EP1853253A1 - Diosmetin derivatives for the treatment and prevention of thrombotic pathologies - Google Patents

Diosmetin derivatives for the treatment and prevention of thrombotic pathologies

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Publication number
EP1853253A1
EP1853253A1 EP06725989A EP06725989A EP1853253A1 EP 1853253 A1 EP1853253 A1 EP 1853253A1 EP 06725989 A EP06725989 A EP 06725989A EP 06725989 A EP06725989 A EP 06725989A EP 1853253 A1 EP1853253 A1 EP 1853253A1
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EP
European Patent Office
Prior art keywords
radical
thrombotic
hydrogen atom
allyl
prevention
Prior art date
Legal status (The legal status 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 status listed.)
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Application number
EP06725989A
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German (de)
French (fr)
Inventor
Tony Verbeuren
Alain Rupin
Patricia Sansilvestri-Morel
Marie-Odile Vallez
Marie-Françoise Boussard
Michel Wierzbicki
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Laboratoires Servier SAS
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Laboratoires Servier SAS
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Publication of EP1853253A1 publication Critical patent/EP1853253A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
    • C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
    • C07D311/32—2,3-Dihydro derivatives, e.g. flavanones
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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

Definitions

  • the subject of the present invention is the use of derivatives of diosmetin for the production of pharmaceutical compositions intended for the prevention and / or treatment of thrombotic pathologies as well as pathologies with thrombotic risk.
  • the invention particularly relates to the use of the diosmetin derivative 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic pathologies.
  • the physiological process of hemostasis is triggered by the alteration of the endothelial cells of the blood vessels, either for accidental reasons or for more complex pathological reasons. It aims at filling and sealing the blood leakage by two distinct but intricate steps that are dependent on each other: primary haemostasis and plasma coagulation.
  • Primary haemostasis is the emergency mechanism involving circulating platelets that adhere to the endothelium to form the white thrombus or platelet nail.
  • the platelet thrombus is consolidated by the constitution of a fibrin network which encloses aggregated platelets in its mesh.
  • Insoluble fibrin is generated from a soluble plasma protein, fibrinogen, by the action of thrombin, the final product of the enzymatic activation cascade of the coagulation system.
  • fibrin-platelet thrombus is resorbed by the phenomenon of fibrinolysis. Indeed, thanks to its ability to reduce the amount of fibrin in the vascular circulation, fibrinolysis allows the body to fight against the occurrence of thrombosis and to destroy the thrombus that initially stopped bleeding.
  • Fibrinolysis mainly implements plasmin, a proteolytic enzyme, which degrades the long chains of fibrin polymerized into D-dimer.
  • This enzyme is present in plasma as a zymogen, plasminogen, which is activated in plasmin by different serine proteases present on the surface of the fibrin-platelet clot.
  • serine proteases are, in particular, tissue-type plasminogen activators (t-PA), essentially released by activated endothelial cells, and of urokinase type (u-PA), the release of which in the form of pro-urokinases is much more ubiquitous. .
  • Fibrinolysis is itself negatively regulated by a three component system comprising plasmin inhibitors (ot 2 -antiplasmin and ⁇ 2 -macroglobulin), plasminogen activating inhibitors (PAI-1 or Plasminogen Activator Inhibitor). 1, PAI-2) and the thrombin activatable fibrinolysis inhibitor (TAFI).
  • plasmin inhibitors ot 2 -antiplasmin and ⁇ 2 -macroglobulin
  • PAI-1 or Plasminogen Activator Inhibitor Plasminogen Activator Inhibitor
  • PAI-2 Plasminogen Activator Inhibitor
  • TAFI thrombin activatable fibrinolysis inhibitor
  • PAI-1 The main inhibitor of plasminogen activators is PAI-1.
  • the PAI-1 protein belonging to the serpin superfamily (Serin Protease Inhibitor), is a 379 amino acid glycoprotein (47KDa) devoid of cysteine but very rich in methionine residues.
  • PAI-1 protein produced by many cell types such as endothelial cells, monocytes, hepatocytes, fibroblasts, adipocytes and megakaryocytes, is present in plasma and platelets.
  • the active site of PAI-1 located at the C-terminus of the protein in position (Arg 346 -Met 347 ), behaves like a pseudo substrate for tissue type plasminogen activators.
  • the main target proteins of PAI-1 are therefore t-PA and u-PA.
  • the plasma level of PAI-1 is high in 30% of patients with venous thromboembolism (Tabernero et al Incidence of increased plasminogen activator inhibitor in patients with deep venous thrombosis and / or pulmonary embolism, 1989, Thromb Res, 56 , 565-570).
  • the consequence is a general dysfunction of the fibrinolytic system and in particular a fall of t-PA.
  • the same observations have been made in patients with pulmonary hypertension following embolism (Huber et al., Fibrinogen, PA-PAI-I and plasma levels in patients with primary pulmonary hypertension, 1994, Am J Crit Care Med, 150, 929-933).
  • the elevation of PAI-1 is due to the endothelial cells located in the thrombosis zone which show an increase in the release of PAI-1 linked to an induction by either thrombin or by a mediator released by platelets.
  • PAI-1 Since a high level of PAI-1 is deleterious in the context of cardiovascular syndromes, it has been imagined to restore normal fibrinolytic activity to prevent the occurrence of thrombotic events in patients at risk.
  • the parenteral anticoagulants are in particular unfractionated heparin (UFH), fractionated heparins (LMWH) and acenocoumarol (Sintrom®).
  • UHF unfractionated heparin
  • LMWH fractionated heparins
  • Sintrom® acenocoumarol
  • fibrinolytic or thrombolytic action More recent treatments with fibrinolytic or thrombolytic action have been developed, they accelerate the dissolution of intra-vascular clots by promoting the conversion of inactive plasminogen into active plasmin (Marder VJ., Thrombolytic therapy: foundations and clinical results in "Haemostasis and Thrombosis” , Fourth Edition 2001, Editors Colman RW et al.).
  • These fibrinolytics or thrombolytics are exclusively administered parenterally, either by general intravenous infusion or bolus, or locally, for example intra-coronary or intra-arterial.
  • these pharmaceutical compositions must be administered as soon as possible after constitution of the thrombus in an attempt to dissolve it and thus lift the obstruction of the vessel.
  • thrombolytics therefore consist of plasminogen or t-PA tissue activator analogs such as for example: genetically engineered alteplase is identical to t-PA, reteplase is a simplified analogue of human t-PA or Tenecteplase is a recombinant protein that is close to endogenous t-PA and has a greater affinity for thrombus fibrin.
  • reteplase is a simplified analogue of human t-PA
  • Tenecteplase is a recombinant protein that is close to endogenous t-PA and has a greater affinity for thrombus fibrin.
  • MAI 12 is a murine antibody specific for human PAI-1 interfering neither with PAI-2 nor with t-PA or r ⁇ 2-antiplasmin.
  • this monoclonal antibody increases fibrinolysis in vitro and in vivo (Levi et al., Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis, 1992, Circulation, 85, 305-312).
  • the monoclonal antibody CLB-2C binds between positions 128 and 145 of the amino acid sequence of vitronectin and thus prevents the binding of PAI-1 protein thereto by accelerating its inactivation.
  • these monoclonal antibodies binds between positions 128 and 145 of the amino acid sequence of vitronectin and thus prevents the binding of PAI-1 protein thereto by accelerating its inactivation.
  • these specific inhibitors of PAI-1 are unusable from a medical point of view given the difficulties related to lack of humanization of antibodies, immunogenicity risks and development costs.
  • the object of the present invention is therefore to propose an alternative strategy to the fibrinolytic modulation mechanisms already available for preventive and curative purposes of thrombotic pathologies, with a view to at least partially overcoming the disadvantages of known treatments for thromboembolic events.
  • This alternative strategy is based on inhibition of PAI-1 gene expression.
  • the invention proposes, as such, the use of diosmetin derivatives for obtaining pharmaceutical compositions intended for the prevention and / or treatment of thrombotic pathologies.
  • the diosmetin derivatives according to the invention are the compounds of general formula (I):
  • R 1 represents a hydrogen atom or a propyl or allyl radical
  • R 2 represents a hydrogen atom or a propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl or 3-acetyloxy-2-hydroxypropyl radical;
  • R 3 represents a hydrogen atom or a propyl or allyl radical
  • R 4 represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl radical or a radical of formula -COR 4; in which R 4 represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical;
  • R 5 represents a hydrogen atom or a propyl or allyl radical
  • - Re represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl radical, a radical of formula -COR ' 6 (wherein R' ⁇ represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical) or a radical of formula (I '):
  • R 1 , R 2 and R 3 each simultaneously represent an atom of hydrogen, then R 4 also represents a hydrogen atom, their diastereoisomers and enantiomers, and their addition salts with a pharmaceutically acceptable acid or base.
  • diosmetin derivatives used according to the invention are the following derivatives, described in patent EP 0 709 383, of formulas (II) to (XVII):
  • the preferred compound of formula (I) according to the invention is 6 ) 8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran. -4-one of formula (XVII):
  • the present invention thus relates to the use of derivatives of diosmetin of formula (I):
  • R 1 represents a hydrogen atom or a propyl or allyl radical
  • R 2 represents a hydrogen atom or a propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl or 3-acetyloxy-2-hydroxypropyl radical;
  • R 3 represents a hydrogen atom or a propyl or allyl radical
  • R 4 represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl or a radical d ⁇ formula -COR 4 in which R 4 represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical;
  • R 5 represents a hydrogen atom or a propyl or allyl radical
  • R ⁇ represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl radical, a radical of formula -COR ' 6 (wherein R ' 6 represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical) or a radical of formula (I 1 ):
  • At least one of the groups R 1, R 2, R 3 , R 4, R 5 and Re is different from a hydrogen atom
  • R 4 also represents a hydrogen atom, their diastereoisomers and enantiomers, and also their addition salts with an acid or a pharmaceutically acceptable base acceptable, for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic pathologies.
  • thrombotic pathologies is defined by any pathology caused by the formation of thrombosis in the cardiovascular system (veins, arteries, heart, microcirculation). ).
  • Local obstruction of the vessel by a clot or obstruction by clot embolization is responsible for the clinical signs of thrombotic disease.
  • Deep vein thromboses appear mainly in the legs; if they become embolized in the lungs, they will cause pulmonary embolism.
  • Arterial thromboses most often occur in association with vascular disease, the most common being atherosclerosis.
  • micro-circulatory thrombosis is usually a complication of disseminated intravascular coagulation in which micro-thrombi produce ischemic necrosis.
  • preventive corresponds to a preventive treatment intended to reduce the risk of developing thrombosis in pre-disposing contexts such as in atherosclerotic diseases, diabetes or metabolic syndrome.
  • preventive may be understood as secondary prevention that is intended to decrease prevalence by reducing the course and duration of the disease. Secondary prevention is essential following cardiovascular events and strokes to reduce the risk of recurrence.
  • treatment means a curative treatment prescribed for the purpose of treating thrombosis complicated or not by embolism.
  • the invention relates preferably to the use of 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4- one of formula (XVII):
  • compositions for the prevention and / or treatment of thrombotic pathologies.
  • the present invention further relates to the use of derivatives of diosmetin for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic risk pathologies.
  • Thrombotic risk diseases means any pathology during which thrombotic events occur.
  • Pathologies aggravating atherosclerosis such as hypercholesterolemia, diabetes, hypertension, obesity, smoking and atrial fibrillation are the main pathologies at arterial thrombotic risk.
  • Genetic abnormalities or acquired coagulation proteins, orthopedic surgery are the main pathologies at risk of venous thrombosis, but we can also mention obesity, certain hormonal treatments, certain cancers and their treatments, pregnancy, causes of immobilization extended.
  • septic shock is the main pathology at risk for micro-circulatory thromboses.
  • a high level of triglycerides such as in hypercholesterolemia is a particularly aggravating marker of cardiovascular risk in general and thrombosis in particular.
  • the invention also extends to the use of diosmetin derivatives, as active principle, in combination with one or more pharmaceutically acceptable excipients for obtaining pharmaceutical compositions intended for the prevention and / or treatment of thrombotic pathologies or at thrombotic risk.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a diosmetin derivative according to the invention in combination with one or more pharmaceutically acceptable excipients intended for the prevention and / or treatment of thrombotic or thrombotic risk disorders.
  • active principle is meant any substance responsible for the pharmacodynamic or therapeutic properties of the pharmaceutical composition.
  • excipients is understood to mean any substance into which the active principle of a medicament is incorporated in order to facilitate the preparation and administration thereof and to condition the consistency thereof, the form and than the volume.
  • compositions intended for the prevention and / or treatment of thrombotic or thrombotic risk conditions are in a form suitable for oral, parenteral, nasal, percutaneous, rectal, perlingual, ocular or respiratory administration and in particular simple or coated tablets, sublingual tablets, sachets, packets, capsules, glossettes, tablets, suppositories, creams, ointments, dermal gels, and oral or injectable ampoules.
  • the diosmetin derivatives according to the invention are used for obtaining pharmaceutical compositions for oral administration intended for the prevention and / or treatment of thrombotic or thrombotic risk disorders.
  • the oral administration of pharmaceutical compositions for thrombotic or thrombotic risk is particularly suitable for preventive treatment because of their ease of use by patients.
  • the dosage varies according to the sex, the age and the weight of the patient, the route of administration, the nature of the therapeutic indication, or possibly associated treatments and ranges between 0.1 mg and 1 g per 24 hours in one or more takes.
  • the present invention is illustrated without being limited by the following examples.
  • the study is carried out on a HepG2 human hepatocyte line (ATCC, No.
  • the cells are cultured in a MEM (minimum essential medium) medium with glutamax-1 (Gibco TM) at 10% FCS (calf serum fetal), 1% non-essential amino acids, 1% sodium pyruvate, and supplemented with penicillin and streptomycin.
  • MEM minimum essential medium
  • FCS calf serum fetal
  • non-essential amino acids 1% sodium pyruvate
  • the set of primers used (Chen et al., Differential mechanisms of PAI-1 gene activation by transforming growth factor beta and tumor necrosis factor alpha in endothelial cells, 2001, Thrmb Haemost, 86, 1563-72) is as follows: -TTACGCGTGGGTTTGGGGCTGGACTTG-S '(SEQ ID NO.1) and ⁇ '-CGAGATCTCAGAGGTGCCTTGCGATTGG-S' (SEQ ID NO.2).
  • the Perkin Elmer 2400 PCR program has high temperature initiation at 94 ° C for 2 min followed by 35 cycle amplification.
  • the PCR product is then precipitated for 1 hour at -80 ° C. in the presence of 7.5 M ammonium acetate and ethanol. After centrifugation at 14,000 rpm at 40 ° C., the pellet is resuspended in 70% ethanol and again centrifuged at 14,000 rpm at 4 ° C. The pellet obtained is then dried and then taken up in water.
  • the amplified sequence is then digested in two stages by the restriction enzymes BgI II and MIu I.
  • the amplified sequence is digested for 1h30 at 37 ° C. in the presence of 1 unit / ⁇ l of enzyme and 100 ⁇ g / ml of BSA (Serum Bovine). Albumin). After each digestion, the product obtained is systematically purified on Micro Bio-Spin columns ® Chromatography (Bio-Rad) to remove the buffer salts.
  • the plasmid pGL3 Basic (Promega), containing the firefly luciferase luciferase gene, is digested with the restriction enzymes BglII and MIuI according to the same protocol as for the insert and then purified on a 1% Low Melting agarose gel.
  • the plasmid PGL3 / PAI-1 is transfected into HepG2 cells.
  • the transfection takes place in plates having cells at 50% confluency, depositing in each of the wells of Lipofectin® (1 mg / ml) and 1.5 ⁇ g of PGL3 / PAI-1 plasmid.
  • Lipofectin® is activated for 30 min in an OPTI-MEM medium (GIBCO TM) and then placed in contact for 15 minutes with the previously diluted plasmids in the medium.
  • the cells are incubated 6 hours at 37 ° C in an atmosphere at 5% CO 2 and 95% O 2 .
  • the transfection medium is removed and replaced overnight with enriched culture medium to stabilize the cells.
  • Induction of the expression of the reporter genes is carried out for 24 hours in a MEM serum-free medium.
  • the cells are scraped and lysed in lysis buffer (Dual-Luciferase® kit, Promega) and then stored at -2O 0 C.
  • the induction phase is 24h for HepG2 cells, in the presence of TGF ⁇ 1 (1 ng / ml ).
  • an inhibitor of PAI-1 expression may be added 4 hours before the TGF ⁇ 1 and kept in contact with the cells until the end of the 24h of inductive phase.
  • the activity of the PAI-1 promoter is determined by a quantification of the luciferase activity produced (Dual-Luciferase® kit, Promega). In each well is added a solution of luciferin, the substrate of firefly luciferase. This results in light emission. The plate is incubated for 10 min in the dark since the luciferase activity is sensitive to light, then starts a reading with the luminometer to quantify the emitted photons (Wallac, Perkin Elmer), the result obtained being the average of cpm (shots per second). minute) over a period of 5s.
  • PAI-1 under basal conditions and under TGF ⁇ L-induced conditions 6,8-Diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4- one (69%) is as potent as T686 (67%) in basal conditions and is significantly more potent in induced conditions (78% inhibition versus
  • the rats are treated with the test compound, for example 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one.
  • the model of arterial thrombosis induced by iron chloride on the abdominal aorta in rats makes it possible to verify the effectiveness of the diosmetin derivatives according to the invention as antithrombotic agents and in particular the diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one (compound A).
  • the activity of 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one is evaluated as a function of weight of the clot removed from the aorta, this activity will be all the more important as the weight of the clot will be low.
  • 6,8-Diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one significantly and significantly reduces clot weight relative to control; this reduction of the clot is of the order of 30%.

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Abstract

The invention relates to the use of diosmetin in order to obtain pharmaceutical compositions that are intended for the prevention and/or treatment of thrombotic pathologies as well as pathologies with thrombotic risk.

Description

DERIVES DE LA DIOSMETINE POUR LE TRAITEMENT ET LA PREVENTION DES PATHOLOGIES THROMBOTIQUESDERIVATIVES OF DIOSMETINE FOR THE TREATMENT AND PREVENTION OF THROMBOTIC PATHOLOGIES
La présente invention a pour objet l'utilisation des dérivés de la diosmétine pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques ainsi que des pathologies à risque thrombotique.The subject of the present invention is the use of derivatives of diosmetin for the production of pharmaceutical compositions intended for the prevention and / or treatment of thrombotic pathologies as well as pathologies with thrombotic risk.
L'invention concerne en particulier l'utilisation du dérivé de diosmétine 6,8- diallyI-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxyphényl)-4H-1- benzopyran-4-one pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques.The invention particularly relates to the use of the diosmetin derivative 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic pathologies.
Le processus physiologique de l'hémostase est déclenché par l'altération des cellules endothéliales des vaisseaux sanguins, soit pour des raisons accidentelles, soit pour des raisons pathologiques plus complexes. Il vise à l'obturation et au colmatage de la fuite sanguine par deux étapes distinctes mais intriquées et dépendantes l'une de l'autre : l'hémostase primaire et la coagulation plasmatique.The physiological process of hemostasis is triggered by the alteration of the endothelial cells of the blood vessels, either for accidental reasons or for more complex pathological reasons. It aims at filling and sealing the blood leakage by two distinct but intricate steps that are dependent on each other: primary haemostasis and plasma coagulation.
L'hémostase primaire est le mécanisme d'urgence mettant en jeu les plaquettes sanguines circulantes qui adhèrent à l'endothélium pour former le thrombus blanc ou clou plaquettaire.Primary haemostasis is the emergency mechanism involving circulating platelets that adhere to the endothelium to form the white thrombus or platelet nail.
Secondairement, le thrombus plaquettaire est consolidé par la constitution d'un réseau de fibrine qui enserre les plaquettes agrégées dans ses mailles. La fibrine insoluble est générée à partir d'une protéine plasmatique soluble, le fibrinogène, sous l'action de la thrombine, produit final de la cascade d'activation enzymatique du système de la coagulation. Enfin, le thrombus fibrino-plaquettaire est résorbé par le phénomène de la fibrinolyse. En effet, grâce à sa capacité à diminuer la quantité de fibrine dans la circulation vasculaire, la fibrinolyse permet à l'organisme de lutter contre la survenue de thromboses ainsi qu'à détruire le thrombus ayant initialement arrêté une hémorragie.Secondarily, the platelet thrombus is consolidated by the constitution of a fibrin network which encloses aggregated platelets in its mesh. Insoluble fibrin is generated from a soluble plasma protein, fibrinogen, by the action of thrombin, the final product of the enzymatic activation cascade of the coagulation system. Finally, the fibrin-platelet thrombus is resorbed by the phenomenon of fibrinolysis. Indeed, thanks to its ability to reduce the amount of fibrin in the vascular circulation, fibrinolysis allows the body to fight against the occurrence of thrombosis and to destroy the thrombus that initially stopped bleeding.
La fibrinolyse met principalement en œuvre la plasmine, enzyme protéolytique, qui dégrade les longues chaînes de fibrine polymérisées en D-dimères. Cette enzyme est présente dans le plasma sous forme d'un zymogène, le plasminogène, qui est activé en plasmine par différentes sérines-protéases présentes à la surface du caillot fibrino-plaquettaire. Ces sérines-protéases sont en particulier les activateurs de plasminogène de type tissulaire (t-PA), essentiellement libérés par les cellules endothéliales activées, et de type urokinase (u-PA) dont la libération sous forme de pro- urokinases est beaucoup plus ubiquitaire.Fibrinolysis mainly implements plasmin, a proteolytic enzyme, which degrades the long chains of fibrin polymerized into D-dimer. This enzyme is present in plasma as a zymogen, plasminogen, which is activated in plasmin by different serine proteases present on the surface of the fibrin-platelet clot. These serine proteases are, in particular, tissue-type plasminogen activators (t-PA), essentially released by activated endothelial cells, and of urokinase type (u-PA), the release of which in the form of pro-urokinases is much more ubiquitous. .
La fibrinolyse est elle-même régulée négativement par un système à trois composantes comprenant les inhibiteurs de la plasmine (ot2-antiplasmine et α2-macroglobuline), les inhibiteurs de l'activation du plasminogène (PAI-1 ou Plasminogen Activator Inhibitor de type 1 , PAI-2) et l'inhibiteur de la fibrinolyse activable par la thrombine (TAFI).Fibrinolysis is itself negatively regulated by a three component system comprising plasmin inhibitors (ot 2 -antiplasmin and α 2 -macroglobulin), plasminogen activating inhibitors (PAI-1 or Plasminogen Activator Inhibitor). 1, PAI-2) and the thrombin activatable fibrinolysis inhibitor (TAFI).
Le principal inhibiteur des activateurs du plasminogène est le PAI-1. La protéine PAI-1 , appartenant à la superfamille des Serpins (Serin Protease Inhibitor), est une glycoprotéine de 379 acides aminés (47KDa) dépourvue en cystéine mais très riche en résidus méthionines. La protéine PAI-1 , produite par de nombreux types cellulaires tels que les cellules endothéliales, les monocytes, les hépatocytes, les fibroblastes, les adipocytes et mégacaryocytes, est présente dans le plasma et les plaquettes. Le site actif du PAI-1 , situé à l'extrémité C-terminale de la protéine en position (Arg346-Met347), se comporte comme un pseudo- substrat pour les activateurs du plasminogène de type tissulaire. Les principales protéines cibles du PAI-1 sont donc le t-PA et l'u-PA.The main inhibitor of plasminogen activators is PAI-1. The PAI-1 protein, belonging to the serpin superfamily (Serin Protease Inhibitor), is a 379 amino acid glycoprotein (47KDa) devoid of cysteine but very rich in methionine residues. PAI-1 protein, produced by many cell types such as endothelial cells, monocytes, hepatocytes, fibroblasts, adipocytes and megakaryocytes, is present in plasma and platelets. The active site of PAI-1, located at the C-terminus of the protein in position (Arg 346 -Met 347 ), behaves like a pseudo substrate for tissue type plasminogen activators. The main target proteins of PAI-1 are therefore t-PA and u-PA.
Compte-tenu des mécanismes de régulations de l'hémostase et de la fibrinolyse à l'échelle de l'organisme, une corrélation positive entre un taux élevé de PAI-1 et une augmentation des risques de thromboses veineuses et artérielles a été démontrée. Cette corrélation positive est rapportée, en particulier, dans les cas des pathologies dont l'origine est la thrombose veineuse ou artérielle tels que l'infarctus du myocarde (Hamsten et al. Plasminogen activator inhibitor in plasma : risk factor for récurrent myocardial infarction, 1987, Lancet 2, 3-9), l'angor (Wieczorek et al. Tissue- type plasminogen activator and tissue plasminogen activator inhibitor activities as predictor of adverse events in unstable angina, 1994, Am J Cardiol, 74, 424-429), la claudication intermittente, les accidents vasculaires cérébraux, la thrombose veineuse profonde (Schulman et al. The significance of hypofibrinolysis for the risk of récurrence of venous thromboembolism, 1996, Thromb Haemost, 75, 307-611), l'embolie pulmonaire ainsi que pour les pathologies dans lesquelles les risques thrombotiques sont augmentés telles que l'hypertension, l'hypercholestérolémie, le diabète, l'obésité, les anomalies génétiques de la coagulation sanguine ou les anomalies acquises de la coagulation.In view of the organism-wide mechanisms of regulation of haemostasis and fibrinolysis, a positive correlation between a high level of PAI-1 and an increased risk of venous and arterial thrombosis has been demonstrated. This positive correlation is reported, in particular, in the case of pathologies whose origin is venous or arterial thrombosis such as myocardial infarction (Hamsten et al., Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction, 1987 Lancet 2, 3-9), angina (Wieczorek et al., Tissue-type plasminogen activator and tissue plasminogen activator inhibitor activities as predictor of adverse events in unstable angina, 1994, Am J Cardiol, 74, 424-429), intermittent claudication, stroke, deep vein thrombosis (Schulman et al., The significance of hypofibrinolysis for the risk of recurrence of venous thromboembolism, 1996, Thromb Haemost, 75, 307-611), pulmonary embolism, and pathologies in which the thrombotic risks are increased such as hypertension, hypercholesterolemia, diabetes, obesity, genetic abnormalities of blood coagulation o u acquired abnormalities of coagulation.
En effet, le niveau plasmatique de PAI-1 est élevé chez 30% des patients atteints de thromboembolisme veineux (Tabernero et al. Incidence of increased plasminogen activator inhibitor in patients with deep venous thrombosis and/or pulmonary embolism, 1989, Thromb Res, 56, 565-570). La conséquence en est un dysfonctionnement général du système fibrinolytique et notamment une chute du t-PA. Les mêmes observations ont été réalisées chez des patients souffrant d'une hypertension pulmonaire consécutive à une embolie (Huber et al. Fibrinogen, t-PA and PAI-I plasma levels in patients with primary pulmonary hypertension, 1994, Am J Crit Care Med, 150, 929-933). Dans ce cas particulier, l'élévation du taux de PAI-1 est due aux cellules endothéliales localisées au niveau de la zone de thrombose qui présentent une augmentation de la libération de PAI-1 liée à une induction soit par la thrombine soit par un médiateur libéré par les plaquettes.Indeed, the plasma level of PAI-1 is high in 30% of patients with venous thromboembolism (Tabernero et al Incidence of increased plasminogen activator inhibitor in patients with deep venous thrombosis and / or pulmonary embolism, 1989, Thromb Res, 56 , 565-570). The consequence is a general dysfunction of the fibrinolytic system and in particular a fall of t-PA. The same observations have been made in patients with pulmonary hypertension following embolism (Huber et al., Fibrinogen, PA-PAI-I and plasma levels in patients with primary pulmonary hypertension, 1994, Am J Crit Care Med, 150, 929-933). In this particular case, the elevation of PAI-1 is due to the endothelial cells located in the thrombosis zone which show an increase in the release of PAI-1 linked to an induction by either thrombin or by a mediator released by platelets.
Etant donné qu'un taux élevé de PAI-1 est délétère dans un contexte de syndromes cardio-vasculaires, il a été imaginé de restaurer une activité fibrinolytique normale afin d'éviter la survenue d'événements thrombotiques chez des patients à risque.Since a high level of PAI-1 is deleterious in the context of cardiovascular syndromes, it has been imagined to restore normal fibrinolytic activity to prevent the occurrence of thrombotic events in patients at risk.
Le contrôle du processus de fibrinolyse constitue donc un problème central dans les pathologies cardio-vasculaires médicales. Les anticoagulants et les antiplaquettaires constituent des traitements classiques dans un large champ de pathologies cardiovasculaires. Leur intérêt dans le cadre de la prévention d'accidents ainsi que pour les urgences cardiovasculaires a été démontré dans des études épidémiologiques. Cependant, le rapport risque/bénéfice doit toujours être pesé car les accidents liés aux anticoagulants et aux antiplaquettaires sont dominés par les hémorragies qui peuvent remettre en question le pronostic fonctionnel ou vital. En effet, la complication principale des traitements par anticoagulants et antiplaquettaires est le saignement.Controlling the process of fibrinolysis is therefore a central problem in medical cardiovascular diseases. Anticoagulants and antiplatelet medications are standard treatments in a wide range of cardiovascular pathologies. Their interest in the prevention of accidents as well as for cardiovascular emergencies has been demonstrated in epidemiological studies. However, the risk / benefit ratio must always be weighed because accidents related to anticoagulants and antiplatelet medications are dominated by haemorrhages that can call into question the functional or vital prognosis. Indeed, the main complication of anticoagulant and antiplatelet therapy is bleeding.
Les anticoagulants administrés par voie parentérale sont en particulier l'héparine non fractionnnée (HNF), les héparines fractionnées (HBPM) et l'acénocoumarol (Sintrom®). Les études épidémiologiques ont montré que la classe thérapeutique des héparines fractionnées et non fractionnées dans le cadre du traitement d'un épisode thromboembolitique induit 0 à 7% de saignements majeurs avec une mortalité allant de 0 à 2%. L'anticoagulant Sintrom®, développé dans Ie traitement prophylactique et curatif des manifestations thromboemboliques, provoque également des événements hémorragiques majeurs engageant le pronostic vital jusqu'à 2,2% des cas. Compte-tenu de l'augmentation critique des risques hémorragiques dans l'association Sintrom® et antiplaquettaire tel que l'aspirine, le rapport risque/bénéfice de cette association ne doit pas être sous-estimé lors des choix thérapeutiques.The parenteral anticoagulants are in particular unfractionated heparin (UFH), fractionated heparins (LMWH) and acenocoumarol (Sintrom®). Epidemiological studies have shown that the therapeutic class of fractionated and unfractionated heparins in the treatment of a thromboembolic episode induces 0 to 7% of major bleeds with a mortality ranging from 0 to 2%. The anticoagulant Sintrom®, developed in the prophylactic and curative treatment of thromboembolic events, also causes major hemorrhagic events that are life-threatening. 2.2% of cases. Given the critical increase in the risk of bleeding in Sintrom® and antiplatelet combination such as aspirin, the risk / benefit ratio of this combination should not be underestimated when making therapeutic choices.
Des traitements plus récents à action fibrinolytique ou thrombolytique ont été développés, ils accélèrent la dissolution des caillots intra-vasculaires en favorisant la transformation du plasminogène inactif en plasmine active (Marder VJ., Thrombolytic therapy : foundations and clinical results in « Haemostasis and Thrombosis », Fourth Edition 2001 , Editors Colman RW et al.). Ces fibrinolytiques ou thrombolytiques sont exclusivement administrés par voie parentérale, soit par voie intraveineuse générale en perfusion ou en bolus, soit par voie locale par exemple intra-coronaire ou intra-artérielle. En outre ces compositions pharmaceutiques doivent être administrées le plus tôt possible après la constitution du thrombus pour tenter de le dissoudre et lever ainsi l'obstruction du vaisseau. Ces nouveaux thrombolytiques consistent donc en des analogues de l'activateur tissulaire du plasminogène ou t-PA comme par exemple : l'altéplase obtenu par génie génétique est identique au t-PA, la rétéplase est un analogue simplifié du t-PA humain ou le ténectéplase est une protéine recombinante proche du t-PA endogène et présentant une plus grande affinité pour la fibrine du thrombus. Ces fibrinolytiques ont rapidement montré leurs limites compte-tenu de leurs utilisations dans l'urgence hospitalière et de leurs difficultés d'administrations, par exemple par introduction d'un cathéter dans l'artère thrombosée.More recent treatments with fibrinolytic or thrombolytic action have been developed, they accelerate the dissolution of intra-vascular clots by promoting the conversion of inactive plasminogen into active plasmin (Marder VJ., Thrombolytic therapy: foundations and clinical results in "Haemostasis and Thrombosis" , Fourth Edition 2001, Editors Colman RW et al.). These fibrinolytics or thrombolytics are exclusively administered parenterally, either by general intravenous infusion or bolus, or locally, for example intra-coronary or intra-arterial. In addition, these pharmaceutical compositions must be administered as soon as possible after constitution of the thrombus in an attempt to dissolve it and thus lift the obstruction of the vessel. These new thrombolytics therefore consist of plasminogen or t-PA tissue activator analogs such as for example: genetically engineered alteplase is identical to t-PA, reteplase is a simplified analogue of human t-PA or Tenecteplase is a recombinant protein that is close to endogenous t-PA and has a greater affinity for thrombus fibrin. These fibrinolytics quickly showed their limits in view of their uses in the hospital emergency and their difficulties of administration, for example by introduction of a catheter into the thrombosed artery.
D'autres perspectives thérapeutiques ont été envisagées afin de moduler le taux de PAI-1 dont l'augmentation est corrélée aux accidents thromboemboliques. En particulier, des anticorps monoclonaux ont été développés en tant qu'inhibiteurs de l'activité du PAI-1. Le MAI 12 est un anticorps murin spécifique du PAI-1 humain n'interférant ni avec le PAI-2 ni avec le t-PA ou rα2-antiplasmine. En bloquant l'interaction du PAI-1 avec le t-PA, cet anticorps monoclonal augmente la fibrinolyse in vitro et in vivo (Levi et al. Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition ofthrombus extension in models of expérimental thrombosis, 1992, Circulation, 85, 305-312).Other therapeutic perspectives have been considered in order to modulate the level of PAI-1 whose increase is correlated with thromboembolic events. In particular, monoclonal antibodies have been developed as inhibitors of PAI-1 activity. MAI 12 is a murine antibody specific for human PAI-1 interfering neither with PAI-2 nor with t-PA or rα2-antiplasmin. By blocking the interaction of PAI-1 with t-PA, this monoclonal antibody increases fibrinolysis in vitro and in vivo (Levi et al., Inhibition of plasminogen activator inhibitor-1 activity results in promotion of endogenous thrombolysis and inhibition of thrombus extension in models of experimental thrombosis, 1992, Circulation, 85, 305-312).
L'anticorps monoclonal CLB-2C se fixe quant à lui entre les positions 128 et 145 de la séquence d'acides aminés de la vitronectine et empêche donc la fixation de la protéine PAI-1 à celle-ci en accélérant son inactivation. En dépit de l'efficacité de ces anticorps monoclonaux dans des modèles in vitro et in vivo, ces inhibiteurs spécifiques de PAI-1 sont inexploitables d'un point de vue médical compte-tenu des difficultés liés au défaut d'humanisation des anticorps, des risques d'immunogénicité et des coûts de développement.The monoclonal antibody CLB-2C binds between positions 128 and 145 of the amino acid sequence of vitronectin and thus prevents the binding of PAI-1 protein thereto by accelerating its inactivation. Despite the efficacy of these monoclonal antibodies in in vitro and in vivo models, these specific inhibitors of PAI-1 are unusable from a medical point of view given the difficulties related to lack of humanization of antibodies, immunogenicity risks and development costs.
La présente invention a donc pour but de proposer une stratégie alternative aux mécanismes de modulation de la fibrinolyse déjà disponibles à des fins préventives et curatives des pathologies thrombotiques, en vue de remédier au moins en partie aux inconvénients de traitements connus des accidents thromboemboliques. Cette stratégie alternative est fondée sur l'inhibition de l'expression du gène PAI-1. L'invention propose à ce titre l'utilisation de dérivés de la diosmétine pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques. Les dérivés de la diosmétine selon l'invention sont les composés de formule générale (I) :The object of the present invention is therefore to propose an alternative strategy to the fibrinolytic modulation mechanisms already available for preventive and curative purposes of thrombotic pathologies, with a view to at least partially overcoming the disadvantages of known treatments for thromboembolic events. This alternative strategy is based on inhibition of PAI-1 gene expression. The invention proposes, as such, the use of diosmetin derivatives for obtaining pharmaceutical compositions intended for the prevention and / or treatment of thrombotic pathologies. The diosmetin derivatives according to the invention are the compounds of general formula (I):
OR, O dans laquelle :OR, O in which :
- Ri représente un atome d'hydrogène ou un radical propyle ou allyle ;R 1 represents a hydrogen atom or a propyl or allyl radical;
- R2 représente un atome d'hydrogène ou un radical propyle, allyle, 2,3- dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle ou 3- acétyloxy-2-hydroxypropyle;R 2 represents a hydrogen atom or a propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl or 3-acetyloxy-2-hydroxypropyl radical;
- R3 représente un atome d'hydrogène ou un radical propyle ou allyle;- R 3 represents a hydrogen atom or a propyl or allyl radical;
- R4 représente un atome d'hydrogène ou un radical méthyle, propyle, allyle, 2,3-dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle ou un radical de formule -COR4 dans laquelle R4 représente un radical alkyle (C-1-C5) linéaire ou ramifié ou un radical phényle;R 4 represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl radical or a radical of formula -COR 4; in which R 4 represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical;
- R5 représente un atome d'hydrogène ou un radical propyle ou allyle, etR 5 represents a hydrogen atom or a propyl or allyl radical, and
- Re représente un atome d'hydrogène ou un radical méthyle, propyle, allyle, 2,3-dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle, un radical de formule -COR'6 (dans laquelle R'β représente un radical alkyle (C1-C5) linéaire ou ramifié ou un radical phényle) ou un radical de formule (I') :- Re represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl radical, a radical of formula -COR ' 6 (wherein R'β represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical) or a radical of formula (I '):
étant entendu que :Being heard that :
- l'un au moins des groupements R-i, R2, R3, R4, R5 et R6 est différent d'un atome d'hydrogène, et - si R1, R2 et R3 représentent chacun simultanément un atome d'hydrogène, alors R4 représente aussi un atome d'hydrogène, leurs diastéréoisomères et énantiomères, ainsi que leurs sels d'addition à un acide ou à une base pharmaceutiquement acceptable.at least one of the groups R 1, R 2 , R 3 , R 4, R 5 and R 6 is different from a hydrogen atom, and if R 1 , R 2 and R 3 each simultaneously represent an atom of hydrogen, then R 4 also represents a hydrogen atom, their diastereoisomers and enantiomers, and their addition salts with a pharmaceutically acceptable acid or base.
La préparation des composés de formule générale (I) est décrite dans le brevet EP 0 709 383.The preparation of the compounds of general formula (I) is described in patent EP 0 709 383.
Plus particulièrement, les dérivés de diosmétine utilisés selon l'invention sont les dérivés suivants, décrits dans le brevet EP 0 709 383, de formules (II) à (XVII) :More particularly, the diosmetin derivatives used according to the invention are the following derivatives, described in patent EP 0 709 383, of formulas (II) to (XVII):
(II) 7-allyloxy-5-hydroxy-2-(3-hydroxy-4-méthoxyphényl)-4H-1-benzopyran- 4-one(II) 7-Allyloxy-5-hydroxy-2- (3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one
(III) 5,7-dihydroxy-2-[3-(2,3-dihydroxypropyloxy)-4-méthoxyphényl]-4/-/-1- benzopyran-4-one(III) 5,7-Dihydroxy-2- [3- (2,3-dihydroxypropyloxy) -4-methoxyphenyl] -4H-1-benzopyran-4-one
(III) (IV) (R1S) 5-hydroxy-2-(3-hydroxy-4-méthoxyphény l)-7-(2,3-dihydroxy propyloxy)-4H-1 -benzopyran-4-one (III) (IV) (R 1 S) 5-hydroxy-2- (3-hydroxy-4-methoxyphenyl) -7- (2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(V) 5,7-di-(2,3-dihydroxypropyloxy)-2-(3-hydroxy-4-méthoxyphényl)-4H-1- benzopyran-4-one(V) 5,7-Di- (2,3-dihydroxypropyloxy) -2- (3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one
(VI) 5-hydroxy-2-[(4-méthoxy-3-pivaloyloxyphényl)-7-(2,3-dihydroxy propyloxy)-4H-1-benzopyran-4-one(VI) 5-hydroxy-2 - [(4-methoxy-3-pivaloyloxyphenyl) -7- (2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(Vl) (VII) 5-allyloxy-2-(3-allyloxy-4-nπéthoxyphényl)-7-[(2,3-dihydroxy propyloxy)-4H-1-benzopyran-4-one (VI) (VII) 5-Allyloxy-2- (3-allyloxy-4-methoxyphenyl) -7 - [(2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(VIII) 6-allyl-5-hydroxy-2-(2-allyl-3-hydroxy-5-méthoxyphényl)-7-(2,3- dihydroxypropyloxy)-4H-1-benzopyran-4-one(VIII) 6-allyl-5-hydroxy-2- (2-allyl-3-hydroxy-5-methoxyphenyl) -7- (2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(IX) 5-hydroxy-2-(3-hydroxy-4-méthoxy-2-propylphényl)-6-propyl-7-(2,3- dihydroxypropyloxy)-4/-/-1-benzopyran-4-one(IX) 5-Hydroxy-2- (3-hydroxy-4-methoxy-2-propylphenyl) -6-propyl-7- (2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(IX) (X) (R1S) 5-hydroxy-2-(3-hydroxy-4-méthoxy-2-propylphényl)-7-(2,3- dihydroxypropyloxy)-4H-1-benzopyran-4-one(IX) (X) (R 1 S) 5-hydroxy-2- (3-hydroxy-4-methoxy-2-propylphenyl) -7- (2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(Xl) (R) 5-hydroxy-2-(3-hydroxy-4-méthoxy-2-propylphényl)-7-(2,3- dihydroxypropyloxy)-4/-/-1-benzopyran-4-one(X1) (R) 5-Hydroxy-2- (3-hydroxy-4-methoxy-2-propylphenyl) -7- (2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(XII) (S) 5-hydroxy-2-(3-hydroxy-4-méthoxy-2-propylphényl)-7-(2,3- dihydroxypropyloxy)-4H-1-benzopyran-4-one(XII) (S) 5-hydroxy-2- (3-hydroxy-4-methoxy-2-propylphenyl) -7- (2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(XIII) (R,S) 5-hydroxy-2-(3-hydroxy-4-méthoxy-2-propylphényl)-7-(3- acétyloxy-2-hydroxypropyloxy)-4H-1-benzopyran-4-one(XIII) (R, S) 5-hydroxy-2- (3-hydroxy-4-methoxy-2-propylphenyl) -7- (3-acetyloxy-2-hydroxypropyloxy) -4H-1-benzopyran-4-one
(XIII) (XIV) 5-hydroxy-2-[4-méthoxy-2-propyl-3-(6-carboxy-3,4,5-trihydroxy tétrahydropyran-2-yl-oxy)-phényl]-7-(2,3-dihydroxypropyloxy)-4H-1- benzopyran-4-one (XIII) (XIV) 5-hydroxy-2- [4-methoxy-2-propyl-3- (6-carboxy-3,4,5-trihydroxytetrahydropyran-2-yl-oxy) -phenyl] -7- (2,3) -dihydroxypropyloxy) -4H-1-benzopyran-4-one
(XV) 5-hydroxy-2-[4-méthoxy-3-(2,3-dihydroxypropyloxy)-phényl]-7-(2,3- dihydroxypropyloxy)-4/-/-1-benzopyran-4-one(XV) 5-Hydroxy-2- [4-methoxy-3- (2,3-dihydroxypropyloxy) -phenyl] -7- (2,3-dihydroxypropyloxy) -4H-1-benzopyran-4-one
(XVI) 5,7- dihydroxy-2-(3-hydroxy-4-méthoxy-2-propylphényl)-6,8-dipropyl- 4/-/-1 -benzopyran-4-one(XVI) 5,7-Dihydroxy-2- (3-hydroxy-4-methoxy-2-propylphenyl) -6,8-dipropyl-4H-1-benzopyran-4-one
(XVl) Plus avantageusement, le composé préféré de formule (I) selon l'invention est le 6)8-diallyl-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxyphényl)-4H-1- benzopyran-4-one de formule (XVII) : (XVI) More preferably, the preferred compound of formula (I) according to the invention is 6 ) 8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran. -4-one of formula (XVII):
La présente invention porte donc sur l'utilisation de dérivés de la diosmétine de formule (I) :The present invention thus relates to the use of derivatives of diosmetin of formula (I):
dans laquelle :in which :
- Ri représente un atome d'hydrogène ou un radical propyle ou allyle ;R 1 represents a hydrogen atom or a propyl or allyl radical;
- R2 représente un atome d'hydrogène ou un radical propyle, allyle, 2,3- dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle ou 3- acétyloxy-2-hydroxypropyle;R 2 represents a hydrogen atom or a propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl or 3-acetyloxy-2-hydroxypropyl radical;
- R3 représente un atome d'hydrogène ou un radical propyle ou allyle;- R 3 represents a hydrogen atom or a propyl or allyl radical;
- R4 représente un atome d'hydrogène ou un radical méthyle, propyle, allyle, 2,3-dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle ou un radical dθ formule -COR4 dans laquelle R4 représente un radical alkyle (Ci-C5) linéaire ou ramifié ou un radical phényle;- R 4 represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl or a radical dθ formula -COR 4 in which R 4 represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical;
- R5 représente un atome d'hydrogène ou un radical propyle ou allyle, etR 5 represents a hydrogen atom or a propyl or allyl radical, and
- Rβ représente un atome d'hydrogène ou un radical méthyle, propyle, allyle, 2,3-dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle, un radical de formule -COR'6 (dans laquelle R'6 représente un radical alkyle (C1-C5) linéaire ou ramifié ou un radical phényle) ou un radical de formule (I1) :- Rβ represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl radical, a radical of formula -COR ' 6 (wherein R ' 6 represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical) or a radical of formula (I 1 ):
étant entendu que :Being heard that :
- l'un au moins des groupements R-i, R2, R3, R4, R5 et Re est différent d'un atome d'hydrogène, etat least one of the groups R 1, R 2, R 3 , R 4, R 5 and Re is different from a hydrogen atom, and
- si R1, R2 et R3 représentent chacun simultanément un atome d'hydrogène, alors R4 représente aussi un atome d'hydrogène, leurs diastéréoisomères et énantiomères, ainsi que leurs sels d'addition à un acide ou à une base pharmaceutiquement acceptable, pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques.if R 1 , R 2 and R 3 each simultaneously represent a hydrogen atom, then R 4 also represents a hydrogen atom, their diastereoisomers and enantiomers, and also their addition salts with an acid or a pharmaceutically acceptable base acceptable, for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic pathologies.
Conformément à l'acception usuelle dans le cadre de l'invention, l'expression « pathologies thrombotiques » se définit par toute pathologie provoquée par la formation d'une thrombose dans le système cardio- vasculaire (veines, artères, cœur, micro-circulation). L'obstruction locale du vaisseau par un caillot ou l'obstruction à distance par embolisation du caillot sont responsables des signes cliniques des pathologies thrombotiques. Les thromboses veineuses profondes apparaissent essentiellement dans les jambes ; si elles s'embolisent dans les poumons, elles vont provoquer l'embolie pulmonaire. Les thromboses artérielles surviennent le plus souvent associées à une pathologie vasculaire, la plus commune étant l'athérosclérose. Elles sont responsables d'une ischémie tissulaire par arrêt du flux sanguin dans l'artère, par exemple l'infarctus du myocarde dans le cas d'une thrombose coronarienne, ou en aval dans la micro-circulation après embolisation, dans le cadre par exemple d'un accident vasculaire cérébral (AVC) après embolisation d'un thrombus intracardiaque ou carotidien. Enfin, la thrombose micro-circulatoire est en général une complication de la coagulation intra-vasculaire disséminée dans laquelle des micro-thrombi produisent une nécrose ischémique.According to the usual meaning of the invention, the expression "thrombotic pathologies" is defined by any pathology caused by the formation of thrombosis in the cardiovascular system (veins, arteries, heart, microcirculation). ). Local obstruction of the vessel by a clot or obstruction by clot embolization is responsible for the clinical signs of thrombotic disease. Deep vein thromboses appear mainly in the legs; if they become embolized in the lungs, they will cause pulmonary embolism. Arterial thromboses most often occur in association with vascular disease, the most common being atherosclerosis. They are responsible for tissue ischemia by stopping the blood flow in the artery, for example myocardial infarction in the case of coronary thrombosis, or downstream in the micro-circulation after embolization, in the context for example of a stroke after embolization of an intracardiac or carotid thrombus. Finally, micro-circulatory thrombosis is usually a complication of disseminated intravascular coagulation in which micro-thrombi produce ischemic necrosis.
Le terme « préventif » selon l'invention correspond à un traitement à visée préventive ayant pour objectif de diminuer le risque de développer une thrombose dans des contextes pré-disposants tels que dans les maladies athérosclérotiques, le diabète ou le syndrome métabolique. En outre, le terme « préventif » peut s'entendre comme la prévention secondaire qui est destinée à diminuer la prévalence en réduisant l'évolution et la durée de la maladie. La prévention secondaire est essentielle suite à des accidents cardio-vasculaires et des accidents vasculaires cérébraux afin de réduire le risque de récidive.The term "preventive" according to the invention corresponds to a preventive treatment intended to reduce the risk of developing thrombosis in pre-disposing contexts such as in atherosclerotic diseases, diabetes or metabolic syndrome. In addition, the term "preventive" may be understood as secondary prevention that is intended to decrease prevalence by reducing the course and duration of the disease. Secondary prevention is essential following cardiovascular events and strokes to reduce the risk of recurrence.
On entend par « traitement », un traitement à visée curative prescrit aux fins de soigner une thrombose compliquée ou non d'une embolie. L'invention porte, de manière préférée, sur l'utilisation du 6,8-diallyl-5,7- dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxyphényl)-4H-1-benzopyran-4-one de formule (XVII) :The term "treatment" means a curative treatment prescribed for the purpose of treating thrombosis complicated or not by embolism. The invention relates preferably to the use of 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4- one of formula (XVII):
pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou le traitement des pathologies thrombotiques.for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic pathologies.
La présente invention porte, en outre, sur l'utilisation de dérivés de la diosmétine pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies à risque thrombotique.The present invention further relates to the use of derivatives of diosmetin for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic risk pathologies.
Par « pathologies à risque thrombotique », on entend toute pathologie au cours de laquelle des événements thrombotiques surviennent. Les pathologies aggravant l'athérosclérose comme l'hypercholestérolémie, le diabète, l'hypertension, l'obésité, le tabagisme ainsi que la fibrillation auriculaire sont les principales pathologies à risque thrombotique artériel. Les anomalies génétiques ou acquises des protéines de la coagulation, la chirurgie orthopédique sont les principales pathologies à risque thrombotique veineux, mais on peut encore citer l'obésité, certains traitements hormonaux, certains cancers et leurs traitements, la grossesse, les causes d'immobilisation prolongée. Enfin, le choc septique est la principale pathologie à risque pour les thromboses micro-circulatoires. Par exemple, un taux élevé de triglycérides tel que dans rhypercholestérolémie est un marqueur particulièrement aggravant du risque cardio-vasculaire en général et de la thrombose en particulier. Il existe une corrélation entre un taux élevé de VLDL et de PAI-1 libéré par les hépatocytes et les cellules endothéliales (Allison et al. Effects of native triglyceride-enriched and oxidatively modified LDL on plasminogen activator inhibitor-1 expression in human endothelial cells, 1999, Arterioscler Thromb Vase Biol, 19, 1354-1360) (Mussoni et al. Hypertriglyceridemia and régulation of fibrinolytic activity, 1992, Arterioscler Thromb, 12, 19-27)."Thrombotic risk diseases" means any pathology during which thrombotic events occur. Pathologies aggravating atherosclerosis such as hypercholesterolemia, diabetes, hypertension, obesity, smoking and atrial fibrillation are the main pathologies at arterial thrombotic risk. Genetic abnormalities or acquired coagulation proteins, orthopedic surgery are the main pathologies at risk of venous thrombosis, but we can also mention obesity, certain hormonal treatments, certain cancers and their treatments, pregnancy, causes of immobilization extended. Finally, septic shock is the main pathology at risk for micro-circulatory thromboses. For example, a high level of triglycerides such as in hypercholesterolemia is a particularly aggravating marker of cardiovascular risk in general and thrombosis in particular. There is a correlation between a high level of VLDL and PAI-1 released by hepatocytes and endothelial cells (Allison et al., Effects of native triglyceride-enriched and oxidatively modified LDL on plasminogen activator inhibitor-1 expression in human endothelial cells, 1999, Arterioscler Thromb Vase Biol, 19, 1354-1360) (Mussoni et al., Hypertriglyceridemia and regulation of fibrinolytic activity, 1992, Arterioscler Thromb, 12, 19-27).
L'invention s'étend également à l'utilisation de dérivés de la diosmétine, comme principe actif, en combinaison avec un ou plusieurs excipients pharmaceutiquement acceptables pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques ou à risque thrombotique.The invention also extends to the use of diosmetin derivatives, as active principle, in combination with one or more pharmaceutically acceptable excipients for obtaining pharmaceutical compositions intended for the prevention and / or treatment of thrombotic pathologies or at thrombotic risk.
De préférence, l'invention concerne une composition pharmaceutique comprenant un dérivé de la diosmétine selon l'invention en combinaison avec un ou plusieurs excipients pharmaceutiquement acceptable destinée à la prévention et/ou au traitement des pathologies thrombotiques ou à risque thrombotique.Preferably, the invention relates to a pharmaceutical composition comprising a diosmetin derivative according to the invention in combination with one or more pharmaceutically acceptable excipients intended for the prevention and / or treatment of thrombotic or thrombotic risk disorders.
Par « principe actif », on entend toute substance responsable des propriétés pharmacodynamiques ou thérapeutiques de la composition pharmaceutique. Dans le contexte de l'invention, on entend par « excipients » toute substance à laquelle on incorpore le principe actif d'un médicament afin d'en faciliter la préparation ainsi que l'administration et d'en conditionner la consistance, la forme ainsi que le volume.By "active principle" is meant any substance responsible for the pharmacodynamic or therapeutic properties of the pharmaceutical composition. In the context of the invention, the term "excipients" is understood to mean any substance into which the active principle of a medicament is incorporated in order to facilitate the preparation and administration thereof and to condition the consistency thereof, the form and than the volume.
De plus, les compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques ou à risque thrombotique sont sous une forme convenant à l'administration orale, parentérale, nasale, per ou transcutanée, rectale, perlinguale, oculaire ou respiratoire et notamment les comprimés simples ou dragéifiés, les comprimés sublinguaux, les sachets, les paquets, les gélules, les glossettes, les tablettes, les suppositoires, les crèmes, les pommades, les gels dermiques, et les ampoules buvables ou injectables.In addition, the pharmaceutical compositions intended for the prevention and / or treatment of thrombotic or thrombotic risk conditions are in a form suitable for oral, parenteral, nasal, percutaneous, rectal, perlingual, ocular or respiratory administration and in particular simple or coated tablets, sublingual tablets, sachets, packets, capsules, glossettes, tablets, suppositories, creams, ointments, dermal gels, and oral or injectable ampoules.
De préférence, les dérivés de la diosmétine selon l'invention sont utilisés pour l'obtention de compositions pharmaceutiques à administration orale destinées à la prévention et/ou au traitement des pathologies thrombotiques ou à risque thrombotiques. L'administration par voie orale des compositions pharmaceutiques destinées aux pathologies thrombotiques ou à risque thrombotique est particulièrement adaptée aux traitements à visée préventive du fait de leur facilité de prise par les patients.Preferably, the diosmetin derivatives according to the invention are used for obtaining pharmaceutical compositions for oral administration intended for the prevention and / or treatment of thrombotic or thrombotic risk disorders. The oral administration of pharmaceutical compositions for thrombotic or thrombotic risk is particularly suitable for preventive treatment because of their ease of use by patients.
Enfin, la posologie varie selon le sexe, l'âge et le poids du patient, la voie d'administration, la nature de l'indication thérapeutique, ou des traitements éventuellement associés et s'échelonne entre 0,1 mg et 1 g par 24 heures en une ou plusieurs prises. La présente invention est illustrée sans pour autant être limitée par les exemples suivants.Finally, the dosage varies according to the sex, the age and the weight of the patient, the route of administration, the nature of the therapeutic indication, or possibly associated treatments and ranges between 0.1 mg and 1 g per 24 hours in one or more takes. The present invention is illustrated without being limited by the following examples.
EXEMPLE 1 : Inhibition in vitro de l'expression du PAMEXAMPLE 1 In Vitro Inhibition of MAP Expression
1. Culture cellulaire1. Cell culture
L'étude est réalisé sur une lignée d'hépatocytes humains HepG2 (ATCC, n°The study is carried out on a HepG2 human hepatocyte line (ATCC, No.
HB-8065). Les cellules sont cultivées dans un milieu MEM (minimal essential médium) with glutamax-1 (Gibco™) à 10% SVF (sérum de veau fœtal), 1 % acides aminés non essentiels, 1 % pyruvate de sodium, et supplémenté en pénicilline et streptomycine.HB-8065). The cells are cultured in a MEM (minimum essential medium) medium with glutamax-1 (Gibco ™) at 10% FCS (calf serum fetal), 1% non-essential amino acids, 1% sodium pyruvate, and supplemented with penicillin and streptomycin.
2) Clonage du promoteur PAI -12) Cloning of the PAI -1 promoter
a) Amplification du promoteur par PCRa) Amplification of the promoter by PCR
Un fragment de 2,5 kb, correspondant au promoteur PAI-1 , s'étendant des nucléotides en position -2442 à +136, a été amplifié par PCR et sous-cloné (Lopez et al., 2000, Atherosclerosis, 152, 359-366). Dans un volume réactionnel final de 50 μl, 2,5 unités de Platinium® Taq DNA polymerase High Fidelity (Invitrogen) sont mis en présence de 300 ng d'ADN génomique humain (Clontech), 300μM de dNTP (deoxynucléotides tri-phosphate) (Clontech),A 2.5 kb fragment, corresponding to the PAI-1 promoter, extending nucleotides at position -2442 to +136, was amplified by PCR and subcloned (Lopez et al., 2000, Atherosclerosis, 152, 359). -366). In a final reaction volume of 50 .mu.l, 2.5 units of Platinum ® Taq DNA Polymerase High Fidelity (Invitrogen) are placed in the presence of 300 ng human genomic DNA (Clontech), 300 .mu.m dNTP (deoxynucleotides triphosphate) ( Clontech)
30OnM d'amorces dans un milieu contenant le tampon spécifique de l'enzyme, supplémenté par 1 ,5 mM de MgC^ et différentes concentrations de PCRx Enhancer System (Invitrogen) qui facilite l'amplification des séquences particulièrement difficiles (0 à 4x). Le jeu d'amorces utilisé (Chen et al. Differential mechanisms of PAI-1 gène activation by transforming growth factor beta and tumor necrosis factor alpha in endothelial cells, 2001 , Thrmb Haemost, 86, 1563-72) est le suivant : δ'-TTACGCGTGGGTTTGGGGCTGGACTTG-S' (SEQ ID NO.1) et δ'-CGAGATCTCAGAGGTGCCTTGCGATTGG-S' (SEQ ID NO.2).30OnM primers in a medium containing the specific enzyme buffer, supplemented with 1.5 mM MgCl 2 and various concentrations of PCRx Enhancer System (Invitrogen) which facilitates the amplification of particularly difficult sequences (0 to 4x). The set of primers used (Chen et al., Differential mechanisms of PAI-1 gene activation by transforming growth factor beta and tumor necrosis factor alpha in endothelial cells, 2001, Thrmb Haemost, 86, 1563-72) is as follows: -TTACGCGTGGGTTTGGGGCTGGACTTG-S '(SEQ ID NO.1) and δ'-CGAGATCTCAGAGGTGCCTTGCGATTGG-S' (SEQ ID NO.2).
Le programme de PCR, sur appareil Perkin Elmer 2400, présente une initiation à température élevée à 94°C pendant 2 min puis une amplification sur 35 cycles. Le produit de PCR est ensuite précipité 1h à -800C en présence d'acétate d'ammonium 7,5 M et d'éthanol. Après centrifugation à 14000 rpm à 40C, le culot est resuspendu dans l'éthanol 70% et de nouveau centrifugé à 14000 rpm à 4°C. Le culot obtenu est alors séché puis repris dans l'eau. b) Digestion de la séquence promotriceThe Perkin Elmer 2400 PCR program has high temperature initiation at 94 ° C for 2 min followed by 35 cycle amplification. The PCR product is then precipitated for 1 hour at -80 ° C. in the presence of 7.5 M ammonium acetate and ethanol. After centrifugation at 14,000 rpm at 40 ° C., the pellet is resuspended in 70% ethanol and again centrifuged at 14,000 rpm at 4 ° C. The pellet obtained is then dried and then taken up in water. b) Digestion of the promoter sequence
La séquence amplifiée est ensuite digérée en deux étapes par les enzymes de restriction BgI II et MIu I. La séquence amplifiée est digérée 1h30 à 37°C en présence de 1 unité/μl d'enzyme et 100 μg/ml de BSA (Bovine Sérum Albumin). Après chaque digestion, le produit obtenu est systématiquement purifié sur colonnes Micro Bio-Spin® Chromatography (Bio-Rad) afin d'éliminer les sels du tampon.The amplified sequence is then digested in two stages by the restriction enzymes BgI II and MIu I. The amplified sequence is digested for 1h30 at 37 ° C. in the presence of 1 unit / μl of enzyme and 100 μg / ml of BSA (Serum Bovine). Albumin). After each digestion, the product obtained is systematically purified on Micro Bio-Spin columns ® Chromatography (Bio-Rad) to remove the buffer salts.
c) Construction du plasmide PGL3 / PAI-1c) Construction of the plasmid PGL3 / PAI-1
Le plasmide pGL3 Basic (Promega), contenant le gène luciférase de la luciole Firefly, est digéré par les enzymes de restriction BgI II et MIu I selon le même protocole que pour l'insert puis purifié sur gel d'agarose Low Melting 1%. La ligation du vecteur plasmidique pGL3-Basic et de l'insert correspondant au promoteur PAI-1 a été réalisée par la T4 DNA Ligase (LigaFast™ Rapid DNA Ligation System de Promega). Par convention, lors d'une ligation il est utilisé un excèdent d'insert équivalent au triple de la quantité de vecteur. De plus, cet insert étant deux fois plus court que le vecteur (2,5kb contre 4,8kb), le maintien de l'équilibre stœchiométrique nécessite une masse double de vecteur. Il a donc été mis en réaction, 37,5 ng d'insert et 25 ng de vecteur pGL3 Basic en présence de 3 unités de T4 ligase, à température ambiante.The plasmid pGL3 Basic (Promega), containing the firefly luciferase luciferase gene, is digested with the restriction enzymes BglII and MIuI according to the same protocol as for the insert and then purified on a 1% Low Melting agarose gel. The ligation of the pGL3-Basic plasmid vector and of the insert corresponding to the PAI-1 promoter was carried out by the T4 DNA Ligase (LigaFast ™ Rapid DNA Ligation System of Promega). By convention, when ligation is used an excess of insert equivalent to three times the amount of vector. Moreover, this insert being twice as short as the vector (2.5kb against 4.8kb), maintaining the stoichiometric equilibrium requires a double mass of vector. 37.5 ng of insert and 25 ng of pGL3 Basic vector were then reacted in the presence of 3 units of T4 ligase at room temperature.
3) Transfection de cellules d'hépatocytes3) Transfection of hepatocyte cells
Le plasmide PGL3/PAI-1 est transfecté dans des cellules HepG2. La transfection a lieu dans des plaques présentant des cellules à 50% de confluence, en déposant dans chacun des puits de la Lipofectin® (1 mg/ml) et 1 ,5 μg de plasmide PGL3/PAI-1. La lipofectin® est activée durant 30 min dans un milieu OPTI-MEM (GIBCO™ ) puis mis en contact 15 min avec les plasmides préalablement dilués dans le milieu. Les cellules sont incubées 6 heures à 37°C dans une atmosphère à 5% CO2 et 95 % O2. Le milieu de transfection est retiré et remplacé pendant la nuit par un milieu de culture enrichi afin de stabiliser les cellules.The plasmid PGL3 / PAI-1 is transfected into HepG2 cells. The transfection takes place in plates having cells at 50% confluency, depositing in each of the wells of Lipofectin® (1 mg / ml) and 1.5 μg of PGL3 / PAI-1 plasmid. Lipofectin® is activated for 30 min in an OPTI-MEM medium (GIBCO ™) and then placed in contact for 15 minutes with the previously diluted plasmids in the medium. The cells are incubated 6 hours at 37 ° C in an atmosphere at 5% CO 2 and 95% O 2 . The transfection medium is removed and replaced overnight with enriched culture medium to stabilize the cells.
L'induction de l'expression des gènes rapporteurs est réalisée durant 24h dans un milieu sans sérum MEM. Les cellules sont grattées et lysées dans un tampon de lyse (kit Dual- Luciferase®, Promega) puis conservées à -2O0C. La phase d'induction est de 24h pour les cellules HepG2, en présence de TGFβi (1 ng/ml). Durant cette phase inductive, il peut être ajouté un inhibiteur d'expression du PAI-1 , 4h avant le TGFβi et resté au contact des cellules jusqu'à la fin des 24h de phase inductive.Induction of the expression of the reporter genes is carried out for 24 hours in a MEM serum-free medium. The cells are scraped and lysed in lysis buffer (Dual-Luciferase® kit, Promega) and then stored at -2O 0 C. The induction phase is 24h for HepG2 cells, in the presence of TGFβ1 (1 ng / ml ). During this inductive phase, an inhibitor of PAI-1 expression may be added 4 hours before the TGFβ1 and kept in contact with the cells until the end of the 24h of inductive phase.
4) Détermination de l'activité du promoteur4) Determination of promoter activity
L'activité du promoteur PAI-1 est déterminée par une quantification de l'activité luciferase produite (kit Dual- Luciferase®, Promega). Dans chaque puits est ajouté une solution de luciférine, le substrat de la luciferase Firefly. II en résulte une émission de lumière. La plaque est incubée 10 min à l'obscurité puisque l'activité luciferase est sensible à la lumière, puis débute une lecture au luminomètre pour quantifier les photons émis (Wallac, Perkin Elmer), le résultat obtenu étant la moyenne de cpm (coups par minute) sur une période de 5s.The activity of the PAI-1 promoter is determined by a quantification of the luciferase activity produced (Dual-Luciferase® kit, Promega). In each well is added a solution of luciferin, the substrate of firefly luciferase. This results in light emission. The plate is incubated for 10 min in the dark since the luciferase activity is sensitive to light, then starts a reading with the luminometer to quantify the emitted photons (Wallac, Perkin Elmer), the result obtained being the average of cpm (shots per second). minute) over a period of 5s.
5) Résultats5) Results
Le 6)8-diallyl-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxyphényl)-4H-1- benzopyran-4-one nommé arbitrairement composé A et le T686 de formule 3(E)-benzylidène-4(E)-(3,4,5-triméthoxybenzylidène)pyrrolidine-2,5-dione (inhibiteur de référence de l'expression du PAI-1 de Tanabé) ont été testés à la concentration de 10μM sur les cellules HepG2 à l'état basai et après induction avec le TGFβi . L'activité du promoteur PAI-1 est mesurée en condition contrôle et en présence d'inhibiteurs en état basai et induit. Les activités, exprimées en cpm et en pourcentage des observations contrôles, sont illustrées dans le tableau 1.6 ) 8-Diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one, named arbitrarily compound A, and T686 of formula 3 (E ) -benzylidene-4 (E) - (3,4,5-trimethoxybenzylidene) pyrrolidine-2,5-dione (Tanabé PAI-1 expression reference inhibitor) were tested at a concentration of 10 μM on the HepG2 cells in the basal state and after induction with TGFβ1. The activity of the PAI-1 promoter is measured under control conditions and in the presence of inhibitors in the basal and induced state. The Activities, expressed in cpm and as a percentage of control observations, are shown in Table 1.
Tableau 1 : Mesure de l'activité du promoteur PAI-1 en présence de 6,8- diallyl-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxyphényl)-4H-1- benzopyran-4-one ou T686 (24h) en condition basale ou induite par TGFβi (1 ng/ml). ** p<0.01 par rapport au contrôle à l'état basai, oo p<0.01 par rapport au contrôle à l'état induit. ANOVA 1 facteur, post test Dunnett (n=6). Le 6,8-diallyl-5,7-dihydroxy-2-(2-aIlyl-3-hydroxy-4-méthoxyphényl)-4H-1 - benzopyran-4-one et le T686 (10μM) inhibent l'expression du gène PAI-1 en condition basale et en condition induite par le TGFβL Le 6,8-diallyl-5,7- dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxyphényl)-4H-1-benzopyran-4-one (69%) est aussi puissant que le T686 (67%) en condition basale en outre il est nettement plus puissant en condition induite (78% d'inhibition versusTable 1: Measurement of PAI-1 promoter activity in the presence of 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran 4-one or T686 (24h) under basal conditions or induced by TGFβ1 (1 ng / ml). ** p <0.01 compared to the baseline control, oo p <0.01 compared to the control in the induced state. ANOVA 1 factor, Dunnett post test (n = 6). 6,8-Diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one and T686 (10 μM) inhibit gene expression. PAI-1 under basal conditions and under TGFβL-induced conditions 6,8-Diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4- one (69%) is as potent as T686 (67%) in basal conditions and is significantly more potent in induced conditions (78% inhibition versus
52% pour le T686; p<0.01 , ANOVA 1 facteur, post test Newman Keuls). EXEMPLE 2 : Evaluation in vivo de l'activité antithrombotique de dérivés de la diosmétine52% for the T686; p <0.01, ANOVA 1 factor, Newman Keuls post test). EXAMPLE 2 In Vivo Evaluation of the Antithrombotic Activity of Diosmetin Derivatives
1) Animaux1) Animals
Les études ont été réalisées sur des rats mâles CD pesant entre 250 et 400g (Charles River Laboratories). Le protocole expérimental, pratiqué sur des lots de 6 à 8 rats, comprend un lot pour chaque composé testé à comparer à un lot contrôle dans lequel les rats sont traités avec un solvant.The studies were performed on CD male rats weighing between 250 and 400 g (Charles River Laboratories). The experimental protocol, performed on batches of 6 to 8 rats, includes one lot for each test compound compared to a control lot in which the rats are treated with a solvent.
Ces rats sont utilisés pour étudier les effets d'un antithrombotique sur un modèle de thrombose artérielle induite par le FeCI3 sur l'aorte abdominale (Tanaka et al. Z-335, a new thromboxane A2 receptor antagonist, prevents arterial thrombosis indυced by ferrie chloride in rats, 2000, Eur. J.These rats are used to study the effects of an antithrombotic on a model of FeCI 3- induced arterial thrombosis on the abdominal aorta (Tanaka et al., Z-335, a new thromboxane A2 receptor antagonist, prevents arterial thrombosis induced by ferrous chloride in rats, 2000, Eur.
PharmacoL 401 , 413-418).Pharmacol 401, 413-418).
2) Modèle de thrombose artérielle induite2) Model of induced arterial thrombosis
Les rats sont traités par le composé à tester, par exemple le 6,8-diallyl-5,7- dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxyphényl)-4H-1-benzopyran-4-oneThe rats are treated with the test compound, for example 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one.
(30mg/kg/j) par administration per os par gavage pendant 5 jours avant d'induire la thrombose artérielle chez ces rats. Les rats sont anesthésiés au pentobarbital 50mg/kg par administration intra-péritonéale et leurs aortes abdominales sont dégagées après laparatomie. Une pastille de 8mm de diamètre saturée en chlorure de fer (FeCI3 50%), comme agent causal, est déposée sur l'aorte pendant 10 minutes de manière à endommager l'endothélium et induire la formation d'un caillot. Dans les 20 minutes suivants la création du modèle de thrombose artérielle, le caillot formé est retiré de l'aorte et pesé. 3) Résultats(30mg / kg / day) by oral administration by gavage for 5 days before inducing arterial thrombosis in these rats. The rats are anesthetized with pentobarbital 50 mg / kg by intraperitoneal administration and their abdominal aortas are cleared after laparotomy. An 8 mm diameter ferric chloride (50% FeCl 3 ) pellet, as a causative agent, is deposited on the aorta for 10 minutes to damage the endothelium and induce clot formation. Within 20 minutes of creating the arterial thrombosis model, the clot formed is removed from the aorta and weighed. 3) Results
Le modèle de thrombose artérielle induite par le chlorure de fer sur l'aorte abdominale chez le rat permet de vérifier l'efficacité des dérivés de la diosmétine selon l'invention en tant qu'agents anti-thrombotiques et en particulier du 6,8-diallyl-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxy phényl)-4/-/-1-benzopyran-4-one (composé A). L'activité du 6,8-diallyl-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxy phényl)-4/-/-1-benzopyran-4-one est évalué en fonction du poids du caillot retiré de l'aorte, cette activité sera d'autant plus importante que le poids du caillot sera faible.The model of arterial thrombosis induced by iron chloride on the abdominal aorta in rats makes it possible to verify the effectiveness of the diosmetin derivatives according to the invention as antithrombotic agents and in particular the diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one (compound A). The activity of 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one is evaluated as a function of weight of the clot removed from the aorta, this activity will be all the more important as the weight of the clot will be low.
Tableau 2 : Mesure de l'activité du 6,8-diallyl-5,7-dihydroxy-2-(2-allyl-3- hydroxy-4-méthoxyphényl)-4H-1-benzopyran-4-one (composé A) en fonction du poids du caillot par rapport au contrôle (sans gavage) (n=6).Table 2: Measurement of the activity of 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one (compound A) according to the weight of the clot compared to the control (without force-feeding) (n = 6).
Le 6,8-diallyl-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxyphényl)-4H-1 - benzopyran-4-one permet de nettement et significativement diminuer le poids du caillot par rapport au contrôle ; cette diminution du caillot est de l'ordre de 30%. 6,8-Diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one significantly and significantly reduces clot weight relative to control; this reduction of the clot is of the order of 30%.

Claims

REVENDICATIONS
1. Utilisation de dérivés de la diosmétine de formule (I)1. Use of derivatives of diosmetin of formula (I)
dans laquelle :in which :
- Ri représente un atome d'hydrogène ou un radical propyle ou allyle ;R 1 represents a hydrogen atom or a propyl or allyl radical;
- R2 représente un atome d'hydrogène ou un radical propyle, allyle, 2,3- dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle ou 3- acétyloxy-2-hydroxypropyle;R 2 represents a hydrogen atom or a propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl or 3-acetyloxy-2-hydroxypropyl radical;
- R3 représente un atome d'hydrogène ou un radical propyle ou allyle;- R 3 represents a hydrogen atom or a propyl or allyl radical;
- R4 représente un atome d'hydrogène ou un radical méthyle, propyle, allyle, 2,3-dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle ou un radical de formule -COR4 dans laquelle R4 représente un radical alkyle (C1-C5) linéaire ou ramifié ou un radical phényle;R 4 represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl radical or a radical of formula -COR 4; wherein R 4 represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical;
- R5 représente un atome d'hydrogène ou un radical propyle ou allyle, etR 5 represents a hydrogen atom or a propyl or allyl radical, and
- R6 représente un atome d'hydrogène ou un radical méthyle, propyle, allyle, 2,3-dihydroxypropyle, (2,2-diméthyl-1 ,3-dioxol-4-yl)méthyle, un radical de formule -COR'6 (dans laquelle R'6 représente un radical alkyle (C-1-C5) linéaire ou ramifié ou un radical phényle) ou un radical de formule (I1) : - R 6 represents a hydrogen atom or a methyl, propyl, allyl, 2,3-dihydroxypropyl, (2,2-dimethyl-1,3-dioxol-4-yl) methyl radical, a radical of formula -COR ' 6 (in which R ' 6 represents a linear or branched (C 1 -C 5 ) alkyl radical or a phenyl radical) or a radical of formula (I 1 ):
étant entendu que :Being heard that :
- l'un au moins des groupements R-i, R2, R3, R4, R5 et R6 est différent d'un atome d'hydrogène, etat least one of the groups R 1, R 2 , R 3 , R 4, R 5 and R 6 is different from a hydrogen atom, and
- si Ri, R2 et R3 représentent chacun simultanément un atome d'hydrogène, alors R4 représente aussi un atome d'hydrogène, leurs diastéréoisomères et énantiomères, ainsi que leurs sels d'addition à un acide ou à une base pharmaceutiquement acceptable,if R 1, R 2 and R 3 each simultaneously represent a hydrogen atom, then R 4 also represents a hydrogen atom, their diastereoisomers and enantiomers, and also their addition salts with a pharmaceutically acceptable acid or base ,
pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques.for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic pathologies.
2. Utilisation du 6,8-diallyl-5,7-dihydroxy-2-(2-allyl-3-hydroxy-4-méthoxy phényl)-4H-1-benzopyran-4-one de formule (XVII) :2. Use of 6,8-diallyl-5,7-dihydroxy-2- (2-allyl-3-hydroxy-4-methoxyphenyl) -4H-1-benzopyran-4-one of formula (XVII):
pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques. for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic pathologies.
3. Utilisation de dérivés de la diosmétine selon l'une des revendications 1 ou 2 pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies à risque thrombotique.3. Use of derivatives of diosmetin according to one of claims 1 or 2 for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic risk pathologies.
4. Utilisation de dérivés de la diosmétine selon l'une des revendications 1 à 3 pour l'obtention de compositions pharmaceutiques à administration orale destinées à la prévention et/ou au traitement des pathologies thrombotiques ou à risque thrombotique.4. Use of derivatives of diosmetin according to one of claims 1 to 3 for obtaining pharmaceutical compositions for oral administration for the prevention and / or treatment of thrombotic or thrombotic risk conditions.
5. Utilisation de dérivés de la diosmétine selon l'une des revendications 1 à5. Use of derivatives of diosmetin according to one of claims 1 to
4 en combinaison avec un ou plusieurs excipients pharmaceutiquement acceptables pour l'obtention de compositions pharmaceutiques destinées à la prévention et/ou au traitement des pathologies thrombotiques ou à risque thrombotique.4 in combination with one or more pharmaceutically acceptable excipients for obtaining pharmaceutical compositions for the prevention and / or treatment of thrombotic or thrombotic risk.
6. Composition pharmaceutique comprenant un dérivé de la diosmétine selon l'une des revendications 1 à 3 en combinaison avec un ou plusieurs excipients pharmaceutiquement acceptable destinée à la prévention et/ou le traitement des pathologies thrombotiques ou à risque thrombotique. 6. A pharmaceutical composition comprising a diosmetin derivative according to one of claims 1 to 3 in combination with one or more pharmaceutically acceptable excipients for the prevention and / or treatment of thrombotic or thrombotic risk.
EP06725989A 2005-03-01 2006-02-28 Diosmetin derivatives for the treatment and prevention of thrombotic pathologies Withdrawn EP1853253A1 (en)

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