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WO1999064062A1 - Nouvelle utilisation therapeutique d'amplificateurs de l'activite de la proteine kinase b (picb) - Google Patents

Nouvelle utilisation therapeutique d'amplificateurs de l'activite de la proteine kinase b (picb) Download PDF

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Publication number
WO1999064062A1
WO1999064062A1 PCT/SE1999/000989 SE9900989W WO9964062A1 WO 1999064062 A1 WO1999064062 A1 WO 1999064062A1 SE 9900989 W SE9900989 W SE 9900989W WO 9964062 A1 WO9964062 A1 WO 9964062A1
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WO
WIPO (PCT)
Prior art keywords
pkb
heart
insulin resistance
use according
cells
Prior art date
Application number
PCT/SE1999/000989
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English (en)
Inventor
Thomas Olin
Stephen James
Harriet RÖNNHOLM
Antek Wielburski
Christina Reuterdahl
Axel STÅHLBOM
Original Assignee
Pharmacia & Upjohn Ab
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SE9802023A external-priority patent/SE9802023D0/xx
Application filed by Pharmacia & Upjohn Ab filed Critical Pharmacia & Upjohn Ab
Priority to AU46694/99A priority Critical patent/AU4669499A/en
Publication of WO1999064062A1 publication Critical patent/WO1999064062A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the treatment of chronic or acute heart failure, e.g. the treatment or protection of the heart from complications derivable from insulin resistance.
  • the treatment involves administration of a drug which enhances protein kinase B (PKB) activity.
  • PPK protein kinase B
  • Heart failure is traditionally viewed as a clinical syndrome in which the impaired ability of the heart to pump blood decreases cardiac output and increases venous pressure.
  • Endstage heart failure patients have transitioned from a normal phenotype with myocardial damage at a cellular level to a state of fatigue, marked dyspnea on exertion, edema and high incidence of sudden death. This transition, can be driven by a variety pathological conditions including ischemia, hypertension, inflammation or be a result of a monogenic disease.
  • most clinical courses are characterized by a compensatory cardiac growth, to sustain an adequate tissue blood supply, followed by a progressive deterioration of cardiac function eventually precipitating chronic heart failure. It is also widely acknowledged that patients sufferings from diabetes mellitus are at risk of acquiring heart disease, see Cardiovasc. Res., 1997, Vol. 34, pp. 1-2.
  • insulin resistance is also apparent in the heart challenged by stress, extending the need for a treatment of insulin resistance in the heart to patients suffering from both acute and chronic heart failure.
  • Insulin resistance will diminish intracellular signaling mandatory for preserving normal cellular function examplified by intact glucose uptake and metabolism, protection against mediators of cell death (e.g. apoptosis) and cardiac myocyte differentiation which is necessary for proper contractile function (see Cardiovascular Research, Vol. 34(1) pp. 3-55, 1997). Accordingly, insulin resistance in the heart can precipitate heart failure by itself or predispose for serious complication in the stressed heart, which may follow traumatic events such as acute coronary events, congestive heart failure and coronary surgery.
  • the insulin resistance is treated by applying supra- physiological levels of circulating insulin, by enhancers of insulin action or by dietary restrictions.
  • Such therapies have severe drawbacks or limitations in efficacy with regard to insulin resistance in general.
  • An increased concentration of circulating insulin can cause hyper-proliferation in vascular cells and thereby disturb tissue blood supply.
  • insulin enhancers tend to increase body fat mass, which in turn is a risk factor in heart disease and dietary restriction is primarily targeted for uncomplicated type 2 diabetes.
  • there is as yet no therapy which has been proven to be efficient in the treatment of complications resulting from insulin resistance in the heart.
  • PI3K phosphoinositide 3- kinase
  • PKB appears to be a key intermediary in the regulation of glucose utilisation and control of protein synthesis by insulin (Cross, D., et al. (1995). Nature 378: 785- 789; Cohen, P. et al. (1997). FEBS Letters 410: 3-10; Peak, M., J. et al. (1998). Diabetologia 41 : 16-25) Gingras, A.-C. et al. (1998). Genes and Development 12: 502- 513).
  • glycogen synthase kinase 3 (GSK3; permitting the synthesis of glycogen from glucose.)
  • GSK3 glycogen synthase kinase 3
  • PKB has been shown to phosphorylate and activate phosphofructo kinase-2 (Deprez, D. et al. (1997) Journal of Biological Chemistry 272: 17269-17275) whose product, fructose 2,6-bisphosphate, acts as an allosteric activator of glycolysis.
  • a third likely substrate for PKB is the type 3B cyclic AMP phosphodiesterase (Wijkander, J. et al. (1998). Endocrinology 139: 219-227), which in insulin-responsive tissues is activated by phosphorylation, leading to the inactivation of adrenergic-stimulated processes.
  • Human growth hormone is a native signaling substance, which has been reported to be of potential benefit in several aspects of cardiac therapy. Reference is here given to the Swedish patent application 9702595-1, filed 1997-07-04. It is highly desirable to find a therapy with which cardiac cellular function can be retained in a normal state or adjusted into improved function. Such therapy applies e.g. to diabetic patients at risk of developing heart failure, chronic heart failure patients with apparent insulin resistance and patients suffering from an acute cardiac trauma, who are often characterized by an inability to respond to insulin. We have now found that the activation of PKB has a clear positive increasing effect on the number of surviving cells, which have been stressed, and on metabolism of glucose in heart cells.
  • a drug which activates PKB can thus be used for improving compromised heart function common in acute or chronic heart failure, caused by e.g. myocardial infarction, ischemic heart diseases, hypertension, genetic disorders or insulin resistance (all examples of cardiac stress) and thereby treating or protecting the heart from complications derivable from this condition.
  • the present invention is based on the finding that a drug which enhances PKB activity can be used in the preparation of an agent for the treatment of chronic or acute heart failure.
  • Such a drug can be used against disturbed glucose metabolism of the heart cells so that the glucose metabolism is normalized or the disturbance is prevented. It can also be used to prevent the heart from complications derived from insulin resistance, e.g. induced by stress or diabetes mellitus.
  • the drug can further improve contractile function of the heart e.g. by improving the contractile efficiency.
  • contractile efficiency is here meant the mechanical work executed per oxygen molecule consumed.
  • the drug can improve the coronary blood flow.
  • the present invention relates to the utility of an effective amount of such a drug in the preparation of an agent for the administration to a patient, having or being at risk of acquiring insulin resistance in the heart. More particularly, the present invention relates to the use of an effective dose of the drug in the preparation of an agent for retaining an adequate metabolism and resistance to stress in the heart challenged by insulin resistance and to prevent the heart from complications derived from insulin resistance.
  • the insulin resistance may be induced by chronic stress or traumatic events including surgery, but it may also derive from conditions related to diabetes.
  • the inventive therapy is specifically targeted to normalize a deranged glucose metabolism, including glucose oxidation and to prevent cell-death in order to achieve a contractile function sufficient to secure an appropriate blood perfusion of tissues.
  • the present invention constitutes a suitable treatment especially for patients suffering from insulin resistance and have, or are at risk of acquiring, cardiac complications. Patients suffering from chronic and acute heart failure will benefit from the therapy.
  • an individual who has or is at risk of acquiring cardiac complications, is eligible for a treatment with a drug which enhances PKB activity by subjecting the patient to an insulin resistance test, for example oral glucose tolerance tests, glucose/insulin clamp or analysis of the fasting blood glucose level.
  • an insulin resistance test for example oral glucose tolerance tests, glucose/insulin clamp or analysis of the fasting blood glucose level.
  • a test allowing for a specific determination of the insulin resistance in the heart is also conceivable when applicable, as for example in connection with bypass surgery.
  • Such tests will provide a simple and prognostic diagnostic tool for determining if the patient will benefit from a drug therapy, in which the drug enhances PKB activity, for treating or preventing the heart from further complications.
  • the inventive utility of such a claimed drug will consequently be especially powerful in pathological states where insulin is prevented from exerting its full action (insulin resistance) and where cardiac complications are clearly developing.
  • the cardiac complications associated with insulin resistance can be clinically defined as chronic ischemia, contractile dysfunction or congestion.
  • a threatening cardiac failure could be identified as progressing cardiac remodeling, by cardiac enzyme leakage into the blood such as cardiac isoforms of creatine kinase and troponin, an inadequate blood perfusion of the heart, a deranged metabolism exemplified by a low glucose up-take and high lactate production, a malfunctioning calcium regulation or an increase in cell death.
  • the effective amount of the drug necessary to administer to treat the above mentioned complications will vary according to the individual patient and the method of administration.
  • the clinically skilled person will be able adjust doses and select the appropriate administration route in order to reach the desired, mentioned therapeutic effects and thereby survey the patients response with conventional methods and tests.
  • Fig. 1 shows PKB activity in H9C2 clones mock-transfected or transfected with w.t. PKB or gag-PKB.
  • Fig. 2 shows Glucose uptake in H9C2 clones transfected with mock, w.t. PKB or gag- PKB.
  • Fig. 3 shows the number of surviving cells in H9C2 clones mock-transfected or transfected with w.t. PKB or gag-PKB.
  • Example 1 Measurement of PKB activity in H9C2 cells transfected with bovine HA- tagged PKBa or GAG-PKBa fusion protein. Methods A series of H9C2 cardiac myocyte clones have been established, transfected with wild type HA-tagged PKBa (W), GAG-PKB a fusion protein (G) or vector alone (neo), (equal to mock and equal to control).
  • the cells were cultivated and kept below complete confluence in Dulbecco's MEM (with Glutamax, 1000 mg/1 glucose, Gibco Cat. No 21885-025) supplemented with 10% Fetal Calf Serum (Gibco) and PEST (penicillin and streptomycin). Thereafter the cells were starved and then lysed and the protein concentration of lysates determined. Lysates were matched for protein and volume, and the ⁇ isoform of PKB was immunoprecipitated using specific antibodies pre-coupled to protein G beads. After rigorous washing, immunoprecipitates were assayed for PKB activity for 30 minutes.
  • H9C2 cardiac myocyte clones cells as in example 1, were incubated in serum free medium for 3 hours. Cell monolayers were then rinsed with Krebs Ringer Bicarbonate buffer (KRB) pH 7.4. Glucose transport was quantified by incubating the cells in the presence of 3H-2-deoxy-glucose in KRB for 4 minutes. Non-specific uptake was determined by quantifying cell associated radioactivity in the presence of 10 ⁇ M cytochalasin B. Uptake of 2-deoxy-glucose was terminated by rapidly aspirating the radioactive medium, followed by three successive washes of cell monolayers with cold PBS. The cells were lysed in 0.5 M NaOH before liquid scintillation counting. Rates of glucose transport were normalized for protein content in each well.
  • Example 3 Method for measurement of survival effect of PKB Assay for chemical induction of cell-death.
  • the cells as in Example 1, were trypsinised and seeded out in 6 well plates at 4 x
  • the cells were cultivated over night and then washed 3 times with serum-free MEM.
  • Mitomycin a DNA crosslinker inducing stress, was added at 20 ug per ml in serum- free MEM for 6 hours. The cells were washed as above and further incubated for 16-
  • Control cells were kept in serum-free MEM throughout the procedure.
  • FITC apoptosis kit Oncogene Research Products, Cat. No PF0302. Briefly, the cells were incubated in an Annexin-FITC solution for 15 minutes at room temperature, collected by centrifugation and suspended in a solution of Propidium Iodide and further kept on ice. Detection and quantification of living cells (unstained), apoptotic cells (Annexin V stained) and necrotic cells (Propidium Iodide stained without
  • the chemical cell death assay described above induced 80-90 % cell death in wild- type H9c2 cells and in mock-transfected cells. See Figure 3. Rate of survival (SD) is normalized to control-transfected (set to 100) in Figure 3. Two independent experiments, performed on each of the PKB-clones, showed increased survival rate in this assay. The protective effect was more pronounced in cells transfected with the Gag construct as compared to the wild-type construct. The results were normalised against mock-transfected cells. Transfection using the wild- type construct of PKB increased survival 4-6 times. Transfection using the Gag construct of PKB increased survival 7-10 times as compared to mock-transfected cells.

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  • Health & Medical Sciences (AREA)
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  • Engineering & Computer Science (AREA)
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  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Organic Chemistry (AREA)
  • Endocrinology (AREA)
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  • Hospice & Palliative Care (AREA)
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Abstract

L'invention concerne le traitement d'une insuffisance cardiaque chronique ou grave, notamment le traitement ou la protection contre le coeur des complications pouvant découler de l'insulinorésistance. Ce traitement consiste à administrer un médicament qui améliore l'activité de la protéine kinase B (PKB).
PCT/SE1999/000989 1998-06-08 1999-06-08 Nouvelle utilisation therapeutique d'amplificateurs de l'activite de la proteine kinase b (picb) WO1999064062A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU46694/99A AU4669499A (en) 1998-06-08 1999-06-08 New therapeutic use of pkb (proteine kinase b) enhancers

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
SE9802023-3 1998-06-08
SE9802023A SE9802023D0 (sv) 1998-06-08 1998-06-08 New therapeutic use
US8970198P 1998-06-18 1998-06-18
US60/089,701 1998-06-18

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WO1999064062A1 true WO1999064062A1 (fr) 1999-12-16

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003102206A3 (fr) * 2002-06-04 2005-02-24 Florian Lang Sgk et nedd utilises comme cibles diagnostiques et therapeutiques

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022360A2 (fr) * 1995-12-20 1997-06-26 Medical Research Council Regulation de synthese de proteines et procede de criblage pour agents pharmaceutiques
WO1999001151A1 (fr) * 1997-07-04 1999-01-14 Pharmacia & Upjohn Ab Utilisation d'hormone de croissance dans des compositions traitant la resistance du coeur a l'insuline, et renforçant l'action de la proteine kinase b (pkb)
WO1999012559A1 (fr) * 1997-09-09 1999-03-18 The Regents Of The University Of California Inhibition de l'apoptose au moyen d'agonistes du recepteur de la prosaposine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022360A2 (fr) * 1995-12-20 1997-06-26 Medical Research Council Regulation de synthese de proteines et procede de criblage pour agents pharmaceutiques
WO1999001151A1 (fr) * 1997-07-04 1999-01-14 Pharmacia & Upjohn Ab Utilisation d'hormone de croissance dans des compositions traitant la resistance du coeur a l'insuline, et renforçant l'action de la proteine kinase b (pkb)
WO1999012559A1 (fr) * 1997-09-09 1999-03-18 The Regents Of The University Of California Inhibition de l'apoptose au moyen d'agonistes du recepteur de la prosaposine

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Title
ANNA KROOK ET AL: "Improved Glucose Tolerance Restores Insulin-stimulated Akt Kinase Activity and Glucose Transport in skeletal Muscle From Diabetic Goto-Kakizaki Rats", DIABETES, vol. 46, December 1997 (1997-12-01), pages 2110 - 2114, XP002919871 *
ASHWANI MALHOTRA ET AL: "Regulation of contractile proteins in diabetic heart", CARDIOVASCULAR RESEARCH, vol. 34, 1997, pages 34 - 40, XP002919868 *
BROWNSEY R.W.ET AL: "Actions of insulin on the mammalian heart:metabolism,patology and biochemical mechanisms", CARDIOVASCULAR RESEARCH, vol. 34, 1997, pages 3 - 24, XP002919866 *
DANIELLE FEUVRAY: "The regulation of intracellular pH in the diabetic myocardium", CARDIOVASCULAR RESEARCH, vol. 34, 1997, pages 48 - 54, XP002919870 *
DATABASE DIALOG MEDLINE 1 January 1900 (1900-01-01), XP002919873, Database accession no. 09272771 *
GRANT N.PIERCE ET AL: "Regulation of intracellular Ca2+ in the heart during diabetes", CARDIOVASCULAR RESEARCH, vol. 34, 1997, pages 41 - 47, XP002919869 *
KOHJIRO UEKI: "Potential Role of Protein Kinase B in Insulin-induced Glucose Transport", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 9, February 1998 (1998-02-01), pages 5315 - 5322, XP002919872 *
WILLIAM C. STANLEY: "Regulation of energy substrate metabolism in the diabetic heart", CARDIOVASCULAR RESEARCH, vol. 34, 1997, pages 25 - 33, XP002919867 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003102206A3 (fr) * 2002-06-04 2005-02-24 Florian Lang Sgk et nedd utilises comme cibles diagnostiques et therapeutiques
CN100406570C (zh) * 2002-06-04 2008-07-30 弗洛里安·朗 用作诊断和治疗靶标的Sgk和Nedd

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