WO2010103040A1 - Activateurs de protéine kinases activées par le 5'-adénosine monophosphate (ampk), destinés au traitement de l'hypertension pulmonaire - Google Patents

Activateurs de protéine kinases activées par le 5'-adénosine monophosphate (ampk), destinés au traitement de l'hypertension pulmonaire Download PDF

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WO2010103040A1
WO2010103040A1 PCT/EP2010/053034 EP2010053034W WO2010103040A1 WO 2010103040 A1 WO2010103040 A1 WO 2010103040A1 EP 2010053034 W EP2010053034 W EP 2010053034W WO 2010103040 A1 WO2010103040 A1 WO 2010103040A1
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metformin
pulmonary hypertension
ampk activator
pulmonary
ampk
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PCT/EP2010/053034
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English (en)
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Pierre Pacaud
Gervaise Loirand
Malvyne Derkinderen
Christian Agard
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Chu Nantes
Universite De Nantes
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • 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
    • A61P9/12—Antihypertensives

Definitions

  • the invention relates to 5 '-adenosine monophosphate-activated protein kinase
  • AMPK pulmonary hypertension
  • Pulmonary hypertension is a serious disease characterized by a progressive increase of pulmonary vascular resistance and pulmonary arterial pressure (PAP), leading to right ventricle hypertrophy and death (HUMBERT, M. (2008). Update in pulmonary arterial hypertension 2007.
  • PAP pulmonary vascular resistance
  • UMBERT right ventricle hypertrophy and death
  • the 3 rd World Symposium on Pulmonary Arterial Hypertension was convened in Venice to attempt classification of pulmonary hypertension based on new understandings of disease mechanisms. Classification system can be summarized as follows (WHO stands for World Health Organization):
  • WHO Group II Pulmonary hypertension associated with left heart disease o Atrial or ventricular disease o Valvular disease (e.g. mitral stenosis) • WHO Group III - Pulmonary hypertension associated with lung diseases and/or hypoxemia o Chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD) o Sleep-disordered breathing, alveolar hypoventilation o Chronic exposure to high altitude o Developmental lung abnormalities
  • COPD chronic obstructive pulmonary disease
  • ILD interstitial lung disease
  • pulmonary hypertension can also be classified according to their ability to function (provided by the New York Heart Association): • Class I: Patients with pulmonary hypertension but without resulting limitation of physical activity. Ordinary physical activity does not cause undue dyspnea or fatigue, chest pain or near syncope.
  • Class II Patients with pulmonary hypertension resulting in slight limitation of physical activity. These patients are comfortable at rest, but ordinary physical activity causes undue dyspnea or fatigue, chest pain or near syncope.
  • Class III Patients with pulmonary hypertension resulting in marked limitation of physical activity. These patients are comfortable at rest, but less than ordinary physical activity causes undue dyspnea or fatigue, chest pain or near syncope.
  • Class IV Patients with pulmonary hypertension resulting in inability to perform any physical activity without symptoms. These patients manifest signs of right heart failure. Dyspnea and/or fatigue may be present at rest, and discomfort is increased by any physical activity.
  • PAH mortality is estimated at about
  • pulmonary hypertension Although major advances in the understanding of disease development and treatment have been achieved over the last 15 years, the pathogenesis of pulmonary hypertension remains not clearly understood. All forms of pulmonary hypertension, including idiopathic (IPAH), familial (FPAH) and connective tissue disease- associated forms of pulmonary hypertension (APAH), display similar pathologic changes and are considered to share common pathogenic mechanisms, which involve endothelial dysfunction, endothelial and smooth muscle cell proliferation and increased vasoconstriction (HUMBERT, 2008, ibid ).
  • IIPAH idiopathic
  • FPAH familial
  • APAH connective tissue disease- associated forms of pulmonary hypertension
  • Current therapies based on the use of drugs that improve endothelial function, through the endothelin-1, the NO, or the prostacyclin pathways have shown benefits for this group of patients.
  • Current therapies may be:
  • Prostacycline or Prostaglandine 12 for example epoprostrerone (Flolan®);
  • - Prostacycline analog for example iloprost (Ventavis®), treprostinil (Remodulin®);
  • - endothelin receptor antagonist for example bosentan (Tracleer®), sitaxentan; - phosphodiesterase-5 inhibitors: for example sildenafil (Revatio®).
  • Biguanide derivative drugs for instance metformin
  • metformin have been widely used for more than 40 years for the treatment of hyperglycemia in patients with type II diabetes mellitus. Their action consists in reducing hyperglycemia by improving peripheral sensitivity to insulin, reducing gastrointestinal glucose absorption, and decreasing hepatic glucose production (BORST, S.E. & SNELLEN, H.G. (2001). Metformin, but not exercise training, increases insulin responsiveness in skeletal muscle of Sprague-Dawley rats. Life Sci, 69, 1497-1507 ; CASPARY, W.F. & CREUTZFELDT, W. (1971). Analysis of the inhibitory effect of biguanides on glucose absorption: inhibition of active sugar transport.
  • Diabetologia, 7, 379-385 HUNDAL, R.S., KRSSAK, M., DUFOUR, S., LAURENT, D., LEBON, V., CHANDRAMOULI, V., iNzuccHi, S.E., SCHUMANN, W.C, PETERSEN, K.F., LANDAU, B.R. & SHULMAN, G.I. (2000).
  • metformin has recently been shown to lower other cardiovascular risk factors (MCALISTER, F. A., EURICH, D.T., MAJUMDAR, S. R. & JOHNSON, J. A. (2008). The risk of heart failure in patients with type 2 diabetes treated with oral agent monotherapy. Eur J Heart Fail, 10, 703-708).
  • the cardioprotective effect of metformin has very recently been shown (GUNDEWAR, S., CALVERT, J. W., JHA, S., TOEDT-PINGEL, L, Ji, S.Y., NUNEZ, D., RAMACHANDRAN, A., ANAYA-CISNEROS, M., TlAN, R. & LEFER, D.J. (2009).
  • metformin Activation of AMP-activated protein kinase by metformin improves left ventricular function and survival in heart failure. Circulation Research, 104, 403-411).
  • metformin at therapeutic concentration, has anti-hypertensive properties in hypertensive obese women and in patients with type II diabetes (GiUGLIANO, D., DE ROSA, N., DI MARO, G., MARFELLA, R., ACAMPORA, R., BUONINCONTI, R. & D'ONOFRIO, F. (1993).
  • Metformin improves glucose, lipid metabolism, and reduces blood pressure in hypertensive, obese women. Diabetes Care, 16, 1387-1390).
  • the word "hypertension" without a qualifier normally refers to systemic arterial hypertension. Hypertension and pulmonary hypertension are not the same diseases, and their causes are different. It was therefore not obvious that metformin presents a protective effect against pulmonary hypertension.
  • AMPK activators present (have, possess, or display) a protective and curative effect against pulmonary hypertension.
  • the present invention pertains to AMPK activators that prevent and/or treat pulmonary hypertension.
  • the invention relates to a method for preventing and/or treating pulmonary hypertension comprising the administration to a patient of a composition comprising one or more AMPK activators in a suitable pharmaceutical medium.
  • composition comprising one or more AMPK activators in a suitable pharmaceutical medium for the manufacture of a medicament for preventing and/or treating pulmonary hypertension.
  • the present invention provides methods for preventing and/or treating pulmonary hypertension by administering one or more AMPK activators to a patient in need of such prevention or treatment.
  • AMPK activators include but are not limited to various biguanide derivatives as described herein, and the compound AICAR, or suitable derivatives thereof.
  • AICAR is "5-aminoimidazole-4- carboxamide riboside", and is also known as “5-aminoimidazole-4-carboxamide-l- ⁇ - 4-ribofuranoside”.
  • AICAR is also referred to as "acadesine".
  • one or more of these AMPK activators including combinations of biguanide derivatives and AICAR (or AICAR derivatives), are used to make pharmaceutical formulations that are acceptable for administration.
  • Biguanide derivatives are a group of drugs that are useful in the treatment of hyperglycemia, and which comprise a substituted biguanide structure.
  • the biguanide derivatives have the following formula (I):
  • Rl and R2 are independently selected from hydrogen or a linear, branched or cyclic, saturated or unsaturated (C1-C4) alkyl group, or an aralkyl group;
  • a and B are independently selected from NH or N-CHR3-N, wherein R3 is selected from hydrogen or a linear, branched or cyclic, saturated or unsaturated (Cl- C4) alkyl group; and also the tautomeric, enantiomeric, diastereoisomeric and epimeric forms, the solvates and the pharmaceutically acceptable salts thereof.
  • the linear, branched or cyclic, saturated or unsaturated (C1-C4) alkyl group is methyl, ethyl, n-propyl, i-propyl, n-butyl or i-butyl.
  • the aralkyl group is benzyl or phenethyl.
  • preferred biguanide derivatives suitable for use in the present invention include, but are not limited to, those substituted on one or both of their primary amine groups (Rl and/or R2) by at least one alkyl or aralkyl group such as methyl, ethyl, n-propyl, n-butyl or phenethyl, and preferably include metformin, phenformin and buformin. More preferably, the biguanide derivative according to the invention is metformin.
  • the biguanide derivatives have the following formula (II) as described in the international patent application WO 02/074740, the complete contents of which is hereby incorporated by reference:
  • Rl and R2 are independently selected from a branched or unbranched (C1-C6) alkyl chain, or Rl and R2 together form a 3-to 8-membered ring including the nitrogen atom to which they are attached,
  • R3 and R4 together form a ring selected from the group aziridine, pyrrolyl, imidazolyl, pyrazolyl, indolyl, indolinyl, pyrrolidinyl, piperazinyl and piperidyl including the nitrogen atom to which they are attached, and also the tautomeric, enantiomeric, diastereoisomeric and epimeric forms, the solvates and the pharmaceutically acceptable salts thereof.
  • the biguanide derivatives have the following formula (III) as described in the international patent application WO 01/55122, the complete contents of which is hereby incorporated by reference:
  • Rl, R2, R3 and R4 are independently selected from the groups:
  • (Cl-C20)alkyl optionally substituted with halogen, (Cl-C5)alkyl, (Cl- C5)alkoxy, (C3-C8)cycloalkyl,
  • R5 and R6 are independently selected from the groups:
  • (Cl-C13)heteroaryl carrying one or more heteroatoms chosen from N, O, S and substituted or otherwise with amino, hydroxyl, thio, halogen, (Cl-C5)alkyl, (Cl- C5)alkoxy, (Cl-C5)alkylthio, (Cl-C5)alkylamino, (C6-C14) aryloxy, (C6-C14)aryl- (C1-C5) alkoxy, cyano, trifluoromethyl, carboxyl, carboxymethyl or carboxyethyl,
  • this compound, or derivatives (analogs, precursors, etc.) thereof that retain the ability to activate AMPK are intended to be encompassed by the invention, as are various isomeric and stereoisomeric forms and pharmaceutically acceptable salts thereof.
  • examples of such derivatives are described, for example, in WO2005/98465, WO2005/038068, WO2004/043957, US2009/0105293 and US2007/0265223, the complete contents of each of which are hereby incorporated by reference.
  • tautomeric forms is meant to include isomers that are interconverted by a tautomerization reaction.
  • enantiomeric forms is meant to include stereoisomers that are mirror images of each other, i.e.
  • diastereoisomeric forms is meant to include stereoisomers that are not enantiomers. When two diastereoisomers differ from each other at only one stereocenter, they are called epimeric forms or epimers.
  • solvates is meant to include the biguanide derivative dissolved in a solvent.
  • salts are meant to include salts of the active compound which are prepared with relatively non toxic acids. Acid addition salts can be obtained by contacting the neutral form of the compound with a sufficient amount of the corresponding acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, those derived from inorganic acids like hydrochloric, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulphuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively non-toxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, p- chlorophenoxyacetic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, embonic (or pamoic) and the like.
  • inorganic acids like hydrochloric, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric
  • the metformin salt is chosen in the group consisting of metformin hydrochloride, metformin p-chlorophenoxyacetate and metformin embonate.
  • the term "patient” denotes an animal, such as birds (e.g. poultry), and mammals (e.g. a rodent, a feline, a canine, or a primate).
  • a patient according to the invention is a human.
  • the term “treating” or “treatment”, as used herein, refers to a method that is aimed at reversing, alleviating, inhibiting, slowing down or stopping the progression, aggravation or deterioration of the symptoms of the pathology, at bringing about ameliorations of the symptoms of the pathology, and/or at curing the pathology.
  • the terms “preventing” or “prevention”, as used herein, refer to a method that is aimed at delaying or preventing the onset of the pathology.
  • composition of the present invention is suitable for use in a variety of drug delivery systems, and is intended for enteral, parenteral, topical, oral or local administration.
  • the composition can be formulated in a solid form.
  • the AMPK activator is administered orally.
  • Solid form preparations include but are not limited to powders, tablets, pills, capsules, cachets, lozenges, and dispersible granules.
  • suitable pharmaceutical medium is meant to include solid carriers, but also substances that may act as diluents, flavouring agents, binders, preservatives, tablets disintegrating agents, or encapsulating material, etc.
  • the powder and tablets preferably contain from about 5 % to about 95 % of an AMPK activator.
  • Suitable carriers include but are not limited to magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatine, tragacanth, methylcellulose, a low melting wax, cocoa butter, and their mixtures.
  • the composition is administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • suitable pharmaceutical medium in this aspect is meant to include an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers may be used including, for example, water, buffered water, 0.4 % saline, 0.3 % glycine, hyaluronic acid and their mixtures.
  • compositions may be sterilized by conventional, well-known sterilization techniques or may be sterile filtered.
  • the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • composition may also be administered in other forms, for example, as suppositories, aerosols, transdermal patches, etc.
  • composition comprising an AMPK activator in a suitable pharmaceutical medium may be in the form of a film-coated tablet.
  • film-coated tablet we mean a tablet or pill covered or coated with one or more layers of substances (such as various sugars, cellulose ethers, cellulose acetates, various polymers and copolymers, etc., as known to those of skill in the art), the layers typically dissolving upon contact with an aqueous environment such as within the body, thereby releasing the active agents in the tablet.
  • an AMPK activator may be administered in a composition in the form of a film-coated tablet.
  • composition comprising an AMPK activator in a suitable pharmaceutical medium in the form of a film-coated tablet may comprise, for example, metformin chlorhydrate, polyvinylpyrrolidone, magnesium stearate, hydroxypropyl methylcellulose, polyethylene glycol and optionally a colouring agent.
  • the composition used according to the invention can be GLUCOPHAGE® of Merck-Lipha.
  • the medium may include polyvinylpyrrolidone (Povidone K30®), magnesium stearate, hydroxypropyl methylcellulose (Hypromellose®), polyethylene glycol (Macrogol 400®, macrogol 8000®) and colouring agent (Opadry®).
  • metformin is typically administered at the initial dosage of about 0.001 mg/kg to about 500 mg/kg daily.
  • a daily dose range of about 0.1 mg/kg to about 150 mg/kg is preferred.
  • the dosage however may be varied depending upon the requirements of the patient and the severity of the condition being treated. For instance, a human being may be treated with an initial dose of 500 mg, and this dose may be increased to 3 g/day.
  • the composition comprising an AMPK activator (e.g. a biguanide derivative and/or AICAR or AICAR derivative) in a suitable pharmaceutical medium is administered with an initial daily dosage of about 0.001 mg/kg to about 500 mg/kg.
  • an AMPK activator e.g. a biguanide derivative and/or AICAR or AICAR derivative
  • the invention pertains to all pulmonary hypertension types from any origins.
  • the invention consists in (provides) an AMPK activator for treating and/or preventing pulmonary hypertension, wherein pulmonary hypertension is pulmonary arterial hypertension (PAH).
  • PAH corresponds to pulmonary hypertension belonging to the group I of the WHO clinical classification of pulmonary hypertension.
  • the invention consists in an AMPK activator for treating and/or preventing pulmonary hypertension, wherein pulmonary hypertension is pulmonary hypertension associated with left heart disease.
  • pulmonary hypertension associated with left heart disease represents group II within WHO clinical classification system.
  • the invention consists in an AMPK activator for treating and/or preventing pulmonary hypertension, wherein pulmonary hypertension is pulmonary hypertension associated with lung diseases and/or hypoxia.
  • pulmonary hypertension associated with lung diseases and/or hypoxia represents group III within WHO clinical classification system.
  • the invention consists in an AMPK activator for treating and/or preventing pulmonary hypertension, wherein pulmonary hypertension is pulmonary hypertension due to chronic thrombotic and/or embolic disease.
  • pulmonary hypertension is pulmonary hypertension due to chronic thrombotic and/or embolic disease.
  • "Pulmonary hypertension due to chronic thrombotic and/or embolic disease” represents group IV within WHO clinical classification system. According to the classification provided by the New York Heart Association, patients with pulmonary hypertension can be classified according to their ability to function (class I to class IV). The invention pertains to the treatment and/or the prevention of pulmonary hypertension to patients belonging to any of these classes.
  • the medicament is used in association with another treatment against pulmonary hypertension, preferably involving the administration of another active agent suitable for preventing and/or treating pulmonary hypertension.
  • the composition of the invention further comprises an additional active agent suitable for preventing and/or treating pulmonary hypertension.
  • two or more AMPK activators may be used (e.g. together or in sequence) to treat pulmonary hypertension.
  • the additional active agent is an endothelin receptor antagonist.
  • the endothelin receptor antagonist is selected from the group consisting of bosentan or sitaxentan.
  • the additional active agent is a phosphodiesterase-5 inhibitor.
  • the phosphodiesterase-5 inhibitor is sildenafil.
  • the additional active agent is a prostacycline or a prostacycline analog.
  • the prostacycline is epoprostenol and the prostacycline analog is selected from the group consisting of iloprost or treprostinil.
  • three (or more) active agents may also be combined such as at least one AMPK activator, an endothelin receptor antagonist and a phosphodiesterase-5 inhibitor (for example metformin, sitaxentan and sildenafil).
  • said additional active agents may be contained in the same composition or administrated separately.
  • the invention relates preferably to a combination for simultaneous or separate administration comprising metformin and bosentan in a suitable pharmaceutical medium.
  • the invention relates to a product comprising metformin and bosentan as a combined preparation for simultaneous, separate or sequential use in the treatment of pulmonary hypertension.
  • the invention further relates to a combination for simultaneous or separate administration comprising metformin, sitaxentan and sildenafil in a suitable pharmaceutical medium.
  • the invention relates to a product comprising metformin, sitaxentan and sildenafil as a combined preparation for simultaneous, separate or sequential use in the treatment of pulmonary hypertension.
  • compositions may be administered to the patient at the same time, or successively, or alternatively.
  • Another aspect of the invention relates to a method for preventing and/or treating pulmonary hypertension comprising administering to a subject in need thereof an AMPK activator such as a derivative biguanide as described above.
  • the method for preventing and/or treating pulmonary hypertension comprises administering to a subject in need thereof an AMPK activator in combination with an additional active agent suitable for preventing and/or treating pulmonary hypertension, as described above.
  • Figure 2 Metformin limits progression of PAH in hypoxic rats, (a) Mean PAP, (b) right ventricular wall thickness, (c) pulmonary artery flow acceleration and (d) [RV/(LV+S)] ratio determined in control rats (normoxia), rats chronically treated for
  • FIG. 4 Metformin prevents pulmonary hypertension-associated pulmonary arterial wall remodelling.
  • Representative sections of lung tissue (a) and quantification of the relative thickness of small pulmonary artery (20-60 ⁇ m) wall (b) and percentages of distal muscularization of normally nonmuscular arteries (c) in samples from control, hypoxic (21 days) and MCT-injected rats (day 30), untreated and treated by metformin ( # P ⁇ 0.001 vs control, *P ⁇ 0.001 vs hypoxia, ⁇ P ⁇ 0.001 vs MCT).
  • FIG. 5 Metformin improves endothelial function and reduces pulmonary artery contractility, (a) ACC phosphorylation and expression, eNOS phosphorylation and expression, Rho kinase activity (assessed by the extent of phosphorylation of MYPT) and RhoA expression have been analyzed by western blot in pulmonary artery from control rats and rats exposed to hypoxia for 21 days, untreated and treated with metformin (3 different samples representative of each condition), ⁇ -actin amounts were also assessed in each sample, (b) Cumulative concentration-response curves for CCh-induced relaxation of PhE (1 ⁇ M)-contracted pulmonary artery rings from hypoxic (21 days, ⁇ ) and metformin-treated hypoxic rats (•).
  • Figure 6 Metformin decreased pulmonary arterial cell proliferation associated with hypoxic PH.
  • Figure 7 Metformin inhibits PDGF-induced pulmonary artery smooth muscle cell proliferation, (a) Analysis by western blot of the expression of PCNA in rat pulmonary artery smooth muscle cells under basal condition and stimulated by
  • Example 1 hypoxic pulmonary hypertension
  • mice Male Wistar rats (250 g) were used. The normoxic rats were housed in room air at normal atmospheric pressure. Chronic hypoxic pulmonary hypertension was obtained by housing animals in a hypobaric chamber at 480 mmHg (Vacucell 11 IL,
  • Metformin Calbiochem was administered by daily intraperitoneal (IP) injections at the dosage of 100 mg.kg ⁇ .day "
  • hypoxic rats receiving saline
  • hypoxic rats treated by metformin were used: normoxic rats (control group), normoxic rats receiving metformin, hypoxic rats receiving saline, and hypoxic rats treated by metformin.
  • Cardiac ultrasonography Cardiac ultrasonography (SONOS 5500 echocardiograph, Philips, 12-MHz sector scare transducer) was performed by the same operator on anesthetized rats, placed in the left lateral decubitus position after thorax epilation. The wall thickness of the right ventricle was measured. The peak tricuspid regurgitation velocity was measured by continuous-wave. Pulmonary artery flow was recorded by pulsed-wave Doppler echocardiography and the acceleration time of pulmonary artery blood flow was measured from the onset of ejection to the time of peak velocity.
  • SONOS 5500 echocardiograph Philips, 12-MHz sector scare transducer
  • Haemodynamic measures were made as previously described (GUILLUY, C, SAUZEAU, V., ROLLI-DERKINDEREN, M., GUERIN, P., SAGAN, C, PACAUD, P. & LOIRAND, G. (2005). Inhibition of RhoA/Rho kinase pathway is involved in the beneficial effect of sildenafil on pulmonary hypertension. Br J Pharmacol, 146, 1010-8.). Rats were anaesthesized by IP injection of ketamine and xylazine. Haemodynamic parameters were measured using a venous catheter inserted in the right jugular vein.
  • the catheter was introduced in the right atrium, the right ventricle and then in the pulmonary artery.
  • Systolic right ventricle pressure, systolic, diastolic and mean PAP were then measured (Hewlett-Packard, Ml 106B).
  • RV hypertrophy was measured using the ratio of RV weight to left ventricle (LV) plus interventricular septum weight (RV/LV+S).
  • Muscularity of distal pulmonary arteries was assessed as previously described (CHASSAGNE, C, EDDAHIBI, S., ADAMY, C, RIDEAU, D., MAROTTE, F., DUBOIS-RANDE, J.L., ADNOT, S., SAMUEL, JX. & TEIGER, E. (2000). Modulation of angiotensin II receptor expression during development and regression of hypoxic pulmonary hypertension.
  • PCNA cell nuclear antigen
  • Rats maintained in hypobaric chamber for 21 days displayed and increased hematocrit (66.0 ⁇ 1.4 % vs 45.4 ⁇ 1.9 in controls, n 10, P ⁇ 0.001) attesting the hypoxic condition.
  • Example 2 monocrotaline induced pulmonary hypertension
  • MCT monocrotaline induced pulmonary hypertension
  • rats received a subcutaneous injection of saline (controls) or MCT (Sigma, Saint Quentin Fallavier, France; 60 mg/kg/rat) to pulmonary hypertension.
  • Experimental groups were further divided in untreated rats (receiving saline), and treated rats that received daily intraperitoneal IP injections of metformin (100 mg.kg ⁇ .day "1 ), starting at the day of MCT injection (day 0). Rats were examined at day 30. Survival curves were established from day 0 to day 55. The same analyses as in Example 1 have been run.
  • Results are presented in Figure 3.
  • the rats challenged with MCT developed severe pulmonary hypertension characterized by an elevated mean PAP (Figure 3a) and an increase in the RV/LV+S ratio ( Figure 3b).
  • MCT-induced pulmonary hypertension is associated with a low survival rate (30 % at day 30, Figure 3c).
  • Metformin treatment improved all the parameters of MCT-induced pulmonary hypertension with a significant decrease of mean PAP at day 30 ( Figure 2a), decrease of the RV/LV+S ratio ( Figure 3b), and a strong improvement of the survival rate to 61 % at day 30 ( Figure 3c).
  • Example 3 Effect of metformin on pulmonary arterial cell proliferation and excessive vasoconstriction
  • Figure 4 shows a comparison of the effect of metformin on both hypoxic pulmonary hypertension and MCT-induced PH, according to the histological analysis of pulmonary arteries.
  • lung specimens from MCT-treated rats (30 days) displayed severe thickening and muscularization of the arterial wall and metformin treatment also significantly reduced pulmonary arterial remodeling in MCT-treated rats ( Figure 4a and b).
  • the progressive arterial wall thickening occurring in pulmonary hypertension resulted from both pulmonary arterial cell proliferation and excessive vasoconstriction.
  • Pulmonary arteries were harvested and put in NETF buffer (100 mM NaCl, 2 mM EGTA, 50 mM Tris-Cl, pH 7.4 and 50 mM NaF) containing 1 % NP-40, 2 mM orthovanadate, protease and phosphatase inhibitor cocktails (Sigma). Lysates were obtained using Polytron material and then sonicated. Protein concentration of each sample was determined and equal amounts of protein were loaded in each lane of polyacrylamide/SDS gels.
  • NETF buffer 100 mM NaCl, 2 mM EGTA, 50 mM Tris-Cl, pH 7.4 and 50 mM NaF
  • samples were analysed by western blot with antibodies against acetyl CoA carboxylase (ACC) and phosphor-ACC (Cell Signaling, Cell Signaling Technology, Inc., Danvers, MA, U.S.A.), eNOS and phospho-eNOS (Cell Signaling, Cell Signaling Techynology, Inc., Danvers, MA, U.S.A.), RhoA (Santa Cruz Biotechnology, Santa cruz, CA, U.S.A.), phospho-ERK (Santa Cruz), phospho- JNK (Cell Signaling), phospho-p38 (Cell Signaling) and PCNA (proliferating cell nuclear antigen).
  • ACC acetyl CoA carboxylase
  • phosphor-ACC Cell Signaling, Cell Signaling Technology, Inc., Danvers, MA, U.S.A.
  • eNOS and phospho-eNOS Cell Signaling, Cell Signaling Techynology, Inc., Danvers, MA, U.S.A.
  • Rho kinase activity was quantified by western blot analysis for phosphorylated MYPT using a rabbit polyclonal anti-phospho-MYPT (Thr696) (Upstate, Euromedex, Munolsheim, France). Equal loading was confirmed by reprobing the membrane with anti-beta-actin antibody (Sigma, Saint Quentin Fallavier, France). Immunoreactive bands were visualized using horseradish peroxidase-conjugated secondary antibody and subsequent ECL kit detection (Amersham Pharmacia Biotech, Orsay, France). Quantification was made by densitometric analysis with QuantyOne (Biorad, Hercules, CA, U.S.A.).
  • metformin has been shown to stimulate AMPK activity (ZHOU, G., MYERS, R., LI, Y., CHEN, Y., SHEN, X., FENYK-MELODY, J., WU, M., VENTRE, J., DOEBBER, T., FUJII, N., MUSI, N., HIRSHMAN, M.F., GOODYEAR, L.J. & MOLLER, D.E. (2001).
  • AMPK activity ZHOU, G., MYERS, R., LI, Y., CHEN, Y., SHEN, X., FENYK-MELODY, J., WU, M., VENTRE, J., DOEBBER, T., FUJII, N., MUSI, N., HIRSHMAN, M.F., GOODYEAR, L.J. & MOLLER, D.E. (2001).
  • metformin has antiproliferative properties.
  • Example 4 ex vivo contraction assay Pulmonary arteries from rats of example 1 and 2 were collected in physiological saline solution (in mM; 130 NaCL, 5.6 KCl, 1 MgCl 2 , 2 CaCl 2 , 11 glucose, 10 Tris, pH 7.4 with HCl) and cut into rings. Rings of pulmonary arteries were suspended under isometric conditions, connected to a force transducer (Pioden controls Ltd, Canterbury, UK) and set up at a tension of 300-500 mg in Krebs- Henseleit solution at 37°C bubbled with 95 % O 2 -5 % CO 2 . After equilibration, the response to KCl 60 mM was determined.
  • physiological saline solution in mM; 130 NaCL, 5.6 KCl, 1 MgCl 2 , 2 CaCl 2 , 11 glucose, 10 Tris, pH 7.4 with HCl
  • Endothelial-dependent relaxation was tested by adding increasing concentrations of carbachol (CCh) to rings precontracted by phenylephrine (PhE) (1 ⁇ M).
  • Concentration-response curves to PhE were obtained by measuring the amplitude of the contractile responses to increasing PhE concentrations.
  • CCh carbachol
  • PhE phenylephrine
  • Concentration-response curves to PhE were obtained by measuring the amplitude of the contractile responses to increasing PhE concentrations.
  • pulmonary arterial rings from normoxic rats were treated by metformin (4 mM, 2.5 h) and contraction measurements were performed in the continuous presence of 4 mM metformin. Amplitude of the PhE -induced contraction was expressed in mg per mg of tissue (mg/mg). Results are presented in Figures 5b, c and d.
  • Example 5 in vitro proliferation assay
  • Smooth muscle cells from rat pulmonary artery explants were cultured in DMEM with 10 % foetal calf serum, 100 U.ml "1 penicillin and 100 ⁇ g.ml "1 streptomycin. Secondary cultures were obtained by serial passages after the cells were harvested with 0.5 g.l "1 trypsin and 0.2 g.l "1 EDTA and reseeded in fresh medium. After dissociation, cells at passage 2 were seeded on a 6 well-plate, washed and maintained in serum-free medium for 24 h, and then stimulated by platelet derived growth factor (PDGF) (Peprotech France, Levallois Perret) during 24 h, with or without 4 rnM metformin. Proliferation was assessed using both Western blot on cell lysates with antibody against PCNA and BrdU (bromodeoxyuridine) proliferation assay.
  • PDGF platelet derived growth factor
  • PDGF (20 ng ml "1 ) induced pulmonary artery smooth muscle cell proliferation, was attested by Western blot by a 3 -fold increase in PCNA expression (Figure 6a) and an increased number of BrDU positive cells (Figure 7a). Both PCNA expression and the number of BrdU positive cells were strongly reduced by metformin (4 mM), indicating that metformin directly inhibited pulmonary artery smooth muscle cell proliferation ( Figures 7a and b).
  • metformin could thus partly account for its beneficial effect on pulmonary artery remodeling and hypertension.

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Abstract

L'invention concerne des activateurs de protéine kinases activées par le 5'-adénosine monophosphate (AMPK), tels que des dérivés du biguanide, et le 5-aminoimidazole-4-carboxamide riboside (AICAR) ou leurs dérivés, qui sont destinés à être utilisés pour la prévention et/ou le traitement d'une hypertension pulmonaire.
PCT/EP2010/053034 2009-03-10 2010-03-10 Activateurs de protéine kinases activées par le 5'-adénosine monophosphate (ampk), destinés au traitement de l'hypertension pulmonaire WO2010103040A1 (fr)

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WO2013086002A1 (fr) * 2011-12-05 2013-06-13 Cellworks Research India Private Limited Compositions, procédé de préparation de ladite composition et méthode de traitement du cancer
WO2020002718A1 (fr) 2018-06-27 2020-01-02 Silio Oliver Ivan Nouveaux dérivés d'adénine 9n substitués, compositions pharmaceutiques les contenant et utilisation de ces dernières

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013086002A1 (fr) * 2011-12-05 2013-06-13 Cellworks Research India Private Limited Compositions, procédé de préparation de ladite composition et méthode de traitement du cancer
WO2020002718A1 (fr) 2018-06-27 2020-01-02 Silio Oliver Ivan Nouveaux dérivés d'adénine 9n substitués, compositions pharmaceutiques les contenant et utilisation de ces dernières

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