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WO2009149931A2 - Protéine de liaison aux acides gras dans la pathogenèse de l’insuffisance cardiaque - Google Patents

Protéine de liaison aux acides gras dans la pathogenèse de l’insuffisance cardiaque Download PDF

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WO2009149931A2
WO2009149931A2 PCT/EP2009/004206 EP2009004206W WO2009149931A2 WO 2009149931 A2 WO2009149931 A2 WO 2009149931A2 EP 2009004206 W EP2009004206 W EP 2009004206W WO 2009149931 A2 WO2009149931 A2 WO 2009149931A2
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protein
diseases associated
fatty acid
fabp4
acid binding
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WO2009149931A3 (fr
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Valéria LAMOUNIER-ZEPTER
Christiane Look
Stefan R. Bornstein
Ingo Morano
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Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Technische Universitaet Dresden
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Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
Technische Universitaet Dresden
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5061—Muscle cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to an inhibitor of fatty acid binding protein (FABP) function and/or expression and/or secretion from fat tissue and/or adipocytes and/or binding and/or uptake of FABP protein into cells or tissue for the treatment and/or prevention of diseases selected from the group consisting of cardiac insufficiency, diseases associated with cardiac hypertrophy, diseases associated with a dysfunction of heart contractility, diseases associated with myocardial ischemia secondary to coronary artery spasm, and diseases associated with blood vessel contraction.
  • the invention also relates to methods of identifying compounds suitable as lead compounds or as medicaments for the treatment of these diseases as well as methods of determining a predisposition of an overweight and obese patient for these diseases.
  • Obesity is a well-known risk factor for systemic hypertension and diabetes mellitus. It has been suggested that also the development of heart failure in obese patients may be explained by the cardiac effect of these co-morbidities secondary to obesity. However, the Framingham study also revealed a relationship between obesity and left ventricular mass and diastolic chamber size, independent of hypertension (Lauer, M. S. et al. (1991) J. Am. Med. Assoc. 266, 231 ). Similarly, the risk of developing heart failure was independent of diabetes mellitus in overweight and obese subjects (Kenchaiah, S. ef al. (2002) N. Engl. J. Med. 347, 305). Thus, hypertension and diabetes mellitus may have an additive deleterious effect on myocardial function, but they do not fully explain the mechanisms underlying obesity-associated heart failure.
  • Another proposed mechanism refers to the hemodynamic changes secondary to obesity.
  • Obesity is associated with an increased hemodynamic load, which leads to left ventricular remodeling and systolic dysfunction; the latter is due to prolonged inadequate left ventricular stress (Alpert, M. A. (2001 ) Am. J. Med. Sci. 321 , 225).
  • increased lipid accumulation into cardiac myocytes is another proposed cause for cardiac dysfunction in obesity (Zhou, Y. ef al. (2000) Proc. Natl. Acad. Sci. U. S A. 97, 1784).
  • Zhou et al. demonstrated a statistical correlation between impaired contractile function and intramyocardial lipid accumulation in an animal model of obesity.
  • the management of chronic heart failure includes general measures, pharmacological therapy, mechanical devices and surgery.
  • General measures in patients with heart failure include moderate sodium and fluids restriction, control of alcohol intake, weight reduction in overweight and obese patients, smoking cessation and regular exercise training.
  • the pharmacological agents recommended in patients with heart failure include the angiotensin-converting enzyme inhibitors, beta-adrenoceptor antagonists, diuretics, aldosterone receptos antagonists and cardiac glycosides.
  • Ventricular resynchronization therapy using bi-ventricular pacing should be considered in patients with reduced ejection fraction and ventricular dyssynchrony. At least, patients with end stage heart failure should be considered for heart transplantation (Swedberg, K. et al (2005) Eur. H.eart J. 26, 1115).
  • the technical problem underlying the present invention thus was the provision of improved means and methods useful in the treatment of cardiac dysfunction.
  • the present invention relates to an inhibitor of fatty acid binding protein (FABP) function and/or expression and/or secretion from fat tissue and/or adipocytes and/or binding and/or uptake of FABP protein into cells or tissue for the treatment and/or prevention of diseases selected from the group consisting of i) cardiac insufficiency; ii) diseases associated with a dysfunction of heart contractility; iii) diseases associated with cardiac hypertrophy; iv) diseases associated with myocardial ischemia secondary to coronary artery spasm; and v) diseases associated with blood vessel contraction.
  • FABP fatty acid binding protein
  • fatty acid binding protein refers to a family of highly homologous cytosolic proteins with a molecular mass of 14 - 15 kDa found in different cell types showing a high affinity for long-chain fatty acids and other hydrophobic ligands (Hertzel, A. V. and Bemlohr, D.A. (2000) Trends Endocrinol. Metab. 11, 175). Fatty acid binding proteins serve to protect cells from excess free fatty acids and also facilitate the transport of fatty acids and other lipid mediators throughout the cell. So far, nine tissue-specific cytoplasmic fatty acid binding proteins have been identified, termed FABP1 to FABP9.
  • fatty acid binding protein function (interchangeably used herein with the term “fatty acid binding protein activity”) as used in the present invention relates to the above outlined functions known in the art, including but not limited to protection of cells from excess free fatty acids as well as facilitation of the transport of fatty acids. Furthermore, the term “fatty acid binding protein function” also refers to the surprising finding of the present inventors that fatty acid binding proteins possess a highly potent cardio-depressant activity by depressing the shortening amplitude and intracellular calcium transients in cardiomyocytes as well as the cardiac force generation and coronary flow due to contraction of coronary vessels in whole hearts.
  • Methods to determine whether a protein has such a function are well known to the person skilled in the art and include, without being limited, the methods outlined in the Examples.
  • functional studies of isolated cardiomyocytes and isolated perfused heart preparations may be carried out following standardized protocols as outlined in the Examples.
  • Cell contractility and intracellular Ca 2+ transients of isolated adult cardiomyocytes can, for example, be measured by using an lonoptix Contractility and Fluorescence System (lonoptix, Milton, USA).
  • Isovolumetric left ventricular pressure LVP
  • maximal rate of isovolumetric pressure decrease (-dP/dtmax) and coronary flow can be analysed in isolated adult heart by using, for example, a Langendorff perfusion system.
  • fatty acid binding protein expression in accordance with the present invention refers to the transcription of the gene encoding the corresponding fatty acid binding protein as well as to the translation of the mRNA encoding the fatty acid binding protein.
  • adipocytes relates to specialised cells of the adipose tissue that store energy as fat.
  • adipose tissue (or fat) defines the loose connective tissue composed of adipocytes that serves the main role of storing energy in the form of fat but also of cushioning and insulating the body.
  • WAT white adipose tissue
  • BAT brown adipose tissue
  • Methods to determine fatty acid binding protein secretion are well known to the skilled person and include, but are not limited to, ELISA kits, radioimmunoassays or immunoassays.
  • inhibitor designates a compound lowering the activity of a target molecule, preferably by performing one or more of the following effects: (i) the transcription of the gene encoding the protein to be inhibited is lowered, (ii) the translation of the mRNA encoding the protein to be inhibited is lowered, (iii) the protein performs its biochemical function with lowered or abolished efficiency in presence of the inhibitor, and (iv) the protein performs its cellular function with lowered or abolished efficiency in presence of the inhibitor.
  • Compounds falling in class (i) include compounds interfering with the transcriptional machinery and/or its interaction with the promoter of said gene and/or with expression control elements remote from the promoter such as enhancers.
  • Compounds of class (ii) comprise antisense constructs and constructs for performing RNA interference (e.g. siRNA, shRNA, miRNA) well known in the art (see, e.g. Zamore (2001 ) Nat. Struct. Biol. 8(9), 746; Tuschl (2001) Chembiochem. 2(4), 239).
  • Class (iii) interferes with molecular function of the protein to be inhibited, in the present case of a fatty acid binding protein with its fatty acid -binding and -transport activity as well as its cardio-depressant activity and its activity on blood vessel and muscle contractility. Accordingly, active site binding compounds, in particular compounds capable of binding to the fatty acid binding pocket of FABP isoforms or to the plasma membrane receptor domain(s) of FABPs are envisaged.
  • Class (iv) includes compounds which do not necessarily bind directly to fatty acid binding proteins, but still interfere with fatty acid binding protein activity, for example by binding to and/or inhibiting the function or expression of members of a pathway which comprises fatty acid binding proteins. These members may be either upstream or downstream of the fatty acid binding protein within said pathway. As non-limiting examples, FABP- binding domain(s) of the plasma membrane receptor(s) or cellular uptake mechanisms may be inhibited.
  • the level of activity is less than 90%, more preferred less than 80%, 70%, 60% or 50% of the activity in absence of the inhibitor. Yet more preferred are inhibitors lowering the level to less than 25%, less than 10%, less than 5% or less than 1 % of the activity in absence of the inhibitor.
  • the efficiency of the inhibitor can be quantified by comparing the level of activity in the presence of the inhibitor to that in the absence of the inhibitor.
  • an activity measure may be used: the change in amount of mRNA formed, the change in amount of protein formed, the change in amount of fatty-acid binding and transport or effect on contractility of cells, and/or the change in the cellular phenotype or in the phenotype of an organism.
  • An inhibitor of "binding and/or uptake of FABP protein into cells or tissue” in accordance with the present invention relates to a compound suitable to inhibit FABP activity on target cells/tissues by preventing either binding of FABP to the target cell/tissue, for example by specifically competing with FABP/receptor interaction, and/or by preventing that FABP is taken up by the target cell/tissue, e.g. by specifically competing with FABP/transporter interaction.
  • Methods for measuring the binding and/or uptake of FABP4 protein into cells or tissue are well known to the skilled person and include, but are not limited to, ELISA tests and confocal microscopy.
  • determination of expression levels can, for example, be carried out on the nucleic acid or protein level.
  • Methods for determining the expression of FABPs on the nucleic acid level include, but are not limited to, northern blotting, PCR, RT-PCR or real RT-PCR. PCR is well known in the art and is employed to make large numbers of copies of a target sequence. This is done on an automated cycler device, which can heat and cool containers with the reaction mixture in a very short time.
  • the PCR generally, consists of many repetitions of a cycle which consists of: (a) a denaturing step, which melts both strands of a DNA molecule and terminates all previous enzymatic reactions; (b) an annealing step, which is aimed at allowing the primers to anneal specifically to the melted strands of the DNA molecule; and (c) an extension step, which elongates the annealed primers by using the information provided by the template strand.
  • PCR can be performed, for example, in a 50 ⁇ l reaction mixture containing 5 ⁇ l of 10 x PCR buffer with 1.5 mM MgCI 2 , 200 ⁇ M of each deoxynucleoside triphosphate, 0.5 ⁇ l of each primer (10 ⁇ M), about 10 to 100ng of template DNA and 1 to 2.5 units of Taq Polymerase.
  • the primers for the amplification may be labeled or be unlabeled.
  • DNA amplification can be performed, e.g., with a model 2400 thermal cycler (Applied Biosystems, Foster City, CA): 2 min at 94°C, followed by 30 to 40 cycles consisting of annealing (e. g. 30 s at 50 0 C), extension (e.
  • Suitable polymerases for use with a DNA template include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, Tth polymerase, Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus Vent, Amplitaq, Pfu and KOD, some of which may exhibit proof-reading function and/or different temperature optima.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • reverse transcriptase refers to an enzyme that catalyzes the polymerization of deoxyribonucleoside triphosphates to form primer extension products that are complementary to a ribonucleic acid template. The enzyme initiates synthesis at the 3'-end of the primer and proceeds towards the 5'-end of the template until synthesis terminates.
  • RNA target sequence into a complementary, copy-DNA (cDNA) sequence examples include avian myeloblastosis virus (AMV) reverse transcriptase and Thermus thermophilus DNA polymerase, a thermostable DNA polymerase with reverse transcriptase activity marketed by Perkin Elmer.
  • AMV avian myeloblastosis virus
  • Thermus thermophilus DNA polymerase a thermostable DNA polymerase with reverse transcriptase activity marketed by Perkin Elmer.
  • the genomic RNA/cDNA duplex template is heat denatured during the first denaturation step after the initial reverse transcription step leaving the DNA strand available as an amplification template.
  • High-temperature RT provides greater primer specificity and improved efficiency.
  • the RT Reaction can be performed, for example, in a 20 ⁇ l reaction mix containing: 4 ⁇ l of 5x AMV-RT buffer, 2 ⁇ l of Oligo dT (100 ⁇ g/ml), 2 ⁇ l of 10 mM dNTPs, 1 ⁇ l total RNA, 10 Units of AMV reverse transcriptase, and H 2 O to 20 ⁇ l final volume.
  • the reaction may be, for example, performed by using the following conditions: The reaction is held at 70 C° for 15 minutes to allow for reverse transcription. The reaction temperature is then raised to 95 C 0 for 1 minute to denature the RNA-cDNA duplex.
  • reaction temperature undergoes two cycles of 95 0 C for 15 seconds and 60 C° for 20 seconds followed by 38 cycles of 90 C° for 15 seconds and 60 C° for 20 seconds. Finally, the reaction temperature is held at 60 C 0 for 4 minutes for the final extension step, cooled to 15 C 0 , and held at that temperature until further processing of the amplified sample. Any of the above mentioned reaction conditions may be scaled up according to the needs of the particular case.
  • the resulting products are loaded onto an agarose gel and band intensities are compared after staining the nucleic acid molecules with an intercalating dye such as ethidiumbromide or SybrGreen.
  • a lower band intensity of the sample derived from the cell treated with the test compound as compared to a non-treated cell indicates a compound that inhibits expression.
  • Real-time PCR employs a specific probe, in the art also referred to as TaqMan probe, which has a reporter dye covalently attached at the 5' end and a quencher at the 3' end. After the TaqMan probe has been hybridized in the annealing step of the PCR reaction to the complementary site of the polynucleotide being amplified, the 5' fluorophore is cleaved by the 5' nuclease activity of Taq polymerase in the extension phase of the PCR reaction.
  • Methods for the determination of the expression of FABPs on the amino acid level include but are not limited to western blotting or polyacrylamide gel electrophoresis in conjunction with protein staining techniques such as Coomassie Brilliant blue or silver-staining.
  • the total protein is loaded onto a polyacrylamide gel and separated by electrophoresis. Afterwards, the separated proteins are transferred onto a membrane, e.g. a polyvinyldifluoride (PVDF) membrane, by applying an electrical current.
  • PVDF polyvinyldifluoride
  • the proteins on the membrane are exposed to an antibody specifically recognizing the protein of interest, here the fatty acid binding protein. After washing, a second antibody specifically recognizing the first antibody and carrying a readout system such as a fluorescent dye is applied.
  • the amount of the protein of interest is determined by comparing the fluorescence intensity of the protein derived from the cell treated with the test compound with the fluorescence intensity of the protein derived from a non- treated cell. A lower fluorescence intensity of the protein derived from the cell treated with the test compound indicates an inhibitor of the protein. Also of use in protein quantification is the Agilent Bioanalyzer technique.
  • assays based on protein detection include without limitation ion exchange chromatography, gel filtration chromatography, affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, disc gel electrophoresis, Western blot analysis, immunoprecipitation, see, for example, Soejima and Koda, Transfusion 45 (2005) 1934-1939; Yeh et al., Anesth. Analg. 101 (2005) 1401-1406; Chou et a!., Am. J. Clin. Pathol.. 124 (2005) 330-338.
  • the efficiency of the inhibitor on fatty acid binding protein mediated transport of fatty acids across a membrane can also be measured in an artificial membrane system.
  • Such systems include liposomes, artificial bilayers of phospholipids, isolated plasma membrane such as cell membrane fragments, cell membrane fractions, or cell membrane vesicles.
  • a labeled (e.g., radioactively labeled) fatty acid substrate can be incubated with one side of a bilayer or in a suspension of liposomes and the accumulation of fatty acids with time can be measured, using appropriate means to detect the label (e.g., scintillation counting of medium on each side of the bilayer, or of the contents of liposomes isolated from the surrounding medium).
  • Methods for the determination of the activity of FABPs on cell contractility include, but are not limited to in vitro studies with isolated cardiomyocytes by using an lonoptix Contractility and Fluorescence System (lonoptix, Milton, USA), in vitro studies in isolated adult heart by using a Langendorff perfusion system or in vivo studies with haemodynamic and functional analysis of heart function the methods as outlined in the Examples.
  • the inhibitor in accordance with the method of the present invention may act extracellularly or intracellular ⁇ .
  • Inhibitors acting intracellular ⁇ may for example be a siRNA down-regulating the expression level of the protein or inhibitors acting via inhibition of fatty acid binding.
  • Inhibitors exerting their effects extracellularly may, for example, be antibodies or aptamers binding either to the circulating FABPs or interfering with binding and uptake of fatty acid binding proteins into cells, for examples as substances eliminating FABP-containing microparticles, i.e. small particles which are derived from cells by microparticle shedding (Zwaal et al. (1993) Biochem. Soc.
  • diseases associated with a dysfunction of heart contractility relates to diseases associated with a dysfunction of the myocardium contractile function leading to impairment of myocardial mechanical function.
  • diseases include, but are not restricted to cardiac insufficiency, acute and chronic myocarditis or cardiomyopathies.
  • cardiac insufficiency also termed “heart failure” in accordance with the present invention relates to a clinical syndrome in which an abnormality of cardiac heart structure or function is responsible for the inability of the heart to fill with or eject blood at a rate commensurate with the requirements of the metabolising tissues (E. Braunwald, Heart failure and cor pulmonale, in Harrison's Principles of Internal Medicine, 16 th ed (2005) 1367).
  • Cardiac insufficiency includes abnormalities during systole (systolic heart failure) and/or diastole (diastolic heart failure), abnormalities in the left and/or right ventricle, chronic and/or acute heart insufficiency, low-output and/or high-output heart failure.
  • Cardiac insufficiency includes, but is not limited to, conditions and diseases such as hypertrophic cardiomyopathy; arrhythmogenic right ventricular dysplasia cardiomyopathy; left ventricular noncompaction cardiomyopathy; cardiomyopathies associated with conduction defect and/or ion channel disorders; primary and secondary dilated cardiomyopathy; primary restrictive nonhypertrophied cardiomyopathy, idiopathic cardiomyopathy; stress ("Tako-Tsubo") cardiomyopathy; tachycardia- induced cardiomyopathy; pregnancy-related cardiomyopathy; toxic cardiomyopathy such as caused by alcohol abuse, cocaine use, ephedrine use, chemotherapeutic agent, radiation; restrictive cardiomyopathy secondary to myocardial infiltrative or storage diseases, such as amyloidosis, Gaucher disease, Hurler's disease, Hunter's disease, hemochromatosis, Fabry's disease, glycogen storage disease, Niemann-Pick disease; cardiomyopathy related to endocrine and/or metabolic disorders such as thyroid disorders, hyper
  • cardiac insufficiency may result from a large number of diseases or conditions, including, but not limited to, ischemic heart disease; valvular heart disease; hypertensive cardiomyopathy; inflammatory and/or infectious myocarditis, such as viral myocarditis, bacterial myocarditis, rickettsial myocarditis, fungal myocarditis, parasitic myocarditiy (Chagas disease, toxoplasmosis), sarcoidosis; pulmonary hypertension or obstructive sleep apnea (Ivlaron, et al (2006), Circulation 113:1807; E. Braunwald, E. Heart failure and cor pulmonale, in Harrison's Principles of Internal Medicine, 16 th ed (2005) 1367;.
  • diseases or conditions including, but not limited to, ischemic heart disease; valvular heart disease; hypertensive cardiomyopathy; inflammatory and/or infectious myocarditis, such as viral myocardit
  • those forms of cardiac insufficiency that are directly and exclusively secondary to diabetes mellitus, metabolic disorders or atherosclerosis are excluded.
  • those forms of cardiac insufficiency that are secondary to diabetes, metabolic disorders or atherosclerosis are excluded.
  • FABP4 inhibitors of FABP4 such as indol derivatives have been suggested to have a potential use in the treatment of diseases such as diabetes or atherosclerosis.
  • novel indole derivatives are disclosed as FABP4 inhibitors that reduce the amount of plasminogen activator inhibitor-1 (PAI-1 ) secretion from macrophages activated by Ac-LDL.
  • PAI-1 plasminogen activator inhibitor-1
  • fatty acid binding proteins have a direct contractile-depressant activity on heart muscle. Therefore, the present invention now allows for new therapeutic approaches suitable for a new group of patients, i.e. patients with heart dysfunction being for example independent of macrophage activation underlying atherosclerosis.
  • the inhibitor is not an indole derivative.
  • diseases associated with myocardial ischemia secondary to coronary artery spasm refers to conditions with a reduction of coronary blood flow caused by a spasm of coronary arteries, such as for example Prinzmetal's angina.
  • diseases associated with blood vessel contraction refers to conditions with pathological contraction of coronary arteries, arterial hypertension, Raynaud's disease or conditions associated with Raynaud's phenomenon, such as collagen vascular diseases, acrocyanosis as well as non-occlusive mesenteric ischemia caused by vasospasm of mesenteric vessels.
  • Obesity is a major risk factor for metabolic syndrome and cardiovascular disease and seems to be directly related to heart failure, independently of others risk factors (Kenchaiah, S et al. (2002) N. Engl. J. Med. 347:305).
  • Several mechanisms to explain this correlation have been discussed, including hemodynamic changes with cardiac overload and left ventricular remodeling, and lipid accumulation into myocardium leading to lipoapoptosis of the cardiomyocytes (Poirier, P. et al. (2006) Circulation. 113:898).
  • these mechanisms do not fully explain the development of heart dysfunction in obese and especially in overweight subjects.
  • the present inventors now surprisingly found that substances secreted from mature human adipocytes, in particular the adipocyte fatty acid binding protein (FABP4), strongly and acutely suppresses the contraction of cardiomyocytes by attenuating intracellular Ca 2+ levels.
  • FABP4 adipocyte fatty acid binding protein
  • This hitherto unknown acute depressant effect of FABP4 on cardiac contraction suggests a new direct role of adipose tissue in the pathogenesis of myocardium dysfunction, thus contributing to increased heart failure risk in overweight patients.
  • the present inventors have extended these findings to other members of the fatty acid binding protein family (Example 6), showing that the cardio-depressant activity is not restricted to the adipocyte specific fatty acid binding protein.
  • a depressant activity on the contraction of coronary blood vessels has also been shown, thus supporting a role of fatty acid binding proteins in a wide variety of muscle tissues.
  • FABP4 has recently been linked to obesity and metabolic syndrome, a cluster of clinical manifestations including type 2 diabetes, hypertension, dyslipidemia, proinflammatory and prothrombotic states, hyperuricemia and cardiovascular diseases.
  • Mice jacking FABP4 exhibit a protective phenotype against the development of insulin resistance associated with genetic or diet-induced obesity (Hotamisligil.G.S. et al. (1996) Science 274:1377; Uysal.K.T. (2000) Endocrinology 141 :3388).
  • FABP4 is also expressed in macrophages, which seem to be a critical site of action of FABP4 in the development of atherosclerosis (Makowski and Hotamisligil (2005) Curr. Opin. Lipidol.
  • the link between the various features of the metabolic syndrome is considered to be due to the distinct actions of FABP4 in adipocytes and macrophages, with expression by macrophages influencing foam-cell formation in blood vessels and, thereby, atherosclerotic processes and expression by adipocytes protecting from hyperinsulinemia and insulin resistance and improving systemic glucose and lipid metabolism (Makowski ef al. (2001 ) Nature 7(6):699). More recently, the use of an inhibitor of FABP4 that competitively inhibits the binding of endogenous fatty acids was effective against severe atherosclerosis and type 2 diabetes in a mouse model of atherosclerosis and obesity (Furuhashi ef al. (2007) Nature 447:959).
  • the present inventors now show for the first time that fatty acid binding proteins directly inhibit cardiomyocyte contraction acutely, in a doses-dependent manner and similar to the previously related adipocyte cardio-depressant factor by reducing intracellular Ca 2+ -transient.
  • a direct bioactive role of fatty acid binding proteins on heart function independently of their role of a transport protein has been shown.
  • the cardio-depressant effect of the adipocyte conditioned media tested strongly correlated with the concentration of fatty acid binding proteins found in the media. Supporting the specificity of this correlation, there was no correlation between the total protein concentration and the cardio-depressant effect of the adipocyte conditioned medium on cardiomyocyte contraction.
  • the mechanical impairment of the heart that is associated with this newly identified direct function of fatty acid binding proteins and that is independent from atherosclerotic or metabolic developments, may be treated by inhibiting fatty acid binding proteins.
  • fatty acid binding proteins of different origins exert a suppressive activity on muscle cells such as heart muscle and blood vessels.
  • diseases that are caused by the contractile-depressant activity of fatty acid binding proteins for example diseases caused by the activity of adipocyte secreted FABP4 on cardiomyocytes, can be treated by inhibition of fatty acid binding proteins.
  • diseases associated with cardiac dysfunction not caused by fatty acid binding proteins but caused by other factors such as primary or secondary cardiomyopathy (e.g. toxic cardiomyopathy and myocarditis), can be ameliorated by inhibiting the contractile-depressant activity of fatty acid binding proteins endogenous to the affected tissue (such as FABP3 in muscle cells, such as heart muscle).
  • the present invention also relates to the use of an inhibitor of fatty acid binding protein (FABP) function an/or expression for the preparation of a pharmaceutical composition for the treatment and/or prevention of diseases selected from the group consisting of i) cardiac insufficiency; ii) diseases associated with a dysfunction of heart contractility; iii) diseases associated with cardiac hypertrophy; iv) diseases associated with myocardial ischemia secondary to coronary artery spasm; and v) diseases associated with blood vessel contraction.
  • FABP fatty acid binding protein
  • the term "pharmaceutical composition” relates to a composition for administration to a patient, preferably a human patient.
  • the pharmaceutical composition of the invention comprises the compounds recited above. It may, optionally, comprise further molecules capable of altering the characteristics of the compounds of the invention thereby, for example, stabilizing, modulating and/or activating their function.
  • the composition may be in solid, liquid or gaseous form and may be, inter alia, in the form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an)
  • the pharmaceutical composition of the present invention may, optionally and additionally, comprise a pharmaceutically acceptable carrier.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration,
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 5 g units per day. However, a more preferred dosage might be in the range 0.01 mg to 100 mg, even more preferably 0.01 mg to 50 mg and most preferably 0.01 mg to 10 mg per day.
  • the inhibitor of the invention is an inhibitor of fatty acid binding protein (FABP) function or expression and/or secretion from fat tissue and/or adipocytes and/or binding of FABP protein to cells or tissue for the treatment and/or prevention of i) diseases associated with a dysfunction of heart contractility and ii) diseases associated with blood vessel contraction, wherein the inhibitor inhibits the contractile-depressant activity of fatty acid binding proteins on muscle cells or tissue.
  • FABP fatty acid binding protein
  • the present invention also relates to an inhibitor of binding of fatty acid binding protein (FABP) to muscle cells or tissue for the treatment and/or prevention of diseases selected from the group consisting of i) diseases associated with a dysfunction of heart contractility; and ii) diseases associated with blood vessel contraction.
  • FABP fatty acid binding protein
  • an inhibitor suitable to inhibit FABP activity on target cells/tissues by preventing binding of FABP to the target cell/tissue provides a novel means for targeting diseases associated with an impairment of either heart or blood vessel muscle contraction.
  • Methods for measuring the binding of FABP4 protein to cells or tissue are well known to the skilled person and include, but are not limited to, the methods described in the examples as well as ELISA tests, confocal microscopy, co- immunoprecipitation, bimolecular fluorescence complementation, affinity electrophoresis, pull-down assays, label transfer, yeast two-hybrid assay, in vivo protein crosslinking, in vitro chemical protein crosslinking, tandem affinity purification, dual polarization interferometry, surface Plasmon resonance, fluorescence resonance energy transfer, nuclear magnetic resonance spectroscopy, fluorescence polarization assay, blot overlay, glutathione-s-transferase based fusion assay.
  • the inhibitor specifically inhibits the function of the N-terminal end of the fatty acid binding protein.
  • the first 20 amino acids at the N- terminus of FABP4 mainly confer the contractile-depressant activity of FABP4. Therefore, by targeting the activity of the fatty acid binding protein mediated by the N-terminal region, and in particular by targeting the activity of the fatty acid binding protein mediated by the first 20 amino acids at the N- terminus, or alternatively by targeting the interaction of FABP4 with a surface receptor, an inhibitor may be particularly suitable for the treatment of the above mentioned diseases.
  • the inhibitor can bind directly to the N-terminal region of the fatty acid binding protein.
  • the inhibitor can interact with other sites of the fatty acid binding protein as long as said interaction leads to a conformational change of the protein that results in an impairment of the function mediated by this region, i.e. an impairment of the contractile-depressant activity of the fatty acid binding protein.
  • the inhibitor specifically interacts with the N-terminal end of FABP. More preferably, the inhibitor interacts with the fatty acid binding protein in the region comprising amino acids 1 to 20 from the N-terminus.
  • Methods to determine whether an inhibitor interacts with the N-terminus of fatty acid binding protein include, without being limiting, the methods shown in the examples as well as ELISA tests, confocal microscopy, co-immunoprecipitation, bimolecular fluorescence complementation, affinity electrophoresis, pull-down assays, label transfer, yeast two-hybrid assay, in vivo protein crosslinking, in vitro chemical protein crosslinking, tandem affinity purification, dual polarization interferometry, surface Plasmon resonance, fluorescence resonance energy transfer, nuclear magnetic resonance spectroscopy, fluorescence polarization assay, blot overlay, glutathione-s-transferase based fusion assay
  • the fatty acid binding protein is selected from the group consisting of FABP3 and FABP4.
  • FABP4 is the major cytosolic protein of mature adipocytes (accounting for ⁇ 6% of the total cellular protein), which also secrete FABP4 into the blood circulation. In addition, FABP4 is also expressed in macrophages. FABP3 is predominantly expressed in skeletal muscle and in the heart. Heart FABP is rapidly released into the circulation upon injury to the myocardium. Thus, the use of FABP3 as an early diagnostic marker in acute myocardial infarction has been proposed.
  • the amino acid sequence of FABP4 is shown in SEQ ID NO: 1 (Accession number NP 001433) while the amino acid sequence of FABP3 is shown in SEQ ID NO:2 (Accession number NP 004093).
  • the nucleic acid sequences of FABP4 and FABP3 are given in SEQ ID NOs: 3 and 4 (Accession numbers NM 001442 and NM 004102), respectively.
  • the fatty acid binding protein is FABP4.
  • the present invention also relates to a method of identifying a compound suitable as a lead compound and/or as a medicament for the treatment and/or prevention of diseases selected from the group consisting of i) cardiac insufficiency; ii) diseases associated with a dysfunction of heart contractility; iii) diseases associated with cardiac hypertrophy; iv) diseases associated with myocardial ischemia secondary to coronary artery spasm; and v) diseases associated with blood vessel contraction, comprising the steps of (a) contacting a test compound with a cell or tissue comprising and/or secreting a protein, wherein said protein (1 ) comprises or consists of the amino acid sequence of any one of SEQ
  • NP 001433 and NP 004093 (2) is encoded by a nucleic acid molecule comprising or consisting of the sequence of any one of SEQ ID NOs: 3 or 4 (NM 001442 and NM 004102); (3) is a fragment of the protein according to (1 ) or (2) and exhibits fatty acid binding protein activity; or (4) has a sequence at least 75% identical with the protein according to (1) or (2) or with the fragment according to (3) and exhibits fatty acid binding protein activity; and (b) determining whether said test compound, upon contacting in step (a) inhibits the activity, expression and/or secretion of said protein wherein said inhibition indicates that said compound is suitable as a lead compound and/or as a medicament for the treatment and/or prevention of diseases selected from the group consisting of cardiac insufficiency, diseases associated with a dysfunction of heart contractility, diseases associated with cardiac hypertrophy, diseases associated with myocardial ischemia secondary to coronary artery spasm, and diseases associated with blood vessel
  • the term "compound” denotes, in one alternative a small molecule.
  • a small molecule may be, for example, an organic molecule.
  • Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds.
  • the original definition of the term organic related to the source of chemical compounds with organic compounds being those carbon-containing compounds obtained from plant or animal or microbial sources, whereas inorganic compounds were obtained from mineral sources.
  • Organic compounds can be natural or synthetic.
  • the compound may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates).
  • the compound may be a macromolecule of natural or synthetic origin. Natural macromolecules are, for example (poly)peptides such as antibodies or nucleic acid molecules such as DNA, RNA or aptamers or polysaccharides. Synthetic macromolecules are for example polymers consisting of covalently linked small organic molecules.
  • Small molecules have a molecular weight below 10.000 Da, preferably less than 1.000 Da, more preferably less than 500 Da and most preferably between 200 Da and 400 Da.
  • Macromolecules have a molecular weight in the range of a few thousand up to several million Da. Preferably their molecular weight is more than 10.000 Da, more preferably more than 100.000 Da and most preferably between 150.000 Da and 250.000 Da.
  • (poly)peptide accordance with the present invention describes a group of molecules which comprises the group of peptides, consisting of up to 30 amino acids, as well as the group of polypeptides, consisting of more than 30 amino acids. Also encompassed by the term “(poly)peptide” are proteins as well as fragments of proteins.
  • fragment of the protein in accordance with the present invention refers to a portion of the protein comprising at least the amino acid residues necessary to maintain the biological activity of the protein. Preferably, these amino acid chains are linear. (Poly)peptides may further form multimers consisting of at least two identical or different molecules.
  • fusion proteins giving rise or corresponding to proteins of the present invention also fall under the definition of the term "(poly)peptide".
  • Those components of said fusion proteins which are not FABP sequences or fragments or variants thereof as defined herein, include amino acid sequence which confer desired properties such as modified/enhanced stability, modified/enhanced solubility and/or the ability of targeting one or more specific cell types or could confer a different biological activity.
  • (poly)peptides where amino acid(s) and/or peptide bond(s) have been replaced by functional analogues are also encompassed by the invention.
  • Such functional analogues include all known amino acids other than the 20 gene-encoded amino acids, such as selenocysteine.
  • the term "(poly)peptide” also refers to naturally modified (poly)peptides where the modification is effected e.g. by glycosylation, acetylation, phosphorylation and similar modifications which are well known in the art. It is also well known that (poly)peptides are not always entirely linear.
  • (poly)peptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of post-translation events, including natural processing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular (poly)peptides may be synthesized by non-translational natural processes and by synthetic methods.
  • the modifications can be a function of how the protein is made.
  • the modifications will be determined by the host cells posttranslational modification capacity and the modification signals in the protein amino acid sequence.
  • a (poly)peptide when glycosylation is desired, a (poly)peptide should be expressed in a glycosylating host, generally an eukaryotic cell, for example Cos7, HELA or others.
  • a glycosylating host generally an eukaryotic cell, for example Cos7, HELA or others.
  • the same type of modification may be present in the same or varying degree at several sites in a given (poly)peptide.
  • a given (poly)peptide may contain more than one type of modification.
  • antibody in accordance with the present invention, comprises polyclonal and monoclonal antibodies as well as derivatives or fragments thereof which still retain the binding specificity. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999.
  • the antibody of the invention also includes embodiments such as chimeric, single chain and humanized antibodies, as well as antibody fragments, like, inter alia, Fab fragments, as well as fusion proteins.
  • Antibody fragments or derivatives further comprise F(ab') 2 , Fv or scFv fragments; see, for example, Harlow and Lane (1988) and (1999), loc. cit.
  • F(ab') 2 , Fv or scFv fragments see, for example, Harlow and Lane (1988) and (1999), loc. cit.
  • the (antibody) derivatives can be produced by peptidomimetics.
  • techniques described for the production of single chain antibodies see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specific for polypeptide(s) and fusion proteins of this invention.
  • transgenic animals may be used to express humanized antibodies specific for polypeptides and fusion proteins of this invention.
  • the antibody of this invention is a monoclonal antibody.
  • any technique which provides antibodies produced by continuous cell line cultures, can be used.
  • examples for such techniques include the hybridoma technique (Kohler and Milstein (1975) Nature 256, 495), the trioma technique, the human B-cell hybridoma technique (Kozbor (1983) Immunology Today 4, 72) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 77).
  • antibody comprises antibody constructs, which may be expressed in cells, e.g. antibody constructs which may be transfected and/or transduced via, amongst others, viruses or plasmid vectors.
  • the antibody described in the context of the invention is capable to specifically bind/interact with an epitope of a fatty acid binding protein.
  • the term "specifically binding/interacting with” as used in accordance with the present invention means that the antibody does not or essentially does not cross-react with an epitope of similar structure.
  • Cross-reactivity of a panel of antibodies under investigation may be tested, for example, by assessing binding of said panel of antibodies under conventional conditions to the epitope of interest as well as to a number of more or less (structurally and/or functionally) closely related epitopes. Only those antibodies that bind to the epitope of interest in its relevant context (e.g. a specific motif in the structure of a protein) but do not or do not essentially bind to any of the other epitope are considered specific for the epitope of interest and thus to be antibodies in accordance with this invention.
  • a conformational or discontinuous epitope is characterized for polypeptide antigens by the presence of two or more discrete amino acid residues which are separated in the primary sequence, but come together on the surface of the molecule when the polypeptide folds into the native protein/antigen (SeIa (1969) Science 166, 1365; Laver (1990) Cell 61 , 553).
  • the two or more discrete amino acid residues contributing to the epitope are present on separate sections of one or more polypeptide chain(s).
  • a continuous or linear epitope consists of two or more discrete amino acid residues, which are present in a single linear segment of a polypeptide chain.
  • Nucleic acid molecules include DNA, such as cDNA or genomic DNA, and RNA. It is understood that the term “RNA” as used herein comprises all forms of RNA including mRNA, ncRNA (non-coding RNA), tRNA and rRNA.
  • RNA comprises all forms of RNA including mRNA, ncRNA (non-coding RNA), tRNA and rRNA.
  • non-coding RNA includes siRNA (small interfering RNA), miRNA (micro RNA), rasiRNA (repeat associated RNA), snoRNA (small nucleolar RNA), and snRNA (small nuclear RNA). Further included are nucleic acid mimicking molecules known in the art such as synthetic or semisynthetic derivatives of DNA or RNA and mixed polymers, both sense and antisense strands.
  • nucleic acid mimicking molecules or nucleic acid derivatives include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2'-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA) and locked nucleic acid (LNA) (see Braasch and Corey (2001 ) Chem Biol. 8, 1).
  • LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2'- oxygen and the 4'-carbon. They may contain additional non-natural or derivatised nucleotide bases, as will be readily appreciated by those skilled in the art.
  • aptamer in accordance with the present invention refers to DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, proteins, small organic compounds, and even entire organisms. A database of aptamers is maintained at http://aptamer.icmb.utexas.edu/. More specifically, aptamers can be classified as DNA or RNA aptamers or peptide aptamers. Whereas the former consist of (usually short) strands of oligonucleotides, the latter consist of a short variable peptide domain, attached at both ends to a protein scaffold.
  • Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalent ⁇ , SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.
  • Peptide aptamers are proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range).
  • the variable loop length is typically comprised of 10 to 20 amino acids, and the scaffold may be any protein which have good solubility properties.
  • the bacterial protein Thioredoxin-A is the most used scaffold protein, the variable loop being inserted within the reducing active site, which is a -Cys-Gly-Pro-Cys- loop in the wild protein, the two cysteins lateral chains being able to form a disulfide bridge.
  • Peptide aptamer selection can be made using different systems, but the most used is currently the yeast two-hybrid system. Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies.
  • aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamer's inherently low molecular weight.
  • Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging.
  • the term "lead compound” in accordance with the present invention refers to a compound discovered with step (b) of the method of the invention which will be e.g. further optimised, in particular to be pharmaceutically more acceptable.
  • the identified lead compounds may be optimised to arrive at a compound, which may be for example used in a pharmaceutical composition. Methods for the optimisation of the pharmacological properties of compounds identified in screens, the lead compounds, are known in the art and are detailed further below.
  • the term “fragment of the protein” in accordance with the present invention has been define above. In particular, the term “fragment of the protein .... and exhibits fatty acid binding protein activity” refers to a portion of the protein comprising at least the amino acid residues necessary to maintain the "fatty acid binding protein activity" as defined above.
  • the fragment has a length of at least 35 amino acids, more preferably between 50 and 120 amino acids and most preferably between 75 and 100 amino acids. Furthermore, said fragment exhibits fatty acid binding protein activity.
  • fatty acid binding protein activity is essentially retained, if at least 60% of the biological activity of the fatty acid binding protein activity are retained. Preferably, at least 75% or at least 80% of the fatty acid binding protein activity are retained. More preferred is that at least 90% such as at least 95%, even more preferred at least 98% such as at least 99% of the biological activity of the fatty acid binding protein are retained. Most preferred is that the biological activity is fully, i.e. to 100%, retained.
  • proteins having increased biological activity compared to fatty acid binding protein i.e. more than 100% enzyme activity.
  • Methods of assessing biological activity of FABPs are well known to the person skilled in the art as outlined above and include, without being limiting, measuring fatty acid binding and transport activity as well as determining fatty acid binding protein activity on cardiac or blood vessel contractility. Methods for measuring fatty acid binding and transport are well known to the skilled person and are described, for example in (Richieri, G.V. ef a/ (1996) J. Biol. Chem. 271 :11291 ; Zimmerman, A. W. er a/. (2001 ) Int. J. Biochem. & Cell Biol.
  • sequences that at least exhibit 75% identity with the above-recited protein are sequences that at least exhibit 75% identity with the above-recited protein.
  • identity is between 75 and 98% such as at least 80%, more preferred at least 90%, more preferred 95% and most preferred the identity is at least 98%.
  • Such molecules may be splice forms, homologous molecules from other species, such as orthologs, or mutated sequences from the same species to mention the most prominent examples.
  • the term "% sequence identity" describes the number of matches ("hits") of identical amino acids of two or more aligned amino acid sequences as compared to the number of amino acid residues making up the overall length of the amino acid sequences (or the overall compared part thereof).
  • the percentage of amino acid residues that are the same may be determined, when the (sub)sequences are compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected.
  • Preferred proteins in accordance with the invention are those where the described identity exists over a region that is at least about 15 to 25 amino acids in length, more preferably, over a region that is at least about 50 to 100 amino acids in length. More preferred proteins in accordance with the present invention are those having the described sequence identity over the entire length of the protein.
  • the BLASTP programme uses as default a word length (W) of 3, and an expectation (E) of 10.
  • the "cell” as recited throughout this specification refers to a primary cell or a cell from a cell line.
  • Primary cells are cells which are directly obtained from an organism and which are not immortalized. Suitable primary cells are, for example, from species such as mouse, rat, human, guinea pig, hamster, pig, dog, sheep, goat, donkey or cow.
  • the cells or cell lines may be, for example, selected from adipocytes, cardiomyocytes or or non-human embryonic stem cells.
  • the inhibitor or the pharmaceutical composition of the invention comprises or is to be administered in conjunction with further agents, such as for example inhibitors of 5,6- Epoxyeicosatrienoic acid (5,6-EET).
  • further agents such as for example inhibitors of 5,6- Epoxyeicosatrienoic acid (5,6-EET).
  • the present invention relates to a method of identifying a compound suitable as a lead compound and/or as a medicament for the treatment and/or prevention of diseases selected from the group consisting of i) diseases associated with a dysfunction of heart contractility; and ii) diseases associated with blood vessel contraction, comprising the steps of (a) contacting a tissue or cell with a protein, wherein said protein (1 ) comprises or consists of the amino acid sequence of SEQ ID NOs: 1; or (2) is encoded by a nucleic acid molecule comprising or consisting of the sequence of SEQ ID NO: 3; or (3) is a fragment of the protein according to (1 ) or (2) and exhibits FABP4 activity, or (4) has a sequence at least 75% identical with the protein according to (1) or (2) or with the fragment according to (3) and exhibits FABP4 activity; in the presence and absence of a test compound; and (b) determining the level of binding of said protein to the tissue or cell in the presence and absence of the test compound; and (c) comparing the amount of binding
  • the present invention relates to a method of identifying a compound suitable as a lead compound and/or as a medicament for the treatment and/or prevention of diseases selected from the group consisting of i) cardiac insufficiency; ii) diseases associated with a dysfunction of heart contractility; iii) diseases associated with cardiac hypertrophy; iv) diseases associated with myocardial ischemia secondary to coronary artery spasm; and v) diseases associated with blood vessel contraction, comprising the steps of (a) contacting a tissue or cell with a protein, wherein said protein (1) comprises or consists of the amino acid sequence of SEQ ID NOs: 1 ; or (2) is encoded by a nucleic acid molecule comprising or consisting of the sequence of SEQ ID NO: 3; or (3) is a fragment of the protein according to (1) or (2) and exhibits FABP4 activity, or (4) has a sequence at least 75% identical with the protein according to (1 ) or (2) or with the fragment according to (3) and exhibits FABP4 activity; in the presence
  • the cell or tissue is a muscle cell or muscle tissue.
  • muscle cell in accordance with the present invention refers to cells that are defined by containing contractile filaments that move past each other and change the size of the cell.
  • muscle tissue refers to the tissue consisting of muscle cells and is classified as skeletal, cardiac, or smooth muscle tissue.
  • the function of muscles is to produce force and cause motion. Muscles can cause either locomotion of the organism itself or movement of internal organs. Cardiac and smooth muscle contraction occurs without conscious thought and is necessary for survival, such as the contraction of the heart and peristalsis which pushes food through the digestive system. Voluntary contraction of the skeletal muscles is used to move the body and can be finely controlled.
  • said cell or tissue is an adipocyte, pre-adipocyte or macrophage or is adipose tissue.
  • pre-adipocytes refers to undifferentiated fibroblasts that can be stimulated to form adipocytes.
  • monocytes in accordance with the present invention describes cells that originate from specific white blood cells called monocytes.
  • Monocytes and macrophages are phagocytes, acting in both innate immunity as well as cell-mediated immunity of vertebrate animals. Their role is to phagocytose cellular debris and pathogens either as stationary or mobile cells, and to stimulate lymphocytes and other immune cells to respond to the pathogen.
  • the inhibitor is a small molecule.
  • small molecule has been defined above.
  • the inhibitor is selected from the group consisting of an antibody or phage or aptamer or from the group consisting of an antisense nucleic acid molecule, a ribozyme, an miRNA, an siRNA or an shRNA.
  • antibody and "aptamer” have been defined above.
  • Phages in accordance with the present invention are well known in the art and are described, for example, in Griffiths, A.D. et a/.: EMBO J. 1994, 13:3245.
  • antisense nucleic acid molecule in accordance with the present invention relates to a nucleic acid molecule that has a base sequence complementary to a given gene's messenger RNA (mRNA), i.e. the "sense" sequence.
  • mRNA messenger RNA
  • mRNA messenger RNA
  • ribozymes refers to ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA, that are RNA molecules that are capable of catalysing chemical reactions. Many naturally occurring ribozymes catalyse either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyse the aminotransferase activity of the ribosome. Examples of well-characterized small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead- dependent ribozymes. The principle of catalytic self-cleavage has become well established in the last 10 years.
  • the hammerhead ribozymes are characterized best among the RNA molecules with ribozyme activity. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it appears that catalytic antisense sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site.
  • RNA which contains the GUC (or CUC) triplet
  • GUC GUC
  • CUC CUC
  • Molecules of this type were synthesized for numerous target sequences. They showed catalytic activity in vitro and in some cases also in vivo. The best results are usually obtained with short ribozymes and target sequences.
  • siRNA in accordance with the present invention refers to small interfering RNA, also known as short interfering RNA or silencing RNA.
  • siRNAs are a class of 18 to 30, preferably 20 to 25, most preferred 21 to 23 or 21 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome.
  • RNAi RNA interference
  • siRNAs have a well defined structure: a short double-strand of RNA (dsRNA), advantageously with one RNA strand having anoverhang. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group.
  • dsRNA short double-strand of RNA
  • Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group.
  • This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs.
  • siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest.
  • any gene of which the sequence is known can in principle be targeted based on sequence complementarity with an appropriately tailored siRNA.
  • the double-stranded RNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation.
  • at least one RNA strand has a 5'- and/or 3'-overhang.
  • one end of the double-strand has a 3'-overhang from 1-5 nucleotides, more preferably from 1-3 nucleotides and most preferably 2 nucleotides.
  • the other end may be blunt-ended or has up to 6 nucleotides 3'-overhang.
  • any RNA molecule suitable to act as siRNA is envisioned in the present invention.
  • siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3'- overhang.
  • the sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et al. 2001).
  • 2'-deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant.
  • r ⁇ iRNA refers to microRNAs, which are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression. miRNAs are encoded by genes that are transcribed from DNA but not translated into protein (non-coding RNA); instead they are processed from primary transcripts known as pri-miRNA to short stem-loop structures called pre-miRNA and finally to functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression. Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs.
  • mRNA messenger RNA
  • miRNAs regulate gene expression post- transcriptionally at the level of translational inhibition at P-bodies in the cytoplasm.
  • miRNAs may also guide mRNA cleavage in a manner similar to siRNAs.
  • miRNAs typically differ from siRNA because they are processed from single stranded RNA precursors and show only partial complementarity to mRNA targets. They have been implicated in a wide range of functions such as cell growth and apoptosis, development, neuronal plasticity and remodeling, and even insulin secretion.
  • a “shRNA” in accordance with the present invention is a short hairpin RNA, which is a sequence of RNA that makes a tight hairpin turn that can also be used to silence gene expression via RNA interference.
  • shRNA utilizes the U6 promoter for its expression.
  • the shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it.
  • RISC RNA-induced silencing complex
  • siRNAs and shRNAs of the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • Suppliers of RNA synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL , USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK).
  • said cell or tissue is comprised in a non-human animal.
  • the non-human animals may, for example, be mice, rats, hamsters, dogs, monkeys, rabbits, pigs, or cows.
  • said non-human animal is a mouse.
  • the method further comprises optimising the pharmacological properties of a compound identified as lead compound.
  • the optimisation comprises modifying the compound to achieve i) modified spectrum of activity, organ specificity, and/or ii) improved potency, and/or iii) decreased toxicity (improved therapeutic index), and/or iv) decreased side effects, and/or v) modified onset of therapeutic action, duration of effect, and/or vi) modified pharmacokinetic parameters (resorption, distribution, metabolism and excretion), and/or vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or viii) improved general specificity, organ/tissue specificity, and/or ix) optimised application form and route by (a) esterification of carboxyl groups, or (b) esterification of hydroxyl groups with carboxylic acids, or (c
  • phosphates, pyrophosphates or sulfates or hemi-succinates or (d) formation of pharmaceutically acceptable salts, or (e) formation of pharmaceutically acceptable complexes, or (f) synthesis of pharmacologically active polymers, or (g) introduction of hydrophiiic moieties, or (h) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (i) modification by introduction of isosteric or bioisosteric moieties, or (j) synthesis of homologous compounds, or (k) introduction of branched side chains, or (k) conversion of alkyl substituents to cyclic analogues, or (I) derivatisation of hydroxyl groups to ketales, acetales, or (m) N- acetylation to amides, phenylcarbamates, or (n) synthesis of Mannich bases, imines, or (o) transformation of ketones or aldehydes to Schiffs bases,
  • the present invention also relates to a method of determining a predisposition of an overweight and obese patient for diseases selected from the group consisting of i) cardiac insufficiency; ii) diseases associated with a dysfunction of heart contractility; iii) diseases associated with cardiac hypertrophy; iv) diseases associated with myocardial ischemia secondary to coronary artery spasm; and v) diseases associated with blood vessel contraction, comprising the steps of (a) determining, in a sample obtained from the patient, the amount of fatty acid binding protein; and (b) comparing the amount of fatty acid binding protein determined in (a) with the amount of fatty acid binding protein present in samples obtained from non-obese or non-overweight patients; wherein the presence of increased amounts of fatty acid binding protein in (a) compared to (b) is indicative of a predisposition for diseases selected from the. group consisting of cardiac insufficiency, diseases associated with a dysfunction of heart contractility, diseases associated with cardiac hypertrophy, diseases associated with my
  • the present invention relates to a method of predicting the responsiveness of an overweight and obese patient to a treatment with an inhibitor of the invention for diseases selected from the group consisting of i) diseases associated with a dysfunction of heart contractility; and ii) diseases associated with blood vessel contraction, comprising the steps of (a) determining, in a sample obtained from the patient, the amount of fatty acid binding protein 4; and (b) comparing the amount of fatty acid binding protein 4 determined in (a) with the amount of fatty acid binding protein 4 present in samples obtained from non-obese or non-overweight patients; wherein the presence of increased amounts of fatty acid binding protein 4 in (a) compared to (b) is predicative for the responsiveness of an overweight and obese patient to a treatment with an inhibitor of the invention for diseases selected from the group consisting of diseases associated with a dysfunction of heart contractility and diseases associated with blood vessel contraction.
  • fatty acid binding proteins act directly on heart muscle cells to depress contractility of these cells and on blood vessels causing vaso-spasm it is now possible to determine whether a person has a predisposition for cardiac insufficiency, diseases associated with a dysfunction of heart contractility, diseases associated with cardiac hypertrophy, diseases associated with myocardial ischemia secondary to coronary artery spasm, and diseases associated with blood vessel contraction by determining the amount of fatty acid binding proteins in a sample of said person.
  • fatty acid binding proteins have been identified herein as agents having a contractile-depressant activity on muscle cells, the detection of increased amounts of fatty acid binding proteins thus allows for the prediction whether a subject will be responsive to a treatment with the inhibitors of the invention.
  • the fatty acid binding protein is FABP4.
  • the sample is blood, adipose tissue, heart tissue, blood vessel or skeletal muscle.
  • the sample is blood, adipose tissue, heart tissue, blood vessel or skeletal muscle.
  • the present invention also relates to a method of increasing heart contractility and/or suppressing blood vessel spasm by administering to a subject in need thereof a therapeutically active amount of a fatty acid binding protein inhibitor of the invention.
  • FIG. 1 Western blot analysis of lysate from human adipocytes and adipocyte-conditioned medium (AM).
  • AM adipocyte-conditioned medium
  • FIG. 1 1 ⁇ g of proteins from cell lysates or AM was separated by 12% SDS-PAGE and immunoblotted with a polyclonal anti-beta-actin or with a polyclonal anti-FABP4 antibody.
  • Figure 2. Effect of adipocyte-conditioned medium (AM) and FABP4 on fractional shortening (A) and Fura-2 peak fluorescence (B) on adult rat cardiomyocytes. Left, control medium for adipocytes-cell culture (open bars) and adipocyte-conditioned medium (filled bars).
  • FIG. 3 Original chart recording of cardiomyocyte shortening amplitudes before and after incubation with adipocyte-conditioned medium (AM) in a dilution of 1 :6 (A); and FABP4 in a concentration of 100 nM (B).
  • AM adipocyte-conditioned medium
  • A adipocyte-conditioned medium
  • B a concentration of 100 nM
  • FIG. 1 Relationship between negative inotropic activity on cardiomyocytes contraction of adipocyte- conditioned medium (AM) from different adipocytes preparations and concentrations of FABP4 found in each adipocyte-medium (A).
  • FABP4 concentrations in AM vary from 12 to 248 nM.
  • FIG. 6 A) lmmunoblotting of FABP4 in adipocyte-conditioned medium (AM). Conditioned medium was fractionated into microvesicle and supernatant by ultracentrifugation. An aliquot of microvesicle fraction, supernatant and unfractionated adipocyte-conditioned medium were separated by SDS-PAGE and followed by immunoblotting with an anti-FABP4 antibody. FABP4 was mainly found in the supernatant fraction, representing 86% of total amount in unfractioned adipocyte-conditioned medium.
  • B) C) Original chart recording of cardiomyocyte shortening amplitudes before and after incubation with supernatant (B) and microvesicle (C) fractions of adipocyte-conditioned medium. Inotropic negative effect of adipocyte-medium was mainly observed in the supernatant fraction.
  • FIG. 7 Effect of FABP3 and FABP4 on fractional shortening on adult rat cardiomyocytes.
  • FABP3 in a concentration of 6OnM and 10OnM significantly decreased shortening amplitude of cardiomyocytes. This effect was similar to the cardiodepressant effect of FABP4.
  • FIG 8. Effect of FABP4 on pressure development (top) and coronary perfusion (bottom) in a Langendorff perfusion mode.
  • FABP4 acutely and reversibly reduced both left ventricular pressure development and coronary flow by contraction of coronary artery.
  • FIG. 11 Labelled FABP4 were directly added for 5 min to the medium of freshly isolated cardiomyocytes. After the incubation period, confocal images of the cells were acquired without exchange of the medium. No internalization of labelled FABP4 occurs into living adult cardiomyocytes.
  • Example 1 Material and Methods
  • Adipocytes were isolated from subcutaneous human adipose tissue as described before (Ehrhart- Bornstein, M. et al. (2003) Proc. Natl. Acad. Sci. U. S. A. 100:14211 ). Briefly, after surgical removal, adipose tissue samples of 20-60 g weight were immediately transported to the laboratory in Dulbecco's modified Eagle's medium/Nutrient Mix F12 (DMEM/F12, Life Technologies, Düsseldorf, Germany) with 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • DEM/F12 Dulbecco's modified Eagle's medium/Nutrient Mix F12
  • adipose tissue was digested in Kreb's Ringer Bicarbonate buffer containing 120 units/ml collagenase type I and type Il from Clostridium histolyticum (Serva, Heidelberg, Germany). Isolated mature adipocytes were kept at 37°C in a humidified atmosphere of 5% CO 2 , and cultured for 24 hrs. The adipocyte-conditioned medium (AM) containing all the factors released by the adipocytes was then collected and used for further experiments with isolated adult rat cardiomyocytes. Culture medium without adipocytes was incubated in the same way and used as control medium (CM).
  • AM adipocyte-conditioned medium
  • CM control medium
  • cardiomyocytes were resuspended in M199 medium completed with 0.2% bovine serum albumin, 5% fetal calf serum, 5 mmol/L creatine, 5 mmol/L taurine, 2 mmol/L carnitine, 10 ⁇ mol/L cytosine-D-arabinofuranoside, and antibiotics. Cardiomyocytes were seeded in laminin-coated 4-well chamber slides (Nunc, Wiesbaden-Schierstein, Germany) specialized for fluorescence microscopy for contractility and fluorescence measurement. Experiments were approved by the institutional animal care body in the state of Berlin, Germany.
  • HBSS Hank's balanced salts solution
  • AM from 6 different preparations were pooled together and concentrated 16 fold to a protein concentration of 372.3 ⁇ g/ml on a centrifuge cartridge (Centricon®, Millipore GmbH, Schwalbach, Germany) with a cutoff of 1 kDa. 9.0 ⁇ g protein were separated on 10% polyacrylamide gel (Invitrogen GmbH, Düsseldorf, Germany) under reducing conditions. Proteins were identified at the Mass Spectrometry Facility, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Protein bands were visualized by staining with Coomassie and full lane was cut into 17 slices, while visible bands were always sliced separately. Excised gel plugs were cut into ca.
  • Microvesicles were prepared from adipocyte-conditioned media as previously described (Aoki, N. ef al. (2007) Endocrinology 148:3850). Briefly, conditioned medium was first centrifuged at 1 ,000 x g for 5 min and then at 15,000 x g for 15 min to remove cell debris and aggregates. The supernatant was ultracentrifuged at 100,000 x g for 1 h. Pelleted vesicles were suspended in phosphate-buffered saline and ultracentrifuged again for washing at 100,000 x g for 15 min. The obtained pelleted vesicles and supernatant were then subjected directly to Western blotting or resuspended in HBSS-Solution and tested on the cardiomyocytes.
  • FABP4 concentrations in the AM were determined by ELISA for human FABP4 (BioVendor Laboratory Medicine, Inc., Modrice, Czech Republic) according to the manufacturer's instructions.
  • a calibration curve was constructed by plotting the absorbance values at 450 nm vs. the FABP4 concentrations of the calibrators, and concentrations of unknown samples were determined by using this calibration curve.
  • Protein concentrations in the AM were quantified by using a Bradford protein assay kit (Bio-Rad, Kunststoff, Germany).
  • Adipocytes were lysed with lysis buffer containing 20 mM Hepes (pH 7.9), 350 mM NaCI, 20% Glycerol, 1 mM MgCI 2, 0.5 mM EDTA, 0.1 mM EGTA, 1 % Tergitol, 4% protease inhibitor cocktail (Sigma-Aldrich, Kunststoff, Germany), 1 % phosphatase inhibitor cocktail (Sigma-Aldrich, Kunststoff, Germany).
  • the blots were incubated with an affinity purified specific FABP4 polyclonal antibody (Cayman Chemical, Ann Arbor, Ml, USA) as a first antibody at a concentration of 1 ⁇ g/ml overnight at 4°C or with a specific polyclonal antibody against ⁇ -Actin (Cell Signaling Technology, Danvers, MA, USA) as control, and subsequently with a goat anti-rabbit-HRP conjugate (BioRad, Kunststoff, Germany). Immunodetection was performed with an enhanced chemiluminescence detection kit (Pierce SuperSignal Kit, Pierce, Bonn, Germany) and the protein bands were analyzed by using Syngene Gene Tools (Syngene, Cambridge, UK).
  • an affinity purified specific FABP4 polyclonal antibody (Cayman Chemical, Ann Arbor, Ml, USA) as a first antibody at a concentration of 1 ⁇ g/ml overnight at 4°C or with a specific polyclonal antibody against ⁇ -Actin (Cell Signaling Technology
  • Example 2 Human adipocytes release FABP4 into extracellular medium
  • Mass spectrometry-based fingerprinting identified 386 proteins in the conditioned medium from isolated differentiated human adipocytes. 80 of these proteins were proteins with typical signaling sequences including previously described adipokines such as adiponectin and complement factor 3 or constituents of extra cellular matrix such as laminin or collagen (data not shown). Interestingly, human fatty acid binding protein 4 was identified as the major component of the band of ⁇ 15 kD. Non-classical secretion of FABP4 is predicted by Secretome 2.0 Server (NN-score 0.765). We next measured the concentration of FABP4 in conditioned medium from adipocytes obtained from different individuals by using a commercial sandwich ELISA assay.
  • Example 4 FABP4 concentrations are closely related to inotropic negative activity of adipocyte medium
  • Microvesicles are vesicles released from the plasma membrane of various cell types, containing cell surface proteins and some cytoplasmic components of the original cell. Recently it has been reported that the murine adipocyte 3T3-L1 cells secrete such microvesicles containing a variety of secreted, integral and cytosolic proteins (Aoki, N. ef a/. (2007) Endocrinology 148:3850). In order to analyze whether FABP4 is released by adipocytes via this mechanism, microvesicle fractions of adipocyte- conditioned medium were prepared by ultracentrifugation as previously described (Aoki, N. ef a/.
  • Fatty acid binding proteins are also expressed in the liver, heart, intestine, skeletal muscle and brain.
  • the human muscle FABP (FABP3) is reported to be identical to human heart FABP.
  • FABP3 muscle/heart FABP
  • FABP4 adipocyte-specific FABP
  • FABP3 in a concentration of 6OnM and 10OnM significantly decreased shortening amplitude of cardiomyocytes ( Figure 7). This effect was similar to the cardiodepressant effect of FABP4.
  • Example 7 Effect of FABP4 on pressure development (top) and coronary perfusion (bottom) (Langendorff perfusion)
  • Isolated rat heart (Langendorff mode) perfused with FABP4 revealed a (reversible) strong decrease of force generation as well as coronary flow due to contraction of the coronary vessels (Figure 8).
  • Example 8 FABP4 does not modulate L-Ty pe Ca 2+ channel activity or action potential duration
  • Cardiomyocytes were incubated with synthetic peptides (100 nmol/L each) which overlap and cover the complete FABP4 molecule.
  • peptides could be grouped into three clusters; namely, a) peptides with pronounced inhibitory action, b) peptides with more stimulating effect, and c) peptides with a smaller or no inhibitory activity (Fig. 10).
  • the N-terminus expressed more cluster a), while clusters b) and c) distributed along the C-terminus.
  • the most prominent inhibitory activity could be observed with the N-terminal peptide 1-20 (mcdafvgtwklvssenfddy) which revealed an approximately
  • Example 10 Primary rat cardiomyocytes do not take up FABP4
  • FABP4 was labelled with the fluorescein-5-isothiocyanate (FITC) (BioTeZ, Berlin, Germany). To analyze an uptake capacity of FABP4 into living primary rat cardiomyocytes, the cells were plated into a laminin-coated ⁇ -slide eight-well ibiTreat (ibidi, Kunststoff, Germany) microscopy observation chamber.
  • FITC fluorescein-5-isothiocyanate
  • FABP4 significantly decreased the shortening amplitude by -24.3 + 14%.
  • the depressive effect on cardiomyocyte contraction of FABP4 was further potentiated by 5,6-EET to -53.9 ⁇ 8.2% (p ⁇ .001 ; Fig. 12).

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Abstract

La présente invention concerne un inhibiteur de la fonction et/ou de l’expression et/ou de la sécrétion de la protéine de liaison aux acides gras (FABP) des tissus adipeux et/ou des adipocytes et/ou de la liaison et/ou de l’absorption de la protéine FABP dans les cellules ou les tissus pour le traitement et/ou la prévention de maladies choisies dans le groupe constitué par l’insuffisance cardiaque, les maladies associées à l’hypertrophie cardiaque, les maladies associées à un trouble de la contractilité cardiaque, les maladies associées à une ischémie du myocarde secondaire à un spasme des artères coronaires, et les maladies associées à la contraction des vaisseaux sanguins. L’invention concerne également des procédés d’identification de composés adaptés en tant que têtes de série ou en tant que médicaments destinés au traitement de ces maladies ainsi que des procédés de détermination d’une prédisposition à ces maladies chez un patient en surpoids et obèse.
PCT/EP2009/004206 2008-06-11 2009-06-10 Protéine de liaison aux acides gras dans la pathogenèse de l’insuffisance cardiaque Ceased WO2009149931A2 (fr)

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CN116966176A (zh) * 2023-06-12 2023-10-31 北京市心肺血管疾病研究所 脂肪酸结合蛋白4抑制剂在治疗射血分数保留型心力衰竭中的应用
WO2024040025A3 (fr) * 2022-08-19 2024-04-25 University Of Washington Prévention et traitement basés sur un vaccin th2 d'une inflammation due à l'obésité

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WO2003025579A2 (fr) * 2001-09-18 2003-03-27 Medigene Ag Utilisation d'une proteine de liaison des acides gras du coeur
WO2004063156A1 (fr) * 2003-01-08 2004-07-29 Biovitrum Ab Nouveaux derives d'indole utiles comme inhibiteurs de fabp-4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040025A3 (fr) * 2022-08-19 2024-04-25 University Of Washington Prévention et traitement basés sur un vaccin th2 d'une inflammation due à l'obésité
CN116966176A (zh) * 2023-06-12 2023-10-31 北京市心肺血管疾病研究所 脂肪酸结合蛋白4抑制剂在治疗射血分数保留型心力衰竭中的应用

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