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EP0779814A1 - Antithrombotic and non-hemorrhagic heparin-based compositions, method for their preparation and therapeutic applications - Google Patents

Antithrombotic and non-hemorrhagic heparin-based compositions, method for their preparation and therapeutic applications

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Publication number
EP0779814A1
EP0779814A1 EP95918118A EP95918118A EP0779814A1 EP 0779814 A1 EP0779814 A1 EP 0779814A1 EP 95918118 A EP95918118 A EP 95918118A EP 95918118 A EP95918118 A EP 95918118A EP 0779814 A1 EP0779814 A1 EP 0779814A1
Authority
EP
European Patent Office
Prior art keywords
heparin
protamine
composition according
activity
fractions
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.)
Withdrawn
Application number
EP95918118A
Other languages
German (de)
French (fr)
Inventor
Christian Raymond Doutremepuich
François Eugène Pierre Marie SAUDUBRAY
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Debiopharm SA
Original Assignee
Debiopharm SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Debiopharm SA filed Critical Debiopharm SA
Publication of EP0779814A1 publication Critical patent/EP0779814A1/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K31/727—Heparin; Heparan
    • 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

Definitions

  • the present invention relates to heparin-based compositions as well as their preparation process and their therapeutic applications.
  • compositions based on heparins neutralized by protamine exhibiting antithrombotic activity but substantially devoid of hemorrhagic and anticoagulant activities.
  • Heparin has been known and used for many decades for the preparation of drugs with antithrombotic and / or anticoagulant activity intended in particular for the preventive and curative treatment of venous and arterial thromboses or also to prevent the coagulation in the extra circulation bodily.
  • heparin which are in particular contraindicated in patients predisposed to haemorrhage, patients suffering from duodenal or gastric ulcers or patients having undergone recent intervention. in which antithrombotic treatment with heparin can cause hemorrhages.
  • the treatment uses protamine sulphate which causes the heparin to neutralize in vivo.
  • the object of the invention is more precisely to eliminate as much as possible the risk of hemorrhage from heparins while retaining their main properties, in particular the antithrombotic activity.
  • the object of the present invention is to provide heparin compositions which have very advantageous pharmacological properties, in particular antithrombotic, substantially equivalent to those of the heparins used hitherto, without having the major drawback which lies in the significant bleeding risk.
  • Another object of the present invention is to provide a process for the preparation of such compositions which is simple to carry out and inexpensive, making it possible moreover to develop their therapeutic applications.
  • the invention also relates to the therapeutic applications of these compositions.
  • heparin compositions according to the invention which exhibit antithrombotic activity and are practically devoid of hemorrhagic activity. These compositions are characterized in that they essentially consist of heparin fractions as obtained by neutralization in vi- heparin by protamine.
  • heparin fraction neutralized by protamine means any fraction originating from a native or already fractionated heparin, or from a synthetic heparin, the haemorrhagic power of which has been neutralized by the action of protamine or any analog or equivalent thereof having a similar ability to reduce hemorrhagic power.
  • compositions according to the invention advantageously consist of heparin fractions as obtained by the in vitro neutralization of an unfractionated heparin or of a low molecular weight heparin with protamine.
  • the composition consists of heparin fractions of which 25% have a molecular weight of less than 2.5 kDa and 40% have a molecular weight of more than 20 kDa,
  • the composition consists only of heparin fractions having a molecular mass of less than 2.5 kDa.
  • the heparin fractions have a spectrum of molecular weights depending on the protamine neutralization modes that are used.
  • compositions according to the invention are substantially free of protamine.
  • the invention also provides a process for the preparation of the above-mentioned compositions, characterized in that it comprises a step of in vitro neutralization of hepatic rine by protamine.
  • the inventors have discovered that, surprisingly, it is possible to neutralize in vitro, in particular with the aid of protamine, the hemorrhagic activity of heparin while preserving its antithrombotic properties.
  • the method according to the invention consists in reacting, in solution, a heparin with protamine, in particular in the form of the protamine salt, according to variable heparin / protamine ratios.
  • a heparin solution is mixed with a protamine salt solution, preferably at room temperature, the mixture obtained is centrifuged and the supernatant is recovered.
  • heparin solution is meant a solution of native or already fractionated heparin, or synthetic heparin.
  • the protamine salt advantageously consists of protamine sulfate.
  • any analog or equivalent of protamine having a similar capacity, neutralizing heparin and therefore reducing hemorrhagic power, can be used.
  • the supernatant can then be lyophilized.
  • the heparin to be treated and the protamine can be used in different reports which essentially lead to the elimination of the risk of haemorrhage linked to heparins.
  • the method comprises the step of neutralizing a heparin with protamine or an equivalent, preferably in heparin / protamine proportions of 2/1 to 1/2.
  • the heparin / protamine ratio is of the order of 1/1.
  • heparin compositions are obtained comprising fractions of which at least 25% have a molecular weight of less than 2.5 kDa and at least 40% have a molecular weight of more than 20 kDa.
  • the heparin / protamine ratio is of the order of 1/2.
  • Heparin compositions are then obtained essentially comprising fractions having a molecular mass of less than 2.5 kDa,
  • heparin compositions free of protamine are obtained.
  • heparin compositions of the invention have made it possible to demonstrate, in a surprising manner, that they are practically devoid of hemorrhagic activity and at the same time retain their antithrombotic property.
  • compositions according to the invention were capable of inhibiting the hydrolytic activity of human leukocyte elastase, more effectively than unfractionated heparin.
  • the elimination of the risk of haemorrhage, in accordance with the invention makes it possible to envisage administration by the parenteral route or by bronchopulmonary aerosol, in the treatment of certain bronchopulmonary conditions which may involve an excess of leukocytic elastase, such as acute respiratory distress syndromes, cystic fibrosis, chronic obstructive pulmonary disease.
  • heparin compositions according to the invention which are stable and non-toxic, can be used for the preparation of medicaments useful in various therapeutic applications. These applications are those of heparin and its conventional derivatives, including cases where heparin is contraindicated because of the risk of bleeding that the patient presents. They can be used in particular for the preparation of medicaments for the treatment and prevention of venous or arterial thromboses or else to prevent activation of coagulation in the extracorporeal circulation.
  • the invention therefore also relates to pharmaceutical compositions comprising an amount therapeutically effective heparin composition according to the invention as described above, in combination with a pharmaceutically acceptable vehicle.
  • They may, for example, be antithrombotic pharmaceutical compositions or else for the inhibition of the hydrolytic activity of human leukocyte elastase.
  • heparin fractions of these compositions can be in the form of a pharmaceutically acceptable salt according to conventional methods.
  • compositions according to the invention are advantageously injectable formulations intended in particular for administration by the parenteral route.
  • formulations suitable for administration by bronchopulmonary route are advantageously provided.
  • FIG. 1 is a comparative graph of the hemorrhagic activity of unfractionated heparin, low molecular weight heparin and non hemorrhagic heparin compositions according to the invention (S1, S2, S3);
  • FIG. 2 is a comparative graph of the antithrombotic activity of unfractionated heparin, low molecular weight heparin and heparin compositions according to the invention (S1, S2, S3).
  • the mixture thus obtained is centrifuged for 10 minutes and the supernatant is recovered which is lyophilized.
  • a solution diluted to 1/20 has two absorption peaks at the following wavelengths:
  • each bottle contains 0.7 ml of supernatant solution. 12 identical assays were carried out to verify the reproducibility: the results are given in Table III below:
  • the assay of the proteins in the supernatants S1 and S2, respectively prepared according to Examples 1 and 2, is carried out according to the Pierce method (Pierce laboratory reagent kit).
  • T0 ligation of the vena cava
  • the supernatant S1 has no effect on blood cells.
  • the supernatant S2 obtained by neutralization according to a heparin / protamine ratio of 1/2, does not exert any hemorrhagic activity but has a reduced antithrombotic activity.
  • FIG. 2 shows that the heparin fractions obtained according to the invention exhibit an interesting antithrombotic activity.
  • SI fraction heparin / protamine ratio of 1/1
  • this activity is comparable to that of heparins which are not neutralized in accordance with the invention.
  • results obtained show that, as a preventive measure, SI exerts an antithrombotic activity comparable to that of heparin and LOVENOX, 24 hours after the induction of thrombosis.
  • the excitation time is fixed at 2 minutes and the observation time at 10 minutes.
  • Duration embolization duration between the first and the embolus embolus ernier of detaching the clot.
  • Number of emboli number of emboli coming off the clot.
  • the supernatant S1 (example 1) exerts a significant antithrombotic activity compared to the placebo group, which persists after 90 minutes (TO + 90 min). This activity is more important than that of heparin injected at the same dose, after 30 and 60 minutes (TO + 30 and TO + 60 min).
  • the observation time is fixed at 10 minutes.
  • SI exerts an antithrombotic activity comparable to that of non-neutralized heparin injected at the same dose and reduces the number of embolisms as well as the duration of embolization in a statistically significant manner, b. Study 2
  • T0 + 6h induction of the third arterial thrombosis.
  • the observation time is fixed at 10 minutes.
  • SI exerts an antithrombotic activity comparable to that of heparin which is not neutralized by protamine.
  • the studies described above show that the antithrombotic activity is observed in the three experimental thrombosis models, namely the venous model induced by stasis, the arterial thrombosis model induced by free radicals and the arterial thrombosis model induced by endothelial laser injury.
  • the fraction of heparin obtained from a low molecular weight heparin has a greater antithrombotic activity than that of the same low molecular weight heparin not treated in in vitro by protamine and also greater than that of the fractions obtained from unfractionated heparins and not treated in vitro with protamine, while no longer presenting any risk of haemorrhage.
  • the method according to the invention allows, in a simple and inexpensive manner, practically to suppress the hemorrhagic activity of heparins while retaining their antithrombotic activity.

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  • General Health & Medical Sciences (AREA)
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  • General Chemical & Material Sciences (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

Heparin compositions having an antithrombotic activity and virtually no hemorrhagic activity. The object of the invention is to eliminate the risk of bleeding associated with heparins while retaining their main properties. The compositions of the invention (S1, S2, S3) therefore consist of heparin moieties such as those obtainable by the in vitro neutralization of a heparin with a protamine. The invention also concerns a method for the preparation of these compositions which are useful in preparing medicaments.

Description

COMPOSITIONS ANTITHROMBOTIQUES ET NON HEMORRAGIQUES A BASE D'HEPARINE, PROCEDE POUR LEUR PREPARATION ET APPLICATIONS ANTITHROMBOTIC AND NON-BLEEDING COMPOSITIONS BASED ON HEPARIN, PROCESS FOR THEIR PREPARATION AND APPLICATIONS
THERAPEUTIQUES La présente invention concerne des compositions à base d'héparine ainsi que leur procédé de préparation et leurs applications thérapeutiques.  THERAPEUTICS The present invention relates to heparin-based compositions as well as their preparation process and their therapeutic applications.
Elle concerne plus particulièrement des compositions à base d'héparines neutralisées par la protamine, présentant une activité antithrombotique mais sensiblement dépourvues d'activités hémorragique et anticoagulante.  It relates more particularly to compositions based on heparins neutralized by protamine, exhibiting antithrombotic activity but substantially devoid of hemorrhagic and anticoagulant activities.
Les héparines sont connues et utilisées depuis de nombreuses décennies pour la préparation de médicaments à activité antithrombotique et/ou anticoagulante destinés en particulier au traitement préventif et curatif des thromboses veineuses et artérielles ou encore pour prévenir l'actication de la coagulation dans les circulations extra-corporelles.  Heparin has been known and used for many decades for the preparation of drugs with antithrombotic and / or anticoagulant activity intended in particular for the preventive and curative treatment of venous and arterial thromboses or also to prevent the coagulation in the extra circulation bodily.
Depuis quelques années, on sait préparer des héparines de bas poids moléculaire, qui présentent toujours une activité antithrombotique mais dont les propriétés anticoagulantes sont réduites.  In recent years, it has been known to prepare heparins of low molecular weight, which still exhibit antithrombotic activity but whose anticoagulant properties are reduced.
Néanmoins, qu'il s'agisse d'héparines non fractionnées ou d'héparine de bas poids moléculaire, le risque hémorragique reste la complication majeure des traitements à base d'héparine. De ce fait, il existe une limitation importante à l'utilisation d'héparine qui sont en particulier contre-indiquées chez des patients prédisposés aux hémorragies, les patients souffrant d'ulcères duodé- naux ou gastriques ou encore des patients ayant subi récemment une intervention chirurgicale chez lesquels le traitement antithrombotique par héparine peut entraîner des hémorragies. However, whether it is unfractionated heparins or low molecular weight heparin, the risk of bleeding remains the major complication of heparin-based treatments. Therefore, there is a significant limitation to the use of heparin which are in particular contraindicated in patients predisposed to haemorrhage, patients suffering from duodenal or gastric ulcers or patients having undergone recent intervention. in which antithrombotic treatment with heparin can cause hemorrhages.
Par suite, les propriétés avantageuses des héparines, à savoir leur activité antithrombotique ou anticoagulante, ne peuvent être correctement mises à profit en raison de leurs effets secondaires importants liés à ce risque hémorragique permanent.  Consequently, the advantageous properties of heparins, namely their antithrombotic or anticoagulant activity, cannot be properly exploited because of their significant side effects linked to this permanent haemorrhagic risk.
Lorsque surviennent des hémorragies au cours des traitements par héparine, le traitement fait appel au sulfate de protamine qui provoque la neutralisation de l'héparine in vivo.  When hemorrhages occur during heparin treatments, the treatment uses protamine sulphate which causes the heparin to neutralize in vivo.
Bien, que la protamine soit ainsi utilisée depuis plusieurs années, on ne connaît pas bien le mécanisme de la neutralisation de l'héparine par la protamine. Une étude relativement récente a simplement montré que les héparines de bas poids moléculaire étaient neutralisées à un degré moindre par rapport aux héparines non fractionnées ("In Vitro Protamine Neutralization Profiles of Héparine Differing in Source and Molecular Weight", SEMINARS IN THROMBOSIS AND IN HEMOSTASIS, vol. 15 N°4, 1989), Le problème que vise à résoudre la présente invention est donc de réduire le risque hémorragique important exposé ci-dessus, limitant l'utilisation thérapeutique des héparines. Although protamine has been used in this way for several years, the mechanism of heparin neutralization by protamine is not well known. A relatively recent study simply showed that low molecular weight heparins were neutralized to a lesser degree compared to unfractionated heparins ("In Vitro Protamine Neutralization Profiles of Heparin Differing in Source and Molecular Weight", SEMINARS IN THROMBOSIS AND IN HEMOSTASIS, vol. 15 No. 4, 1989), The problem which the present invention aims to solve is therefore to reduce the significant bleeding risk set out above, limiting the therapeutic use of heparins.
Le but de l'invention est plus précisément de supprimer le plus possible le risque hémorragique des héparines tout en conservant leurs principales propriétés, en particulier l'activité antithrombotique.  The object of the invention is more precisely to eliminate as much as possible the risk of hemorrhage from heparins while retaining their main properties, in particular the antithrombotic activity.
Aussi, le but de la présente invention est de fournir des compositions d'héparines qui présentent des propriétés pharmacologiques très intéressantes, en particulier antithrombotique, sensiblement équivalentes à celles des héparines utilisées jusqu'alors, sans en présenter l'inconvénient majeur qui réside dans le risque hémorragique important.  Also, the object of the present invention is to provide heparin compositions which have very advantageous pharmacological properties, in particular antithrombotic, substantially equivalent to those of the heparins used hitherto, without having the major drawback which lies in the significant bleeding risk.
Un autre but de la présente invention est encore de fournir un procédé de préparation de telles compositions qui soit simple à mettre en oeuvre et peu coûteux, permettant en outre de développer leurs applications thérapeuti- ques.  Another object of the present invention is to provide a process for the preparation of such compositions which is simple to carry out and inexpensive, making it possible moreover to develop their therapeutic applications.
L'invention vise également les applications thérapeutiques de ces compositions.  The invention also relates to the therapeutic applications of these compositions.
Ces buts sont atteints à l'aide des compositions d'héparines selon l'invention qui présentent une activité antithrombotique et sont pratiquement dépourvues d'activité hémorragique. Ces compositions sont caractérisées par le fait qu'elles sont constituées essentiellement de fractions d'héparine telles qu'obtenues par la neutralisation in vi- tro d'héparine par la protamine. These aims are achieved using the heparin compositions according to the invention which exhibit antithrombotic activity and are practically devoid of hemorrhagic activity. These compositions are characterized in that they essentially consist of heparin fractions as obtained by neutralization in vi- heparin by protamine.
Par fraction d'héparine neutralisée par la protamine, on entend toute fraction provenant d'une héparine native ou déjà fractionnée, ou d'une héparine de synthèse, dont le pouvoir hémorragique a été neutralisé par l'action de la protamine ou de tout analogue ou équivalent de celle-ci ayant une capacité similaire de réduction du pouvoir hémorragique.  The expression heparin fraction neutralized by protamine means any fraction originating from a native or already fractionated heparin, or from a synthetic heparin, the haemorrhagic power of which has been neutralized by the action of protamine or any analog or equivalent thereof having a similar ability to reduce hemorrhagic power.
Les compositions selon l'invention sont avantageuse- ment constituées de fractions d'héparine telle qu'obtenues par la neutralisation in vitro d'une héparine non fractionnée ou d'une héparine de bas poids moléculaire, par la protamine.  The compositions according to the invention advantageously consist of heparin fractions as obtained by the in vitro neutralization of an unfractionated heparin or of a low molecular weight heparin with protamine.
Selon un mode de réalisation de l'invention, la composition est constituée de fractions d'héparine dont 25 % présentent une masse moléculaire inférieure à 2,5 kDa et 40 % présentent une masse moléculaire supérieure à 20 kDa, According to one embodiment of the invention, the composition consists of heparin fractions of which 25% have a molecular weight of less than 2.5 kDa and 40% have a molecular weight of more than 20 kDa,
Selon un autre mode de réalisation de l'invention, la composition est constituée uniquement de fractions d'hépa- rine présentant une masse moléculaire inférieure à 2,5 kDa. According to another embodiment of the invention, the composition consists only of heparin fractions having a molecular mass of less than 2.5 kDa.
Dans d'autres modes de réalisation les fractions d'héparine ont un spectre de masses moléculaires dépendant des modes de neutralisation par la protamine qu'on utilise.  In other embodiments the heparin fractions have a spectrum of molecular weights depending on the protamine neutralization modes that are used.
Les compositions conformes à l'invention sont sensi- blement exemptes de protamine.  The compositions according to the invention are substantially free of protamine.
L'invention fournit également un procédé pour la préparation des compositions précitées caractérisé par le fait qu'il comprend une étape de neutralisation in vitro d'hépa- rine par la protamine. The invention also provides a process for the preparation of the above-mentioned compositions, characterized in that it comprises a step of in vitro neutralization of hepatic rine by protamine.
Les inventeurs ont découvert que, d'une manière surprenante, on pouvait neutraliser in vitro, notamment à l'aide de la protamine, l'activité hémorragique de l'héparine tout en préservant ses propriétés antithrombotiques.  The inventors have discovered that, surprisingly, it is possible to neutralize in vitro, in particular with the aid of protamine, the hemorrhagic activity of heparin while preserving its antithrombotic properties.
D'une manière plus précise, le procédé selon l'invention consiste à faire réagir, en solution, une héparine avec la protamine, notamment sous la forme de sel de protamine, selon des rapports héparine/protamine variables.  More specifically, the method according to the invention consists in reacting, in solution, a heparin with protamine, in particular in the form of the protamine salt, according to variable heparin / protamine ratios.
Selon un mode de réalisation préférentiel de l'invention, on mélange une solution d'héparine avec une solution de sel de protamine, de préférence à la température ambiante, on centrifuge le mélange obtenu et on récupère le surnageant.  According to a preferred embodiment of the invention, a heparin solution is mixed with a protamine salt solution, preferably at room temperature, the mixture obtained is centrifuged and the supernatant is recovered.
Selon l'invention, par solution d'héparine, on entend une solution d'héparine native ou déjà fractionnée, ou d'héparine de synthèse.  According to the invention, by heparin solution is meant a solution of native or already fractionated heparin, or synthetic heparin.
Le sel de protamine consiste avantageusement en du sulfate de protamine.  The protamine salt advantageously consists of protamine sulfate.
Selon l'invention, on peut utiliser tout analogue ou équivalent de la protamine ayant une capacité similaire, neutralisation de l'héparine et donc de réduction du pouvoir hémorragique.  According to the invention, any analog or equivalent of protamine having a similar capacity, neutralizing heparin and therefore reducing hemorrhagic power, can be used.
Le surnageant peut être ensuite lyophilisé.  The supernatant can then be lyophilized.
L'héparine à traiter et la protamine peuvent être utilisées selon différents rapports qui conduisent essentiellement à l'élimination du risque hémorragique lié aux héparines. The heparin to be treated and the protamine can be used in different reports which essentially lead to the elimination of the risk of haemorrhage linked to heparins.
Le procédé comprend l'étape de neutralisation d'une héparine par la protamine ou un équivalent, de préférence dans des proportions héparine/protamine de 2/1 à 1/2.  The method comprises the step of neutralizing a heparin with protamine or an equivalent, preferably in heparin / protamine proportions of 2/1 to 1/2.
Selon un mode de réalisation de ce procédé, le rapport héparine/protamine est de l'ordre de 1/1. Dans ce cas, on obtient des compositions d'héparine comprenant des fractions dont au moins 25 % présentent une masse moléculaire inférieure à 2,5 kDa et au moins 40 % présentent une masse moléculaire supérieure à 20 kDa.  According to one embodiment of this method, the heparin / protamine ratio is of the order of 1/1. In this case, heparin compositions are obtained comprising fractions of which at least 25% have a molecular weight of less than 2.5 kDa and at least 40% have a molecular weight of more than 20 kDa.
Selon un autre mode de réalisation de l'invention, le rapport héparine/protamine est de l'ordre de 1/2. On obtient alors des compositions d'héparines comprenant essentiellement des fractions présentant une masse moléculaire inférieure à 2,5 kDa,  According to another embodiment of the invention, the heparin / protamine ratio is of the order of 1/2. Heparin compositions are then obtained essentially comprising fractions having a molecular mass of less than 2.5 kDa,
Selon le procédé conforme à la présente invention, on obtient des compositions d'héparines exemptes de protamine.  According to the process according to the present invention, heparin compositions free of protamine are obtained.
L'étude pharmacologique des compositions d'héparine de l'invention a permis de mettre en évidence, d'une manière surprenante, qu'elles sont pratiquement dépourvues d'activité hémorragique et conservent parallèlement leur propriété antithrombotique.  The pharmacological study of the heparin compositions of the invention has made it possible to demonstrate, in a surprising manner, that they are practically devoid of hemorrhagic activity and at the same time retain their antithrombotic property.
Cette étude pharmacoloqique a également mis en évidence, d'une manière surprenante, que les fractions d'héparines obtenues par neutralisation par la protamine conformément à l'invention, exercent une activité antithrombotique qui croît avec l'administration de doses crois- santés, sans augmentation parallèle de leur activité hémorragique ou anticoagulante. This pharmacological study also surprisingly demonstrated that the heparin fractions obtained by protamine neutralization in accordance with the invention exert an antithrombotic activity which increases with the administration of increasing doses. health, without parallel increase in their haemorrhagic or anticoagulant activity.
Un autre protocole expérimental a permis de montrer que les compositions selon l'invention étaient capables d'inhiber l'activité hydrolytique de l'élastase leucocytaire humaine, de manière plus efficace que l'héparine non fractionnée. La suppression du risque hémorragique, conformément à l'invention, permet d'envisager une administration par voie parentérale ou par voie broncho-pulmonaire en aérosol, dans le traitement de certaines affections broncho-pulmonaires pouvant impliquer un excès d'élastase leucocytaire, telles que les syndromes de détresse respiratoire aigϋe, la mucoviscidose, les bronchopneumopathies chroniques obstructives.  Another experimental protocol made it possible to show that the compositions according to the invention were capable of inhibiting the hydrolytic activity of human leukocyte elastase, more effectively than unfractionated heparin. The elimination of the risk of haemorrhage, in accordance with the invention, makes it possible to envisage administration by the parenteral route or by bronchopulmonary aerosol, in the treatment of certain bronchopulmonary conditions which may involve an excess of leukocytic elastase, such as acute respiratory distress syndromes, cystic fibrosis, chronic obstructive pulmonary disease.
Les compositions d'héparine selon l'invention, qui sont stables et non toxiques, peuvent être employées pour la préparation de médicaments utiles dans diverses applications thérapeutiques. Ces applications sont celles de l'héparine et de ses dérivés classiques y compris les cas où l'héparine est contre-indiquée en raison du risque hémorragique que présente le patient. Elles peuvent servir en particulier à la préparation de médicaments pour le traitement et la prévention des thromboses veineuses ou artérielles ou encore pour prévenir l'activation de la coagulation dans la circulation extracorporelle, L'invention est donc également relative à des compositions pharmaceutiques comprenant une quantité thérapeutiquement efficace d'une composition d'héparine selon l'invention telle que décrite précédemment, en combinaison avec un véhicule pharmaceutiquement acceptable. The heparin compositions according to the invention, which are stable and non-toxic, can be used for the preparation of medicaments useful in various therapeutic applications. These applications are those of heparin and its conventional derivatives, including cases where heparin is contraindicated because of the risk of bleeding that the patient presents. They can be used in particular for the preparation of medicaments for the treatment and prevention of venous or arterial thromboses or else to prevent activation of coagulation in the extracorporeal circulation. The invention therefore also relates to pharmaceutical compositions comprising an amount therapeutically effective heparin composition according to the invention as described above, in combination with a pharmaceutically acceptable vehicle.
Il peut s'agir, par exemple, de compositions pharmaceutiques antithrombotiques ou encore pour l'inhibi- tion de l'activité hydrolytique de l'élastase leucocytaire humaine.  They may, for example, be antithrombotic pharmaceutical compositions or else for the inhibition of the hydrolytic activity of human leukocyte elastase.
Les fractions d'héparine de ces compositions peuvent être mises sous la forme de sel pharmaceutiquement acceptable selon les procédés classiques.  The heparin fractions of these compositions can be in the form of a pharmaceutically acceptable salt according to conventional methods.
Les compositions pharmaceutiques selon l'invention sont avantageusement des formulations injectables destinées en particulier à une administration par voie parentérale.  The pharmaceutical compositions according to the invention are advantageously injectable formulations intended in particular for administration by the parenteral route.
Pour d'autres applications telles que l'inhibition de l'élastase leucocytaire, on prévoit avantageusement des formulations appropriées à une administration par voie broncho-pulmonaire.  For other applications such as the inhibition of leukocyte elastase, formulations suitable for administration by bronchopulmonary route are advantageously provided.
D'autres caractéristiques et avantages de l'invention apparaîtront à la lecture des exemples donnés ci¬- après à titre indicatif et non limitatif, en référence aux dessins annexés dans lesquels ;  Other characteristics and advantages of the invention will become apparent on reading the examples given below by way of indication and without limitation, with reference to the appended drawings in which;
- la figure 1 est un graphe comparatif de l'activité hémorragique d'héparine non fractionnée, d'héparine de bas poids moléculaire et non hémorragique des compositions d'héparines selon l'invention (S1, S2, S3) ;  - Figure 1 is a comparative graph of the hemorrhagic activity of unfractionated heparin, low molecular weight heparin and non hemorrhagic heparin compositions according to the invention (S1, S2, S3);
- la figure 2 est un graphe comparatif de l'activité antithrombotique d'héparine non fractionnée, d'héparine de bas poids moléculaire et de compositions d'héparines selon l'invention (S1, S2, S3). - Figure 2 is a comparative graph of the antithrombotic activity of unfractionated heparin, low molecular weight heparin and heparin compositions according to the invention (S1, S2, S3).
EXEMPLES  EXAMPLES
Produits utilisés : Héparine standard (LEO), Sulfate de protamine (CHOAY) et Héparine de bas poids moléculaire, "Enoxaparine", commercialisée sous le nom "LOVENOX"  Products used: Standard heparin (LEO), Protamine sulfate (CHOAY) and Low molecular weight heparin, "Enoxaparin", marketed under the name "LOVENOX"
(PHARMUKA).  (PHARMUKA).
- EXEMPLE 1 : Préparation du surnageant SI  - EXAMPLE 1: Preparation of the supernatant SI
On prépare 14,4 ml d'une solution d'héparine standard présentant un titre de 72000 UI (480 mg) et 48 ml d'une solution de sulfate de protamine présentant un titre de 48000 UAH. On mélange ces solutions à temparature ambiante. Le rapport héparine/protamine est alors de 1/1, c'est-à-dire qu'on neutralise 1 mg d'héparine par 1 mg de sulfate de protamine.  14.4 ml of a standard heparin solution having a titer of 72,000 IU (480 mg) and 48 ml of a protamine sulfate solution having a titer of 48,000 UAH are prepared. These solutions are mixed at room temperature. The heparin / protamine ratio is then 1/1, that is to say that 1 mg of heparin is neutralized with 1 mg of protamine sulfate.
On centrifuge le mélange ainsi obtenu pendant 10 minutes et on récupère le surnageant qu'on lyophilise.  The mixture thus obtained is centrifuged for 10 minutes and the supernatant is recovered which is lyophilized.
- EXEMPLE 2 : Préparation du surnageant S2  - EXAMPLE 2: Preparation of the supernatant S2
On procède comme décrit à l'exemple 1 à partir de 9 ml d'une solution d'héparine standard (soit 45000 UI, 300 mg) et de 60 ml de sulfate de protamine (soit 60000 UAH). Le rapport héparine/protamine est alors de 1/2, c'est-à-dire qu'on neutralise 1 mg d'héparine par 2 mg de sulfate de protamine.  The procedure is as described in Example 1 from 9 ml of a standard heparin solution (i.e. 45,000 IU, 300 mg) and 60 ml of protamine sulfate (i.e. 60,000 UAH). The heparin / protamine ratio is then 1/2, that is to say that 1 mg of heparin is neutralized with 2 mg of protamine sulfate.
- EXEMPLE 3 : Préparation du surnageant S3  - EXAMPLE 3: Preparation of the supernatant S3
On procède comme décrit à l'exemple 1 à partir de The procedure is as described in Example 1 starting from
4 ml d'une solution d'héparine de bas poids moléculaire, "Enoxaparine" (LOVENOX), (soit 400 mg) et de 40 ml de sulfate de protamine (soit 40000 UAH), Le rapport hépa- rine/protamine est alors de 1/1, c'est-à-dire qu'on neutralise 1 mg d'héparine de bas poids moléculaire par 1 mg de sulfate de protamine. 4 ml of a low molecular weight heparin solution, "Enoxaparin" (LOVENOX), (i.e. 400 mg) and 40 ml of protamine sulfate (i.e. 40,000 UAH), The hepatic ratio rine / protamine is then 1/1, that is to say that 1 mg of low molecular weight heparin is neutralized with 1 mg of protamine sulfate.
CARACTERISATION BIOLOGIQUE C A BIOLOGICAL RACTERIZATION
- Distribution des masses moléculaires  - Distribution of molecular weights
TABLEAU I TABLE I
Surnageant S1 obtenu selon l'exemple 1  S1 supernatant obtained according to Example 1
- Distribution des masses moléculaires exprimées en pourcentage  - Distribution of molecular weights expressed as a percentage
TABLEAU II TABLE II
Surnageant S2 obtenu selon l'exemple 2  Supernatant S2 obtained according to Example 2
- Distribution des masses moléculaires exprimées en pourcentage  - Distribution of molecular weights expressed as a percentage
- Spectre d'absorption en ultra-violet pour le surnageant S1 (exemple 1) - Ultraviolet absorption spectrum for the supernatant S1 (example 1)
Une solution diluée au 1/20 présente deux pics d'absorption aux longueurs d'onde suivantes :  A solution diluted to 1/20 has two absorption peaks at the following wavelengths:
212 nm : BO=3,47 et 271,5 nm : DO=2,22  212 nm: BO = 3.47 and 271.5 nm: DO = 2.22
- Titrage du surnageant SI (exemple 1)  - Titration of the supernatant SI (example 1)
Avant lyophilisation du surnageant SI préparé selon l'exemple 1, chaque flacon c1ntient 0,7 ml de solution surnageante. 12 dosages identiques ont été réalisés pour vérifier la reproductibilité : les résultats sont donnés dans le Tableau III ci-dessous : Before lyophilization of the supernatant SI prepared according to example 1, each bottle contains 0.7 ml of supernatant solution. 12 identical assays were carried out to verify the reproducibility: the results are given in Table III below:
AZURE A : Méthode de Klein M.D, et al.  AZURE A: Klein's method M.D, et al.
A-Xa : Dosage chronométrique de l'héparine (Hépadot Laboratoire Stago)  A-Xa: Chronometric determination of heparin (Hépadot Laboratoire Stago)
A-lIa : Méthode amidolytique  A-lIa: Amidolytic method
- Dosage des protéines  - Protein dosage
Le dosage des protéines dans les surnageants S1 et S2, respectivement préparés selon les exemples 1 et 2, est effectué selon la méthode de Pierce (Coffret réactif Laboratoire Pierce).  The assay of the proteins in the supernatants S1 and S2, respectively prepared according to Examples 1 and 2, is carried out according to the Pierce method (Pierce laboratory reagent kit).
Les résultats sont donnés dans le Tableau IV ci-dessous :  The results are given in Table IV below:
- Composition en électrolytes (mEq/l) - Composition of electrolytes (mEq / l)
La composition en électrolytes des surnageants S1 et S2 est donnée dans le tableau V ci-après :  The electrolyte composition of the supernatants S1 and S2 is given in table V below:
Détermination du pH PH determination
ETUDE PHARMACOLOGIQUE PHARMACOLOGICAL STUDY
A. Etudes expérimentales chez le rat dans un modèle de thrombose veineuse induite par stase et un modèle d'hémorragie provoquée :  A. Experimental studies in rats in a model of venous thrombosis induced by stasis and a model of induced hemorrhage:
Des études ont été menées selon la méthode décrite par C. DOUTREMEPUICH et coll., "Expérimental venous throm- bosis in rats treated with heparin and a low molecular weight heparin fraction", Haemostasis, 13, 109-112 (1983). a. Modèle curatif (injections par voie sous-cutanée deux heures après l'induction de la thrombose).  Studies have been carried out according to the method described by C. DOUTREMEPUICH et al., "Experimental venous thrombosis in rats treated with heparin and a low molecular weight heparin fraction", Haemostasis, 13, 109-112 (1983). at. Curative model (injections subcutaneously two hours after the induction of thrombosis).
Deux études ont été réalisées selon le protocole suivant :  Two studies were carried out according to the following protocol:
T0 : ligature de la veine cave  T0: ligation of the vena cava
T0+2H : injection des solutions par voie sous-cutanée TO+5H30 : induction de l'hémorragie T0 + 2H: injection of the solutions subcutaneously TO + 5H30: induction of hemorrhage
T0+6H : prélèvements (sang et caillot)  T0 + 6H: samples (blood and clot)
Les résultats obtenus à l'issue de la première étude sont rassemblés dans les tableaux VII et VIII ci-dessous :  The results obtained at the end of the first study are collated in Tables VII and VIII below:
T.H.P. : Temps d'Hémorragie Provoquée T.H.P. : Time of Provoked Hemorrhage
T.C.K. : Temps de Céphaline Kaolin  T.C.K. : Time of Cephalin Kaolin
T.T.D. : Temps de Thrombine Diluée  T.T.D. : Diluted Thrombin Time
* = p<0,05 (test de Mann Whitney)  * = p <0.05 (Mann Whitney test)
Cette première étude montre que l'héparine neutralisée in vitro par la protamine dans un rapport héparine/ protamine de 1/1 conduisant au surnageant S1 exerce, à une dose de 2 mg, une activité antithrombotique assez importante qui est comparable à celle de l'héparine non neutralisée et à celle du LOVENOX (héparine de bas poids moléculaire), tandis que l'activité anticoagulante et l'activité hémorragique ne sont que faiblement augmentées. This first study shows that heparin neutralized in vitro by protamine in a heparin / protamine ratio of 1/1 leading to the supernatant S1 exerts, at a dose of 2 mg, a fairly significant antithrombotic activity which is comparable to that of non-neutralized heparin and that of LOVENOX (low molecular weight heparin), while the anticoagulant activity and the hemorrhagic activity are only slightly increased.
Le surnageant S1 n'a aucun effet sur les cellules sanguines.  The supernatant S1 has no effect on blood cells.
Par ailleurs, le surnageant S2, obtenu par neutralisation selon un rapport héparine/protamine de 1/2, n'exerce aucune activité hémorragique mais possède une activité antithrombotique réduite.  Furthermore, the supernatant S2, obtained by neutralization according to a heparin / protamine ratio of 1/2, does not exert any hemorrhagic activity but has a reduced antithrombotic activity.
Les résultats de la seconde étude sont donnés dans le tableau IX ci-après :  The results of the second study are given in Table IX below:
P.C. : Poids du Caillot expérimental PC: Weight of experimental clot
T.H. P. : Temps d'Hémorragie Provoqué  T.H. P.: Provoked Hemorrhage Time
T.C.K. : Temps de Céphaline-Kaolin  T.C.K. : Cephalin-Kaolin time
T.T.D. : Temps de Thrombine Diluée  T.T.D. : Diluted Thrombin Time
il résulte des résultats obtenus que l'activité antithrombotique des fractions S1, S2 et S3 selon l'invention croît avec des doses administrées croissantes.  it results from the results obtained that the antithrombotic activity of the fractions S1, S2 and S3 according to the invention increases with increasing administered doses.
Si l'on se réfère aux courbes dose-effet, établies pour des doses allant de 2 mg/kg à 10 mg/kg, on voit, sur la figure 1, que quel que soit le type d'héparine traitée conformément à l'invention, la fraction d'héparine obtenue présente une activité hémorragique similaire à celle du groupe contrôle, même aux doses les plus élevées. L'héparine non fractionnée et l'héparine de bas poids moléculaire (LOVENOX) , non neutralisée par la protamine, selon l'invention, présentent une activité hémorragique considérable par rapport aux fractions d'héparine de l'invention.  Referring to the dose-effect curves, established for doses ranging from 2 mg / kg to 10 mg / kg, it can be seen in FIG. 1 that whatever the type of heparin treated in accordance with invention, the heparin fraction obtained has a hemorrhagic activity similar to that of the control group, even at the highest doses. Unfractionated heparin and low molecular weight heparin (LOVENOX), not neutralized by protamine, according to the invention, exhibit considerable haemorrhagic activity compared to the heparin fractions of the invention.
La figure 2 montre que les fractions d'héparine ob- tenues selon l'invention présentent une activité antithrombotique intéressante. Dans le cas de la fraction SI (rapport héparine/protamine de 1/1), cette activité est comparable à celle des héparines non neutralisées conformément à l'invention.  FIG. 2 shows that the heparin fractions obtained according to the invention exhibit an interesting antithrombotic activity. In the case of the SI fraction (heparin / protamine ratio of 1/1), this activity is comparable to that of heparins which are not neutralized in accordance with the invention.
b. Modèle préventif (administration par voie souscutanée une heure avant l'induction de la thrombose).  b. Preventive model (subcutaneous administration one hour before induction of thrombosis).
L'étude a été menée avec le surnageant SI (exemple 1) selon le protocole suivant : TO : injection des solutions par voie sous-cutanée TO+1 Heure : induction de la stase The study was carried out with the supernatant SI (example 1) according to the following protocol: TO: injection of solutions by subcutaneous route TO + 1 Hour: induction of stasis
TO+24 Heures : prélèvement (sang et caillot)  TO + 24 Hours: sample (blood and clot)
Les résultats obtenus sont donnés dans le Tableau X ci-dessous :  The results obtained are given in Table X below:
T.CK. : Temps de Céphaline Kaolin  T.CK. : Time of Cephalin Kaolin
T.T.D. : Temps de Thrombine Diluée  T.T.D. : Diluted Thrombin Time
Ti : Temps de Titrarine (Laboratoire Stago)  Ti: Titrarin Time (Stago Laboratory)
* : p<0,05 (test de Mann Whitney)  *: p <0.05 (Mann Whitney test)
Les résultats obtenus montrent, qu'à titre préventif, SI exerce une activité antithrombotique comparable à celle de l'héparine et du LOVENOX, 24 heures après l'induction de la thrombose.  The results obtained show that, as a preventive measure, SI exerts an antithrombotic activity comparable to that of heparin and LOVENOX, 24 hours after the induction of thrombosis.
B. Etude expérimentale chez le rat dans un modèle de thrombose induite par génération de radicaux libres  B. Experimental study in rats in a model of thrombosis induced by generation of free radicals
(référence : DOUTREMEPUICH - In prèss - Annales de Cardiologie et Angiologie)  (reference: DOUTREMEPUICH - In prèss - Annales de Cardiologie et Angiologie)
L'étude a été menée avec S1 (exemple 1) selon le protocole suivant :  The study was carried out with S1 (example 1) according to the following protocol:
(TO : injection des solutions par voie sous-cutanée) (TO: injection of the solutions by subcutaneous route)
TO+25 min : injection de rose bengale à une dose de TO + 25 min: injection of bengal rose at a dose of
5 mg/kg TO+30 min : induction des radicaux libres au niveau de la première artériole par réaction photo-chimique 5 mg / kg TO + 30 min: induction of free radicals at the level of the first arteriole by photochemical reaction
TO+55 min injection de rose bengale à la même dose TO+60 min induction des radicaux libres au niveau de la deuxième artériole  TO + 55 min injection of rose bengal at the same dose TO + 60 min induction of free radicals in the second arteriole
TO+85 min injection de rose bengale à la même dose TO+90 min induction des radicaux libres au niveau d'une veinule  TO + 85 min injection of Bengal rose at the same dose TO + 90 min induction of free radicals in a vein
Après la dernière thrombose, on effectue un prélèvement sanguin par voie intra-cardiaque.  After the last thrombosis, an intra-cardiac blood sample is taken.
Le temps d'excitation est fixé à 2 minutes et le temps d'observation à 10 minutes.  The excitation time is fixed at 2 minutes and the observation time at 10 minutes.
On obtient les résultats donnés dans le tableau XI ci-dessous :  The results given in Table XI below are obtained:
Durée d'embolisation : durée entre le premier embole et le dernier embole se détachant du caillot. Duration embolization: duration between the first and the embolus embolus ernier of detaching the clot.
Nombre d'emboles : nombre d'emboles se détachant du caillot.  Number of emboli: number of emboli coming off the clot.
Dans ce modèle de thrombose induite par radicaux libres, le surnageant S1 (exemple 1) exerce une activité antithrombotique significative par rapport au groupe placebo, qui persiste après 90 minutes (TO+90 min). Cette activité est plus importante que celle de l'héparine injectée à la même dose, après 30 et 60 minutes (TO+30 et TO+60 min).  In this model of thrombosis induced by free radicals, the supernatant S1 (example 1) exerts a significant antithrombotic activity compared to the placebo group, which persists after 90 minutes (TO + 90 min). This activity is more important than that of heparin injected at the same dose, after 30 and 60 minutes (TO + 30 and TO + 60 min).
C. Etude expérimentale chez le rat dans un modèle de thrombose induite par lésion endothëliale au laser (Réf. : Vesvres, Haemostasis 1993, 23, 8-12)  C. Experimental study in rats in a model of thrombosis induced by laser endothelial injury (Ref.: Vesvres, Haemostasis 1993, 23, 8-12)
a. Etude 1  at. Study 1
L'étude a été menée selon le protocole suivant :  The study was conducted according to the following protocol:
TO : injection de la substance à tester à une dose de 2 mg/kg par voie sous cutanée  TO: injection of the test substance at a dose of 2 mg / kg subcutaneously
TO+35 min : induction de la thrombose artérielle par rayon laser.  TO + 35 min: induction of arterial thrombosis by laser beam.
Le temps d'observation est fixé à 10 minutes.  The observation time is fixed at 10 minutes.
Les résultats obtenus sont donnés dans le Tableau XII ci-dessous :  The results obtained are given in Table XII below:
SI exerce une activité antithrombotique comparable à celle de l'héparine non neutralisée injectée à la même dose et réduit le nombre d'embols ainsi que la durée d'embolisation de manière statistiquement significative, b. Etude 2 SI exerts an antithrombotic activity comparable to that of non-neutralized heparin injected at the same dose and reduces the number of embolisms as well as the duration of embolization in a statistically significant manner, b. Study 2
L'étude a été menée selon le protocole suivant :  The study was conducted according to the following protocol:
TO : injection des substances à tester à une dose de TO: injection of the substances to be tested at a dose of
2 mg/kg par voie sous-cutanée. 2 mg / kg subcutaneously.
TO+1h : induction de la première thrombose artérielle  TO + 1h: induction of the first arterial thrombosis
T0+3h : induction de la deuxième thrombose artérielle  T0 + 3h: induction of the second arterial thrombosis
T0+6h : induction de la troisième thrombose artérielle.  T0 + 6h: induction of the third arterial thrombosis.
Le temps d'observation est fixé à 10 minutes.  The observation time is fixed at 10 minutes.
Les résultats obtenus sont donnés dans le Tableau XIII ci-dessous :  The results obtained are given in Table XIII below:
SI exerce une activité antithrombotique comparable à celle de l'héparine non neutralisée par la protamine.  SI exerts an antithrombotic activity comparable to that of heparin which is not neutralized by protamine.
En conclusion, les études décrites ci-dessus montrent que l'activité antithrombotique est observée dans les trois modèles de thrombose expérimentale à savoir le modèle veineux induit par stase, le modèle de thrombose artérielle induite par radicaux libres et le modèle de thrombose artérielle induite par lésion endothëliale au laser. In conclusion, the studies described above show that the antithrombotic activity is observed in the three experimental thrombosis models, namely the venous model induced by stasis, the arterial thrombosis model induced by free radicals and the arterial thrombosis model induced by endothelial laser injury.
Selon l'invention, la fraction d'héparine obtenue à partir d'une héparine de bas poids moléculaire, l'"Enoxaparine" (LOVENOX), présente une activité antithrombotique plus importante que celle de la même héparine de bas poids moléculaire non traitée in vitro par la protamine et plus importante également que celle des fractions obtenues à partir des héparines non fractionnées et non traitées in vitro par la protamine, tout en ne présentant plus aucun risque hémorragique.  According to the invention, the fraction of heparin obtained from a low molecular weight heparin, "Enoxaparin" (LOVENOX), has a greater antithrombotic activity than that of the same low molecular weight heparin not treated in in vitro by protamine and also greater than that of the fractions obtained from unfractionated heparins and not treated in vitro with protamine, while no longer presenting any risk of haemorrhage.
Le procédé selon l'invention permet, d'une manière simple et peu coûteuse, pratiquement de supprimer l'activité hémorragique des héparines tout en conservant leur activité antithrombotique.  The method according to the invention allows, in a simple and inexpensive manner, practically to suppress the hemorrhagic activity of heparins while retaining their antithrombotic activity.

Claims

REVENDICATIONS
1. Composition d'héparines présentant une activité antithrombotique et pratiquement dépourvue d'activité hémorragique, caractérisée en ce qu'elle est constituée de fractions d'héparine telle qu'obtenues par la neutralisation in vitro d'une héparine par la protamine.  1. A composition of heparins exhibiting antithrombotic activity and practically devoid of hemorrhagic activity, characterized in that it consists of heparin fractions as obtained by the in vitro neutralization of a heparin by protamine.
2. Composition selon la revendication 1, caractérisée en ce qu'elle est constituée de fractions d'héparine telle qu'obtenues par la neutralisation in vitro d'une héparine non fractionnée par la protamine.  2. Composition according to claim 1, characterized in that it consists of heparin fractions as obtained by the in vitro neutralization of a heparin not fractionated by protamine.
3. Composition selon la revendication 1, caractérisée en ce qu'elle est constituée de fractions d'héparine telle qu'obtenues par la neutralisation in vitro d'une héparine de bas poids moléculaire par la protamine.  3. Composition according to claim 1, characterized in that it consists of heparin fractions as obtained by the in vitro neutralization of a low molecular weight heparin with protamine.
4. Composition selon l'une des revendications précédentes, caractérisée en ce qu'elle est constituée de fractions dont au moins 25 % présentent un poids moléculaire inférieur à 2,5 kDa.  4. Composition according to one of the preceding claims, characterized in that it consists of fractions of which at least 25% have a molecular weight of less than 2.5 kDa.
5. Composition selon la revendication 4, caractéri- sée en ce qu'elle est constituée de fractions dont au moins 40 % présentent un poids moléculaire supérieur à 20 kDa.  5. Composition according to claim 4, characterized in that it consists of fractions of which at least 40% have a molecular weight greater than 20 kDa.
6. Composition selon l'une des revendications 1 à 4, caractérisée en ce qu'elle est constituée de fractions de poids moléculaire inférieur à 2,5 kDa.  6. Composition according to one of claims 1 to 4, characterized in that it consists of fractions of molecular weight less than 2.5 kDa.
7. Composition selon la revendication 5, caractérisée en ce qu'elle est constituée d'une fraction présentant la distribution des masses moléculaires données au Tableau I. 7. Composition according to claim 5, characterized in that it consists of a fraction having the distribution of the molecular masses given in Table I.
8. Composition selon la revendication 7, caractérisée en ce qu'elle est constituée d'une fraction présentant la distribution des masses moléculaires donnée au Tableau II. 8. Composition according to claim 7, characterized in that it consists of a fraction having the distribution of molecular weights given in Table II.
9. Composition selon l'une quelconque des revendications précédentes, caractérisée en ce qu'elle est sensiblement exempte de protamine.  9. Composition according to any one of the preceding claims, characterized in that it is substantially free of protamine.
10. Composition selon l'une quelconque des revendications 1 à 9, caractérisée eh ce qu'elle présente des propriétés inhibitrices de l'activité hydrolytique de l'élastase leucocytaire humaine.  10. Composition according to any one of claims 1 to 9, characterized in that it has inhibitory properties of the hydrolytic activity of human leukocyte elastase.
11. Procédé de préparation d'une composition selon l'une quelconque des revendications 1 à 10, caractérisé en ce qu'il comprend la neutralisation in vitro d'une héparine par la protamine.  11. A method of preparing a composition according to any one of claims 1 to 10, characterized in that it comprises the in vitro neutralization of a heparin with protamine.
12. Procédé selon la revendication 11, caractérisé en ce qu'il comprend les étapes consistant à mélanger une solution d'héparine et une solution d'un sel de protamine, à centrifuger le mélange obtenu et à récupérer le surnageant.  12. The method of claim 11, characterized in that it comprises the steps of mixing a heparin solution and a solution of a protamine salt, centrifuging the mixture obtained and recovering the supernatant.
13. Procédé selon l'une des revendications 11 ou 12, caractérisé en ce que le sel de protamine est le sulfate de protamine.  13. Method according to one of claims 11 or 12, characterized in that the protamine salt is protamine sulfate.
14. Procédé selon l'une des revendications 11 à 13, caractérisé en ce que l'héparine et la protamine sont utilisées dans un rapport de 1 pour 1.  14. Method according to one of claims 11 to 13, characterized in that heparin and protamine are used in a ratio of 1 to 1.
15. Procédé selon l'une des revendications 11 à 13, caractérisé en ce que l'héparine et la protamine sont utilisées dans un rapport de 1 pour 2. 15. Method according to one of claims 11 to 13, characterized in that heparin and protamine are used in a ratio of 1 to 2.
16. Procédé selon l'une des revendications 11 à 15, caractérisé en ce que l'on neutralise de l'héparine non fractionnée. 16. Method according to one of claims 11 to 15, characterized in that unfractionated heparin is neutralized.
17. Procédé selon l'une des revendications 11 à 16, caractérisé en ce que l'on neutralise de l'héparine de bas poids moléculaire.  17. Method according to one of claims 11 to 16, characterized in that low molecular weight heparin is neutralized.
18. Utilisation d'une composition d'héparine selon l'une quelconque des revendications 1 à 10 pour la préparation d'un médicament présentant une activité anti- thrombotique et pratiquement dépourvue d'activité hémorragique.  18. Use of a heparin composition according to any one of claims 1 to 10 for the preparation of a medicament exhibiting anti-thrombotic activity and practically devoid of hemorrhagic activity.
19. Composition pharmaceutique caractérisée en ce qu'elle comprend une quantité efficace d'une composition selon l'une quelconque des revendications 1 à 10, en combinaison avec un véhicule pharmaceutiquement acceptable.  19. Pharmaceutical composition characterized in that it comprises an effective amount of a composition according to any one of claims 1 to 10, in combination with a pharmaceutically acceptable vehicle.
20. Composition pharmaceutique selon la revendication 19, caractérisée en ce qu'elle se présente sous la forme d'une solution injectable.  20. Pharmaceutical composition according to claim 19, characterized in that it is in the form of an injectable solution.
21. Composition pharmaceutique pour l'inhibition de l'activité hydrolytique de l'élastase leucocytaire humaine, caractérisée en ce qu'elle comprend, à titre de principe actif, une quantité efficace d'une composition selon l'une quelconque des revendications 1 à 10, en combinaison avec un véhicule pharmaceutiquement acceptable.  21. Pharmaceutical composition for the inhibition of the hydrolytic activity of human leukocyte elastase, characterized in that it comprises, as active principle, an effective amount of a composition according to any one of claims 1 to 10, in combination with a pharmaceutically acceptable vehicle.
22. Composition pharmaceutique selon la revendication 21, caractérisée en ce qu'elle se présente sous une forme appropriée pour une administration par voie broncho-pulmonaire.  22. Pharmaceutical composition according to claim 21, characterized in that it is in a form suitable for administration by bronchopulmonary route.
EP95918118A 1994-08-29 1995-05-29 Antithrombotic and non-hemorrhagic heparin-based compositions, method for their preparation and therapeutic applications Withdrawn EP0779814A1 (en)

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FR9410380 1994-08-29
FR9410380A FR2723847A1 (en) 1994-08-29 1994-08-29 HEPARIN - BASED ANTITHROMBOTIC AND NON - HEMORRHAGIC COMPOSITIONS, PROCESS FOR THEIR PREPARATION AND THERAPEUTIC APPLICATIONS.
PCT/IB1995/000405 WO1996006623A1 (en) 1994-08-29 1995-05-29 Antithrombotic and non-hemorrhagic heparin-based compositions, method for their preparation and therapeutic applications

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JP (1) JPH09510736A (en)
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US7056504B1 (en) 1998-08-27 2006-06-06 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase I and II
CA2341412A1 (en) * 1998-08-27 2000-03-09 Massachusetts Institute Of Technology Rationally designed heparinases derived from heparinase i and ii
WO2000065521A2 (en) 1999-04-23 2000-11-02 Massachusetts Institute Of Technology System and method for polymer notation
AU4351201A (en) * 2000-03-08 2001-09-17 Massachusetts Inst Technology Heparinase iii and uses thereof
AU2001292618A1 (en) 2000-09-12 2002-03-26 Massachusetts Institute Of Technology Methods and products related to low molecular weight heparin
EP1328260A2 (en) * 2000-10-18 2003-07-23 Massachusetts Institute Of Technology Methods and products related to pulmonary delivery of polysaccharides
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RU2151602C1 (en) 2000-06-27
FR2723847A1 (en) 1996-03-01
CA2198722A1 (en) 1996-03-07
AU696954B2 (en) 1998-09-24
US5922358A (en) 1999-07-13
CN1159162A (en) 1997-09-10
JPH09510736A (en) 1997-10-28
AU2417195A (en) 1996-03-22

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