WO2000066132A1 - Use of fragments of hyaluronic acid - Google Patents

Use of fragments of hyaluronic acid Download PDF

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Publication number
WO2000066132A1
WO2000066132A1 PCT/FR2000/001178 FR0001178W WO0066132A1 WO 2000066132 A1 WO2000066132 A1 WO 2000066132A1 FR 0001178 W FR0001178 W FR 0001178W WO 0066132 A1 WO0066132 A1 WO 0066132A1
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glcua
glcnac
hyaluronic acid
fragments
formula
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PCT/FR2000/001178
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French (fr)
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Abdesslam Chajara
Hervé LEVESQUE
Bertrand Delpech
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Laboratoire L. Lafon
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Publication of WO2000066132A1 publication Critical patent/WO2000066132A1/en

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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
    • A61K31/728—Hyaluronic acid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the prevention of neointimal proliferation consecutive to a vascular trauma and in particular to a transluminal angioplasty.
  • Transluminal angioplasty an operation aimed at treating arterial stenoses, particularly coronary, has known immense success since its appearance in 1979; it is currently used routinely, with an immediate success rate of around 95%.
  • the solution brought by angioplasty to the problem of coronary stenosis has given rise to a new problem: restenosis occurring in 30 to 40% of patients within 6 months after angioplasty.
  • Restenosis is the end result of all the mechanisms triggered by angioplasty and whose starting point is the abrasion of the endothelium associated with the trauma of the underlying arterial media induced by the passage of the inflatable balloon. Endothelial denudation and damage to the media induce a cascade of events which involve, on the one hand, the blood elements (leukocytes, platelets, red blood cells and plasma factors) and, on the other hand, the constituents of the arterial wall. (endotheiial cells, arterial smooth muscle cells (CMLA) and the extracellular matrix [ECM]).
  • CMLA arterial smooth muscle cells
  • ECM extracellular matrix
  • CMLA chemotactic and mitogenic cytokines
  • the CMLA are also capable, once activated, of locally releasing factors which stimulate their own proliferation and migration.
  • the arterial lesion also induces the formation of oxygenated free radicals, the latter being capable of stimulating the passage of AMCs from the contractile phenotype to the proliferative and secretory phenotype.
  • migration cell plays a capital role in the formation of intai thickening which leads to restenosis.
  • the migration of cells from the media allows those who do not migrate to escape contact inhibition and proliferate, and those who colonize the intima to have rapid and close contact with plasma growth factors , platelets and leukocytes.
  • CMLA complex mechanisms induced by angioplasty therefore mainly target CMLA.
  • CMLA activity results in the formation of intimal hyperpiasia (called neointimal overgrowth), which combined with reshaping of the arterial media causes restenosis after angioplasty.
  • hyaluronic acid is a high molecular weight glycosaminoglycan (MW ⁇ 10 5 to 10 7 Daltons) present in many tissues of the body.
  • This linear polymer of variable length consists of a chain of disaccharide units composed of D-glucuronic acid (GIcUA) and N-acetyl-D-glucosamine (GIcNAc) whose general structure is: [D-glucuronic acid ⁇ 1 ⁇ 3 N-acetyl-D-glucosamine ⁇ 1 ⁇ 4] n
  • the subject of the present invention is a medicament intended to limit the neointimal proliferation consecutive to a vascular trauma and which comprises an effective dose of a fragment (or of a mixture of fragments) of hyaluronic acid comprising from 4 to 100 units monosaccharide or a pharmaceutically acceptable salt thereof.
  • the present invention also relates to the use, for the manufacture of a medicament intended to limit the neointimal proliferation consecutive to a vascular trauma, of a fragment (or of a mixture of fragments) of hyaluronic acid comprising from 4 to 100 monosaccharide units or one of its pharmaceutically acceptable salts.
  • oligosaccharides constituted by fragments of hyaluronic acid and which can be used in the invention, there may be mentioned: - oligosaccharides of formula:
  • the pharmaceutically acceptable salts are those obtained with pharmaceutically acceptable bases and are in particular the sodium, potassium or calcium salts.
  • the hyaluronic acid fragments can be obtained according to known methods.
  • the hyaluronic acid degradation technique used is conventional and uses enzymatic digestion with hyaluronidase. This technique, and the oligosaccharides which it makes it possible to prepare, were described, in 1985, in the publication of P. Bertrand and B. Delpech (J. Neurochem. 1985; 45 (2): 434-439).
  • the hyaluronic acid extracted from the umbilical cord is dissolved in pH 5 buffer (0.1 M sodium acetate / 0.15 M NaCl), before the addition of hyaluronidase extracted from bovine testes (EC 3.2.1.35). The mixture is incubated at 37 ° C for 6 hours, then the hydrolysis reaction is stopped by heating the reaction medium at 100 ° C for 3 minutes.
  • the cooled reaction medium is then centrifuged at 40,000 g for 10 minutes.
  • the precipitate containing very high molecular weight oligosaccharides and proteins - is eliminated.
  • the mixture of low molecular weight oligosaccharides contained in the supernatant is separated by chromatography on a column of AcA 202 with as elution solvent a mixture of 0.25M glacial acetic acid and 0.28M pyridine of pH 5.
  • L The identification of each fraction of interest is carried out by measuring the content of glueronic acid determined by the carbazol method of T. Bitter and HM Muir (Anal. Biochem. 1962; 4: 330-334). This method allows the preparation of hyaluronic acid fragments comprising from 2 to 8 disaccharide units of formula I.
  • Fragments of the same general structure but of higher molecular mass, comprising 9 to 50 disaccharide units, are obtained by enzymatic digestion with hyaluronidase by modifying the experimental conditions described above, in particular by reducing the duration of the enzymatic incubation to 37 ° C.
  • the nonasaccharide GlcNAc- (GlcUA-GlcNAc) 4 the non-reducing end of which is an N-acetyl Glucosamine residue (GIcNAc) is prepared by the method initially described by VC. Hascall and D. Heinegard (J. Biol. Chem 1974; 249: 4520-56).
  • Decasaccharide (GlcUA-GlcNAc) 5 is subjected to the action of ⁇ -glucuronidase (EC 3.1.3.1) for 3 hours at 37 ° C in 0.05M sodium acetate / 0.1% bovine albumin pH 5.5 buffer, then purified by chromatography on a column of AcA 202.
  • the nonasaccharide (GIcUA-GlcNAc) 4- GIcUA, the reducing end of which is a glueronic acid residue (GIcUA) is prepared according to the method described by JE. Christner et al. (J. Biol. Chem. 1974; 254: 4624-30) per action for 4 h at 37 ° C, 0.25M sodium carbonate on the starting decasaccharide (GlcUA-GlcNAc). The reaction is stopped by the mixture (0.25 M glacial acetic acid / 0.28 M pyridine) before chromatographic separation on a column of AcA 202.
  • GlcNAc- (GlcUA-GlcNAc) n 5, 6, 7, 8 or 9.
  • Fragments of the same general structure but of higher molecular mass, comprising 10 to 49 disaccharide units, are obtained by enzymatic or chemical digestion according to the same principles.
  • the incubation with bovine testicular hyaluronidase (10 mg - EC 3.2.1.35) makes it possible to obtain a mass of mixture of fragments, comprising from 3 to 8 disaccharide units - equal to 36.33 mg - or a useful yield of around 36%.
  • This set is characterized by the following distribution, expressed in number of disaccharide units (D-glucuronic acid + N-acetyl D- glucosamine) [either (GlcUA-GlcNAc) n ]:
  • the subset considered to have the best bioavailability, and therefore the greatest incorporation into arterial tissue, is the subset of fragments with 3, 4, 5 and 6 disaccharide units.
  • this subset represents 80.54% of the total, the fragments with 7 and 8 disaccharide units representing only 10.3 and 7.7% of the total, respectively.
  • the average molecular mass (MW ⁇ 2000 Da) of this mixture results from the preponderant contribution in the mixture of fragments with 3, 4, 5 and 6 disaccharide units.
  • the effect of the low molecular weight fragments of hyaluronic acid obtained after degradation by hyaluronidase has been studied on the stenosis of the rat aorta which has undergone angioplasty.
  • the protocol adopted was as follows: 2 injections of 30 mg / kg on the day of the angioplasty (the first by the venous route bolus 10 min before the endoluminal procedure and the second by the subcutaneous route), then a subcutaneous injection every two days from D2 to D12.
  • these applications can therefore be implemented either using the mixture of fragments of hyaluronic acid described above, or of a subset such as, for example, that constituted by the fragments comprising 5 and 6 disaccharide units or by the nonasaccharides used individually.
  • the preferred therapeutic indication for fragments of hyaluronic acid as defined above will therefore be the prevention of restenotic phenomena consecutive to angioplasty, the medical benefit of which is perfectly demonstrated today, alongside surgical techniques for aortic bypass coronaries, as a non-surgical myocardial revascularization technique ensuring dilation of one (or more) coronary arterial stenosis (s) after percutaneous approach.
  • Subcutaneous injection of fragments of low molecular weight hyaluronic acid will therefore be useful for patients with stable exertional angina, those suffering from unstable angina or those whose infarction is in the process of being formed, as soon as the indication for coronary transluminal angioplasty is made.
  • Treatment with fragments of low molecular weight hyaluronic acid will also be effective after placement of a stent, to avoid neointimal proliferation following abrasion of the intestinal artery, and after endarterectomy.
  • the hyaluronic acid fragments of the present invention are generally administered in dosage units established either per m 2 of body surface area, or per kg of weight.
  • Said dosage units are preferably formulated in pharmaceutical compositions in which the active principle is mixed with one (or more) pharmaceutical excipient (s).
  • the present invention relates to pharmaceutical compositions containing, as active principle, fragments of hyaluronic acid under one of its pharmaceutically acceptable salts (sodium, potassium, calcium).
  • the above hyaluronic acid fragments can be used according to the pathology of the subject to be treated at doses of between 0.5 and 50 mg / kg of body weight per day or 20 to 2000 mg / m 2 of body surface per day .
  • the dosage may advantageously be adjusted according to the degree of urgency of the interventional cardiology procedure [emergency intervention (eg percutaneous transluminal coronary angioplasty) or adjusted (placement of a stent-type arterial stent)].
  • treatment may combine an intravenous bolus before the intervention, a continuous intravenous infusion during the intervention and the following 24 hours, then daily subcutaneous injection for 1 to 4 weeks after the intervention.
  • the doses may be from 15 to 50 mg / kg / day (ie 600 to 2000 mg / m 2 / day); for maintenance treatment, the fragments of hyaluronic acid will be used at doses of 0.5 to 15 mg / kg / day (i.e. 20 to 600 mg / m 2 / day), preferably at doses of 1 to 10 mg / kg / day (i.e. 40 to 400 mg / m 2 / day).
  • the active principles can be administered in unit administration forms, in mixture with conventional pharmaceutical carriers suitable for human therapy.
  • Suitable unit administration forms include intravenous administration forms (ready-to-use solutions for iv bolus and / or se; solution for iv infusion).
  • parenteral administration intravenous infusion at constant flow rate
  • isotonic and sterile aqueous saline solutions are used which may contain pharmacologically compatible excipients.

Abstract

The invention relates to the use of a fragment (or mixture of fragments) of hyaluronic acid comprising 4-100 monosaccharide motifs or motifs of one of the pharmaceutically acceptable salts thereof in the production of a medicament which is designed to limit neo-intimal proliferation following vascular trauma.

Description

Utilisation de fragments d' acide hyaluronique . Use of hyaluronic acid fragments.
La présente invention concerne la prévention de la prolifération néo- intimale consécutive à un trauma vasculaire et notamment à une angioplastie transluminale.The present invention relates to the prevention of neointimal proliferation consecutive to a vascular trauma and in particular to a transluminal angioplasty.
L'angioplastie transluminale, opération visant à traiter les sténoses artérielles notamment coronaires, a connu depuis son apparition en 1979 un immense succès; elle est utilisée actuellement de manière courante, avec un taux de réussite immédiate avoisinant les 95%. Malheureusement, la solution apportée par l'angioplastie au problème des sténoses coronaires a fait naître un nouveau problème : une resténose survenant chez 30 à 40% des patients dans les 6 mois qui suivent l'angioplastie.Transluminal angioplasty, an operation aimed at treating arterial stenoses, particularly coronary, has known immense success since its appearance in 1979; it is currently used routinely, with an immediate success rate of around 95%. Unfortunately, the solution brought by angioplasty to the problem of coronary stenosis has given rise to a new problem: restenosis occurring in 30 to 40% of patients within 6 months after angioplasty.
La resténose est le résultat final de l'ensemble des mécanismes déclenchés par l'angioplastie et dont le point de départ est l'abrasion de l'endothélium associée au traumatisme de la média artérielle sous-jacente induite par le passage du ballonnet gonflable. La dénudation endothéliale et la lésion de la média induisent une cascade d'événements qui mettent en jeu d'une part, les éléments sanguins (leucocytes, plaquettes, globules rouges et facteurs plasmatiques) et d'autre part, les constituants de la paroi artérielle (cellules endothéiiales, cellules musculaires lisses artérielles (CMLA) et la matrice extracellulaire [MEC]). L'apport de cytokines chimiotactiques et mitogènes par les plaquettes et les leucocytes qui adhèrent à la paroi lésée vont activer les CMLA, ces dernières migrant alors depuis la média jusqu'à l'intima, où elles prolifèrent et synthétisent de manière abondante la MEC (JS. Forrester et al. J. Am. Coll. Cardiol. 1991 ;17 :758-769). La réponse plaquettaire induit également la formation d'un thrombus sur le site de lésion, ce dernier facilitant la stimulation des CMLA. En effet, la thrombine générée lors de la formation du thrombus stimule la prolifération des CMLA, active les plaquettes et attire les monocytes, source supplémentaire de facteurs de croissance, vers le site de la lésion. Les CMLA sont par ailleurs capables, une fois activées, de libérer localement des facteurs qui stimulent leur propre prolifération et leur migration. La lésion artérielle induit également la formation de radicaux libres oxygénés, ces derniers étant capables de stimuler le passage des CMLA du phénotype contractile au phénotype proliférant et sécrétoire. En plus de la prolifération, la migration cellulaire joue un rôle capital dans la formation de l'épaississement intimai qui aboutit à la resténose. La migration des cellules de la média permet à celles qui ne migrent pas d'échapper à l'inhibition de contact et de proliférer, et à celles qui vont coloniser l'intima d'avoir un contact rapide et proche avec les facteurs de croissance plasmatiques, plaquettaires et leucocytaires.Restenosis is the end result of all the mechanisms triggered by angioplasty and whose starting point is the abrasion of the endothelium associated with the trauma of the underlying arterial media induced by the passage of the inflatable balloon. Endothelial denudation and damage to the media induce a cascade of events which involve, on the one hand, the blood elements (leukocytes, platelets, red blood cells and plasma factors) and, on the other hand, the constituents of the arterial wall. (endotheiial cells, arterial smooth muscle cells (CMLA) and the extracellular matrix [ECM]). The supply of chemotactic and mitogenic cytokines by the platelets and the leukocytes which adhere to the damaged wall will activate the CMLA, the latter then migrating from the media to the intima, where they proliferate and synthesize abundantly CEM ( JS. Forrester et al. J. Am. Coll. Cardiol. 1991; 17: 758-769). The platelet response also induces the formation of a thrombus at the site of injury, the latter facilitating stimulation of CMLA. In fact, the thrombin generated during the formation of the thrombus stimulates the proliferation of AMCs, activates platelets and attracts monocytes, an additional source of growth factors, to the site of the lesion. The CMLA are also capable, once activated, of locally releasing factors which stimulate their own proliferation and migration. The arterial lesion also induces the formation of oxygenated free radicals, the latter being capable of stimulating the passage of AMCs from the contractile phenotype to the proliferative and secretory phenotype. In addition to proliferation, migration cell plays a capital role in the formation of intai thickening which leads to restenosis. The migration of cells from the media allows those who do not migrate to escape contact inhibition and proliferate, and those who colonize the intima to have rapid and close contact with plasma growth factors , platelets and leukocytes.
L'ensemble de ces mécanismes complexes induits par l'angioplastie a donc pour cible principale les CMLA. L'activité anormale des CMLA qui en résulte aboutit à la formation d'une hyperpiasie intimale (appelée prolifération néointimale), laquelle associée à un remodelage de la média artérielle provoque la resténose après l'angioplastie.All of these complex mechanisms induced by angioplasty therefore mainly target CMLA. The resulting abnormal CMLA activity results in the formation of intimal hyperpiasia (called neointimal overgrowth), which combined with reshaping of the arterial media causes restenosis after angioplasty.
Pour prévenir la resténose, plusieurs substances ont été utilisées chez l'homme, choisies en fonction de leurs propriétés à agir sur un ou plusieurs des mécanismes déclenchés par l'angioplastie, qu'il s'agisse de facteurs agissant sur les CMLA ou sur ces cellules elles-mêmes. Parmi les substances utilisées, celles ayant une activité anti-thrombotique ont été particulièrement étudiées (aspirine, dipyridamole, ticlopidine, héparine non fractionnée, coumadine). Malheureusement, toutes ses substances se sont avérées inefficaces en dépit de résultats encourageants lors d'expérimentations animales. Des produits ayant une activité anti-proliférante ont été également testés (stéroides, huiles de poissons, héparines non fractionnée et de bas poids moléculaire, trapidil), mais toujours sans succès. Cependant, quelques études ont montré qu'une inhibition de la prolifération cellulaire (par irradiation locale par exemple) induisait chez l'homme une inhibition de la prolifération néointimale, et de fait, de la resténose, suggérant qu'il s'agissait d'une cible thérapeutique intéressante. L'absence actuelle de molécules efficaces pour diminuer la fréquence de resténose chez l'homme est probablement liée à la complexité des phénomènes mis en jeu, lors de la cicatrisation de la sténose artérielle dilatée. La resténose est la conséquence de plusieurs événements induits par l'angioplastie dont les principaux sont : 1 - l'infiltration des leucocytes dans la paroi artérielle lésée. 2- la migration des CMLA de la média dans l'intima artérielle. 3- la prolifération des CMLA dans l'intima. 4- la synthèse accrue de la MEC dans l'intima. L'inhibition de la resténose nécessite forcément une modulation de chacun de ces principaux phénomènes.To prevent restenosis, several substances have been used in humans, chosen based on their properties to act on one or more of the mechanisms triggered by angioplasty, whether these are factors acting on AMCs or on these. cells themselves. Among the substances used, those with anti-thrombotic activity have been particularly studied (aspirin, dipyridamole, ticlopidine, unfractionated heparin, coumadin). Unfortunately, all of its substances have proven ineffective despite encouraging results in animal experiments. Products with anti-proliferative activity have also been tested (steroids, fish oils, unfractionated and low molecular weight heparins, trapidil), but still without success. However, some studies have shown that an inhibition of cell proliferation (by local irradiation for example) induces in humans an inhibition of neointimal proliferation, and indeed, restenosis, suggesting that it was an interesting therapeutic target. The current absence of molecules effective in reducing the frequency of restenosis in humans is probably linked to the complexity of the phenomena involved, during the healing of dilated arterial stenosis. Restenosis is the consequence of several events induced by angioplasty, the main ones being: 1 - the infiltration of leukocytes into the damaged arterial wall. 2- the migration of CMLA from the media into the arterial intima. 3- the proliferation of CMLA in the intima. 4- the increased synthesis of MEC in the intima. The inhibition of restenosis necessarily requires modulation of each of these main phenomena.
Dans EP-A-0 661 981 et WO 95/26 193, on a décrit l'utilisation d'acide hyaluronique ou de fragments de haut poids moléculaire (150 000 à 225 000 daltons) pour éviter la resténose. L'acide hyaluronique naturel est un glycosaminoglycane de haut poids moléculaire (MW ≈ 105 à 107 Daltons) présent dans de nombreux tissus de l'organisme. Ce polymère linéaire de longueur variable est constitué d' un enchaînement d'unités disaccharidiques composées d'acide D-glucuronique (GIcUA) et de N-acétyl-D-glucosamine (GIcNAc) dont la structure générale est : [Acide D-glucuronique β 1→3 N-acétyl-D-glucosamine β 1→4]nIn EP-A-0 661 981 and WO 95/26 193, the use of hyaluronic acid or fragments of high molecular weight (150,000 to 225,000 daltons) has been described to avoid restenosis. Natural hyaluronic acid is a high molecular weight glycosaminoglycan (MW ≈ 10 5 to 10 7 Daltons) present in many tissues of the body. This linear polymer of variable length consists of a chain of disaccharide units composed of D-glucuronic acid (GIcUA) and N-acetyl-D-glucosamine (GIcNAc) whose general structure is: [D-glucuronic acid β 1 → 3 N-acetyl-D-glucosamine β 1 → 4] n
Par ailleurs, RC. Savani et EA. Turley (Int. J. Tiss. Reac. 1995; XVII (4) : 141-145) ont étudié les effets d'injections intraveineuses (iv) et sous-cutanées (se) d'acide hyaluronique natif (dose = 30 mg/kg) sur l'hyperplasie néointimale artérielle induite, chez le rat, lors d'une angioplastie carotidienne expérimentale. Ils ont montré que l'administration d'acide hyaluronique non fractionné (iv ou se) permettait d'obtenir une concentration sérique maximale de 5000 μg/ml (boius iv) ou de 1500 μg/ml (injection se) ainsi qu'une réduction significative (environ 50%) de l'épaississement intimai induit par l'abrasion causée par la dilatation de la carotide instrumentée par le ballonnet. Cependant, ces résultats n' ont pas pu être reproduits par la Demanderesse.In addition, RC. Savani and EA. Turley (Int. J. Tiss. Reac. 1995; XVII (4): 141-145) studied the effects of intravenous (iv) and subcutaneous (se) injections of native hyaluronic acid (dose = 30 mg / kg) on induced arterial neointimal hyperplasia, in rats, during an experimental carotid angioplasty. They showed that the administration of unfractionated hyaluronic acid (iv or se) made it possible to obtain a maximum serum concentration of 5000 μg / ml (boius iv) or 1500 μg / ml (se injection) as well as a reduction significant (approximately 50%) of the intai thickening induced by the abrasion caused by the dilation of the carotid instrumented by the balloon. However, these results could not be reproduced by the Applicant.
La présente invention a pour objet un médicament destiné à limiter la prolifération néo-intimale consécutive à un trauma vasculaire et qui comprend une dose efficace d'un fragment (ou d'un mélange de fragments) d'acide hyaluronique comprenant de 4 à 100 motifs monosaccharidiques ou d'un de ses sels pharmaceutiquement acceptables.The subject of the present invention is a medicament intended to limit the neointimal proliferation consecutive to a vascular trauma and which comprises an effective dose of a fragment (or of a mixture of fragments) of hyaluronic acid comprising from 4 to 100 units monosaccharide or a pharmaceutically acceptable salt thereof.
La présente invention a également pour objet l'utilisation, pour la fabrication d'un médicament destiné à limiter la prolifération néo-intimale consécutive à un trauma vasculaire, d'un fragment (ou d'un mélange de fragments) d'acide hyaluronique comprenant de 4 à 100 motifs monosaccharidiques ou d'un de ses sels pharmaceutiquement acceptables.The present invention also relates to the use, for the manufacture of a medicament intended to limit the neointimal proliferation consecutive to a vascular trauma, of a fragment (or of a mixture of fragments) of hyaluronic acid comprising from 4 to 100 monosaccharide units or one of its pharmaceutically acceptable salts.
Parmi les oligosaccharides constitués par des fragments d'acide hyaluronique et utilisables dans l'invention, on peut citer : - des oligosaccharides de formule :Among the oligosaccharides constituted by fragments of hyaluronic acid and which can be used in the invention, there may be mentioned: - oligosaccharides of formula:
(GlcUA-GlcNAc)n1 I avec n1 de 2 à 8 et notamment (GlcUA-GlcNAc)5 la(GlcUA-GlcNAc) n1 I with n 1 from 2 to 8 and in particular (GlcUA-GlcNAc) 5 la
(GlcUA-GlcNAc)6 lb ainsi que leurs différents mélanges ;(GlcUA-GlcNAc) 6 lb and their various mixtures;
- des nonasaccharides de formule :- nonasaccharides of formula:
GlcNac-(GlcUA-GlcNAc)4 lia (GlcUA-GlcNac)4-GlcUA llbGlcNac- (GlcUA-GlcNAc) 4 lia (GlcUA-GlcNac) 4 -GlcUA llb
- des oligosaccharides de formule :- oligosaccharides of formula:
GlcNac-(GlcUA-GlcNAc)n2 III avec n2 de 5 à 9, ainsi que leurs mélanges ; - des oligosaccharides de formule :GlcNac- (GlcUA-GlcNAc) n2 III with n 2 from 5 to 9, as well as their mixtures; - oligosaccharides of formula:
(GlcUA-GlcNAc)n3-GlcUA IV avec n3 de 5 à 9,(GlcUA-GlcNAc) n3 -GlcUA IV with n 3 from 5 to 9,
- des oligosaccharides de formule :- oligosaccharides of formula:
(GlcUA-GlcNAc)n4 V avec n4 = 9 à 50,(GlcUA-GlcNAc) n4 V with n 4 = 9 to 50,
- des oligosaccharides de formule :- oligosaccharides of formula:
GlcNac-(GlcUA-GlcNAc)n5 ViaGlcNac- (GlcUA-GlcNAc) n5 Via
(GlcUA-GlcNac) n5-GlcUA Vlb avec n5 = 10 à 49.(GlcUA-GlcNAc) n5 -GlcUA VIb with n 5 = 10-49.
Les sels pharmaceutiquement acceptables sont ceux obtenus avec des bases pharmaceutiquement acceptables et sont notamment les sels de sodium, potassium ou calcium.The pharmaceutically acceptable salts are those obtained with pharmaceutically acceptable bases and are in particular the sodium, potassium or calcium salts.
Les fragments d'acide hyaluronique peuvent être obtenus selon des procédés connus.The hyaluronic acid fragments can be obtained according to known methods.
La technique de dégradation de l'acide hyaluronique utilisée est classique et fait appel à une digestion enzymatique par la hyaluronidase. Cette technique, et les oligosaccharides qu'elle permet de préparer, ont été décrit, en 1985, dans la publication de P. Bertrand et B. Delpech (J. Neurochem. 1985; 45(2):434- 439). L'acide hyaluronique extrait de cordon ombilical est mis en solution dans du tampon pH 5 (acétate de sodium 0,1 M/NaCI 0,15M), avant l'addition de hyaluronidase extraite de testicules de bovin (EC 3.2.1.35). Le mélange est incubé à 37°C pendant 6 heures, puis la réaction d'hydrolyse est arrêtée par chauffage du milieu réactionnel à 100°C pendant 3 minutes. Le milieu réactionnel refroidi est alors centrifugé à 40.000g pendant 10 minutes. Le précipité contenant des oligosaccharides de très haut poids moléculaire et des protéines - est éliminé. Le mélange d'oligosaccharides de bas poids moléculaire contenu dans le surnageant est séparé par chromatographie sur colonne d' AcA 202 avec comme solvant d'élution un mélange d'acide acétique glacial 0,25M et de pyridine 0,28M de pH 5. L'identification de chaque fraction d'intérêt est réalisée par la mesure du contenu en acide glueuronique déterminé par la méthode au carbazol de T. Bitter et H. M. Muir (Anal. Biochem. 1962; 4: 330-334). Cette méthode permet la préparation des fragments d'acide hyaluronique comportant de 2 à 8 unités disaccharidiques de formule I.The hyaluronic acid degradation technique used is conventional and uses enzymatic digestion with hyaluronidase. This technique, and the oligosaccharides which it makes it possible to prepare, were described, in 1985, in the publication of P. Bertrand and B. Delpech (J. Neurochem. 1985; 45 (2): 434-439). The hyaluronic acid extracted from the umbilical cord is dissolved in pH 5 buffer (0.1 M sodium acetate / 0.15 M NaCl), before the addition of hyaluronidase extracted from bovine testes (EC 3.2.1.35). The mixture is incubated at 37 ° C for 6 hours, then the hydrolysis reaction is stopped by heating the reaction medium at 100 ° C for 3 minutes. The cooled reaction medium is then centrifuged at 40,000 g for 10 minutes. The precipitate containing very high molecular weight oligosaccharides and proteins - is eliminated. The mixture of low molecular weight oligosaccharides contained in the supernatant is separated by chromatography on a column of AcA 202 with as elution solvent a mixture of 0.25M glacial acetic acid and 0.28M pyridine of pH 5. L The identification of each fraction of interest is carried out by measuring the content of glueronic acid determined by the carbazol method of T. Bitter and HM Muir (Anal. Biochem. 1962; 4: 330-334). This method allows the preparation of hyaluronic acid fragments comprising from 2 to 8 disaccharide units of formula I.
Des fragments de même structure générale mais de masse moléculaire plus élevée, comprenant 9 à 50 unités disaccharidiques, sont obtenus par digestion enzymatique à la hyaluronidase en modifiant les conditions expérimentales décrites ci-dessus, en particulier en réduisant la durée de l'incubation enzymatique à 37°C.Fragments of the same general structure but of higher molecular mass, comprising 9 to 50 disaccharide units, are obtained by enzymatic digestion with hyaluronidase by modifying the experimental conditions described above, in particular by reducing the duration of the enzymatic incubation to 37 ° C.
Le nonasaccharide GlcNAc-(GlcUA-GlcNAc)4 dont l'extrémité non réductrice est un reste N-acétyl Glucosamine (GIcNAc) est préparé par la méthode initialement décrite par VC. Hascall et D. Heinegard (J. Biol. Chem 1974; 249: 4520-56). Le décasaccharide (GlcUA-GlcNAc)5 est soumis à l'action de la β-glucuronidase (EC 3.1.3.1 ) pendant 3 heures à 37°C en tampon 0.05M acétate de sodium/0.1 % albumine bovine pH 5.5, puis purifié par chromatographie sur colonne d'AcA 202. Le nonasaccharide (GIcUA-GlcNAc)4- GIcUA dont l'extrémité réductrice est un reste acide glueuronique (GIcUA) est préparé selon la méthode décrite par JE. Christner et al. (J. Biol. Chem. 1974; 254: 4624-30) par action pendant 4h à 37°C, de carbonate de sodium 0,25M sur le décasaccharide (GlcUA-GlcNAc)5 de départ. La réaction est arrêtée par le mélange (acide acétique glacial 0,25M/pyridine 0,28M) avant séparation chromatographique sur colonne d'AcA 202.The nonasaccharide GlcNAc- (GlcUA-GlcNAc) 4, the non-reducing end of which is an N-acetyl Glucosamine residue (GIcNAc) is prepared by the method initially described by VC. Hascall and D. Heinegard (J. Biol. Chem 1974; 249: 4520-56). Decasaccharide (GlcUA-GlcNAc) 5 is subjected to the action of β-glucuronidase (EC 3.1.3.1) for 3 hours at 37 ° C in 0.05M sodium acetate / 0.1% bovine albumin pH 5.5 buffer, then purified by chromatography on a column of AcA 202. The nonasaccharide (GIcUA-GlcNAc) 4- GIcUA, the reducing end of which is a glueronic acid residue (GIcUA) is prepared according to the method described by JE. Christner et al. (J. Biol. Chem. 1974; 254: 4624-30) per action for 4 h at 37 ° C, 0.25M sodium carbonate on the starting decasaccharide (GlcUA-GlcNAc). The reaction is stopped by the mixture (0.25 M glacial acetic acid / 0.28 M pyridine) before chromatographic separation on a column of AcA 202.
Selon des procédures analogues, les oligosaccharides à nombre impair d'unités saccharidiques comprenant n = 11 , 13, 15, 17 et 19 résidus monosaccharidiques sont préparés à partir de leurs oiigomères homologues supérieur (n+1 ) comprenant un nombre pair d'unités saccharidiques (c'est à dire n = 12, 14, 16, 18, et 20 unités saccharidiques) :According to analogous procedures, the oligosaccharides with an odd number of saccharide units comprising n = 11, 13, 15, 17 and 19 monosaccharide residues are prepared from their higher homologous oiomomers (n + 1) comprising an even number of saccharide units (i.e. n = 12, 14, 16, 18, and 20 saccharide units):
- soit par digestion enzymatique à la β-glucuronidase (extraite de foie de bovin - EC 3.1.3.1 ) pendant 3 heures à 37°C; la structure des fragments obtenus est :- either by enzymatic digestion with β-glucuronidase (extracted from bovine liver - EC 3.1.3.1) for 3 hours at 37 ° C; the structure of the fragments obtained is:
GlcNAc-(GlcUA-GlcNAc)n avec n = 5, 6, 7, 8 ou 9.GlcNAc- (GlcUA-GlcNAc) n with n = 5, 6, 7, 8 or 9.
- soit par hydrolyse chimique pour obtenir des oligosaccharides de structure : (GlcUA-GlcNAc)n- GIcUA avec n = 5, 6, 7, 8 ou 9.- Either by chemical hydrolysis to obtain oligosaccharides with a structure: (GlcUA-GlcNAc) n- GIcUA with n = 5, 6, 7, 8 or 9.
Des fragments de même structure générale mais de masse moléculaire plus élevée, comprenant 10 à 49 unités disaccharidiques, sont obtenus par digestion enzymatique ou chimique selon les mêmes principes.Fragments of the same general structure but of higher molecular mass, comprising 10 to 49 disaccharide units, are obtained by enzymatic or chemical digestion according to the same principles.
L'exemple suivant illustre la présente invention.The following example illustrates the present invention.
1 ) Préparation des fragments1) Preparation of the fragments
Les fragments d'oligosaccharides obtenus selon la technique décrite par P. Bertrand et B. Delpech (J. Neurochem. 1985; 45(2):434 - 439) au cours d'une hydrolyse de 6 heures à 37°C en présence de hyaluronidase, sont majoritairement des fragments comprenant 3, 4, 5 et 6 unités disaccharidiques. Ainsi, en partant d'une masse d'acide hyaluronique naturel égale à 100 mg, l'incubation avec la hyaluronidase testiculaire bovine (10 mg - EC 3.2.1.35) permet d'obtenir une masse de mélange de fragments, comprenant de 3 à 8 unités disaccharidiques - égale à 36,33 mg - soit un rendement utile d'environ 36 %. Cet ensemble est caractérisé par la répartition suivante, exprimée en nombre d'unités disaccharidiques (acide D-glucuronique + N-acétyl D- glucosamine) [soit (GlcUA-GlcNAc)n] :The oligosaccharide fragments obtained according to the technique described by P. Bertrand and B. Delpech (J. Neurochem. 1985; 45 (2): 434 - 439) during a 6 hour hydrolysis at 37 ° C in the presence of hyaluronidase, are mainly fragments comprising 3, 4, 5 and 6 disaccharide units. Thus, starting from a mass of natural hyaluronic acid equal to 100 mg, the incubation with bovine testicular hyaluronidase (10 mg - EC 3.2.1.35) makes it possible to obtain a mass of mixture of fragments, comprising from 3 to 8 disaccharide units - equal to 36.33 mg - or a useful yield of around 36%. This set is characterized by the following distribution, expressed in number of disaccharide units (D-glucuronic acid + N-acetyl D- glucosamine) [either (GlcUA-GlcNAc) n ]:
• fragments à 3 unités disaccharidiques• fragments with 3 disaccharide units
9,42 mg soit 25,93 % • fragments à 4 unités disaccharidiques :9.42 mg or 25.93% • fragments with 4 disaccharide units:
6,57 mg soit 18,08 %6.57 mg or 18.08%
• fragments à 5 unités disaccharidiques :• fragments with 5 disaccharide units:
7,35 mg soit 20,23 %7.35 mg or 20.23%
• fragments à 6 unités disaccharidiques : 5,92 mg soit 16,30 %• fragments with 6 disaccharide units: 5.92 mg or 16.30%
• fragments à 7 unités disaccharidiques :• fragments with 7 disaccharide units:
3,73 mg soit 10,27 %3.73 mg or 10.27%
• fragments à 8 unités disaccharidiques :• fragments with 8 disaccharide units:
2,80 mg soit 7,71 %2.80 mg or 7.71%
Le sous-ensemble, considéré comme présentant la meilleure biodisponibilité, donc la plus grande incorporation dans le tissu artériel, est le sous-ensemble de fragments à 3, 4, 5 et 6 unités disaccharidiques. Dans le mélange défini ci-dessus, ce sous-ensemble représente 80,54 % du total, les fragments à 7 et 8 unités disaccharidiques ne représentant que 10,3 et 7,7% du total, respectivement. La masse moléculaire moyenne (MW ≈ 2000 Da) de ce mélange résulte de la contribution prépondérante dans le mélange de fragments à 3, 4, 5 et 6 unités disaccharidiques.The subset, considered to have the best bioavailability, and therefore the greatest incorporation into arterial tissue, is the subset of fragments with 3, 4, 5 and 6 disaccharide units. In the mixture defined above, this subset represents 80.54% of the total, the fragments with 7 and 8 disaccharide units representing only 10.3 and 7.7% of the total, respectively. The average molecular mass (MW ≈ 2000 Da) of this mixture results from the preponderant contribution in the mixture of fragments with 3, 4, 5 and 6 disaccharide units.
2) Effet des fragments d' acide hyaluronique sur l'épaississement artériel induit par l'angioplastie2) Effect of fragments of hyaluronic acid on arterial thickening induced by angioplasty
L'effet des fragments de bas poids moléculaire d' acide hyaluronique, obtenus après dégradation par la hyaluronidase a été étudié, sur la sténose de l'aorte de rat qui a subi une angioplastie. Le protocole adopté était le suivant : 2 injections de 30 mg/kg le jour de l'angioplastie (la première par voie veineuse en bolus 10 min avant le geste endoluminal et la seconde par voie sous-cutanée), puis une injection sous-cutanée tous les deux jours dès J2 jusqu'à J12. Trois groupes de rats ont été étudiés : un groupe témoin opération (JO, pas d'angioplastie), un groupe témoin ayant subi l'angioplastie mais ne recevant que du NaCI 0,9% contenant la hyaluronidase préalablement détruite (J14 Placebo) et un groupe ayant subi l'angioplastie et recevant le mélange d'oligosaccharides [acide hyaluronique dégradé par la hyaluronidase] (J14 FRAGAH); la préparation d'oligosaccharides par dégradation enzymatique de I' acide hyaluronique incluant une étape de destruction de la hyaluronidase avant injection aux animaux). Les résultats de cette étude sont résumés dans le tableau I.The effect of the low molecular weight fragments of hyaluronic acid obtained after degradation by hyaluronidase has been studied on the stenosis of the rat aorta which has undergone angioplasty. The protocol adopted was as follows: 2 injections of 30 mg / kg on the day of the angioplasty (the first by the venous route bolus 10 min before the endoluminal procedure and the second by the subcutaneous route), then a subcutaneous injection every two days from D2 to D12. Three groups of rats were studied: an operation control group (OJ, no angioplasty), a control group having undergone angioplasty but receiving only 0.9% NaCl containing the previously destroyed hyaluronidase (D14 Placebo) and a group having undergone angioplasty and receiving the mixture of oligosaccharides [hyaluronic acid degraded by hyaluronidase] (D14 FRAGAH); the preparation of oligosaccharides by enzymatic degradation of hyaluronic acid including a step of destruction of the hyaluronidase before injection into animals). The results of this study are summarized in Table I.
Tableau ITable I
Figure imgf000010_0001
Figure imgf000010_0001
Les valeurs sont exprimées en moyennes ± ESM. J0 : groupe de rats n'ayant pas subi l'angioplastie. J14Placebo : groupe de rats contrôle ayant subi l'angioplastie et ayant reçu du NaCI 0,9% contenant la hyaluronidase préalablement détruite. J14 FragAH : groupe de rats ayant subi l'angioplastie et ayant reçu I' acide hyaluronique dégradé par la hyaluronidase (30 mg/kg toutes les 48 h, la hyaluronidase a été également détruite avant l'injection). Le test de Mann-Whitney a été utilisé. a' b' °' e signalent les différences entre les J14 FragAH et les J14Placebo (a 2p = 0,00005, b 2p = 0,0009, ° 2p = 0,01 , d 2p = 0,017, e2p = 0,03).Values are expressed as means ± ESM. D0: group of rats not having undergone angioplasty. D14Placebo: group of control rats having undergone angioplasty and having received 0.9% NaCl containing the hyaluronidase previously destroyed. D14 FragAH: group of rats having undergone angioplasty and having received hyaluronic acid degraded by hyaluronidase (30 mg / kg every 48 h, the hyaluronidase was also destroyed before the injection). The Mann-Whitney test was used. a ' b ' ° ' e indicate the differences between J14 FragAH and J14 Placebo ( a 2p = 0.00005, b 2p = 0.0009, ° 2p = 0.01, d 2p = 0.017, e 2p = 0.03 ).
Ces résultats montrent que le traitement par des fragments d'acide hyaluronique réduit de manière significative l'épaississement artériel induit par l'angioplastie, comme en témoigne la baisse significative du poids frais de l'aorte et du contenu en ADN de l'intima-média. En effet la prolifération néointimale induite par l'angioplastie est significativement réduite (poids frais de l'aorte : 17 ± 2,4% versus 32,6 ± 3,2%, 2p = 0,0009 ; contenu en ADN : 19,2 ± 3,1 % versus 32,6 ± 4,2%, 2p = 0,017) par administration de fragments d'acide hyaluronique sans modification de l'état général des rats (poids corporel similaire, absence d'effets indésirables).These results show that treatment with fragments of hyaluronic acid significantly reduces arterial thickening induced by angioplasty, as evidenced by the significant reduction in the fresh weight of the aorta and the DNA content of the intestinal media. Indeed the neointimal proliferation induced by angioplasty is significantly reduced (fresh weight of the aorta: 17 ± 2.4% versus 32.6 ± 3.2%, 2p = 0.0009; DNA content: 19.2 ± 3.1% versus 32.6 ± 4.2%, 2p = 0.017) by administration of fragments of hyaluronic acid without modification of the general condition of the rats (similar body weight, absence of undesirable effects).
La mesure du taux sérique d'acide hyaluronique et/ou de ses fragments à J1 confirme la présence d'un taux élevé d'acide hyaluronique dès J1 (x11 par rapport aux animaux témoins), comme l'a montré une étude de pharmacocinétique préalable.Measuring the serum level of hyaluronic acid and / or its fragments on D1 confirms the presence of a high level of hyaluronic acid from D1 (x11 relative to the control animals), as shown by a prior pharmacokinetic study .
Ces résultats montrent que l'utilisation de fragments de bas poids moléculaires d'acide hyaluronique, chez le rat, limite la prolifération néointimale survenant après une angioplastie. L'originalité de cette étude tient au fait que l'on a utilisé des fragments de bas poids moléculaire d'acide hyaluronique (4 à 12 unités saccharidiques) dans cette indication. Les travaux de RC. Savani et EA. Turley (Int. J. Tiss. React.1995, XVII(4) 141-151 ) suggéraient que l'acide hyaluronique non fractionné pourrait être utile dans la prévention de la resténose post-angioplastie. Cependant, leurs résultats n'ont pas pu être reproduits probablement du fait de la variabilité interindividuelle (qui peut-être elle-même liée à la très forte viscosité de l'acide hyaluronique non fractionné) des paramètres pharmacocinétiques lors de l'utilisation de glycosaminoglycanes de haut poids moléculaire. Les résultats comparatifs sont donnés dans le Tableau II. Tableau IIThese results show that the use of low molecular weight fragments of hyaluronic acid, in rats, limits the neointimal proliferation occurring after angioplasty. The originality of this study is due to the fact that low molecular weight fragments of hyaluronic acid (4 to 12 saccharide units) have been used in this indication. RC works. Savani and EA. Turley (Int. J. Tiss. React. 1995, XVII (4) 141-151) suggested that unfractionated hyaluronic acid could be useful in the prevention of post-angioplasty restenosis. However, their results could not be reproduced probably due to the interindividual variability (which may itself be linked to the very high viscosity of unfractionated hyaluronic acid) of the pharmacokinetic parameters when using glycosaminoglycans high molecular weight. The comparative results are given in Table II. Table II
Figure imgf000012_0001
Figure imgf000012_0001
Les valeurs sont exprimées en moyennes ± ESM. J0 : groupe de rats n'ayant pas subi l'angioplastie. J14 Témoins: groupe de rats contrôle ayant subi l'angioplastie. J14 Healon® : groupe de rats ayant subi l'angioplastie et ayant reçu l'acide hyaluronique non fractionné (Healon®). ND = Non Détecté. Le test de Mann-Whitney a été utilisé. * signale la différence entre les groupes angioplastie (J14 et J14Healon®) et le groupe J0 (* 2p < 0,01 ). s signale la différence entre le groupe J14Healon® et le groupe J14 (1t2p = 0,016).Values are expressed as means ± ESM. D0: group of rats not having undergone angioplasty. D14 Witnesses: group of control rats having undergone angioplasty. D14 Healon®: group of rats having undergone angioplasty and having received unfractionated hyaluronic acid (Healon®). ND = Not Detected. The Mann-Whitney test was used. * indicates the difference between the angioplasty groups (D14 and J14Healon®) and the D0 group ( * 2p <0.01). s indicates the difference between the J14Healon® group and the J14 group ( 1t 2p = 0.016).
Les applications thérapeutiques de ces fragments d'acide hyaluronique ont comme principe général, de lutter contre la prolifération néo-intimale et donc de prévenir, en premier lieu, la resténose post-angioplastie transluminale.Therapeutic applications of these fragments of hyaluronic acid have as a general principle, to fight against neointimal proliferation and therefore to prevent, in the first place, post-angioplasty transluminal restenosis.
Si après une angioplastie coronaire - application la plus fréquente de l'angioplastie transluminale - les complications aiguës sont dues à la formation d'un thrombus artériel au site de la dilatation, les resténoses post-angioplasties sont des complications retardées qui apparaissent chez 20 à 30 % des patients traités par angioplastie. Ces resténoses traduisent l'hyperplasie intimale et le remodelage vasculaire qui apparaissent au site de l'angioplastie (T. Kimura et al., Circulation 1997 ; GS. Mintz et al. Am J Cardiol, 1996 ; GS. Mintz et al., Circulation 1996). La réocclusion vasculaire, immédiate ou retardée, touche 6,8 à 8,3 % des patients traités par angioplastie coronaire (PJ. de Feyter et al., Circulation 1991 ; KM. Detre et al., Circulation 1990 ; AM. Lincoff et al., Circulation 1992) et on sait qu'il faut ré-intervenir une seconde fois (par angioplastie par ballonnement intra-coronaire) dans 4,5% des cas (The Epie Investigators, New. Eng. J. Med., 1994). A ce jour, aucun traitement médical n'a permis de réduire la fréquence des resténoses post angioplastie (JJ. Popma et al., Circulation, 1991 ; JR Herrman et al., Drugs, 1993 ; J. Lefkovits et EJ. Topol, Prog Cardiovasc Dis, 1997). Même si la pose d'un stent (endoprothèse artérielle) réduit la fréquence des resténoses (MP. Savage et al., N Engl J Med, 1997 ; DL. Fischman et al., N Engl J Med, 1994), un traitement destiné à bloquer la néo- prolifération intimale pourra améliorer le pronostic global de réussite des procédures de cardiologie interventionnelle. C'est un problème de santé publique important puisque, rien qu'aux Etats-Unis, plus de 600 000 stents coronaires ont été posés en 1998 chez des patients présentant des sténoses des artères coronaires. Sans que cette description soit limitative, ces applications pourront donc être mises en œuvre soit à l'aide du mélange de fragments d'acide hyaluronique décrits ci-dessus, soit d'un sous-ensemble comme, par exemple, celui constitué par les fragments comprenant 5 et 6 unités disaccharidiques ou par les nonasaccharides utilisés individuellement. L'indication thérapeutique privilégiée des fragments d'acide hyaluronique tels que définis ci-dessus sera donc la prévention des phénomènes resténotiques consécutifs à une angioplastie, dont l'intérêt médical est parfaitement démontré aujourd'hui, à côté des techniques chirurgicales de pontage aorto-coronaires, comme technique de revascularisation myocardique non chirurgicale assurant la dilatation d'une (ou de plusieurs) sténose(s) artérielle(s) coronaire(s) après abord percutané. L'injection sous-cutanée de fragments d'acide hyaluronique de bas poids moléculaire sera donc utile aux patients atteints d'angine d'effort stable, à ceux souffrant d'angor instable ou chez ceux dont l'infarctus est en voie de constitution, dès lors que l'indication d'angioplastie transluminale coronaire est posée. Le traitement par les fragments d'acide hyaluronique de bas poids moléculaire sera également efficace après pose d'un stent, pour éviter la prolifération néo-intimale consécutive à l'abrasion de l'intima artérielle, et après endartériectomie.If after coronary angioplasty - the most frequent application of transluminal angioplasty - acute complications are due to the formation of an arterial thrombus at the site of dilation, post-angioplasty restenoses are delayed complications which appear in 20 to 30 % of patients treated with angioplasty. These restenoses reflect intimal hyperplasia and vascular remodeling which appear at the site of the angioplasty (T. Kimura et al., Circulation 1997; GS. Mintz et al. Am J Cardiol, 1996; GS. Mintz et al., Circulation 1996). Vascular reocclusion, immediate or delayed, affects 6.8 to 8.3% of patients treated with coronary angioplasty (PJ. De Feyter et al., Circulation 1991; KM. Detre et al., Circulation 1990; AM. Lincoff et al ., Circulation 1992) and we know that it is necessary to re-intervene a second time (by angioplasty by intra-coronary bloating) in 4.5% of cases (The Epie Investigators, New. Eng. J. Med., 1994) . To date, no medical treatment has reduced the frequency of post-angioplasty restenosis (JJ. Popma and al., Circulation, 1991; JR Herrman et al., Drugs, 1993; J. Lefkovits and EJ. Topol, Prog Cardiovasc Dis, 1997). Even if the placement of a stent (arterial stent) reduces the frequency of restenosis (MP. Savage et al., N Engl J Med, 1997; DL. Fischman et al., N Engl J Med, 1994), a treatment intended blocking intimal neoliferation can improve the overall prognosis for the success of interventional cardiology procedures. It is a major public health problem since, in the United States alone, more than 600,000 coronary stents were placed in 1998 in patients with stenoses of the coronary arteries. Without this description being limiting, these applications can therefore be implemented either using the mixture of fragments of hyaluronic acid described above, or of a subset such as, for example, that constituted by the fragments comprising 5 and 6 disaccharide units or by the nonasaccharides used individually. The preferred therapeutic indication for fragments of hyaluronic acid as defined above will therefore be the prevention of restenotic phenomena consecutive to angioplasty, the medical benefit of which is perfectly demonstrated today, alongside surgical techniques for aortic bypass coronaries, as a non-surgical myocardial revascularization technique ensuring dilation of one (or more) coronary arterial stenosis (s) after percutaneous approach. Subcutaneous injection of fragments of low molecular weight hyaluronic acid will therefore be useful for patients with stable exertional angina, those suffering from unstable angina or those whose infarction is in the process of being formed, as soon as the indication for coronary transluminal angioplasty is made. Treatment with fragments of low molecular weight hyaluronic acid will also be effective after placement of a stent, to avoid neointimal proliferation following abrasion of the intestinal artery, and after endarterectomy.
Les fragments d'acide hyaluronique de la présente invention sont généralement administrés en unités de dosage établies soit par m2 de surface corporelle, soit par kg de poids. Les dites unités de dosage sont de préférence formulées dans des compositions pharmaceutiques dans lesquelles le principe actif est mélangé avec un (ou plusieurs) excipient(s) pharmaceutique(s).The hyaluronic acid fragments of the present invention are generally administered in dosage units established either per m 2 of body surface area, or per kg of weight. Said dosage units are preferably formulated in pharmaceutical compositions in which the active principle is mixed with one (or more) pharmaceutical excipient (s).
Ainsi, selon un autre de ses aspects, la présente invention concerne des compositions pharmaceutiques renfermant, en tant que principe actif, des fragments d'acide hyaluronique sous un de ses sels pharmaceutiquement acceptables (sodium, potassium, calcium).Thus, according to another of its aspects, the present invention relates to pharmaceutical compositions containing, as active principle, fragments of hyaluronic acid under one of its pharmaceutically acceptable salts (sodium, potassium, calcium).
Les fragments d'acide hyaluronique ci-dessus peuvent être utilisés selon la pathologie du sujet à traiter à des doses comprises entre 0.5 et 50 mg/kg de poids corporel et par jour ou 20 à 2000 mg/m2 de surface corporelle et par jour. La posologie pourra être avantageusement modulée en fonction du degré d'urgence de la procédure de cardiologie interventionnelle [intervention d'urgence (ex. angioplastie coronaire transluminale percutanée) ou réglée (pose d'une endoprothèse artérielle de type stent)]. En pratique d'urgence, le traitement pourra combiner un bolus intraveineux avant l'intervention, une perfusion intraveineuse continue pendant l'intervention et les 24 heures suivantes puis l'injection sous-cutanée quotidienne pendant 1 à 4 semaines après l'intervention. Ainsi, pour le traitement dans sa phase aiguë, les doses pourront être de 15 à 50 mg/kg/jour (soit 600 à 2000 mg/m2/jour) ; pour un traitement d'entretien, on utilisera les fragments d' acide hyaluronique à des doses de 0.5 à 15 mg/kg/jour (soit 20 à 600 mg/m2/jour), de préférence, à des doses de 1 à 10 mg/kg/jour (soit 40 à 400 mg/m2/jour).The above hyaluronic acid fragments can be used according to the pathology of the subject to be treated at doses of between 0.5 and 50 mg / kg of body weight per day or 20 to 2000 mg / m 2 of body surface per day . The dosage may advantageously be adjusted according to the degree of urgency of the interventional cardiology procedure [emergency intervention (eg percutaneous transluminal coronary angioplasty) or adjusted (placement of a stent-type arterial stent)]. In emergency practice, treatment may combine an intravenous bolus before the intervention, a continuous intravenous infusion during the intervention and the following 24 hours, then daily subcutaneous injection for 1 to 4 weeks after the intervention. Thus, for treatment in its acute phase, the doses may be from 15 to 50 mg / kg / day (ie 600 to 2000 mg / m 2 / day); for maintenance treatment, the fragments of hyaluronic acid will be used at doses of 0.5 to 15 mg / kg / day (i.e. 20 to 600 mg / m 2 / day), preferably at doses of 1 to 10 mg / kg / day (i.e. 40 to 400 mg / m 2 / day).
Dans les compositions pharmaceutiques de la présente invention pour l'administration par voies intraveineuse ou sous-cutanée les principes actifs peuvent être administrés sous formes unitaires d'administration, en mélange avec des supports pharmaceutiques classiques adaptés à la thérapeutique humaine. Les formes unitaires d'administration appropriées comprennent les formes d'administration intraveineuse (solutions prêtes à l'emploi pour bolus iv et/ou se; soluté pour perfusion iv).In the pharmaceutical compositions of the present invention for administration by the intravenous or subcutaneous routes, the active principles can be administered in unit administration forms, in mixture with conventional pharmaceutical carriers suitable for human therapy. Suitable unit administration forms include intravenous administration forms (ready-to-use solutions for iv bolus and / or se; solution for iv infusion).
Pour une administration parentérale (perfusion intraveineuse à débit constant), on utilise des solutions aqueuses salines isotoniques et stériles qui pourront contenir des excipients pharmacologiquement compatibles. For parenteral administration (intravenous infusion at constant flow rate), isotonic and sterile aqueous saline solutions are used which may contain pharmacologically compatible excipients.

Claims

REVENDICATIONS
1. Utilisation, pour la fabrication d'un médicament destiné à limiter la prolifération néo-intimale consécutive à un . trauma vasculaire, d'un fragment d'acide hyaluronique comprenant de 4 à 100 motifs monosaccharidiques ou d'un de ses sels pharmaceutiquement acceptables.1. Use, for the manufacture of a medicament intended to limit the neointimal proliferation consecutive to one. vascular trauma, of a hyaluronic acid fragment comprising from 4 to 100 monosaccharide units or of a pharmaceutically acceptable salt thereof.
2. Utilisation selon la revendication 1 , pour la fabrication d'un médicament destiné à limiter la prolifération néo-intimale consécutive à une angioplastie transluminale.2. Use according to claim 1, for the manufacture of a medicament intended to limit the neointimal proliferation consecutive to a transluminal angioplasty.
3. Utilisation selon la revendication 1 ou la revendication 2 d'un fragment d'acide hyaluronique de formule :3. Use according to claim 1 or claim 2 of a fragment of hyaluronic acid of formula:
(GlcUA-GlcNAc)n1 I avec n de 2 à 8.(GlcUA-GlcNAc) n1 I with n from 2 to 8.
4. Utilisation selon la revendication 1 ou la revendication 2 d'un fragment d'acide hyaluronique de formule : GlcNac-(GlcUA-GlcNAc)4 lia ou (GlcUA-GlcNac)4-GlcUA llb4. Use according to claim 1 or claim 2 of a hyaluronic acid fragment of formula: GlcNac- (GlcUA-GlcNAc) 4 lia or (GlcUA-GlcNac) 4 -GlcUA llb
5. Utilisation selon la revendication 1 ou la revendication 2 d'un fragment d'acide hyaluronique de formule :5. Use according to claim 1 or claim 2 of a hyaluronic acid fragment of formula:
GlcNac-(GlcUA-GlcNAc)n2 III avec n2 de 5 à 9.GlcNac- (GlcUA-GlcNAc) n2 III with n 2 from 5 to 9.
6. Utilisation selon la revendication 1 ou la revendication 2 d'un fragment d'acide hyaluronique de formule :6. Use according to claim 1 or claim 2 of a fragment of hyaluronic acid of formula:
(GlcUA-GlcNAc)n3-GlcUA IV avec n3 de 5 à 9. (GlcUA-GlcNAc) n3 -GlcUA IV with n 3 from 5 to 9.
7. Utilisation selon la revendication 1 ou la revendication 2 d'un fragment d'acide hyaluronique de formule :7. Use according to claim 1 or claim 2 of a hyaluronic acid fragment of formula:
(GlcUA-GlcNAc)n4 V avec n4 = 9 à 50. (GlcUA-GlcNAc) n4 V with n 4 = 9 to 50.
8. Utilisation selon la revendication 1 ou la revendication 2 d'un fragment d'acide hyaluronique de formule :8. Use according to claim 1 or claim 2 of a hyaluronic acid fragment of formula:
GlcNac-(GlcUA-GlcNAc)n5 Via ou (GlcUA-GlcNac)n5-GlcUA Vlb avec n5 = 10 à 49.GlcNAc (GlcUA-GlcNAc) n5 or Via (GlcUA-GlcNAc) n5 -GlcUA Vlb with n 5 = 10 to 49.
9. Composition pharmaceutique destinée à limiter la prolifération intimale consécutive à un trauma vasculaire, comprenant une dose efficace d'un fragment d'acide hyaluronique de formule : (GlcUA-GlcNAc)n1 I avec n1 = 2 à 8.9. Pharmaceutical composition intended to limit intimal proliferation following a vascular trauma, comprising an effective dose of a fragment of hyaluronic acid of formula: (GlcUA-GlcNAc) n1 I with n 1 = 2 to 8.
10. Composition pharmaceutique destinée à limiter la prolifération intimale consécutive à un trauma vasculaire, comprenant une dose efficace d'un fragment d'acide hyaluronique de formule : (GlcUA-GlcNAc)n3-GlcUA IV avec n3 = 4 à 9.10. Pharmaceutical composition intended to limit intimal proliferation following a vascular trauma, comprising an effective dose of a fragment of hyaluronic acid of formula: (GlcUA-GlcNAc) n3 -GlcUA IV with n 3 = 4 to 9.
1 1. Composition pharmaceutique destinée à limiter la prolifération intimale consécutive à un trauma vasculaire, comprenant une dose efficace d'un fragment d'acide hyaluronique de formule : (GlcUA-GlcNAc)n4 V avec n4 = 9 à 50.1 1. Pharmaceutical composition intended to limit intimal proliferation following a vascular trauma, comprising an effective dose of a fragment of hyaluronic acid of formula: (GlcUA-GlcNAc) n4 V with n 4 = 9 to 50.
12. Composition pharmaceutique destinée à limiter la prolifération intimale consécutive à un trauma vasculaire, comprenant une dose efficace d'un fragment d'acide hyaluronique de formule : GlcNac-(GlcUA-GlcNAc)n5 Via ou (GlcUA-GlcNac)n5-GlcUA Vlb avec n5 = 10 à 49. 12. A pharmaceutical composition for limiting consecutive intimal proliferation in a vascular trauma, comprising an effective dose of a hyaluronic acid fragment of the formula: GlcNAc (GlcUA-GlcNAc) n5 or Via (GlcUA-GlcNAc) n5 -GlcUA Vlb with n 5 = 10 to 49.
PCT/FR2000/001178 1999-05-03 2000-05-02 Use of fragments of hyaluronic acid WO2000066132A1 (en)

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FR9905611A FR2793140B1 (en) 1999-05-03 1999-05-03 USE OF HYALURONIC ACID FRAGMENTS

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Publication number Priority date Publication date Assignee Title
CN108303386A (en) * 2017-12-15 2018-07-20 浙江景嘉医疗科技有限公司 A kind of detection method of the external enzymatic hydrolyzation of medical cross-linking sodium hyaluronate gel

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