WO2004029070A2 - Procedes de blocage de la differentiation d'adipocyte et de l'accumulation de triglyceride a l'aide d'inhibiteurs a double specifite de kinase 4 regulee par phosphorylation de tyrosine (y) (dyrk4) - Google Patents

Procedes de blocage de la differentiation d'adipocyte et de l'accumulation de triglyceride a l'aide d'inhibiteurs a double specifite de kinase 4 regulee par phosphorylation de tyrosine (y) (dyrk4) Download PDF

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WO2004029070A2
WO2004029070A2 PCT/US2003/029889 US0329889W WO2004029070A2 WO 2004029070 A2 WO2004029070 A2 WO 2004029070A2 US 0329889 W US0329889 W US 0329889W WO 2004029070 A2 WO2004029070 A2 WO 2004029070A2
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dyrk4
inhibitors
disease
inhibitor
cell
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WO2004029070A3 (fr
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Eric G. Marcusson
Nicholas M. Dean
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Isis Pharmaceutical, Inc.
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Publication of WO2004029070A3 publication Critical patent/WO2004029070A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense

Definitions

  • Obesity is known to be a major health risk throughout Europe and the United States leading to a number of potentially life threatening diseases. Obesity is usually defined as being about 20% above the mean adiposity. Lifelong obesity is associated with an excess number of adipocytes, presumably a genetically determined phenomenon. On the other hand, the obesity that begins in adult life develops against a background of larger, i.e., hypertrophied, adipocytes, the number of which remains the same. An excessive recruitment and differentiation of preadipocytes into mature adipocytes is a characteristic of human obesity, which is a strong risk factor for Type 2 diabetes, certain cancers, and cardiovascular disease, including hypertension, atherosclerosis, and coronary artery disease.
  • Obesity and insulin resistance share a complex relationship that gives rise to a range of metabolic disorders, including Type 2 diabetes.
  • Obesity can itself engender insulin resistance.
  • the most important consequence of obesity is type II (maturity-onset) diabetes, which is associated with normal or high level of circulating insulin and peripheral resistance to insulin's action.
  • Most human obesity is associated with insulin resistance and leptin resistance. In fact obesity may have an even greater impact on insulin action than does diabetes itself. Sindelka et al., Physiol Res . , 2002, 51 , 85-91.
  • Weight reduction usually ameliorates the glucose intolerance of type II diabetes, presumably owing to a decrease in the stimulus for insulin secretion by the pancreatic beta cells. Furthermore, it is believed that as the fat cells (adipocytes) accumulate triglycerides, they release free fatty acids. A flux of these fatty acids to the liver may be important in the cause of diabetes . In addition to diet control, several methods of chemically treating obesity with pharmacologically active substances have been identified. However, these methods may cause other health problems. For example, caffeine- and amphetamine- based diet aids may be addictive and adversely affect other areas of health. The combination of fenfluramine and phentermine has been proven to cause heart valve disease .
  • Hyperlipidemia is an abnormally high concentration of lipids in the blood serum.
  • the composition of the lipid pool in the circulation consists mostly of triglyceride (fatty acid esters of glycerol) , cholesterol, and fatty acid esters of cholesterol. It is believed that as the fat cells (adipocytes) accumulate triglycerides, they release free fatty acids. Fatty acids are precursors to cholesterols . As such, a reduction of triglyceride synthesis effectively reduces cholesterol.
  • Lipid molecules are generally bound to and are carried by specific proteins, known as apoproteins. Various combinations of different and specific lipids and apoproteins form lipoproteins . Lipoproteins can transport lipids and perform specific biological functions.
  • hyperlipidemia characterized by excessively high triglyceride levels in plasma is called hypertriglyceridemia. Elevated triglycerides may be a consequence of other disease, such as untreated diabetes mellitus. Like cholesterol, high in triglyceride levels are detected by plasma measurements. These measurements should be made after an overnight food and alcohol fast.
  • the National Cholesterol Education Program guidelines for triglycerides are (based on fasting triglyceride levels): Normal: Less than 150 mg/dL; Borderline-high: 150-199 mg/dL; High: 200-499 mg/dL; Very High: 500 mg/dL or higher.
  • hyperlipidemia Common pathological sequelae of hyperlipidemia include cardiovascular diseases or conditions including coronary artery disease, atherosclerosis, hypertension, thrombosis, and ischemic events (for example, myocardial infarction, cerebral stroke, and organ insufficiency) . Insulin resistance is also associated with hypertriglyceremia . Sindelka et al . , Physiol Res . , 2002, 51, 85-91.
  • Various drugs are available which can lower serum lipid levels in human patients. For example, LOPIDTM (available from Parke-Davis) , and TRICORTM (available from Abbott) , are effective in treating Type IV and V hyperlipidemias, with triglyceride levels being abnormally high. However, these drugs may cause many side effects, some of which are quite severe.
  • Syndrome X or Metabolic syndrome is a new term for a cluster of conditions, that, when occurring together, may indicate a predisposition to diabetes and cardiovascular disease. These symptoms, including high blood pressure, high triglycerides, decreased HDL and obesity, tend to appear together in some individuals.
  • hypertriglyceridemia hyperlipidemia, obesity, and sequelae of one or more of these conditions, including metabolic syndrome, diabetes, insulin resistance, and cardiovascular diseases and conditions including coronary artery disease, atherosclerosis, hypertension, thrombosis and ischemic events (for example, myocardial infarction, cerebral stroke, and organ insufficiency) .
  • cardiovascular diseases and conditions including coronary artery disease, atherosclerosis, hypertension, thrombosis and ischemic events (for example, myocardial infarction, cerebral stroke, and organ insufficiency) .
  • ischemic events for example, myocardial infarction, cerebral stroke, and organ insufficiency
  • DYRK1A is the human homolog of Drosophila minibrain, a gene involved in the formation of neurons.
  • Drosophila carrying mutations of minibrain have been observed to exhibit specific defects in the central nervous system. Tejedor et al . have proposed that analysis of genes such as minibrain may also be used to study changes in the circuitry of the brain in relation to specific behavioral traits (Tejedor et al., Neuron, 1995, 14, 287-301).
  • mice overexpressing the full-length cDNA of DYRK1A were observed to exhibit delayed craniocaudal maturation, altered motor skill acquisition and impairment in spatial learning and cognitive flexibility. These alterations are comparable to those found in the partial trisomy chromosome 16 murine models of Down syndrome and suggest a causative role for DYRK1A in the mental retardation and motor anomalies of Down syndrome (Altafaj et al., Hum. Mol . Genet . , 2001, 10, 1915-1923).
  • US Patent 6,100,033 Disclosed and claimed in US Patent 6,100,033 is a method for prenatal testing of a child for Down syndrome comprising the steps of preparing labeled nucleic acid probes which hybridize to the human DYRK gene and exposing said probes to a cell from said child and detecting specific binding of three probes in the nucleus of a cell (Smith and Rubin, 2000) .
  • the human DYRK4 gene (also known as dual-specificity tyrosine- (Y) -phosphorylation regulated kinase 4) was identified in the human teratocarcinoma cell line NTERA-2 cl.Dl, a cell line capable of production of postmitotic neurons when induced with retinoic acid and cell aggregation (Leypoldt et al . , J. Neurochem . , 2001, 76, 806-814).
  • Isolated nucleic acid sequences encoding DYRK4 are disclosed and claimed in US Patents 5,965,365 and 6,100,033 (Bandman et al . , 1999; Brun et al . , 1999).
  • DYRK4 is involved in neuronal differentiation (Leypoldt et al . , J " . Neurochem. , 2001, 76, 806-814) . This finding indicates the possibility that overexpression of DYRK4 could lead to human neurological disorders similar to those observed in mice. Thus, modulation of expression of DYRK4 may prove to be an appropriate target for intervention in neurological disorders such as Down syndrome. It is now surprisingly discovered that inhibitors of DYRK4 can be used to block differentiation of preadipocytes to adipocytes and to block triglyceride accumulation in adipocytes.
  • inhibitors of DYRK4 can be used to block differentiation of preadipocytes to adipocytes and to block triglyceride accumulation in adipocytes .
  • Methods for inhibiting the differentiation of an adipocyte cell or for inhibiting lipid accumulation, particularly triglyceride accumulation, in a cell by contacting the cell with an inhibitor of DYRK4 activity or expression are provided.
  • Methods for treating, preventing or delaying the onset of diseases or conditions associated with adipocyte differentiation, excess adipocytes or lipid accumulation, particularly triglyceride accumulation or high triglyceride levels, are also provided.
  • the inhibitor of DYRK4 may be a small molecule, antibody, peptide and/or antisense compound.
  • An adipocyte cell is a connective tissue cell specialized for the synthesis and storage of fat. During differentiation from pre-adipocytes to adipocytes, numerous changes occur, including accumulation of triglycerides as lipid droplets, secretion of several hormones and autocrine factors (e.g., leptin and TNF- ⁇ ) , and changes in gene expression. Mature adipocyte cells are swollen with globules of triglycerides; increased triglyceride content is a well established marker of adipocyte differentiation in culture.
  • Mature adipocytes are also characterized by a number of molecular markers that are not present in pre-adipocytes .
  • "Hallmark" or marker genes for adipocyte differentiation include adipocyte lipid binding protein 2 (aP2) , glucose transporter 4 (GLUT4) and hormone sensitive lipase (HSL) .
  • the products of these genes play important roles in the uptake of glucose and the metabolism and utilization of fats.
  • the presence of one, or preferably more than one, more preferably all of these gene products is indicative of mature adipocytes, i.e., of differentiation of adipocytes from preadipocyte cells.
  • inhibitors of DYRK4 may be administered to reduce or prevent adipocyte differentiation and/or triglyceride accumulation.
  • conditions associated with adipocyte differentiation, triglyceride accumulation and excess adiposity may also be treated by the administration of a DYRK4 inhibitor. These conditions include, for example, obesity, hyperlipidemia, and associated conditions and/or sequelae such as cardiovascular disease, metabolic syndrome, diabetes and/or insulin resistance.
  • treatment includes prophylactic as well as therapeutic use, i.e., treatment of a disease or condition includes prevention as well as delay of onset of the disease or condition.
  • the DYRK4 protein of a mammal may be inhibited by the administering to the mammal a therapeutically effective amount of an inhibitor of DYRK4.
  • a DYRK4 inhibitor is a compound that inhibits DYRK4 expression, levels, or activity.
  • inhibit may be partial or complete reduction in- the amount or activity of DYRK4 to a level at or below that found under normal physiological conditions if used prophylactically, or below the existing (pre-treatment) levels if used in treatment of an active or acute condition.
  • the activity or amount of DYRK4 is inhibited by about 10%.
  • the activity or amount of DYRK4 is inhibited by about 30%. More preferably, the activity or amount of DYRK4 is inhibited by 50% or more.
  • the reduction of the expression of targets may be measured in adipose, liver, blood or other tissue of the mammal.
  • the cells being inhibited contain therein a nucleic acid molecule encoding for a DYRK4 protein and/or the DYRK4 protein itself.
  • a mammal is a warm-blooded vertebrate animal, which includes a human. Any inhibitor of DYRK4 may be employed in accordance with the present invention.
  • DYRK4 inhibitors include compound that act on the DYRK4 protein to directly inhibit DYRK4 function or activity, as well as compounds which indirectly inhibit DYRK4 by reducing amounts of DYRK4 , e.g., by reducing expression of the gene encoding DYRK4 via interference with transcription, translation or processing of the mRNA encoding DYRK4.
  • Inhibitors of DYRK4 also include compounds that bind to DYRK4 and inhibit its function, including ability to bind substrate or receptor molecules and/or any enzymatic or other activity that DYRK4 may have.
  • inhibitors of DYRK4 include small molecules, preferably organic small molecule compounds; antibodies; peptides and peptide fragments, particularly DYRK4 dominant negative peptides and fragments, and the like.
  • Inhibitors of DYRK4 also include compounds which inhibit the expression or reduce the levels of DYRK4, including small molecules, antibodies, peptides and peptide fragments, nucleic acids and the like which are designed to bind to a particular target nucleic acid and thereby inhibiting its expression.
  • DYRK4 inhibitors used in accordance with the present invention are antisense compounds.
  • Non-limiting examples of antisense compounds in accordance with the present invention include ribozymes; short inhibitory RNAs (siRNAs) ; long double-stranded RNAs, antisense oligonucleotides; antisense oligonucleotide mimetics such as peptide nucleic acid (PNA) , morpholino compounds and locked nucleic acids (LNA) ; external guide sequence (EGS) ; oligonucleotides (oligozymes) and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression, and mixtures thereof.
  • Antisense inhibitors of DYRK4 are disclosed in U.S. Application Serial No.
  • small molecules are administered as DYRK4 inhibitors in accordance with the present invention.
  • Libraries of small organic molecules may be obtained commercially, for example from ChemBridge Corp. in San Diego, California or LION Bioscience, Inc. (formerly Trega Biosciences) in San Diego, California. Libraries of small molecules may also be prepared according to standard methods that are well known in the art. An appropriate screening or assaying for inhibitors of the desired molecule is essential to finding inhibitors with the desired selectivity and specificity, and such screening and assaying may be readily practiced by one of ordinary skill in the art.
  • DYRK4 inhibitors are antibodies or fragments thereof. These antibodies or fragments thereof may selectively bind to DYRK4 and in so doing, selectively inhibit or interfere with the DYRK4 polypeptide, preferably with the activity thereof. Standard methods for preparation of monoclonal and polyclonal antibodies and active fragments thereof are well known in the art. Antibody fragments, particularly Fab fragments and other fragments which retain epitope-binding capacity and specificity are also well known, as are chimeric antibodies, such as "humanized” antibodies, in which structural (not determining specificity for antigen) regions of the antibody are replaced with analogous or similar regions from another species. Thus antibodies generated in mice can be "humanized” to reduce negative effects which may occur upon administration to human mammals.
  • Chimeric antibodies are now accepted therapeutic modalities with several now on the market .
  • the present invention therefore includes use of antibody inhibitors of DYRK4 which include F (a ' ) 2 .
  • Fab, Fv and Fd antibody fragments chimeric antibodies in which one or more regions have been replaced by homologous human or non-human portions, and single chain antibodies.
  • U.S. Patent No. 6,150,401 discloses techniques for antibodies specific for a protein, for example DYRK4. These techniques may be employed to produce inhibiting antibodies specific for DYRK4.
  • the disclosure of U.S. Patent No. 6,150,401 is incorporated in its entirety herein by reference.
  • the present invention provides use of DYRK4 inhibitors which are peptides, for example dominant negative DYRK4 polypeptides.
  • a dominant negative polypeptide is an inactive variant or fragment of a protein which competes with or otherwise interferes with the active protein, reducing the function or effect of the normal active protein.
  • the target protein is an enzyme
  • dominant negatives may include polypeptides which have an inactive or absent catalytic domain, so that the polypeptide binds to the substrate but does not phosphorylate it, or polypeptides which have a catalytic domain with reduced enzymatic activity or reduced affinity for the substrate.
  • One of ordinary skill in the art can use standard and accepted mutagenesis techniques to generate dominant negative polypeptides.
  • DYRK4 nucleotide sequence of DYRK4 along with standard techniques for site-directed mutagenesis, scanning mutagenesis, partial deletions, truncations, and other such methods known in the art. For examples, see Sambrook et al . , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY, 1989, pp. 15.3-15.113. U.S. Patent No. 6,150,401, which is incorporated in its entirety herein by reference, also discloses techniques which may readily be adapted to create dominant negative DYRK4 polypeptides.
  • Inhibitors of DYRK4 may be antisense compounds, including antisense oligonucleotides, ribozymes and other catalytic oligonucleotides, and inhibitory RNAs including transfected, intracellularly expressed single stranded antisense RNAs or double stranded RNAs, as well as small intefering RNAs (siRNA) .
  • Ribozymes are catalytic RNAs. A number of labs around the world are now using these ribozymes to study gene function in precisely the manner described above most notably in the study of HIV, the AIDS virus, and in cancer research. Ribozymes may be synthetically engineered via the technologies of Ribozyme Pharmaceuticals, Inc.
  • siRNAs are short double stranded RNAs (dsRNA) which may be designed to inhibit a specific mRNA, for example the mRNA encoding DYRK4.
  • dsRNA short double stranded RNAs
  • Such method comprises introducing an oligoribonucleotide with double stranded structure (dsRNA) or a vector coding for the dsRNA into the cell, where a strand of the dsRNA is at least in part complementary to the target gene.
  • dsRNA double-stranded structure
  • U.S. patent 6,506,559 discloses and claims gene-specific inhibition of gene expression by double-stranded ribonucleic acid (dsRNA) and is incorporated herein by reference in its entirety. See also PCT publications WO 01/48183, WO 00/49035, WO 00/63364, WO 01/36641, WO 01/36646, WO 99/32619 and WO 00/44914, and Elbashir et al .
  • RNAi RNAi or siRNA compound
  • a vector coding for the inhibitory RNA which is capable of inhibiting the nucleotide sequence encoding the DYRK4 protein.
  • Antisense oligonucleotides and antisense oligonucleotide mimetics such as peptide nucleic acid (PNA) and morpholino compounds are preferred antisense compounds.
  • Antisense compounds specifically hybridize with one or more nucleic acids encoding DYRK4. Examples of antisense inhibitors of DYRK4, as well as various chemical modifications and methods for making and using them are disclosed in U.S. Application Serial No. 10/144,140, filed May 10, 2002, the contents of which are incorporated herein in their entirety.
  • inhibitors used in the present invention may also admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • the compounds used in the present invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • the methods of the present invention may also use pharmaceutical compositions and formulations of one or more DYRK4 inhibitors.
  • the pharmaceutical compositions may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery) , pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal) , oral or parenteral .
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial , e.g., intrathecal or intraventricular, administration.
  • Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • compositions may conveniently be presented in unit dosage form and may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier (s) or excipient(s) . In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product .
  • the compositions used in the methods of the invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • compositions include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations.
  • the pharmaceutical compositions and formulations used may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.
  • formulations are routinely designed according to their intended use, i.e. route of administration.
  • Preferred formulations for topical administration may include those in which the compounds to be administered are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearoylphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidyl ethanolamine (DOTMA) .
  • neutral e.g. di
  • DYRK4 inhibitors used in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes.
  • inhibitors may be complexed to lipids, in particular to cationic lipids.
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non- aqueous media, capsules, gel capsules, sachets, tablets or minitablets .
  • Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • inhibitors are administered in conjunction with one or more penetration enhancers, surfactants and chelators .
  • surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Combinations of penetration enhancers may also be used.
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or ex ⁇ ipients .
  • compositions containing one or more inhibitors of DYRK4 and one or more other agents that function by a non-DYRK4 mechanism include but are not limited to cancer chemotherapeutic drugs, anti- inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs.
  • the other agent (s) may be an anti-diabetes drug.
  • insulin which may typically be porcine or human and is typically given by needle injection or pump, but may be given orally or via aerosol delivery via the mouth or nose, there are several types of orally administered treatments for diabetes.
  • Oral hypoglycemics, starch blockers, insulin sensitizers and drugs which decrease the production of glucose by the liver and increase glucose utilization by the tissues are all comprehended by the present invention.
  • Common orally administered drugs for diabetes include insulin, pioglitazone, glimepiride, metformin, rosiglitazone, rosiglitazone/metformin, sulfonylurea, glyburide, glyburide/metformin, glipizide, miglitol, glipizide/metformin, repaglinide, acarbose, troglitazone, and nateglinide.
  • the DYRK4 inhibitor and the additional agent may be used individually, sequentially or in combination.
  • compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual inhibitors, and can generally be estimated based on EC 50 s found to be effective in in vitro and in vivo animal models.
  • This assay measures the accumulation of triglyceride by newly differentiated adipocytes.
  • the in vitro triglyceride assay model used here is a good representation of an in vivo model because a time-dependent increase in triglyceride accumulation by the adipocytes has been shown to increase concomitantly with increasing leptin secretion. Furthermore, an increased in triglyceride content is a well established marker for adipocyte differentiation.
  • Triglyceride accumulation is measured using the INFINITYTM Triglyceride reagent kit (Sigma-Aldrich, St. Louis, MO).
  • Human white preadipocytes (Zen-Bio Inc., Research Triangle Park, NC) are grown in preadipocyte media (ZenBio Inc.)
  • 96-well plates are seeded with 3000 cells/well.
  • Cells are treated according to standard published procedures with DYRK4 inhibitor (in this experiment, 250nM oligonucleotide) in lipofectin (Gibco) . Monia et al . , J " . Biol . Chem. , 1993, 268, 14514-22.
  • Inhibitors are tested in triplicate on each 96-well plate, and the effects of TNF- ⁇ , a positive drug control that inhibits adipocyte differentiation, are also measured in triplicate. Negative controls and transfectant-only controls may be measured up to six times per plate. After the cells have reached confluence (approximately three days) , they are exposed to differentiation media (Zen-Bio, Inc.; differentiation media contains a PPAR- ⁇ agonist, IBMX, dexamethasone and insulin) for three days. Cells are then fed adipocyte media (Zen-Bio, Inc.), which is replaced at 2 to 3 day intervals.
  • differentiation media Zen-Bio, Inc.
  • differentiation media contains a PPAR- ⁇ agonist, IBMX, dexamethasone and insulin
  • Horseradish peroxidase uses H 2 0 2 to oxidize 4-aminoantipyrine and 3,5 dichloro-2-hydroxybenzene sulfonate to produce a red-colored dye.
  • Dye absorbance which is proportional to the concentration of glycerol, is measured at 515nm using an UV spectrophotometer .
  • Glycerol concentration is calculated from a standard curve for each assay, and data are normalized to total cellular protein as determined by a Bradford assay (Bio-Rad Laboratories, Hercules, CA) . Results are expressed as a percent ⁇ standard deviation relative to transfectant-only control.
  • the DYRK4 inhibitor employed in this assay was an antisense oligomer, ISIS 206820; SEQ ID NO: l,and the control (or negative control) employed in this assay was a nonsense oligomer, ISIS
  • DYRK4 inhibitor At 250 nM of DYRK4 inhibitor, the triglyceride accumulation was reduced by 72% as compared to control. This indicates that differentiation of preadipocytes to adipocytes was inhibited by treatment with DYRK4 inhibitor.
  • Leptin is a marker for differentiated adipocytes.
  • leptin secretion into the media above the newly differentiated adipocytes is measured by protein ELISA.
  • Cell growth, treatment with DYRK4 inhibitor and differentiation procedures are carried out as described for the triglyceride accumulation assay (see above) .
  • 96-well plates are coated with a monoclonal antibody to human leptin (R&D Systems, Minneapolis, MN) and are left at 4°C overnight . The plates are blocked with bovine serum albumin (BSA) , and a dilution of the media is incubated in the plate at room temperature for 2 hours . After washing to remove unbound components, a second monoclonal antibody to human leptin
  • HRP horseradish peroxidase
  • adipocyte differentiation the gene expression patterns in adipocytes change considerably. This gene expression pattern is controlled by several different transcription factors, including glucose transporter-4 (GLUT4) , hormone-sensitive lipase (HSL) and adipocyte lipid binding protein (aP2) . These genes play important roles in the uptake of glucose and the metabolism and utilization of fats.
  • GLUT4 glucose transporter-4
  • HSL hormone-sensitive lipase
  • AP2 adipocyte lipid binding protein
  • the DYRK4 inhibitor employed in this assay was an antisense oligomer, ISIS 206820; SEQ ID NO. 1; and the control (or negative control) employed in this assay was an nonsense oligomer, ISIS 29848, NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN, SEQ ID NO: 2; where N is a mixture of A, C, G and T.
  • Other antisense inhibitors of DYRK4 are disclosed in U.S. Application Serial No. 10/144,140, filed May 10, 2002.
  • HSL was reduced by 51% as compared to control. This indicates that differentiation of preadipocytes to adipocytes was inhibited by treatment with DYRK4 inhibitor.

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Abstract

L'invention concerne des procédés de blocage de la différentiation d'adipocyte et de l'accumulation de triglycéride à l'aide d'inhibiteurs de DYRK4. Les inhibiteurs de DYRK4 de l'invention comprennent de petites molécules, des anticorps, des peptides (y compris des peptides négatifs dominants) et des composés antisens, y compris des ribozymes, des molécules D'ARN inhibitrices comprenant des molécules ARNsi et des oligonucléotides antisens.
PCT/US2003/029889 2002-09-24 2003-09-23 Procedes de blocage de la differentiation d'adipocyte et de l'accumulation de triglyceride a l'aide d'inhibiteurs a double specifite de kinase 4 regulee par phosphorylation de tyrosine (y) (dyrk4) WO2004029070A2 (fr)

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AU2003273351A AU2003273351A1 (en) 2002-09-24 2003-09-23 Methods for blocking adipocyte differentiation and triglyceride accumulation with dual-specificity tyrosine- (y) - phosphorylation regulated kinase 4 (dyrk4) inhibitors

Applications Claiming Priority (2)

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US41321802P 2002-09-24 2002-09-24
US60/413,218 2002-09-24

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WO2004029070A2 true WO2004029070A2 (fr) 2004-04-08
WO2004029070A3 WO2004029070A3 (fr) 2004-09-30

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015097504A (ja) * 2013-11-19 2015-05-28 学校法人 中央大学 緑藻の脂質蓄積変異体およびその利用
US9279127B2 (en) 2006-11-01 2016-03-08 The Medical Research Fund At The Tel-Aviv Sourasky Medical Center Adipocyte-specific constructs and methods for inhibiting platelet-type 12 lipoxygenase expression
US20160303089A1 (en) * 2013-12-05 2016-10-20 Kyoto University Compound pertaining to neuropoiesis and drug composition
WO2017115798A1 (fr) * 2015-12-28 2017-07-06 北海道公立大学法人 札幌医科大学 Peptide antigénique tumoral

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BECKER ET AL: 'Sequence characteristics, subcellular localization and substrate specificity of CYRK-related kinases, a novel family of dual specificity protein kinases' J. BIOL. CHEM. vol. 273, no. 40, 02 October 1998, pages 25893 - 25902, XP002927376 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9279127B2 (en) 2006-11-01 2016-03-08 The Medical Research Fund At The Tel-Aviv Sourasky Medical Center Adipocyte-specific constructs and methods for inhibiting platelet-type 12 lipoxygenase expression
US9663790B2 (en) 2006-11-01 2017-05-30 The Medical Research, Infrastructure, And Health Services Fund Of The Tel Aviv Medical Center Adipocyte-specific constructs and methods for inhibiting platelet-type 12 lipoxygenase expression
JP2015097504A (ja) * 2013-11-19 2015-05-28 学校法人 中央大学 緑藻の脂質蓄積変異体およびその利用
WO2015075881A1 (fr) * 2013-11-19 2015-05-28 株式会社デンソー Mutant accumulant les lipides d'algues vertes, et son utilisation
US20160303089A1 (en) * 2013-12-05 2016-10-20 Kyoto University Compound pertaining to neuropoiesis and drug composition
WO2017115798A1 (fr) * 2015-12-28 2017-07-06 北海道公立大学法人 札幌医科大学 Peptide antigénique tumoral
JPWO2017115798A1 (ja) * 2015-12-28 2018-10-25 北海道公立大学法人 札幌医科大学 腫瘍抗原ペプチド

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WO2004029070A3 (fr) 2004-09-30
AU2003273351A1 (en) 2004-04-19
AU2003273351A8 (en) 2004-04-19

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