WO2002085396A1 - Inhibition de la kinase jun - Google Patents

Inhibition de la kinase jun Download PDF

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Publication number
WO2002085396A1
WO2002085396A1 PCT/US2002/012687 US0212687W WO02085396A1 WO 2002085396 A1 WO2002085396 A1 WO 2002085396A1 US 0212687 W US0212687 W US 0212687W WO 02085396 A1 WO02085396 A1 WO 02085396A1
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jnk
jnkl
mice
insulin resistance
inhibitor
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PCT/US2002/012687
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English (en)
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Gokhan S. Hotamisligil
Michael Karin
Lufen Chang
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President And Fellows Of Harvard College
Regents Of The University Of California
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Priority to CA002477842A priority Critical patent/CA2477842A1/fr
Priority to US10/475,505 priority patent/US7232897B2/en
Priority to EP02764295A priority patent/EP1390052A4/fr
Publication of WO2002085396A1 publication Critical patent/WO2002085396A1/fr
Priority to US11/799,397 priority patent/US20070207137A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin

Definitions

  • This invention relates to insulin resistance. BACKGROUND OF THE INVENTION
  • NIDDM non-insulin dependent diabetes mellitus
  • the invention is based on the discovery that reduced expression of a NH2-terminal Jun Kinase (JNK), e.g., JNKl, leads to reduced weight and improved insulin sensitivity. Accordingly, the invention features a method of treating a metabolic disorder associated with insulin resistance by administering to a mammal an inhibitor of JNK.
  • the mammal e.g., a human patient, is identified as being obese or at risk of becoming obese.
  • obese is meant having an excess amount of adipose tissue.
  • Standard clinical tests are used to determine whether an individual is obese, e.g., by calculating relative weight or body mass index (BMI) for an individual and comparing the values to a predetermined standard of ideal or desirable relative weight or BMI.
  • BMI body mass index
  • assessment of skin fold thickness over various areas of the body is taken into consideration together with height, weight, and age to determine the amount of adipose tissue content in an individual.
  • Excess of adipose tissue content is determined by comparing the value against average (or standard) values for an individual of comparable age. For example, a 20% increase in mean relative weight or a BMI above the 85 th percentile for young adults constitutes a health risk and may indicate therapeutic intervention, e.g., treatment with a JNK inhibitor.
  • the inhibitors are also administered to individuals who are not obese, but wish to reduce their weight.
  • the mammal is identified as suffering from diabetes, is at risk of developing diabetes, suffering from insulin resistance, or at risk of developing insulin resistance.
  • diabetes includes both insulin-dependent diabetes mellitus (i.e., IDDM, also known as type I diabetes) and non-insulin-dependent diabetes mellitus (i.e., NIDDM, also known as Type II diabetes).
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin-dependent diabetes mellitus
  • the mammal is suffering from or at risk of developing Type II diabetes.
  • JNK inhibitors are compounds, which reduce the enzymatic activity of a JNK, e.g., JNKl or JNK2, or expression of a JNK isotype.
  • compounds, which inhibit JNK enzymatic activity bind to an ATP binding site in JNK or bind to a catalytic domain of JNK.
  • the compound preferentially inhibits JNKl compared to JNK2 or other JNK isotypes.
  • the compound inhibits JNK2 or both JNKl and JNK2.
  • the compound is SP600125.
  • Compounds, e.g., polypeptides, organic compounds, or inorganic compounds are isolated or purified.
  • An "isolated" or “purified” composition is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • a polypeptide that is substantially free of cellular material includes preparations of the polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated, e.g., the polypeptide is recombinantly produced.
  • a preparation of a therapeutic compound e.g., a JNK inhibitor, is at least 75%, more preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, and most preferably 99 or 100% of the dry weight of the preparation.
  • the invention also includes a method of improving insulin sensitivity or alleviating a symptom of insulin resistance, reducing the severity of insulin resistance, diabetics, or an associated metabolic disorder, by administering to a mammal an inhibitor of JNK expression or activity.
  • Methods of treating or preventing the development of obesity are also within the invention.
  • Metabolic conditions associated with insulin resistance include high blood glucose levels, markedly elevated serum insulin concentrations, and insensitivity to intravenously administered insulin. Insulin resistance is defined as the requirement of 200 or more units of insulin per day to control hyperglycemia and prevent ketosis.
  • Compounds are administered at a dose that is therapeutically effective.
  • therapeutically effective amount means that the amount of a compound(s) or pharmaceutical composition elicits a beneficial biological or medicinal response in a tissue, system, animal or human.
  • a therapeutically effective amount of a JNK inhibitory compound is a dose which leads to a clinically detectable improvement in insulin sensitivity, weight loss, or a reduction in hepatic fat content.
  • a method of identifying an individual that is at risk of developing insulin resistance is carried out by measuring the level of JNK activity in a tissue of a mammal. Measuring the level of JNK activity in a tissue of a mammal is also useful to diagnose insulin resistance, diabetes, or a predisposition to develop the disorders. An increase in activity compared to a normal control indicates that the mammal is suffering from or is predisposed to developing insulin resistance. Insulin resistance or a predisposition thereto is also diagnosed by measuring the level of JNK expression in a tissue of a mammal.
  • JNK expression is measured by detecting a gene product, e.g., using an antibody or other specific ligand, or by detecting gene transcription, e.g., using a standard Northern blot assay or reverse transcriptase polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the invention also includes a method of inhibiting fat accumulation in liver tissue by contacting the tissue with an inhibitor of a JNK.
  • the JNK inhibitor reduces JNK enzymatic activity as described above, e.g., SP600125.
  • the inhibitor preferentially reduces enzymatic activity of JNKl compared to JNK2.
  • the inhibitor reduces JNKl activity by at least 10%, more preferably 20%, 50%, 100%, and 200% compared to the level of reduction of JNK2 activity.
  • the method is useful to prevent the development or slow the progression of fatty liver disease or hepatosteosis.
  • the method is carried out by identifying an individual who is at risk of developing fatty liver disease, e.g., by identifying one who consumes excessive amounts or alcohol, one who is at least 10% above ideal body weight, one who is obese, or one who has a family history of liver disease, and administering to the individual an inhibitor of JNKl activity.
  • Liver tissue is contacted directly in situ, e.g., by direct injection into the liver, or systemically, e.g., by oral or intravenous administration. Contacting liver tissue with a compound which preferentially inhibits JNKl activity leads to reduced accumulation of fat in hepatic cells.
  • Fig. 1 is a line graph showing body weight in JNK-deficient (JNK -/-) mice compared to wild type (JNK +/+) control mice. JNK-deficient and control mice were put on a high fat diet for 12 weeks. The body weight was of JNK-deficient mice was consistently and significantly reduced compared to control mice fed the same diet.
  • Fig. 2A is a bar graph showing blood glucose levels in JNK-deficient mice compared to control mice. Both groups of mice were fed a high fat diet for 12 weeks. Blood glucose levels of JNK-deficient mice were significantly reduced compared to wild type mice.
  • Fig. 2B is a bar graph showing blood insulin levels in JNK-deficient mice compared to control mice. Both groups of mice were fed a high fat diet for 12 weeks. Blood insulin levels of JNK-deficient mice were significantly reduced compared to wild type mice. The reduction in blood glucose and blood insulin levels shown in Figs. 2A-B indicate improved insulin sensivity in JNK-deficient mice compared to control mice.
  • Fig. 4 is a bar graph showing increased JNK activity in obesity.
  • Figs. 5 A is a photograph of an electrophoretic gel showing the results of a solid- phase JNK assay measuring total JNK activity.
  • Fig. 5B is an photograph of an immunoblot showing different JNKl/2 isoforms.
  • Fig. 5C is a bar graph showing means ⁇ SEM of the quantitated and normalized activity. All mice assayed were male, 16- week- old and on C57B1/6 background. Total JNK activity and protein levels was measured in liver, muscle and adipose tissues of lean and obese [dietary (obese-HF) and genetic (ob/ob)] mice.
  • Figs 6A and 6C are line graphs showing weight gain over time.
  • Figs. 8A and 8C are bar graphs showing blood glucose levels (a measure of glucose homeostasis) by fasting plasma glucose.
  • Figs 8B and 8D are bar graphs showing blood insulin levels.
  • Figs. 8G/K are line graphs and Figs. 8H /L are bar graphs showing the results of glucose toleranc tests in lean and obese Jnkl-/-, Jnk2-/- and control male mice at 16-weeks of age.
  • Figs.8E/I are line graphs and Figs. 8F/J are bar graphs showing the results of insulin tolerance tests in lean and obese Jnkl-/-, Jnk2-/- and control male mice at 16-weeks of age.
  • Figs. 8F, 8H, 8J, and 8L "AUC" designates the area under curve for the glucose disposal curves in Figs. 8F, 8H, 8J and 8L.
  • Investigation of the dynamics of the responses to the tolerance tests were done by ANOVA repeated measures analysis (Statview 4.01, Abacus Concepts, Berkeley, CA) and demonstrated statistically significant differences between Jnkl-/- and Jnkl+/+ mice indicated by * (p ⁇ 0.001).
  • Figs. 9A and 9B are bar graphs showing a reduction in hepatomegaly in Jnk-/- mice.
  • Figs. 10A-F are photomicrographs of liver tissue sections. Tissue sections shown in Figs. 10A-C were stained with a standard eosin hematoxylin stain to visual tissue architecture. Tissue sections shown in Figs. 10D-F were stained with Oil-Red-O to visualize fat deposits. Dark areas in the images shown in Figs. 10D-F represent fat deposits. The amount of fat accumulation in liver tissue of JNKl -deficient mice was greated reduced compared to the amount observed in WT or JNK2-deficient mice.
  • Figs. 11 A-C are bar graphs showing body weight and glucose homeostasis in Jnkl+/+and Jnkl-/-ob/ob mice.
  • Fig. 11A shows weight gain over time.
  • Fig. 11B shows plasma glucose levels, and
  • Fig. 11 C shows plasma insulin levels.
  • Body weight measurements and blood sampling in the ob/ob group were performed at 4 and 8 weeks of age and following a 6-h daytime food withdrawal. Statistical significance (p ⁇ 0.05) is indicated by *.
  • Figs. 12A-G show JNK activity and insulin signaling in JNKl deficient mice.
  • Fig. 12A is a photograph of an electrophoretic gel showing JNK kinase activity.
  • Fig. 12A is a photograph of an electrophoretic gel showing JNK kinase activity.
  • FIG. 12B is a photograph of an immunoblot showing JNK protein levels in liver, muscle and adipose tissues of lean and obese, Jnkl+/+ (wt) and Jnkl-/- mice.
  • Fig. 12 C is a bar graph showing JNK kinase activity as means ⁇ SEM of the quantitated and normalized JNK activity based on the data shown in Figs. 12A-B.
  • Fig. 12D is a photograph of an electrophoretic gel showing insulin receptor substrate- 1 (IRS-1) phosphorylation at serine 307; total and serine 307-phosphorylated IRS-1 levels were determined in liver tissues from lean (L) and obese (O) mice.
  • Fig. 1 insulin receptor substrate- 1
  • FIG. 12E is a bar graph showing the serine phosphorylation mean values.
  • FIG. 12F is a photograph of an immunoblots of insulin-stimulated tyrosine phosphorylation (pTyr) of IR and IRS-1 in liver tissues of Jnkl-/- and Jnkl+/+ mice in specific immunoprecipitates. IR and IRS-1 tyrosine phosphorylation (pTyr) and total protein levels after vehicle (-) or insulin (+) stimulation was determined by immunoblot analyses. Each lane represents an individual mouse.
  • Fig. 12G is a bar graph showing IR tyrosine phosphorylation mean values.
  • TNF-alpha leads to serine phosphorylation of insulin receptor substrate- 1 (IRS-1) to induce insulin resistance.
  • JNK phosphorylates IRS-1 at a serine residue. Genetic ablation of JNK was found to result in decreased body weight, increased systemic insulin sensitivity, and reduced glucose and insulin levels.
  • Inhibitors of JNK are useful to treat obesity, insulin resistance, and diabetes. Modulation of expression or activity of JNK influences body weight, insulin resistance, and levels of insulin, glucose, and lipids in vivo.
  • Insulin resistant mammals e.g., humans, include mammals suffering from non- insulin dependent diabetes mellitus (NIDDM) or pre-NIDDM and other insulin resistant states such as glucose intolerance. These conditions may be related to aging and obesity.
  • NIDDM non- insulin dependent diabetes mellitus
  • pre-NIDDM insulin dependent diabetes mellitus
  • glucose intolerance other insulin resistant states
  • Inhibiting JNK kinase activity or expression of JNK is used to treat obesity and other metabolic disorders associated with disregulation of JNK and/or insulin resistance.
  • Treatment includes the management and care of an individual for the purpose of alleviating a symptom of a disease or pathological condition.
  • Treatment includes the administration of a compound to prevent the onset of symptoms or complications of a clinical disorder, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.
  • Treating an insulin resistant mammal includes increasing insulin sensitivity and/or insulin secretion to prevent islet cell failure.
  • JNK inhibition is also useful to alleviate the symptoms of other conditions associated with insulin resistance such as cancer cachexia, HIV-1 infection, polycystic ovarian syndrome, atherosclerosis, and severe burns. In the latter case, acute phase burn victims are given a JNK inhibitor shortly after the burn incident to prevent or decrease the development of insulin resistance.
  • JNK-deficient mice Improvement of conditions related to obesity and insulin resistance JNK-deficient mice were generated using methods known in the art.
  • the mice contain a null mutation in a gene encoding a JNK, and therefore, fail to express the corresponding gene product or express a non-functional gene product.
  • Fig. 1 shows that JNK-deficient mice fed a high fat diet weigh less than wild type control mice fed the same diet.
  • Figs. 2A-B and Figs. 3A-B indicate that JNK-deficient mice have improved insulin sensitivity compared to wild type control mice. In most tissues, inhibition of one isotype of JNK leads to an increase in expression of another isotype.
  • JNK2 does not compensate for a decrease or loss of JNK-1 expression or activity.
  • contacting cells or a tissue of a mammal with an inhibitor of JNK expression or an inhibitor of JNK enzyme activity improves insulin sensitivity.
  • the data also indicate that such compounds are useful to treat or prevent the development of obesity.
  • Therapeutic Administration Mammals such a humans, which are overweight, obese, or at risk of becoming so, are treated with compounds which decrease JNK expression or activity.
  • humans, who are at risk of developing hepatosteosis also benefit by intervention to reduce JNK expression or activity.
  • activity is meant kinase enzyme activity.
  • Body mass index (BMI) is measured (kg/m (or lb/in X 704.5)).
  • waist circumference estimates fat distribution
  • waist-to-hip ratio estimates fat distribution
  • skinfold thickness if measured at several sites, estimates fat distribution
  • bioimpedance based on principle that lean mass conducts current better than fat mass (i.e. fat mass impedes current), estimates % fat
  • Overweight individuals are characterized as having a waist circumferenceof >94 cm for men or >80 cm for women and waist to hip ratios of > 0.95 in men and > 0.80 in women.
  • Obese individuals are characterized as having a BMI of 30 to 34.9, being greater than 20% above "normal" weight for height, having a body fat percentage > 30% for women and 25% for men, and having a waist circumference >102 cm (40 inches) for men or 88 cm (35 inches) for women.
  • Individuals with severe or morbid obesity are characterized as having a BMI of > 35.
  • hepatosteosis Individuals who are at risk of developing hepatosteosis include overweight persons as well as those who consume excessive amounts of alcohol, e.g., greater than two drinks per day for females, and greater than 3 drinks per day for males.
  • Candidates for JNK inhibitory treatment are also identified by examining the liver by ultrasonography to detect hepatomegaly or excessive fat accumulation or by biopsy to detect fat deposits.
  • Administration of a JNK inhibitory compound protects agains the development of hepatosteosis and/or slows its progression. The inhibitor is administered locally or systemically as described below.
  • Inhibitors of JNK kinase activity are known in the art, e.g., SP-600125 (Signal Pharmaceuticals Inc., San Diego, CA). SP-600125 is a selective JNK inhibitor, which inhibits the phosphorylation of c-Jun in a dose-dependent manner. This inhibitor is selective for JNK compared to other kinases and other enzymes.
  • Other compounds, which inhibit JNK activity include genistein, herbimycin A, 4-amino-5-(4-chlorophenol) -7-(t-butyl)pyrazolo[3,4-D]pyrimidine (or PP2).
  • EGFR specific inhibitor, tyrphostin AG1478 also inhibits c-JNK activation.
  • JNK JNK interacting protein
  • a peptide inhibitor includes amino acids 33-79 of c-Jun (U.S. Patent No.
  • This peptide is a competitive inhibitor, which decreases the amount of c-Jun activation by JNK.
  • Antibodies or other ligands, which bind to an ATP binding site or catalytic domain of JNK are used to inhibit JNK kinase activity.
  • the amino acid sequence, nucleotide sequence, and domains of JNKl are described in U.S. Patent No. 6,193,965.
  • Inhibitory compounds are formulated with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral or inhalation) or topical application.
  • suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.
  • Compounds are administered using conventional methods.
  • inhibitors are in the form of injectable, sterile solutions, e.g., oily or aqueous solutions, as well as suspensions, emulsions, or implants.
  • Other formulations suitable for parenteral adminstration include tablets, liquids, drops, suppositories, or capsules.
  • Sustained or directed release compositions can be formulated, e.g., liposomes or those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.
  • Administration by injection e.g. subcutaneous, intramuscular or constant infusion by intravenous drip is also useful.
  • the compounds are also administered by transdermally, e.g., by transdermal patch, to allow administration over a long period of time, e.g., over days or weeks.
  • the compounds are at doses of 50 to about 150 ⁇ g/kg in a pharmaceutically acceptable carrier per unit dosage. Doses are adjusted depending upon the response of the mammal to the drug. Diagnosis of pathological conditions
  • Fig. 4 shows increased JNK activity in tissues derived from obese individuals compared to non-obese individuals.
  • An increase in JNK enzyme activity, e.g., JNKl, in bodily tissues or fluids indicates a diagnosis of insulin resistance, diabetes, or a predisposition thereto.
  • An increase also indicates a predisposition to obesity.
  • Diagnostic assays are carried out by obtaining a tissue sample or sample of bodily fluid from an individual and measuring JNK activity using a standard kinase assay.
  • a standard kinase assay For example, the data in Fig. 4 was generated using a standard solid phase kinase assay.
  • the assay was carried out by contacting tissue or a cell suspension (e.g., 600 ⁇ g tissue) with 20 ⁇ l of glutathione S-transferase (GST)-agarose resin suspension to which 5 ⁇ g of GST-c- Jun (amino acids 1-79) is bound.
  • GST glutathione S-transferase
  • the mixture is agitated at 4°C overnight, pelleted by centrifugation, and washed in a buffer containing 10 mM HEPES pH 7.7, 50 mM NaCl, 2.5 mM MgCl 2 .
  • the pelleted beads were subjected to an in vitro kinase assay described by Hibi et al. Upon staining with Coomassie Blue R250 and autoradiography, the bands corresponding to GST-c-Jun were quantified by Phosphorlmager.
  • This assay measures total JNK enzyme activity.
  • An increase in JNK activity of at least 10% compared to a normal control indicates a diagnosis of diabetes, insulin resistance, or a risk of developing diabetes, insulin resistance, or obesity.
  • a greater increase over a normal level e.g., 20%, 25%, 30%, 40%, or 50% indicates a greater risk of developing a disorder or greater severity of disease.
  • the increase in activity is at least 2-fold that of a normal control value.
  • JNKl independent of other JNK isotypes such as JNKl or JNK2
  • a tissue sample is obtained from a test subject.
  • Cells are lysed, and proteins are extracted from the tissue sample.
  • the membrane fraction is removed by centrifugation.
  • the supernatant is subjected to immunoprecipitation using a JNKl specific antibody.
  • JNKl specific antibodies are known in the art and commercially available (e.g., MAb 333.8 from Pharmingen, Inc., La Jolla, CA).
  • MAb 333.8 from Pharmingen, Inc., La Jolla, CA
  • a standard kinase assay is performed as described above.
  • a preferential increase in JNKl (relative to other JNK isotypes) compared to a normal control JNKl value indicates a diagnosis of diabetes, insulin resistance, or predisposition to develop diabetes, insulin resistance, or obesity.
  • Fatty liver disease or a risk of developing the disease is carried out by measuring the level of JNKl expression or activity in a liver tissue sample.
  • the tissue sample is obtained by biopsy.
  • An increase in the amount of JNKl expression indicates that the individual from which the tissue was obtained is suffering from or at risk of developing a condition of excessive fat accumulation in the liver.
  • Inhibitors of JNK enzymatic activity are identified by contacting a JNK with a candidate compound.
  • a control assay is run in parallel; the control assay include a JNK in the absence of the candidate compound.
  • Kinase activity is measured using methods known in the art (e.g., as described by Hibi et al., 1993, Genes Dev. 7:2135-2148).
  • a decrease in enzyme activity in the presence of the compound compared to the level in the absence of the compound indicates that the compound is a JNK inhibitor.
  • an in-gel kinase assay may be used.
  • the in-gel assay is carried out using known methods, e.g., as described by
  • c-Jun binding proteins were isolated from whole cell extracts by using GSH-agarose beads containing GST-c-Jun. Proteins are eluted in a standard SDS-PAGE sample buffer and resolved on 10% SDS-polyacrylamide gel, which was polymerized in the absence or presence of GST-c-Jun. After electrophoresis, the gel was washed and incubated in 200 ml of 6M urea.
  • the gel was washed and incubated with kinase buffer containing 32 P- ATP. After the reaction, the gel was washed with 100 ml of 5% tricholoroacetic acid and 1% sodium pyrophosphate at room temperature several times, followed by drying and autoradiography.
  • Inhibitors of JNK expression are identified by incubating a JNK promoter region operably linked to a reporter sequence with a candidate compound. An decrease in transcription of the reporter gene (or an increase in the amount of the reporter gene product) in the presence of the candidate compound compared to the level in the absence of the compound indicates that the compound decreases JNK expression.
  • JNKl plays a central role in obesity and insulin resistance
  • Obesity and type 2 diabetes are associated with a state of chronically enhanced inflammatory response characterized by abnormal cytokine production, increased circulating acute-phase reactants and other stress-induced molecules. Many of these alterations seem to be initiated and reside within adipose tissue, an unusual site for inflammatory responses. Elevated production of TNF ⁇ by adipose tissue was found in a variety of experimental obesity models and in obese humans, and free fatty acids (FFAs), are also implicated in the etiology of obesity-induced insulin resistance. Since both TNFa and FFAs are potent JNK activators, experiments were carried out to determine whether obesity is associated with alterations in stress-activated and inflammatory responses through in this signaling pathway and whether JNKs are causally linked to aberrant metabolic control in this state.
  • FFAs free fatty acids
  • mice deficient in JNKl and JNK2 were made using known methods (e.g., Davis et al. 2000, Cell 103:239-252). In the studies described herein, Jnkl-/- mice on
  • mice C57BL/6/129 mixed genetic background were backcrossed for 3 generations to C57BL/6 prior to experiments. These mice were intercrossed with Jnk2-/-mice on C57BL/6 background to produce mice heterozygous for mutations in both JNKl and JNK2. All mice were generated from intercrosses between these double heterozygotes and groups were derived from littermates. ob/ob-Jnkl+/+ and ob/ob-Jnkl-/-mice were generated by intercrossing Jnkl-/-and OB/ob animals to generate double heterozygotes and with subsequent crosses with OB/ob breeders to create double homozygous mutant mice.
  • mice of different genotypes were housed in a barrier free facility and placed on a high fat/high carbohydrate diet ad libidum (Diet F3282, Bioserve, NJ) at 4 weeks of age and were followed for a period of twelve weeks. Parallel groups were left on standard rodent chow to serve as controls. Total body weight measurements were initiated at 4- week of age. Blood samples were collected after a 6-hour, daytime fast at indicated ages and biochemical measures were conducted using 12-week-old animals. Standard glucose and insulin tolerance tests were performed on conscious mice following a 6 hour fast. Total JNK enzymatic activity and total JNK protein levels
  • JNK activity was measured in liver, muscle and adipose tissues of various models of obesity compared to the lean controls to determine whether obesity activates this pathway.
  • Measurement of JNK activity and protein levels were carried out as follows. Tissue extracts (600 ⁇ g protein) were mixed with 20 ⁇ l of glutathione S-transferase (GST)-agarose resin suspension (Sigma) to which 5 ⁇ g of GST-c-Junl-79 were bound. The mixture was agitated at 40°C for overnight, pelleted by centrifugation, washed twice and JNK activity was measured using known methods, e.g., as described by Yuan et al., 2001, Immunity 14:217-230. Upon staining with Commassie Brilliant Blue R250 and autoradiography, the bands corresponding to GST-c-Jun were quantified by Molecular Dynamics Phosphorlmager.
  • Jnkl-/- or Jnk2-/- mice and their control littermates were placed on a high fat (50% of total calories derived from fat) and high caloric diet (5286 kcal/kg, Bioserve, NJ) along with a control group in each genotype on standard diet.
  • high fat diet both controls and Jnk2-/- mice developed marked obesity as compared to mice kept on standard diet (Fig 6A and 6B).
  • the weight gain curves of these animals were indistinguishable on either standard or high fat diet.
  • weight gain on both standard and high fat diets was significantly reduced for the Jnkl-/- group (Fig. 6C and 6D).
  • Animals with one targeted allele of Jnkl displayed intermediary body weight between wild type and Jnkl-/- mice maintained on either diet (Fig. 6D).
  • Adipose tissue can have a substantial impact on systemic glucose homeostasis through the production of various bioactive molecules. Serum levels of adipocyte-derived secreted proteins were examined to evaluate their roles in obesity and insulin action. ACRP30/Adiponectin levels in the obese Jnkl-/- mice were found to be significantly higher compared to obese Jnkl+/+ controls (Fig. 7G). In contrast, the levels of resistin protein were lower in Jnkl-/- mice compared to Jnkl+/+ animals (Fig. 7H) . Adiponectin has been shown to act as a mediator of fatty acid oxidation and hepatic insulin sensitivity, and resistin may have a role in insulin resistance. The alterations in adiponectin and resistin could also impact systemic insulin sensitivity.
  • IITT intraperitoneal insulin
  • IGTT glucose
  • mice Genetically obese mice (ob/ob) with targeted mutations in Jnkl gene were generated to test the action of JNKl in a different and more severe model of obesity. This experiment included two additional generations of backcrossing into the C57B1/6 genetic background. Ob/ob mice developed early onset and severe obesity (Fig. 11 A). In contrast, the extent of weight gain was significantly lower in ob/ob-Jnkl-/-mice than in ob/ob-Jnkl+/+animals. Furthermore, at both 4- and 8-weeks of age the blood glucose levels were significantly lower in the ob/ob-Jnkl-/- mice compared to ob/ob- Jnkl+/+ animals (Fig. 1 IB).
  • the ob/ob-Jnkl +/+ animals also displayed a severe and progressive hyperinsulinemia during the course of the study (18.4 ⁇ 6.2 and 26.4 ⁇ 7.1 ng/ml at 4 and 8 weeks of age, respectively; Fig. 11C).
  • the ob/ob-Jnkl-/- displayed significantly lower plasma insulin levels throughout the study (5.7 ⁇ 2.1 and 7.7 ⁇ 2.3 ng/ml at 4 and 8 weeks of age, respectively; Fig. 11C) compared to the ob/ob animals with functional JNKl.
  • IITT analysis also demonstrated significantly increased insulin sensitivity in ob/ob- Jnkl-/- compared to ob/ob-Jnkl +/+ animals.
  • JNKl-/- and Jnk2-/- animals in the context of obesity and type 2 diabetes is intriguing.
  • JNKl isoforms in obesity-related insulin resistance, total JNK activity was measured in liver, muscle and adipose tissues of obese Jnkl-/- and Jnk2-/- mice and compared to obese wild type controls.
  • the JNKl isoforms account for most, if not all, of the increased total JNK activity in the target tissues relevant for obesity-induced insulin resistance.
  • IRS-1 serine phosphorylation A more direct involvement of JNK in insulin signaling was suggested to be at the level of IRS-1 serine phosphorylation, which uncouples this important adaptor protein from insulin receptor thereby reducing IRS-1 tyrosine phosphorylation and insulin receptor signaling.
  • Inhibitory serine phosphorylation of IRS-1 has been shown to be a mechnanism for both TNF ⁇ and FFA-induced insulin resistance.
  • Immune complexes were collected, washed, electrophoresed and transferred to nitrocellulose membranes. Immunoblot analysis was performed using a 1:2000 dilution of a monoclonal anti-phosphotyrosine (Santa Cruz, CA), 1 :2000 dilution of polyclonal anti- IR (Santa Cruz, CA) or 1 ⁇ g/mg polyclonal anti-IRS-1 or anti-IRS-l-pSer307 antibodies (Upstate Biotechnology, Lake Placid, NY), followed by 1 :2000 dilution of horse radish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Amersham Pharmacia Biotech Inc., Piscataway, NJ) for detection. JNK-mediated IRS was phosphorylated in the liver tissue of lean and obese,
  • JNKl provides a critical link between obesity and insulin resistance in the mouse and its ablation prevents obesity-induced insulin resistance in two different models.
  • One mechanism for JNK action involves the phosphorylation of IRS-1 at serine 307, a site where phosphorylation causes the uncoupling of IRS-1 from IR.
  • JNKl is a critical component of the biochemical pathway responsible for obesity-induced insulin resistance in two in vivo models.
  • JNK scaffold protein JIP1 is involved in type 2 diabetes in humans.
  • Selective inhibition of JNKl activity is a novel approach for the treatment of obesity, insulin resistance and type 2 diabetes.
  • fatty liver Excessive fat accumulation in liver tissue is termed fatty liver or steatosis.
  • Fatty liver with liver inflammation is called or steatohepatitis.
  • Steatosis and steatohepatitis can be caused by alcohol and other drugs and can also occur in patients with diabetes mellitus.
  • Steatohepatitis notcaused by alcohol is sometimes referred to as non-alcoholic steatohepatitis or "NASH".
  • Most people who do not abuse alcohol and have fatty liver are obese. The patient is usually 10% or more above ideal body weight.
  • Steatohepatitis can lead to scarring of the liver and cirrhosis, which may be life- threatening.
  • JNKl- and JNK2- deficient mice as well as wild type control mice were fed a high fat diet (55% fat) for 20 weeks. Liver tissue was excised and assayed. Gross liver weight was determined, and tissue sections were stained to visualize fat deposition. Figs. 9A-9B show that liver weight is reduced in JNKl -deficient mice compared to JNK wild type mice. Liver tissue sections were stained with eosin/hematoxylin to visualize the tissue architecture and with Oil-Red O to visualize fat deposits.
  • Figs. 10A-F demonstrate a striking reduction in the amount of fat accumulation in liver tissue from JNKl -deficient mice compared to JNK2- deficient and JNK wild type mice. The data indicate that inhibition of JNKl results in decreased fat accumulation in liver tissue and that contacting liver tissue with a compound that preferentially inhibits JNKl protects against development of fatty liver disease.

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Abstract

L'invention concerne un procédé permettant de traiter un trouble métabolique associé à la résistance à l'insuline. Ce procédé consiste à administrer à un mammifère un inhibiteur d'une kinase JNK (NH2-terminal Jun Kinase), par exemple, un composé ou un peptide qui inhibe l'expression de JNK1 ou l'activité enzymatique.
PCT/US2002/012687 2001-04-24 2002-04-24 Inhibition de la kinase jun WO2002085396A1 (fr)

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WO2004084901A1 (fr) * 2003-03-24 2004-10-07 Signal Pharmaceuticals, Llc Methodes permettant de traiter ou de prevenir un trouble inflammatoire ou metabolique par inhibition de kinases jnk
US6897231B2 (en) 2000-07-31 2005-05-24 Signal Pharmaceuticals, Inc. Indazole derivatives as JNK inhibitors and compositions and methods related thereto
US6987184B2 (en) 2001-02-15 2006-01-17 Signal Pharmaceuticals, Llc Isothiazoloanthrones, isoxazoloanthrones, isoindolanthrones and derivatives thereof as JNK inhibitors and compositions and methods related
US7119114B1 (en) 1999-08-19 2006-10-10 Signal Pharmaceuticals, Llc Pyrazoloanthrone and derivatives thereof as JNK inhibitors and compositions and methods related thereto
US7122544B2 (en) 2000-12-06 2006-10-17 Signal Pharmaceuticals, Llc Anilinopyrimidine derivatives as IKK inhibitors and compositions and methods related thereto
US7129242B2 (en) 2000-12-06 2006-10-31 Signal Pharmaceuticals, Llc Anilinopyrimidine derivatives as JNK pathway inhibitors and compositions and methods related thereto
US7211594B2 (en) 2000-07-31 2007-05-01 Signal Pharmaceuticals, Llc Indazole compounds and compositions thereof as JNK inhibitors and for the treatment of diseases associated therewith
EP1797899A1 (fr) * 2004-08-24 2007-06-20 Yamaguchi University Traitement prophylactique et theraeutique de la perturbation des metabolismes du collagene ou de l'elastine
EP1799263A2 (fr) * 2004-09-15 2007-06-27 The President and Fellows of Harvard College Reduction du stress du re dans le traitement de l'obesite et du diabete
WO2008016995A2 (fr) * 2006-08-01 2008-02-07 Joslin Diabetes Center, Inc. Procédés d'identification de modulateurs de la signalisation de l'insuline
US7335676B2 (en) 1999-08-19 2008-02-26 Signal Pharmaceuticals, Llc Methods for treating inflammatory conditions or inhibiting JNK
WO2008040548A2 (fr) * 2006-10-04 2008-04-10 Ares Trading S.A. Traitement pour l'hépatite stéatosique non alcoolique
EP2065383A1 (fr) 2003-11-19 2009-06-03 Signal Pharmaceuticals, Inc. Composés d'indazoles et procédés d'utilisation associés en tant qu'inhibiteurs de kinase de protéine
CN102676466A (zh) * 2011-03-17 2012-09-19 南京大学 一种突变型jnk1序列及其质粒的应用
US8501812B2 (en) 2005-03-29 2013-08-06 University Of Massachusetts Therapeutic methods for type I diabetes
WO2015138426A1 (fr) * 2014-03-10 2015-09-17 Croce Carlo M Compositions et méthodes permettant de traiter la cachexie
US9956236B2 (en) 2011-02-07 2018-05-01 Cornell University Methods for increasing immune responses using agents that directly bind to and activate IRE-1
US9957506B2 (en) 2013-09-25 2018-05-01 Cornell University Compounds for inducing anti-tumor immunity and methods thereof

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US5498631B1 (en) * 1993-04-16 1998-03-03 Univ School Of Medicine Method for treatment of menopausal and premenstrual symptoms
US5506211A (en) * 1994-05-09 1996-04-09 The Uab Research Foundation Genistein for use in inhibiting osteroclasts

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US7335676B2 (en) 1999-08-19 2008-02-26 Signal Pharmaceuticals, Llc Methods for treating inflammatory conditions or inhibiting JNK
US7119114B1 (en) 1999-08-19 2006-10-10 Signal Pharmaceuticals, Llc Pyrazoloanthrone and derivatives thereof as JNK inhibitors and compositions and methods related thereto
US7208513B2 (en) 2000-07-31 2007-04-24 Signal Pharmaceuticals, Llc Indazole compounds and compositions thereof as JNK inhibitors and for the treatment of diseases associated therewith
US6897231B2 (en) 2000-07-31 2005-05-24 Signal Pharmaceuticals, Inc. Indazole derivatives as JNK inhibitors and compositions and methods related thereto
US7220771B2 (en) 2000-07-31 2007-05-22 Signal Pharmaceuticals, Llc Methods of using indazole derivatives as JNK inhibitors
US7211594B2 (en) 2000-07-31 2007-05-01 Signal Pharmaceuticals, Llc Indazole compounds and compositions thereof as JNK inhibitors and for the treatment of diseases associated therewith
US7429599B2 (en) 2000-12-06 2008-09-30 Signal Pharmaceuticals, Llc Methods for treating or preventing an inflammatory or metabolic condition or inhibiting JNK
US7129242B2 (en) 2000-12-06 2006-10-31 Signal Pharmaceuticals, Llc Anilinopyrimidine derivatives as JNK pathway inhibitors and compositions and methods related thereto
US7122544B2 (en) 2000-12-06 2006-10-17 Signal Pharmaceuticals, Llc Anilinopyrimidine derivatives as IKK inhibitors and compositions and methods related thereto
US7442699B2 (en) 2000-12-06 2008-10-28 Signal Pharmaceuticals, Llc Anilinopyrimidine derivatives as IKK inhibitors and compositions and methods related thereto
US6987184B2 (en) 2001-02-15 2006-01-17 Signal Pharmaceuticals, Llc Isothiazoloanthrones, isoxazoloanthrones, isoindolanthrones and derivatives thereof as JNK inhibitors and compositions and methods related
US7354947B2 (en) 2001-02-15 2008-04-08 Signal Pharmaceuticals, Llc Isothiazoloanthrones, isoxazoloanthrones, isoindolanthrones and derivatives thereof a JNK inhibitors and compositions and methods related thereto
JP2006521394A (ja) * 2003-03-24 2006-09-21 シグナル ファーマシューティカルズ，エルエルシー Ｊｎｋを阻害することにより、炎症または代謝性症状を治療または予防する方法
WO2004084901A1 (fr) * 2003-03-24 2004-10-07 Signal Pharmaceuticals, Llc Methodes permettant de traiter ou de prevenir un trouble inflammatoire ou metabolique par inhibition de kinases jnk
EP2065383A1 (fr) 2003-11-19 2009-06-03 Signal Pharmaceuticals, Inc. Composés d'indazoles et procédés d'utilisation associés en tant qu'inhibiteurs de kinase de protéine
EP1797899A1 (fr) * 2004-08-24 2007-06-20 Yamaguchi University Traitement prophylactique et theraeutique de la perturbation des metabolismes du collagene ou de l'elastine
US7750033B2 (en) 2004-08-24 2010-07-06 Yamaguchi University Preventive and remedy for collagen or elastin metabolic disorder
EP1797899A4 (fr) * 2004-08-24 2009-06-24 Univ Yamaguchi Traitement prophylactique et theraeutique de la perturbation des metabolismes du collagene ou de l'elastine
EP1799263A2 (fr) * 2004-09-15 2007-06-27 The President and Fellows of Harvard College Reduction du stress du re dans le traitement de l'obesite et du diabete
EP1799263A4 (fr) * 2004-09-15 2009-07-29 Harvard College Reduction du stress du re dans le traitement de l'obesite et du diabete
US8501812B2 (en) 2005-03-29 2013-08-06 University Of Massachusetts Therapeutic methods for type I diabetes
WO2008016995A3 (fr) * 2006-08-01 2008-11-27 Joslin Diabetes Center Inc Procédés d'identification de modulateurs de la signalisation de l'insuline
WO2008016995A2 (fr) * 2006-08-01 2008-02-07 Joslin Diabetes Center, Inc. Procédés d'identification de modulateurs de la signalisation de l'insuline
WO2008040548A3 (fr) * 2006-10-04 2009-05-22 Ares Trading Sa Traitement pour l'hépatite stéatosique non alcoolique
JP2010505783A (ja) * 2006-10-04 2010-02-25 アレス トレーディング ソシエテ アノニム 非アルコール性脂肪肝炎の治療
WO2008040548A2 (fr) * 2006-10-04 2008-04-10 Ares Trading S.A. Traitement pour l'hépatite stéatosique non alcoolique
US9956236B2 (en) 2011-02-07 2018-05-01 Cornell University Methods for increasing immune responses using agents that directly bind to and activate IRE-1
CN102676466A (zh) * 2011-03-17 2012-09-19 南京大学 一种突变型jnk1序列及其质粒的应用
US9957506B2 (en) 2013-09-25 2018-05-01 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
US10421965B2 (en) 2013-09-25 2019-09-24 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
US10450566B2 (en) 2013-09-25 2019-10-22 Cornell University Compounds for inducing anti-tumor immunity and methods thereof
WO2015138426A1 (fr) * 2014-03-10 2015-09-17 Croce Carlo M Compositions et méthodes permettant de traiter la cachexie
US10036018B2 (en) 2014-03-10 2018-07-31 Carlo M. Croce Compositions and methods for treating cachexia

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