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WO2001096398A2 - Proteines bruleuses de graisses excedentaires utilisees comme cibles dans le traitement de l'insuffisance cardiaque - Google Patents

Proteines bruleuses de graisses excedentaires utilisees comme cibles dans le traitement de l'insuffisance cardiaque Download PDF

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Publication number
WO2001096398A2
WO2001096398A2 PCT/US2001/019230 US0119230W WO0196398A2 WO 2001096398 A2 WO2001096398 A2 WO 2001096398A2 US 0119230 W US0119230 W US 0119230W WO 0196398 A2 WO0196398 A2 WO 0196398A2
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ucp
heart failure
agent
expression
activity
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PCT/US2001/019230
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WO2001096398A3 (fr
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Charles J. Homcy
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Cor Therapeutics, Inc.
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Priority to AU2001268461A priority Critical patent/AU2001268461A1/en
Publication of WO2001096398A2 publication Critical patent/WO2001096398A2/fr
Publication of WO2001096398A3 publication Critical patent/WO2001096398A3/fr

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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

  • the present invention relates to methods of identifying agents effective for treating heart failure and methods of treating heart failure. More specifically, the agents are identified by determining whether they modulate the expression or activity of an uncoupling protein.
  • Heart failure affects approximately 2 to 3 million Americans, and 400,000 new cases are diagnosed each year. Heart failure is slightly more common among men than women and is twice as common among African Americans as whites.
  • the term "heart failure” suggests a sudden and complete stop of heart activity, but actually, the heart does not suddenly stop. Rather, heart failure usually develops slowly, often over years, as the heart gradually loses its pumping ability and works less efficiently. Some people may not become aware of their condition until symptoms appear years after their heart began its decline.
  • the term “congestive heart failure” is often used to describe all patients with heart failure. However, congestion (the buildup of fluid) is just one feature of the condition and does not occur in all patients.
  • systolic heart failure occurs when the heart's ability to contract decreases. The heart cannot pump with enough force to push a sufficient amount of blood into the circulation. Blood coming into the heart from the lungs may back up and cause fluid to leak into the lungs, a condition known as pulmonary congestion. Diastolic heart failure, on the other hand, occurs when the heart has a problem relaxing. The heart cannot properly fill with blood because the muscle has become stiff, losing its ability to relax. This form of heart failure may lead to fluid accumulation, especially in the feet, ankles, and legs. Some patients may have lung congestion.
  • Congestive heart failure is not a specific disease, but rather a compilation of signs and symptoms, all of which are caused by an inability of the heart to appropriately increase cardiac output during exertion.
  • the cardiac diseases associated with symptoms of congestive failure include dilated cadiomyopathy, restrictive/constrictive cardiomyopathy, and hypertrophyic cardiomyopathy.
  • Dilated cardiomyopathy patients have typical symptoms that are caused by both systolic as well as diastolic dysfunction, although the systolic dysfunction clearly predominates.
  • the cause of the heart dysfunction is ischemic heart disease due to coronary atherosclerosis. That is, patients have had either a single myocardial infarction or multiple myocardial infarctions and the resultant scarring and remodeling has resulted in the development of a dilated and hypofunctional heart.
  • the disease is referred to as idiopathic dilated cardiomyopathy as the causative agent remains undefined.
  • IDC idiopathic
  • ISC ischemic
  • patients with congestive heart failure have a one year survival of nearly 70% and a five year survival of only 20% after referral to a tertiary heart failure center.
  • Morbidity is also significant as the average heart failure patient is hospitalized approximately two times each year with an average length of stay of greater than five days. Approximately half of all patients with congestive failure die suddenly—presumably due to a ventricular arrhythmia and sudden death, while the remaining patients die of worsening congestive failure.
  • Another approach to the therapy of patients with congestive heart failure is based on the recognition that patients with congestive heart failure expressed a group of neurohormonal substances whose plasma concentrations could be inversely associated with morbidity and mortality in large populations of patients with CHF. These neurohormonal agents all share a common finding: when given in vivo or in vitro they can initiate an aladaptive remodeling of the heart and in some cases are cardiotoxic. Additionally, in experimental CHF models, they delay or attenuate the development of the heart failure phenotype. The first neurohormonal agent to successfully serve as a therapeutic target was angiotensin II, a potent vasoconstrictor and activator of aldosterone.
  • beta-adrenergic receptor may be associated with congestive heart failure.
  • beta-blocker metoprolol no difference in survival in patients receiving active drug as versus those receiving placebo is reported.
  • Beta-blockers are not easy to use in patients with CHF, as they require careful up-titration and cannot be utilized as rescue therapy. Carvedilol, for example, cannot be used for rescue therapy as it requires careful up-titration over a minimum of several weeks.
  • beta-adrenergic receptor signal transduction pathway is critical for rapid adjustments to increased cardiovascular demand (e.g., during exercise).
  • beta-adrenergic receptor stimulation may become maladaptive.
  • elevation of circulating catecholamines and depletion of cardiac tissue stores of norepinephrine occur in the failing heart, resulting in desensitization.
  • stimulation or inhibition of the beta-adrenergic receptor signaling pathway is beneficial in heart failure is controversial.
  • Uncoupling protein has been known to uncouple oxidative phosphorylation by moving protons across the mitochondrial inner membrane toward the mitochondrial atrix (Fleury et al, (1997) Nat. Genet. 15, 269-272). In the past, it was thought that UCP was expressed only in mammalian brown and white adipose tissue, since UCP1, the first uncoupling protein identified, is expressed only in these tissues. However, recently, uncoupling proteins expressed in other tissues and species have been reported. In 1995, a plant uncoupling protein was discovered and later sequenced (Vercesi et al (1995) Nature, 375-24; Laloi et al, (1997) Nature 389, 135).
  • UCP2 and UCP3 were cloned (Fleury et al, (1997) Nat. Genet. 15, 269-272; Boss et al, (1997) FEBS Lett. 408, 39-42; Vidal-Puig et al (1997) Biochem. Biophys Res. Commun. 235, 79-82).
  • UCP-3 is expressed in skeletal muscle, whereas UCP-2 is expressed ubiquitously in mammalian tissues. Expression of UCP2 is relatively high in heart tissues (Fleury et al, (1997), supra; Boss et al, (1997), supra).
  • UCP4 has recently been described as a brain-specific UCP (Mao et al. (1999) FEBS Lett. 443-326-330).
  • Van Der Lee et al. provides an animal model for hypertrophied heart
  • the model is an acute hypertrophied heart model, since pressure is placed on the aorta. Accordingly, the model is not applicable to chronic heart failure and the role of uncoupling proteins in congestive heart failure has not been previously elucidated.
  • the present invention is based on the discovery that UCPs are overexpressed in a heart failure animal model.
  • the present invention provides methods of identifying an agent effective for the treatment of heart failure comprising administering the agent, and monitoring expression or activity of an uncoupling protein (UCP) to determine whether the agent modulates the activity or expression of UCP.
  • An agent that modulates the activity or expression of UCP is effective for treating heart failure.
  • the method comprises administering the agent to in vitro cells.
  • the cells are selected from the group consisting of H9C2 cells, hepatocytes, and neonatal cardiomyocytes.
  • the method comprises administering the agent to host cells engineered to express an UCP.
  • the method comprises administering the agent to a Gs ⁇ transgenic animal, preferably a transgenic mouse.
  • the present invention includes monitoring the expression or activity of an UCP selected from the group consisting of UCP- 1 , UCP-2, and UCP-3.
  • the agent is an inhibitor or inducer of UCP expression or activity.
  • the agent is an inhibitor selected from the group consisting of a purine nucleotide, an UCP antibody, and an antisense molecule of an UCP.
  • the agent is an inducer selected from the group consisting of a fatty acid and a fatty acid- activated transcription factor.
  • agents that modulate the concentration of cAMP include but are not limited to phosphodiesterase inhibitors, forskolin, and inhibitors and stimulators of adenylate cyclase.
  • the present invention also provides a method of treating heart failure comprising administering an agent that modulates expression or activity of an UCP.
  • the agent modulates the expression or activity of an UCP selected from the group consisting of UCP-1, UCP-2, and UCP-3.
  • the present invention also contemplates treatment of the underlying causes of heart failure, including but are not limited to, congestive heart failure, cardiomyopathy, and ischemic heart disease.
  • Another aspect of the present invention involves a method of diagnosing heart failure comprising detecting the expression level of an UCP.
  • the expression level of an UCP is detected using a nucleic acid probe that detects an UCP transcript.
  • the expression level of an UCP is detected using an antibody.
  • the present invention is based on the discovery that an UCP is overexpressed in Gs ⁇ transgenic mouse.
  • the Gs ⁇ transgenic mouse which exhibits symptoms of heart failure similar to that found in human patients is a useful animal model for studying heart failure.
  • heart failure refers to a wide variety of underlying disease states, including among others, congestive heart failure, cardiomyopathy, and ischemic heart disease.
  • the present invention is also based on the discovery that a modulator of UCP expression or activity is effective in the treatment of heart failure.
  • the present invention provides methods for identifying agents that modulate the expression or activity of UCP comprising administering the agent and monitoring the expression or activity of UCP.
  • the present invention also provides methods for treating heart failure comprising administering an agent that modulates the expression or activity of an UCP.
  • the present invention is also based in part on the finding that overexpression of a
  • UCP is associated with heart failure.
  • the present invention provides method of diagnosing heart failure, comprising detecting the level of expression of an UCP.
  • Gs ⁇ Transgenic Mouse as an Animal Model for Heart Failure
  • Gs ⁇ the alpha subunit of the stimulatory G protein
  • This mouse exhibits a remarkably enhanced sensitivity to catecholamines, specifically in the heart. This is exhibited by a leftward shift of both the chronotropic and inotropic dose response curves for isoproterenol.
  • the present invention is based in part on obtaining gene expression patterns over the life of the Gs ⁇ animal and comparing them to its wild type litter-mate control at each time point. The gene profile patterns at three months, six months, nine months, twelve mohths, fifteen months and twenty months of age, in the Gs ⁇ animal are compared to the wild type litter-mate.
  • UCP2 is one of several isoforms, (homologous proteins) of uncoupling proteins including UCP1, UCP3, UCP4, and UCP2.
  • UCP1 the first of these to be identified, is expressed in brown and white adipose tissue, particularly in rodents. Its content is thought to be regulated by the ⁇ 3 -adrenergic receptor, specifically through a cyclic AMP response element.
  • UCP1 uncouples substrate utilization in the mitochondria from ATP generation; specifically, its expression promotes dissipation of the proton gradient across the mitochondria that is the responsible driving force for ATP production. It is thought to play an important role in energy metabolism and has been implicated in states of obesity. Unlike UCP 1 , UCP2 is widely expressed in a variety of tissues, and UCP3 is more restricted to skeletal muscle. It is known that UCP2 is expressed in the heart, in cardiomyocytes. Upregulation of UCP2 in the heart of the Gs ⁇ transgenic mouse, may play a direct role in the development of cardiomyopathy in this mouse model.
  • modulators of UCP expression or activity include anything that regulates or adjusts the expression or activity of an UCP and therefore include both inhibitors and inducers of expression or activity of UCP.
  • Inhibitors of UCP expression or activity include but are not limited to purine nucleotides, antibodies that bind UCP2, and antisense molecules.
  • a preferred example of a purine nucleotide is ATP.
  • Inducers of UPC expression and activity include but are not limited to fatty acids and fatty acid-activated transcription factors. Preferred examples of fatty acids are palmitic oil and oleic oil, and preferred example of fatty acid- activated transcription factors include peroxisome proliferator activated receptor (PPAR) ⁇ .
  • PPAR peroxisome proliferator activated receptor
  • UCP2 gene has a cyclic AMP response element in its promoter region. It is known that cyclic AMP generation is turned on in the Gs ⁇ mouse, particularly in response to ⁇ -adrenergic receptor occupancy. Therefore, agents that modulate the concentration of cyclic AMP would be expected to modulate the expression of UCP2.
  • preferred agents that modulate UCP2 expression include but are not limited to phosphodiesterase inhibitors, forskolin, and inhibitors and stimulators of adenylate cyclase.
  • preferred agents that stimulates expression of UCP2 include but are not limited to cyclic AMP analogs such as dibutyryl-cAMP and 8-Br-cAMP. It is also contemplated that other UCPs encoded by genes comprising a cyclic AMP response element in its promoter region can be modulated by the same preferred agents.
  • Nan Der Lee et al. disclose that stimulation of neonatal cardiomyocytes with triiodothyronine increases UCP2 mR ⁇ A levels. Accordingly, triiodothyronine is also contemplated as a modulator for UCP expression or activity to be used in the claimed methods.
  • the present invention provides methods of identifying agents effective for the treatment of heart failure or disease.
  • the method comprises administering a test agent and monitering the expression or activity of an UCP to determine whether the agent is effective for treating heart failure.
  • An effective agent for treating heart failure modulates the expression or activity of an UCP.
  • test agent refers to any molecule that is to be tested.
  • the term encompasses inducers and inhibitors.
  • the method is performed using cells that naturally express UCPs, preferably embryonic rat heart derived H9C2 cells, hepatocytes, or cardiomyoctes.
  • the method is performed using a eukaryotic host cell engineered to express a desired UCP, i.e. transfected with the nucleic acid encoding the desired UCP.
  • eukaryotic host cells include mammalian cells, insect cells, plant cells, or fungal cells.
  • the eukaryotic host cells are mammalian cells and yeast cells.
  • mammalian host cells examples include but are not limited to Chinese hamster ovary cells (CHO), 3T3 cells (derived from Swiss, Balb-c or NIH mice) , COS-1 cells, COS-7 cells, CV-1 cells, HeLa cells, L-929 cells, BHK cells, and HaK cells.
  • the mammalian host cells are COS-1, COS-7, HEK, and 3T3.
  • suitable mammalian host cells and methods for fransfection, culture, amplification, and product production are known in the art.
  • yeast cells Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention.
  • the preferred yeast cell is Saccharomyces cerevisiae.
  • the method is performed using prokaryotic cells.
  • UCP is expressed in bacteria and reconstituted into liposomes to measure activity.
  • Various prokaryotic cells are known in the art. Preferred prokaryotic cells include various strains of E. coli such as HB101, DH5 ⁇ , DH10, and MC10161.
  • the activity of an UCP can be monitored using fluorescent probes to measure the potential across the mitochondrial membrane or using probes to measure the flux activity of an uncoupling protein. This method is routinely used to study the activity of UCP, see for example, Jaburek et al, (1999) J. Biol. Chem. 274, 26003-26007; Simonyan et al, (1998), FEBS Letters 436, 81-84; and Korshunov et al, (1998) FEBS Letters 435, 215- 218. Expression of UCP can be detected by northern blot analysis and quantitated by densitomen .
  • the amount of UCP mRNAs can also be quantitated by reverse transcription-competitive PCR (RT-competitive PCR; Auboeuf et al, (1997) Anal. Biochem., 245, 141-148) or any other nucleic acid detection mean.
  • RT-competitive PCR reverse transcription-competitive PCR; Auboeuf et al, (1997) Anal. Biochem., 245, 141-148) or any other nucleic acid detection mean.
  • the content of the expressed protein can be detected by Western analysis or radioimmunoassay (RIA) using an appropriate antibody.
  • the present invention also provides high throughput assays for identifying agents effective in treating heart failure.
  • assays are performed using native cell types such as cardiocytes or liver cells.
  • the test agent is administered to cardiocytes or liver cells.
  • the UCP is extracted from the native cell and an antibody to the protein is used to measure its level to determine whether the agent modulates the expression of UCP.
  • An agent that modulates the expression of UCP is effective in treating heart failure.
  • high throughput assays are developed for identifying agents that modulate the transcriptional activity of the UCP gene.
  • nucleic acid construct comprising the promoter of the gene of an UCP containing all the important cis-acting elements which drive tissue specific expression and regulation, coupled to a marker protein such as ⁇ -galactosidase, luciferase, placental alkaline phosphatase, or others well known in the art.
  • the assay comprises transfecting the construct into a host cell, adding a test agent to the cell, and measuring the amount of marker protein. The level of the marker protein directly reflects the transcriptional activity of the UCP gene.
  • the present invention also provides methods of identifying agents effective in treating heart failure using the Gs ⁇ transgenic mouse.
  • the method comprises administering the test agent to the Gs ⁇ transgenic mouse and monitoring the expression or activity of an UCP to determine whether the agent is effective for treating heart failure.
  • the test agent can be administered in the drinking water at a concentration from about 0.2 to 0.8 g/L and preferably about 0.5 g/L.
  • the agent can also be administered parenterally, intravenously, intramuscularly, subcutaneously, intraperitoneally, or transdermally at a dosage of 1 ⁇ g to 200 mg/kg/day.
  • the expression level of UCP in transgenic Gs ⁇ mice can be compared with that of control transgenic mice.
  • test agent modulates UCP expression include but are not limited to age of death, echocardiography (e.g. ejection fraction), measuring heart rate, measuring arterial pressure, and histology of the heart, including assessment of cellular apoptosis, necrosis, and fibrosis of the heart.
  • the results from the treated transgenic mice can be compared to a control transgenic mice.
  • the expression profile of UCP is also obtained for the treated and the control transgenic mice for comparison.
  • RIA, Western analysis, and 31 P-NMR spectroscopy of the heart can be used to assess the energy status of the heart which could be altered by UCP modulators.
  • cardiocytes can be isolated from the heart and their UCP activities assessed using methods described previously.
  • heart failure is meant an abnormality of cardiac function where the heart does not pump blood at the rate needed for the requirements of metabolizing tissues.
  • Heart failure includes a wide range of disease states such as congestive heart failure, myocardial infarction, tachyarrhythmia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, and myocarditis.
  • Heart failure can be caused by any number of factors, including ischemic, congenital, rheumatic, or idiopathic forms.
  • Chronic cardiac hypertrophy is a significantly diseased state which is a precursor to congestive heart failure and cardiac arrest.
  • the term "treatment” refers to both therapeutic treatment and prophylactic or preventative measures.
  • the object is to prevent or slow down (lessen) hypertrophy.
  • Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in which the disorder is to be prevented.
  • the hypertrophy may be from any cause including but not limited to congenital, viral, idiopathic, cardiotrophic, or myotrophic causes, or as a result of ischemia or ischemic insults such as myocardial infarction, or pressure or volume overload.
  • the treatment may also be administered to those with liver failure.
  • the treatment is performed to stop or slow the progression of hypertrophy, especially after heart damage, such as from ischemia, has occurred.
  • the agent(s) is given immediately after the myocardial infarction, to prevent or lessen hypertrophy and/or cellular damage or death.
  • the present invention provides composition comprising an agent that is effective to treat heart failure and routes for administering the composition to the patient.
  • An amount of an agent that is effective for treating heart failure is administered to the patient.
  • an effective dosage of an agent for a 50 kg to 100 kg human being is in the range of about 0.1 mg to 3000 mg per day in single or divided doses, and preferably about one mg to about 1000 mg per day in single or divided doses. More preferably, a dosage range is 10 mg to 100 mg per day in single or divided doses.
  • the amount and timing of the agent administered will be dependent upon the subject being treated, on the severity of the affliction, on the manner of administration and upon the judgment of the prescribing physician. Thus, due to patient to patient variability, the dosages given above are intended to be a guideline.
  • the physician may titrate doses of the agent to achieve the treatment, e.g., congestive heart failure improvement, that the physician considers appropriate for the patient. In considering the degree of treatment desired, the physician must balance a variety of factors such as age of the patient, presence of preexisting disease, as well as presence of other diseases.
  • the agent may be administered alone or in combination with other agents such as but not limited to digitalis, thiazide diuretics, other diuretics and ACE inhibitors.
  • the agent is administered in the form of a pharmaceutical composition.
  • pharmaceutical composition refers to a composition comprising an agent together with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutical composition of the present invention is directed to a composition suitable for the treatment of heart failure.
  • a pharmaceutically acceptable carrier includes, but is not limited to, physiological saline, ringers, phosphate buffered saline, and other carriers known in the art.
  • Pharmaceutical compositions may also include stabilizers, anti-oxidants, colorants, and diluents.
  • Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not canceled or inhibited to such an extent that treatment is ineffective.
  • the agent can be administered individually or together in any conventional oral, parenteral, rectal or transdermal dosage form.
  • the agent may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses.
  • suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, oils (e.g., peanut oil, sesame oil) and various organic solvents.
  • the pharmaceutical compositions formed by combining an agent and pharmaceutically acceptable carriers can then be readily administered in a variety of dosage forms such as tablets, powders, lozenges, emulsions, oil soft gels, syrups, injectable solutions and the like.
  • compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like.
  • excipients such as sodium citrate, calcium carbonate and calcium phosphate
  • disintegrants such as starch, methylcellulose, alginic acid and certain complex silicates
  • binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
  • lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules.
  • Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof.
  • parenteral administration in which solutions containing the agent in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be employed.
  • aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • the sterile aqueous media employed are all readily available by standard techniques known to those skilled in the art.
  • transdermal e.g., topical
  • aqueous or partially aqueous solutions are prepared.
  • compositions according to this invention may contain 0.1%-95% of the agent.
  • the composition or formulation to be administered will contain a quantity of the agent in an amount effective to treat the disease/condition of the subject being treated, e.g., heart failure.
  • UCP modulators or agents effective for treating heart failure that are not nucleic acid molecules can be delivered to target sites in patients as discussed above.
  • UCP modulators and agents effective for treating heart failure that are nucleic acid molecules can be delivered to target sites in patients as discussed below.
  • Gene therapy is a method for delivering functionally active therapeutic or other forms of genes into targeted cells. Initial efforts of gene transfer into somatic tissues have relied on indirect means, such as ex vivo gene therapy, wherein target cells are removed from the body, transfected or infected with vectors carrying recombinant genes, and re-implanted into the body.
  • Viral vectors are often the most efficient gene therapy system, and recombinant replication-defective viral vectors have been used to transduce (i.e., infect) cells both ex vivo and in vivo.
  • Such vectors include retroviral, adenovirus and adeno-associated and herpes viral vectors.
  • an UCP modulator such as an UCP antisense molecule, or a nucleic acid molecule effective for treating heart failure can be subloned into an appropriate vector and transferred into a cell or tissue by gene transfer techniques discussed above.
  • an UCP modulator such as an UCP antisense molecule, or a nucleic acid molecule effective for treating heart failure can be provided to the cell or tissue using a fransfection facilitating composition, such as cationic liposomes containing desired polynucleotide.
  • UCP2 upregulation of UCP2 in Gs ⁇ mouse suggests that UCP2 and its homologs can be used as diagnostic marker for detecting heart failure.
  • An antibody that binds to UCP can be used as a probe to detect and quantitate the expression of the UCP in a biological sample to determine whether the UCP is over expressed.
  • the level of expression of the nucleic acid encoding an UCP in a biological sample can be quantitated to determine whether the UCP is overexpressed.
  • Methods of using antibody to detect and to quantitate a specific protein include but not limited to Western analysis and RIA and are well known to the skilled artisan (see for example Sivitz et al, (1999) Endocrinology 140, 1511-9).
  • Measuring the level of expression of a nucleic acid is also well known in the art.
  • the expression level of a nucleic acid is quantitated by northern blot analysis (Yoshitomi et al, (1999) Biochem. J. 340, 397-404), by reverse transcription- competitive PCR (RT-competitive PCR; Millet et al, (1997) J. Clin. Invest., 100, 2665- 2670), or by DNA microarray technology (Voehringer et al, (2000) Proc. Natl. Acad. Sci., 97, 2680-2685).
  • transgenic mice (9.4 + 0.1 months old) and 24 age-matched wild-type (WT) litter-mates (9.5 + 0.1 months old) of either sex from the same genetic background as the TG mice are studied.
  • the transgene consists of a rat ⁇ -myosin heavy chain promoter linked to a Gs minigene coding for the short isoform of Gs (Gaudin et al. (1995) J. Clin. Invest. 95, 1676-1683).
  • Animals used in the present study are maintained in accordance with guidelines from the National Institutes of Health (National Institutes of Health, Office of Science and Health Reports. 1996. Guide for care and use of laboratory animals. Publication no. 83-23. Office of Science and Health Reports, Department of Health and Human Services. Bethesda, MD.).
  • a test agent is administered in the drinking water at a concentration of 0.5 g/L, or tap water was given to the untreated group. Water with freshly added drug is changed 3 times weekly, and mice are treated for 6-7 months.
  • Echocardiographic studies are performed in 19 TG mice (9 test agent-treated and 10 untreated, 14.4 ⁇ 0.1 months old) and 24 WT controls (14 test agent-treated and 10 untreated, 14.8 ⁇ 0.2 months old), hi addition, echocardiographic studies are performed in 10 younger TG mice (8.8 ⁇ 0.1 months old).
  • mice are anesthetized with ketamine (0.065 mg/g), acepromazine (0.002 mg/g), and xylazine (0.013 mg/g) injected intraperitoneally.
  • the procedure for echocardiography has been described in detail in Iwase et al, (1996.) Circ. Res., 78, 517-524 and Iwase et al, (1997) Am. J. Physiol., 272, H585-H589.
  • Heart rate is measured using telemetry techniques in the conscious, unrestrained state in 16 TG mice (9 test agent-treated and 7 untreated, 15.6 + 0.1 months old) and 23 WT mice (14 test agent-treated and 9 untreated, 15.9 ⁇ 0.1 months old).
  • Mice are anesthetized as already described, and a telemetry transducer (TA10EA-F20; Data Science Co., St. Paul, Minnesota, USA) is implanted in the peritoneal cavity with paired electrodes placed subcutaneously over the thorax (chest bipolar electrocardiogram leads) (Uechi et al, (1998) Circ. Res. 82, 416-423). Experiments are initiated 3-5 days after recovery from surgical instrumentation. Mice with implanted telemetry devices are housed in individual cages with free access to food and water, and are exposed to 12-hour light/ 12-hour dark cycles.
  • RNA Total RNA is extracted by RNAzol B, as described in Yoshitomi et al. (1999, supra). Total RNA (15 ⁇ g) is applied per lane on 1.2% agarose gel containing 2.2 M formaldehyde, and electrophoresis is carried out. RNA is then transferred onto nylon membranes (GeneScreen Plus, DuPont, Boston, MA, U.S.A.) for 20 h and crosslinked by UV irradiation. It is confirmed that total RNA is loaded equally for all lanes by hybridization of human 28S probe (Clontech).
  • the probes for detecting UCPs are generated with PCT methodology and labelled by random priming in the presence of [ ⁇ - 32 P]dCTP by BcaBEST random-priming kit (Takara).
  • Hybridization is performed at 68°C for 2-5 h in ExpressHyb Hybridization solution (Clontech), followed by washing twice for 10-30 min at room temperature with 2 x SSC (where 1 x SSC is 0.15 M NaCl / 0.015 M sodium citrate) / 0.05% SDS, twice for 15 min at 50°C with 2 x SSC/ 0.05 % SDS, and finally twice for 5-30 min at 50°C with 0.1 x SSC / 0.1% SDS.
  • the filter is exposed to X-ray film (Konica, Tokyo, Japan) at -80°C for 5-48 h, and each band intensity is quantified with a bioimaging analyser, BAS2000 system (Fuji Photofilm, Tokyo, Japan).
  • Heart sections are also dehydrated and embedded in glycol methacrylate, sectioned at 1- ⁇ m thickness, and stained with methylene blue-basic fuchsin for light-microscopic examination.
  • Methacrylate sections are also stained with silver-gold (Accustain; Sigma Diagnostics, St. Louis, Missouri, USA) for basement membrane to outline cardiac myocytes for cross-sectional area measurement.
  • Myocardial connective tissue is quantitatively analyzed on a cross-section of LV obtained mid-distance from base to apex and stained with picric acid sirius red. Images are obtained from a Sony CCD video camera (Sony Electronics Inc., Park Ridge, New Jersey, USA) attached to a Nikon E800 microscope (Nikon Inc. Melville, New York, USA) with a x60 objective, and are analyzed with MetaMorph image analysis software (Universal Imaging Corp., West Chester, Pennsylvania, USA). Myocyte cross-sectional area is measured from images captured from silver-stained 1- ⁇ m-thick methacrylate sections obtained mid-distance from base to apex.
  • Suitable cross-sections are defined as having nearly circular capillary profiles and circular-to-oval myocyte sections. No correction for oblique sectioning is made.
  • the outline of 100-200 myocytes is traced in the LV of each animal, using the MetaMorph image system software to determine myocyte cross-sectional area.
  • the mean area is calculated for the LV in each animal, and the group mean is calculated for each region and group.
  • DNA fragmentation is detected in situ by using TUNEL (dUTP nick end-labeling) on paraffin sections of the mouse hearts from TG and WT mice, as described previously (Geng et al, (1999) Circ. Res. 84, 34-42).
  • TUNEL dUTP nick end-labeling
  • the slides are washed in PBS, mounted in a Vector DAPI medium, and observed under a fluorescence microscope.
  • the mean number of myocyte nuclei per x40 field in LV regions is determined by manual counting of DAPI-stained nuclei with ultraviolet excitation.
  • a minimum of 20 fields in the LV regions of each heart are examined for TUNEL-positive myocytes. All morphometric measurements are performed by at least 2 independent individuals in a blinded manner.
  • UCP expression of UCP in E. coli
  • Cells from a 700-ml culture are lysed in a French press in 20 ml of lysis buffer (10 mM Tris, pH 7, 1 mM EDTA, 1 mM dithiothreitol); the lysate is centrifuged at 27,000 x g for 15 min; and the pellet is resuspended in 20 ml of lysis buffer and centrifuged at 1000 x g for 3 min.
  • One ml aliquots of the supernatant are centrifuged at 14,000 x g for 15 min in a microcentrifuge, and the resulting pelleted inclusion bodies are stored frozen at 70 °C.
  • the nucleic acids encoding UCPs can also be expressed in mammalian and yeast host cells for production of the encoded protein. Mammalian and yeast host cells are known to the skilled artisan and are described earlier.
  • Solubilization of E. coli inclusion bodies is previously described in Jaburek et al, (1999 J. Biol. Chem. 274, 26003-26007).
  • the pelleted inclusion bodies (about 2 mg of protein) are suspended and washed three times in wash buffer (tetraethylammonium (TEA+) salts of 0.15 M phosphate, 25 mM EDTA, 1 mM ATP, and 1 mM dithiothreitol, pH 7.8).
  • the final pellet is solubilized in 0.4 ml of 50 mM TEA-TES, pH 7.2, containing 1.5% sodium lauroylsarcosinate (SLS).
  • the extract is supplemented with 10 mg/ml asolectin and 3% octylpentaoxyethylene (C8E5 detergent) and then dialyzed for 15 h against 3 x 400 ml of extraction buffer (TEA+ salts of 50 mM TES and 1 mM EDTA, pH 7.2) to remove SLS.
  • extraction buffer TEA+ salts of 50 mM TES and 1 mM EDTA, pH 7.2
  • the extraction buffer is supplemented with 1 mM dithiothreitol and 0.03% sodium azide. These are removed from the final dialysis (1 h). Aliquots of the dialyzed extract, containing about 0.2 mg of protein, are stored at 20 °C.
  • UCPs are reconstituted as described in Jaburek et al, (1999 J. Biol. Chem. 274, 26003-26007).
  • Egg yolk phosphatidylcholine or soybean phospholipids are supplemented with cardiolipin (2 mg/ml), dried, and stored under nitrogen.
  • Internal medium (TEA+ salts of TES (30 mM), SO 4 (80 mM), and EDTA (1 mM), pH 7.2) is added to give a final concentration of 40 mg of phospholipid/ml of proteoliposome stock.
  • the mixture is vortexed and sonicated to clarity in a bath sonicator, and detergent (10% C8E5), protein extract, and fluorescent probe are added.
  • the final mixture (1.1 ml) is applied onto 2 ml of Bio-Bead SM-2 (Bio-Rad) column to remove the detergent. After 2 h of incubation, the column is centrifuged, and the resulting proteoliposomes are applied onto a new 2-ml Bio-Bead column, incubated for 30 min, and centrifuged. The formed vesicles (1 ml) are passed through a Sephadex G-25-300 column to remove external probe.
  • Ion flux in proteoliposomes is measured using ion-specific fluorescent probes and an SLM Aminco 8000C spectrofluorometer. Measurements of H* fluxes are obtained from changes in 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence due to quenching by the anion of TES buffer (Orosz et al, (1993) Anal. Biochem. 210, 1-15). Measurements of K+ fluxes, reflecting the movement of ionic charge across the membrane, are obtained from changes in potassium-binding benzofuran isophthalate fluorescence (Garlid et al, (1995) Methods Enzymol. 266, 331-348; Jeek et al, (1990) J. Biol.

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Abstract

La présente invention porte sur des procédés d'identification d'un agent efficace dans le traitement de l'insuffisance cardiaque et de modulation de l'expression ou de l'activité de la protéine brûleuse de graisses excédentaires. Cette invention porte également sur des compositions et des procédés de traitement de l'insuffisance cardiaque.
PCT/US2001/019230 2000-06-15 2001-06-15 Proteines bruleuses de graisses excedentaires utilisees comme cibles dans le traitement de l'insuffisance cardiaque WO2001096398A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050894A3 (fr) * 2002-11-27 2006-08-10 Artesian Therapeutics Inc Detection des genes lies a l'insuffisance cardiaque et dosage therapeutique
JP2013515488A (ja) * 2009-12-23 2013-05-09 カッパーアールエヌエー，インコーポレイテッド Ｕｃｐ２に対する天然アンチセンス転写産物の阻害による脱共役タンパク質２（ｕｃｐ２）関連疾患の治療
WO2014209905A3 (fr) * 2013-06-26 2015-02-26 Stealth Peptides International, Inc. Méthodes et compositions destinées à détecter et à diagnostiquer des maladies et des affections

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998045313A1 (fr) * 1997-04-04 1998-10-15 Amylin Pharmaceuticals, Inc. Proteine decouplante et procedes d'utilisation

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004050894A3 (fr) * 2002-11-27 2006-08-10 Artesian Therapeutics Inc Detection des genes lies a l'insuffisance cardiaque et dosage therapeutique
JP2013515488A (ja) * 2009-12-23 2013-05-09 カッパーアールエヌエー，インコーポレイテッド Ｕｃｐ２に対する天然アンチセンス転写産物の阻害による脱共役タンパク質２（ｕｃｐ２）関連疾患の治療
EP2515947A4 (fr) * 2009-12-23 2014-01-01 Curna Inc Traitement de maladies associées à la protéine ucp2 (uncoupling protein) par inhibition du produit de transcription antisens naturel en ucp2
US9068183B2 (en) 2009-12-23 2015-06-30 Curna, Inc. Treatment of uncoupling protein 2 (UCP2) related diseases by inhibition of natural antisense transcript to UCP2
KR101793753B1 (ko) 2009-12-23 2017-11-03 큐알엔에이, 인크. 커플링방지 단백질 ２(ｕｃｐ２)에 대한 천연 안티센스 전사체의 저해에 의한 ｕｃｐ２ 관련 질환의 치료
US10221413B2 (en) 2009-12-23 2019-03-05 Curna, Inc. Treatment of uncoupling protein 2 (UCP2) related diseases by inhibition of natural antisense transcript to UCP2
WO2014209905A3 (fr) * 2013-06-26 2015-02-26 Stealth Peptides International, Inc. Méthodes et compositions destinées à détecter et à diagnostiquer des maladies et des affections

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