old male littermate ⁇ and Hif2a Am mice were fed a chow or HFD for 1 or 12 weeks to induce hepatic steatosis.
  • C16:0 ceramide purchased from Avanti Polar Lipids (Alabaster, AL), was suspended in saline with 0.5% sodium carboxymethyl cellulose.
  • Hif2a ⁇ mice fed a HFD were intraperitoneally injected every other day with vehicle or C 16:0 ceramide (10 mg/kg) for 6 weeks.
  • DANA and naringin were purchased from Sigma- Aldrich (St. Louis, MO). DANA was dissolved in saline and naringin was suspended in saline with 0.5% sodium carboxymethyl cellulose and 5% dimethyl sulfoxide.
  • C57B6/N mice fed a HFD were gavaged with vehicle, DANA (20 mg/kg o.p.d.), or naringin (200 mg/kg o.p.d.) for 4 weeks.
  • PT2385 purchased from MedChem Express (Monmouth Junction, NJ), was suspended in saline with 0.5% sodium carboxymethyl cellulose, 2.5% Tween 80, and 2.5% dimethyl sulfoxide.
  • PT2385 For treatment of hepatic steatosis, obese C57BL/6N mice fed a HFD for 8 weeks were administered vehicle or PT2385 (20 mg/kg o.p.d.) by gavage for another 4 weeks.
  • PT2385 was HIF-2a dependent, 8- to 10- weeks-old male littermate ⁇ and Hif2a Am mice, were fed a HFD and administered vehicle or PT2385 (20 mg/kg o.p.d.) by gavage for 12 weeks.
  • mice For the short-term treatment, 8- to 10-weeks-old male littermate Vhl/Hifla ⁇ , Vhl/Hifla Am mice, were fed a chow diet and administered vehicle or PT2385 (20 mg/kg o.p.d.) by gavage for 3 days. All mice were randomly assigned to experimental groups (at least 4 mice per group) and the groups did not present differences in body weights before the treatments. All mouse studies were approved by the NCI Animal Care and Use Committee and performed in accordance with the Institute of Laboratory Animal Resources guidelines. All mice were fed ad-libitum and kept in a 12-h light- dark cycle.
  • ODD-luciferase transgenic mice were obtained from Jackson Laboratories (Bar
  • mice Ten- week-old male littermate mice were fed a chow or HFD for 1 week. Small intestines were collected, extracted with lysis buffer and the luciferase activities were measured by use of the luciferase assay system (Promega).
  • GTT glucose tolerance test
  • ITT insulin tolerance test
  • mice were fasted overnight for 4 hours.
  • Glucose at 2 g/kg, or insulin (Eli Lilly, Washington, DC) at 0.8 U/kg, in saline were injected intraperitoneally to conscious animals and from tail vein blood glucose at was measured at 15, 30, 60, and 90 min post injection using a Glucometer (Bayer, Pittsburgh, PA).
  • Indirect calorimetry was performed on Hif2a and Hif2a mice fed a HFD for 1 week using a 12-chamber Environment Controlled CLAMS (Columbus Instruments, Columbus, OH). After 48-h acclimatization, mice were monitored for 24 h at 22 °C. During testing, food and water were provided ad libitum.
  • Formalin fixed paraffin embedded liver sections was stained by Hematoxylin and eosin (H&E) and OCT embedded frozen liver sections was stained by Oil O Red according to standard protocols followed by microscopic examination. At least three discontinuous liver sections were evaluated for each mouse.
  • Liver injury was evaluated by measuring alanine aminotransferase (ALT) in serum (Catachem Inc., Oxford, CT). Hepatic and serum triglycerides were determined with a triglyceride colorimetric assay kit (Bioassay Systems, Hayward, CA). Hepatic and serum cholesterol were measured using assay kit from Wako Diagnostics (Wako Chemicals USA, Inc., Richmond, VA).
  • ALT alanine aminotransferase
  • Intestine neuraminidase activity was determined in the intestine homogenates using a Neuraminidase Activity Assay kit (Sigma- Aldrich). Inhibition of NEU3 in the intestines may be a treatment strategy for metabolic disorders.
  • NEU3 promoter region was predicted by FANTOM5 mouse promoterome.
  • Hypoxia response elements in the promoter region were further identified by HIF-2a CHIP-seq (see below).
  • the NEU3 promoter and the HRE-lacking promoter fragments were amplified by PCR from mouse genomic DNA. The primer sequences are listed in FIG. 21.
  • the amplified fragments were digested by Kpn I and Xho I restriction enzymes (New England Biolabs), and then cloned into the pGL4. l l luciferase vector (Promega).
  • NEU3 reporter vectors and phRL-TK Renilla luciferase control vector were co-transfected into HCTl 16 cells (ATCC CCL-247) by use of Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific).
  • HIF2aTM expression vector or the empty backbone vector (pcDNA3) were co-transfected into the cells and cobalt (II) chloride hexahydrate (Sigma-Aldrich) was added to culture medium at a 100 ⁇ final concentration to mimic hypoxia.
  • Empty vector pGL4.11 was used as a negative control and the standard.
  • luciferase assays were performed by use of the Dual-luciferase assay system (Promega). Firefly and Renilla luciferase activities were measured by Veritas microplate luminometer (Turner Biosystems).
  • HCT116 cells were seeded in 12-well plates (for gene expression analysis) or 6- well plates (for lipidomics analysis). Cells were treated with vehicle, PT2385 (10 ⁇ ), or DANA (100 ⁇ ), or transfected with siNEU3 (20 nM, Thermo Fisher Scientific, Waltham, MA) and exposed to either vehicle or CoCl 2 (100 ⁇ ) for 24 hours.
  • RNA from frozen intestine and liver was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA).
  • cDNA was synthesized from 1 ⁇ g total RNA using qScriptTM cDNA SuperMix (Gaithersburg, MD). Real-time PCR primer sequences are included in FIG. 21. The relative amounts of each mRNA was calculated after normalizing to their corresponding ⁇ - actin mRNA and the results expressed as fold change relative to the control group.
  • Intestine samples were lysed in RIPA buffer with protease and phosphatase inhibitors, and then the protein extracts were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane.
  • the membrane was incubated overnight at 4°C with antibodies against HIF2a, HIFla (Novus Biologicals, LLC, Littleton, CO), NEU3 (Origene Technologies, Rockville, MD), and ytf-ACTIN (Cell Signaling, Danvers, MA).
  • ChIP was performed as described previously on duodenal epithelium scrapings using 1% formaldehyde in IX PBS as a cross -linker 40 .
  • the primary antibody for HIF2a (Novus Biologicals) was used for immunoprecipitation.
  • the precipitated DNA samples were incubated with RNase A and proteinase K, purified using PCR clean-up column (Qiagen), and 2 ⁇ L ⁇ of sample was used for real-time PCR using primers listed in FIG. 21.
  • the lipidomics analysis was undertaken as previously described 19 .
  • the multivariate data matrix was analyzed by SIMCA-P+14 software (Umetrics, Kinnelon, NJ).
  • TargetLynx software a subroutine of the MassLynx software (Waters Corp.).
  • the ceramide standards including C16:0, C18:0, C18: l, C20:0, C22:0, C24:0 and C24: l were obtained from Avanti Polar Lipids
  • hypoxia- inducible factor HIF

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Abstract

Le procédé selon l'invention concerne un traitement de l'obésité, de la résistance à l'insuline, de la stéatose hépatique non alcoolique et de la stéatohépatite non alcoolique chez un sujet, comprenant la réduction sélective de l'expression ou l'inhibition de l'activité de HIF-2a dans l'intestin, réduisant ainsi l'obésité, la résistance à l'insuline et la stéatose hépatique non alcoolique chez le sujet.
PCT/US2018/020333 2017-02-28 2018-02-28 Procédé de traitement de l'obésité, de la résistance à l'insuline, d'une stéatose hépatique non alcoolique comprenant une stéatohépatite non alcoolique WO2018160772A1 (fr)

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WO2021093724A1 (fr) * 2019-11-15 2021-05-20 武汉光谷亚太医药研究院有限公司 Nouveau composé dérivé de 2,3-hydrindène, procédé de préparation et application
CN113509555A (zh) * 2021-07-19 2021-10-19 中国药科大学 Akt2抑制剂在制备非酒精性脂肪肝病治疗药物中的应用
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EP3982944A4 (fr) * 2019-06-10 2023-09-13 University of Iowa Research Foundation Modulateurs de swell1 pour le traitement d'une stéatose hépatique non alcoolique, de déficiences immunitaires, d'une stérilité masculine et de maladies vasculaires
WO2024078127A1 (fr) * 2022-10-13 2024-04-18 浙江大学医学院附属第一医院 MUTANT DU GÈNE HIF2α ET SON UTILISATION DANS LA CONSTRUCTION D'UN MODÈLE DE SOURIS NAFLD

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