EP1527169A2 - Leech extracts for stents - Google Patents
Leech extracts for stentsInfo
- Publication number
- EP1527169A2 EP1527169A2 EP03756529A EP03756529A EP1527169A2 EP 1527169 A2 EP1527169 A2 EP 1527169A2 EP 03756529 A EP03756529 A EP 03756529A EP 03756529 A EP03756529 A EP 03756529A EP 1527169 A2 EP1527169 A2 EP 1527169A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- destabilase
- activity
- complex
- destabilase complex
- liposome
- 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- Withdrawn
Links
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
- A61K38/57—Protease inhibitors from animals; from humans
- A61K38/58—Protease inhibitors from animals; from humans from leeches, e.g. hirudin, eglin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/005—Ingredients of undetermined constitution or reaction products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8114—Kunitz type inhibitors
- C07K14/8117—Bovine/basic pancreatic trypsin inhibitor (BPTI, aprotinin)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
- A61L2300/254—Enzymes, proenzymes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/30—Compounds of undetermined constitution extracted from natural sources, e.g. Aloe Vera
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/426—Immunomodulating agents, i.e. cytokines, interleukins, interferons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
- A61L2300/626—Liposomes, micelles, vesicles
Definitions
- the invention relates to obtaining biologically active substances with cosmetic or pharmaceutical properties in the form of liposomes.
- these liposomes are natural liposomes extracted from medicinal leeches. These natural liposomes have both anti-coagulant and immunomodulatory properties.
- stents are stents, commonly used in cardiovascular surgery to be implanted in a vascular duct, in particular coronary artery or peripheral artery. It is known to treat vascular atheromatous diseases, which correspond to narrowing of the arteries, by balloon dilation techniques, techniques called angioplasties. The balloon is introduced into the artery, inflated to a pressure such that it crushes the deposit of atheromas.
- a stent more commonly called a stent, typically a metal support acting as a support once it is introduced to inside the artery at the treated area.
- Such stents are for example crimped onto the balloon, in a so-called rest position, prior to the introduction of the balloon into the vascular duct.
- stents for drug delivery is described, for example, in WO 01/01957, US 6,099,561; 6,071,305; 6,063,101; 5,997,468; 5,980,551; 5,980,566; 5,972,027 5,968,092; 5,951,586; 5,893,840; 5,891,108; 5,851,231; 5,843,172; 5,837,008 5,769,883; 5,735,811; 5,700,286; 5,679,400; 5,649,977; 5,637,113; 5,591,227 5,551,954; 5,545,208; 5,500,013; 5,464,450; 5,419,760; 5,411,550; 5342348 5,286,254; and 5,163,952.
- Methods of dressing stents are described in particular in documents US 6,409,716; 6,464,893; and 5,356,433.
- the prior art does not describe substances capable of forming a covering for stents, which are in the form of a liposome, and which have both anti-coagulant and immunomodulatory properties.
- the patient In the uses of the prior art, the patient must be administered two separate drugs, namely an anti-coagulant substance and an immunomodulatory substance.
- liposomes have the great advantage of serving as vectors for both apolar and polar active pharmaceutical compounds. This liposome form thus makes it possible to administer these two types of compounds on the same site.
- the invention aims to overcome the drawbacks of the prior art, and in particular to obtain a composition having both anti-coagulant and immunomodulatory properties, capable of being combined with equipment such as stents, and presenting in the form of liposomes so as to obtain an appropriate release of the substance.
- the inventors have succeeded in obtaining such a substance from medicinal leeches.
- Prior art is known a method for obtaining a destabilase complex, designated prototype, using a step of affinity chromatography on a support insoluble in water (CNBr activated agarose, for example) carrying immobilized lysine ( REF).
- the extract is conveyed by a buffer with a pH lower than the neutral pH, the pH is neutralized, then dialysis and lyophilization take place.
- the elution of the final product from the immobilized lysine using a buffer with a low pH of less than 9 does not make it possible to obtain a destabilase complex capable of the desired storage and the sufficient enzymatic activity sought .
- the destabilase molecule extracted by this process is in the form of an unstable monomeric enzymatic complex which undergoes a change of conformation, which makes it lose its capacity to form an enzymatic complex in polymeric form.
- the enzymatic complex in the form of a monomer no longer has the capacity to structure itself in a desirable manner into a polymer, a polymer which is organized into a liposome.
- the known complexes (prototypes) produced by medicinal leeches form a complex of hirudin, prostaglandin, destabilase, and kallikrein inhibitors of blood plasma in the ratio 1: 1: 1: 1.
- the abstract XP002237190 of patent application RU 2 129429 C describes a method for isolating prostaglandin complexes from leeches medicinal. The method involves purifying the mixture by affinity chromatography with anti-6-ketoprostaglandin-F1 antibodies immobilized on a water-insoluble support.
- the analysis of the eluate is carried out by electrophoresis.
- the fraction obtained is composed of destabilase monomer aggregate and has the properties of micellar proteins and is represented by a stable lipid-protein complex.
- the destabilase monomers can be in the form of a destabilase complex polymer forming a liposome. Said liposomes have the capacity to rapidly penetrate cell membranes and to have anti-thrombotic activity.
- Patent application WO 92 02206 describes lyophilized native collagen sheets comprising cosmetic forms for the treatment of rosacea.
- the active ingredients in the cosmetic formulation may include hirudin, an anti-coagulant protein from leeches.
- Patent application WO 98 16604 describes cleaning compositions, preferably detergent compositions for detergents, comprising an iso-peptidase type enzyme having the capacity to catalytically cut the bonds between glutamine and lysine.
- Patent application WO 01 47572 describes a method for inhibiting the restenosis of a blood vessel comprising: - the supply of a device comprising an active compound having an anti-thrombotic and anti-inflammatory activity, and
- the inventors have given themselves the goal of obtaining a stable destabilase complex having improved biological activities compared to the compositions of the prior art.
- the inventors have succeeded in obtaining a monomeric destabilase complex which is stable and capable of aggregating into a polymer organizing itself into a liposome.
- the subject of the invention is, according to a first aspect, a stable destabilase complex, in the form of a monomer, capable of aggregating into a polymeric destabilase complex forming a liposome, capable of being obtained from medicinal leeches by a process comprising: - affinity chromatography with said antibodies to 6-ketoprostaglandin immobilized on an appropriate column;
- said complex having an antithrombin activity of at least 700 ATU / mg, a plasma recalcification duration of at least 800 APC / mg, a fibrinolytic activity of at least 40 min 2 / mg, and immunomodulatory activity.
- the purification process is done by chromatography, preferably affinity chromatography. This process includes:
- Said destabilase complex obtained by this process is in the form of a monomer and comprises destabilase and at least one compound from the hirudin group, prostaglandin, kallikrein inhibitor. It is also possible to combine the action of anti-6-keto-PGF1 ⁇ antibodies and of lysine sepharose.
- the destabilase liposome complex has, like the monomeric destabilase complex, very useful pharmaceutical properties, typically an antithrombin activity of at least 500, preferably at least 600 or 700 ATU / mg, a duration of plasma recalcification. at least 800 APC / mg, a fibrinolytic activity of at least 40 mm / mg.
- the antithrombic and thrombolytic action is clearly improved compared to the control and the prototype.
- the invention also relates to a pharmaceutical composition comprising a liposome destabilase complex according to the invention and a pharmaceutically acceptable vehicle.
- pharmaceutically acceptable carrier is used to denote a non-toxic, solid or diluent or encapsulating liquid material, which does not react negatively on the active compound for its effectiveness.
- the invention also relates to a cosmetic composition comprising this liposome destabilase complex according to the invention.
- the invention also relates to the liposome destabilase complex as a medicament, and the use of a liposome monomeric or polymeric destabilase complex for the preparation of a medicament having anti-coagulant and immunomodulatory activity.
- the invention also relates to a device for purifying a destabilase complex from medicinal leeches, comprising an affinity column loaded with anti-6-keto-prostaglandin antibodies.
- the invention also relates to an implantable medical prosthesis, in which at least part of the prosthesis is covered with a covering, said covering comprising a destabilase liposome complex according to the invention.
- the implantable prosthesis is a stent support.
- the method for purifying the stable destabilase complex according to the invention comprises a step of affinity chromatography using antibodies of 6-keto-PGFl ⁇ (6-keto-prostaglandin F l ⁇ ), immobilized on a support insoluble in l 'water.
- the principle of immunoaffinity chromatography is known to those skilled in the art and is based on the specificity of mono or polyclonal antibodies to capture specific protein antigens from complex natural extracts.
- the inventors have succeeded in obtaining, quite surprisingly, a destabilase complex with preserved biological activities which is both anti-coagulant and immunomodulatory, and capable of being structured into liposomes.
- the antibodies are coupled to a solid chromatographic phase, typically of agarose, by covalent bonds (cyanogen bromide, for example CNBR), or other chemical couplings targeting the amino, hydroxyl, carboxyl or sulphidryl groups of the immunoglobulins so as to form a solid matrix.
- a solid chromatographic phase typically of agarose
- covalent bonds cyanogen bromide, for example CNBR
- the solid phase coupled to the antibodies is then placed in an affinity chromatography column.
- the mixture of target antigen and contaminants is diluted in a binding buffer and then applied to the column. Non-adsorbed contaminants are removed by washing with different buffers.
- the elution of the target protein antigen is then obtained using for example extreme pH conditions, changes in ionic strengths.
- Hirudin and kallikrein inhibitors have activity only in the aqueous phase.
- Destabilase has activity only in the nonaqueous phase.
- the stable destabilase complex obtained by the inventors exhibits hydrophobic properties conditioned by the prostaglandin component, and hydrophilic properties by the liposome polypeptides.
- the monomeric form of the destabilase complex has a molecular weight of 25 kDa, is stable, has an aggregation capacity which results in the formation of liposomes. It is obtained from total extracts of leeches, or from a fraction in particular of secretions from the salivary glands or from the blood of the digestive tract of the leech. After purification by affinity chromatography, the destabilase complex obtained comprises the following components: hirudin, prostaglandin (substance similar to prostaciclyne), kallikrein inhibitor, destabilase.
- the destabilase complex has the following properties: antithrombotic activity by hirudin, increased time for recalcification of blood plasma by kallikrein inhibitors, blocking of adhesion and platelet aggregation by prostacyclin-like substances, dissolution of fibrins stabilized by destabilase.
- destabilase complex determines its antithrombotic, thrombolytic (2.5 times greater than those of the prototype), immunomodulatory, and hypotensive activity completely absent in the prototype.
- the following are the methods for measuring the biological activity used for the purified leech extract and in the form of a liposome, then four examples of realization demonstrating both the anti-coagulant and immunomodulatory activity of the destabilase complex according to the invention.
- the antithrombin activity is determined by the lengthening of the duration of fibrinogen precipitation by thrombin.
- the duration of the formation of a precipitate is determined in a system comprising 0.2 ml of a 0.3% fibrinogen solution, and 0.1 ml of the destabilase complex after fixing 0.1 ml of a thrombin solution. comprising a thrombin activity unit.
- Hirudin activity is expressed in international antithrombin units (ATU NIH). 2 /
- the time for recalcification of the blood plasma is determined in a system comprising 0.1 ml of citric blood plasma and 0.1 ml of destabilase complex after fixing 0.1 ml of a 0.025 M solution of CaCl 2 . The increase of this parameter by two corresponds to one APC unit.
- the prostaglandin content is determined using a radio immunoassay for 6-keto-PGFl ⁇ obtained from the "Amersham” Company.
- 4 / The antithrombotic action of the destabilase complex is determined on rats using the thromboformation method of Wessler (5). The level of thromboformation blockage is evaluated in relation to the control. For this, we study the results following a 4-hour separation between the injection of the destabilase complex and an injection of cold-activated human blood serum. This degree of blockage is expressed in% relative to the control (equal volume of normal saline solution).
- hypotensive action is determined following the oral administration of 0.2 ml of a destabilase complex in rats of a SHR line (spontaneously hypertensive).
- the initial pressure level was 165 ⁇ 5 mm.
- the pressure level in the rat is measured in the tail vein.
- the level of hypotensive efficiency is expressed as a percentage relative to the control (equal volume administered with normal saline).
- the protocol is as follows:
- the elution is obtained by a buffer comprising 0.2 M glycine with 0.15 M HC1 and
- the volume of the eluate is 35 ml.
- the final product has fibrinolytic, antithrombin activity, increases recalcification time, contains prostaglandin, and in addition to the high antithrombolytic potential, has a hypotensive action reducing blood pressure by 25% ( bringing it back practically to normal).
- Example 2 5 ml of a secretion from the salivary glands of leeches were introduced into an agarose column with immobilized anti-6-keto-PGF1 ⁇ antibodies. The elution is obtained using a 0.5 M phosphate buffer, pH 6.4. The volume of the eluate is 10 ml.
- the final product has fibrinolytic antithrombin activity, increases recalcification time, contains prostaglandin, and in addition to a high antithrombic and thrombolitic potential, has a hypotensive action, reducing blood pressure by 25 % (bringing it back to practically normal).
- the destabilase complex increases the cytophagous index and the percentage of phagocytosis (Table 1), demonstrating the immunostimulatory action.
- the final product has fibrinolytic antithrombin activity, increases the recalcification time, contains prostaglandin, and in addition to a high antithrombic and thrombolytic potential, has a hypotensive action, reducing blood pressure by 25% (bringing it back to practically normal).
- the destabilase complex increases the cytophagous index and the percentage of phagocytosis (Table 1), demonstrating the immunostimulatory action.
- the protocol is as follows: take twenty medicinal leeches (total 21 g), extract the front part of the animal, homogenize, and collect the aqueous extract. 10 ml of the extract are placed in an L-Lysine-Sepharose column. The elution is obtained with a 1 M KC1 solution. The volume of the eluate is 20 ml.
- the final product has fibrinolytic antithrombin activity, increases recalcification time, contains prostaglandin, and in addition to a high antithrombic and thrombolytic potential, has a hypotensive action, reducing blood pressure by 25 %) (bringing it back to almost normal).
- the destabilase complex increases the cytophagous index and the percentage of phagocytosis (Table 1), demonstrating the immunostimulatory action.
- Table 1 definition activities of leech extracts.
- the support stents of the destabilase complex obtained by the inventors can be of very varied types.
- a stent will have an external polymeric surface on which a gelatinous matrix will be placed, the matrix including the destabilase complex in the form of liposomes.
- the polymer surface will be linked by bonds covalent with the gelatinous matrix.
- the polymer gel can, for example, have a thickness of 10 to 50 ⁇ m in the uncompressed state.
- This gel can be chosen, for example, from the group consisting of polycarboxylic acids, cellulosic polymers, gelatin, polyvinylpyrrolidone, maleic anhydride polymers, polyamides, polyvinyl alcohols, polyethylene oxides, polyacrylic acid. .
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Abstract
The invention relates to a stable destabilase complex in the form of a monomer, which is capable of aggregating into a polymer destabilase complex forming a liposome and which can be obtained from medicinal leeches using the following method comprising: affinity chromatography with 6-keto-prostaglandin antibodies which are immobilised on a suitable column; and high ionic strength elution of the destabilase complex, said complex having an antithrombin activity of at least 700 ATU/mg, a plasma recalcification time of at least 800 APC/mg, a fibrinolytic activity of at least 40mm<2>/mg and an immunomodulating activity.
Description
Extraits de sangsues pour stents Leech extracts for stents
L'invention concerne l'obtention de substances biologiquement actives à propriétés cosmétiques ou pharmaceutiques se présentant sous forme de liposomes.The invention relates to obtaining biologically active substances with cosmetic or pharmaceutical properties in the form of liposomes.
Plus précisément ces liposomes sont des liposomes naturels extraits de sangsues médicinales. Ces liposomes naturels présentent des propriétés à la fois anti-coagulantes et immuno-modulatrices.More precisely, these liposomes are natural liposomes extracted from medicinal leeches. These natural liposomes have both anti-coagulant and immunomodulatory properties.
On connaît déjà des composés ayant des propriétés anti-coagulantes, tels que l'héparine, et des composés ayant des propriétés imuno-modulatrices. De tels composés sont utilisés en thérapeutique, et administrés par exemple par l'intermédiaire de supports physiquement acceptables dénommés « stents ». Ces stents sont des endoprothèses, couramment utilisés en chirurgie cardiovasculaire pour être implantés dans un conduit vasculaire, notamment artère coronaire ou artère périphérique. Il est connu de traiter les maladies athéromateuses vasculaires, qui correspondent à des rétrécissements des artères, par des techniques de dilatation par ballonnet, techniques dénommées angioplasties. Le ballonnet est introduit dans l'artère, gonflé à une pression telle qu'il écrase le dépôt d'athéromes. Ces techniques n'étant toutefois pas toujours suffisantes, il est connu d'implanter dans le conduit vasculaire au niveau de la zone traitée par angioplastie, une endoprothèse plus communément appelée stent, typiquement support métallique agissant comme tuteur une fois qu'il est introduit à l'intérieur de l'artère au niveau de la zone traitée. De tels stents sont par exemple sertis sur le ballonnet, dans une position dite de repos, préalablement à l'introduction du ballonnet dans le conduit vasculaire. Une fois le stent acheminé jusqu'à la zone d'implantation à l'intérieur du conduit vasculaire, il est déployé par expansion du ballonnet de manière à être amené au contact de la paroi du conduit vasculaire à élargir. II est connu de couvrir des stents à l'aide de substances thérapeutiquement actives destinées à agir notamment dans la zone d'implantation.Already known are compounds having anti-coagulant properties, such as heparin, and compounds having immunomodulating properties. Such compounds are used in therapy, and administered for example via physically acceptable carriers called "stents". These stents are stents, commonly used in cardiovascular surgery to be implanted in a vascular duct, in particular coronary artery or peripheral artery. It is known to treat vascular atheromatous diseases, which correspond to narrowing of the arteries, by balloon dilation techniques, techniques called angioplasties. The balloon is introduced into the artery, inflated to a pressure such that it crushes the deposit of atheromas. However, these techniques are not always sufficient, it is known to implant in the vascular duct at the level of the area treated by angioplasty, a stent more commonly called a stent, typically a metal support acting as a support once it is introduced to inside the artery at the treated area. Such stents are for example crimped onto the balloon, in a so-called rest position, prior to the introduction of the balloon into the vascular duct. Once the stent is delivered to the implantation zone inside the vascular duct, it is deployed by expansion of the balloon so as to be brought into contact with the wall of the vascular duct to be widened. It is known to cover stents with therapeutically active substances intended to act in particular in the implantation area.
Un très grand nombre de dispositifs stents sont connus de l'art antérieur. Des méthodes et appareils pour libérer des substances actives à partir de dispositifs de type stents sont décrits par exemple dans les brevets US 6,096,070; 5,824,049; 5,624,411; 5,609,629; 5,569,463; 5,447,724; et 5,464,650. L'utilisation de stents pour la libération de médicaments est décrite par exemple dans les documents WO 01/01957, US 6,099,561; 6,071,305; 6,063,101; 5,997,468; 5,980,551; 5,980,566; 5,972,027 5,968,092; 5,951,586; 5,893,840; 5,891,108; 5,851,231; 5,843,172; 5,837,008 5,769,883; 5,735,811; 5,700,286; 5,679,400; 5,649,977; 5,637, 113; 5,591,227 5,551,954; 5,545,208; 5,500,013; 5,464,450; 5,419,760; 5,411,550; 5,342,348
5,286,254; et 5,163,952. Des méthodes d'habillage de stents sont décrites notamment dans les documents US 6,409,716; 6,464,893; et 5,356,433.A very large number of stent devices are known from the prior art. Methods and apparatuses for releasing active substances from stent-type devices are described for example in US Patents 6,096,070; 5,824,049; 5,624,411; 5,609,629; 5,569,463; 5,447,724; and 5,464,650. The use of stents for drug delivery is described, for example, in WO 01/01957, US 6,099,561; 6,071,305; 6,063,101; 5,997,468; 5,980,551; 5,980,566; 5,972,027 5,968,092; 5,951,586; 5,893,840; 5,891,108; 5,851,231; 5,843,172; 5,837,008 5,769,883; 5,735,811; 5,700,286; 5,679,400; 5,649,977; 5,637,113; 5,591,227 5,551,954; 5,545,208; 5,500,013; 5,464,450; 5,419,760; 5,411,550; 5342348 5,286,254; and 5,163,952. Methods of dressing stents are described in particular in documents US 6,409,716; 6,464,893; and 5,356,433.
Toutefois l'art antérieur ne décrit pas de substances capables de former un habillage pour des stents, se présentant sous forme de liposome, et présentant des propriétés à la fois anti-coagulantes et immuno-modulatrices. Dans les utilisations de l'art antérieur, il faut administrer au patient deux médicaments distincts à savoir une substance anti-coagulante et une substance immuno-modulatrice.However, the prior art does not describe substances capable of forming a covering for stents, which are in the form of a liposome, and which have both anti-coagulant and immunomodulatory properties. In the uses of the prior art, the patient must be administered two separate drugs, namely an anti-coagulant substance and an immunomodulatory substance.
On rappelle que les liposomes ont le grand intérêt de servir de vecteurs à la fois pour des composés pharmaceutiquernent actifs apolaires et polaires. Cette forme liposome permet ainsi d'administrer sur un même site ces deux types de composés.It will be recalled that liposomes have the great advantage of serving as vectors for both apolar and polar active pharmaceutical compounds. This liposome form thus makes it possible to administer these two types of compounds on the same site.
L'invention vise à pallier les inconvénients de l'art antérieur, et en particulier à obtenir une composition ayant des propriétés à la fois anti-coagulantes et immuno- modulatrices, capable d'être associée à des équipements tels que des stents, et se présentant sous la forme de liposomes de manière à obtenir une libération appropriée de la substance.The invention aims to overcome the drawbacks of the prior art, and in particular to obtain a composition having both anti-coagulant and immunomodulatory properties, capable of being combined with equipment such as stents, and presenting in the form of liposomes so as to obtain an appropriate release of the substance.
Les inventeurs ont réussi à obtenir une telle substance à partir de sangsues médicinales.The inventors have succeeded in obtaining such a substance from medicinal leeches.
De l'art antérieur est connue une méthode d'obtention d'un complexe destabilase, désigné prototype, utilisant une étape de chromatographie par affinité sur un support insoluble dans l'eau (agarose activée CNBr, par exemple) portant de la lysine immobilisée (REF). L'extrait est véhiculé par un tampon à pH inférieur au pH neutre, le pH est neutralisé, puis ont lieu une dialyse et une lyophilisation. Cependant, l' elution du produit final à partir de la lysine immobilisée à l'aide d'un tampon à faible pH inférieur à 9, ne permet pas d'obtenir un complexe destabilase apte au stockage souhaité et à l'activité enzymatique suffisante recherchée. En effet, la molécule de destabilase extraite par ce procédé se présente sous forme d'un complexe enzymatique monomérique instable qui subit un changement de conformation, qui lui fait perdre sa capacité à former un complexe enzymatique sous forme polymérique. Le complexe enzymatique sous forme de monomère n'a plus la capacité de se structurer de manière souhaitable en polymère, polymère qui s'organise en liposome. Les complexes connus (prototypes) produits par les sangsues médicinales forment un complexe d'hirudine, de prostaglandine, de destabilase, et d'inhibiteurs de kallikréine du plasma sanguin dans le rapport 1 :1 :1 :1.Prior art is known a method for obtaining a destabilase complex, designated prototype, using a step of affinity chromatography on a support insoluble in water (CNBr activated agarose, for example) carrying immobilized lysine ( REF). The extract is conveyed by a buffer with a pH lower than the neutral pH, the pH is neutralized, then dialysis and lyophilization take place. However, the elution of the final product from the immobilized lysine using a buffer with a low pH of less than 9, does not make it possible to obtain a destabilase complex capable of the desired storage and the sufficient enzymatic activity sought . Indeed, the destabilase molecule extracted by this process is in the form of an unstable monomeric enzymatic complex which undergoes a change of conformation, which makes it lose its capacity to form an enzymatic complex in polymeric form. The enzymatic complex in the form of a monomer no longer has the capacity to structure itself in a desirable manner into a polymer, a polymer which is organized into a liposome. The known complexes (prototypes) produced by medicinal leeches form a complex of hirudin, prostaglandin, destabilase, and kallikrein inhibitors of blood plasma in the ratio 1: 1: 1: 1.
L'abrégé XP002237190 de la demande de brevet RU 2 129429 C décrit un procédé pour isoler des complexes de prostaglandine provenant de sangsues
médicinales. Le procédé implique la purification du mélange par chromatographie d'affinités avec des anticorps anti 6-cétoprostaglandine-Fl immobilisés sur un support insoluble dans l'eau.The abstract XP002237190 of patent application RU 2 129429 C describes a method for isolating prostaglandin complexes from leeches medicinal. The method involves purifying the mixture by affinity chromatography with anti-6-ketoprostaglandin-F1 antibodies immobilized on a water-insoluble support.
Le document de Nikonov G I et al. (« Destabilase complexes - natural liposome produced by médicinal leeches hirudo medicinalis » Fundamental & Clinical Pharmacology, Elsevier, Paris, FR, vol.13, n°.l, 1999, pages 102-106) décrit un procédé pour produire des liposomes naturels à base de complexes de destabilase obtenus à partir de sangsues médicinales. Afin d'obtenir des complexes de destabilase ayant des propriétés de type micelle, il est procédé à : - une extraction par des solvants organiques à partir de la tête de sangsues,The document by Nikonov G I et al. (“Destabilase complexes - natural liposome produced by medicinal leeches hirudo medicinalis” Fundamental & Clinical Pharmacology, Elsevier, Paris, FR, vol.13, n ° .l, 1999, pages 102-106) describes a process for producing natural liposomes with base of destabilase complexes obtained from medicinal leeches. In order to obtain destabilase complexes having micelle-type properties, it is carried out: - extraction with organic solvents from the head of leeches,
- une chromatographie d'affinités comprenant des anticorps anti-céto-PFG, immobilisés sur un substrat de sépharose 4B activé par du CNBr.- an affinity chromatography comprising anti-keto-PFG antibodies, immobilized on a 4B sepharose substrate activated by CNBr.
L'analyse de l'éluat est effectuée par électrophorèse. La fraction obtenue est composée d'agrégat de monomère de destabilase et présente les propriétés des protéines micellaires et est représentée par un complexe stable lipides-protéines.The analysis of the eluate is carried out by electrophoresis. The fraction obtained is composed of destabilase monomer aggregate and has the properties of micellar proteins and is represented by a stable lipid-protein complex.
Les monomères de destabilase peuvent se mettre sous forme de polymère de complexe de destabilase formant un liposome. Lesdits liposomes présentent la capacité de pénétrer rapidement les membranes cellulaires et d'avoir une activité anti- thrombotique. La demande de brevet WO 92 02206 décrit des feuilles de collagène natives lyophilisées comprenant des formulaires cosmétiques pour le traitement de la couperose. Les ingrédients actifs de la formulation cosmétique peuvent comprendre de l'hirudine, une protéine anti-coagulante provenant de sangsues.The destabilase monomers can be in the form of a destabilase complex polymer forming a liposome. Said liposomes have the capacity to rapidly penetrate cell membranes and to have anti-thrombotic activity. Patent application WO 92 02206 describes lyophilized native collagen sheets comprising cosmetic forms for the treatment of rosacea. The active ingredients in the cosmetic formulation may include hirudin, an anti-coagulant protein from leeches.
La demande de brevet WO 98 16604 décrit des compositions nettoyantes, de préférence des compositions détergentes pour lessives, comprenant une enzyme de type iso-peptidase ayant la capacité de couper de manière catalytique les liaisons entre la glutamine et la lysine.Patent application WO 98 16604 describes cleaning compositions, preferably detergent compositions for detergents, comprising an iso-peptidase type enzyme having the capacity to catalytically cut the bonds between glutamine and lysine.
La demande de brevet WO 01 47572 décrit une méthode pour inhiber la resténose d'un vaisseau sanguin comprenant : - la fourniture d'un dispositif comportant un composé actif ayant une activité anti-thrombotique et anti-inflammatoire, etPatent application WO 01 47572 describes a method for inhibiting the restenosis of a blood vessel comprising: - the supply of a device comprising an active compound having an anti-thrombotic and anti-inflammatory activity, and
- l'implantation du dispositif dans le vaisseau sanguin pour inhiber la resténose dudit vaisseau sanguin.
Les inventeurs se sont donnés pour but d'obtenir un complexe de destabilase stable présentant des activités biologiques améliorées par rapport aux compositions de l'art antérieur.- implantation of the device in the blood vessel to inhibit the restenosis of said blood vessel. The inventors have given themselves the goal of obtaining a stable destabilase complex having improved biological activities compared to the compositions of the prior art.
Les inventeurs ont réussi à obtenir un complexe monomérique de destabilase qui est stable et capable de s'agréger en polymère s'organisant en liposome.The inventors have succeeded in obtaining a monomeric destabilase complex which is stable and capable of aggregating into a polymer organizing itself into a liposome.
Ainsi l'invention a pour objet selon un premier aspect un complexe de destabilase stable, sous forme de monomère, capable de s'agréger en complexe destabilase polymère formant un liposome, susceptible d'être obtenu à partir de sangsues médicinales par un procédé comprenant : - une chromatographie d'affinité avec lesdits anticorps de la 6-keto- prostaglandine immobilisés sur une colonne appropriée ;Thus, the subject of the invention is, according to a first aspect, a stable destabilase complex, in the form of a monomer, capable of aggregating into a polymeric destabilase complex forming a liposome, capable of being obtained from medicinal leeches by a process comprising: - affinity chromatography with said antibodies to 6-ketoprostaglandin immobilized on an appropriate column;
- P elution à force ionique élevée du complexe destabilase, ledit complexe présentant une activité antithrombine d'au moins 700 ATU/mg, une durée de recalcification du plasma d'au moins 800 APC/mg, une activité fibrinolytique d'au moins 40mrn2/mg, et une activité immunomodulatrice.- P elution at high ionic strength of the destabilase complex, said complex having an antithrombin activity of at least 700 ATU / mg, a plasma recalcification duration of at least 800 APC / mg, a fibrinolytic activity of at least 40 min 2 / mg, and immunomodulatory activity.
Le procédé de purification est fait par chromatographie, de préférence chromatographie par affinité. Ce procédé comprend :The purification process is done by chromatography, preferably affinity chromatography. This process includes:
- une chromatographie d'affinité avec lesdits anticorps de la 6-keto- prostaglandine immobilisés sur une colonne appropriée, de préférence de la 6-keto- PGFlα ;- Affinity chromatography with said antibodies to 6-keto-prostaglandin immobilized on an appropriate column, preferably 6-keto-PGFl α ;
- l'élution à force ionique élevée du complexe destabilase.- elution at high ionic strength of the destabilase complex.
Ledit complexe de destabilase obtenu par ce procédé est sous forme de monomère et comprend de la destabilase et au moins un composé du groupe hirudine, prostaglandine, inhibiteur de kallikréine. On peut également combiner l'action d'anticorps anti 6-keto-PGFlα et de lysine sépharose.Said destabilase complex obtained by this process is in the form of a monomer and comprises destabilase and at least one compound from the hirudin group, prostaglandin, kallikrein inhibitor. It is also possible to combine the action of anti-6-keto-PGF1 α antibodies and of lysine sepharose.
Le complexe destabilase liposome présente, tout comme le complexe destabilase monomérique, des propriétés pharmaceutiques très utiles, à savoir typiquement une activité antithrombine d'au moins 500, de préférence d'au moins 600 ou 700 ATU/mg, une durée de recalcification du plasma d'au moins 800 APC/mg, une activité fibrinolytique d'au moins 40 mm /mg. L'action antithrombique et thrombolytique est nettement améliorée par rapport au contrôle et au prototype.
L'invention concerne également une composition pharmaceutique comprenant un complexe destabilase liposome selon l'invention et un véhicule pharmaceutiquement acceptable.The destabilase liposome complex has, like the monomeric destabilase complex, very useful pharmaceutical properties, typically an antithrombin activity of at least 500, preferably at least 600 or 700 ATU / mg, a duration of plasma recalcification. at least 800 APC / mg, a fibrinolytic activity of at least 40 mm / mg. The antithrombic and thrombolytic action is clearly improved compared to the control and the prototype. The invention also relates to a pharmaceutical composition comprising a liposome destabilase complex according to the invention and a pharmaceutically acceptable vehicle.
Le terme transporteur pharmaceutiquement acceptable est utilisé pour désigner un matériau non toxique, solide ou liquide diluant ou encapsulant, qui ne réagit pas sur le composé actif de manière négative pour son efficacité.The term pharmaceutically acceptable carrier is used to denote a non-toxic, solid or diluent or encapsulating liquid material, which does not react negatively on the active compound for its effectiveness.
L'invention concerne également une composition cosmétique comprenant ce complexe de destabilase liposome selon l'invention.The invention also relates to a cosmetic composition comprising this liposome destabilase complex according to the invention.
L'invention concerne également le complexe de destabilase liposome en tant que médicament, et l'utilisation d'un complexe de destabilase monomère ou polymère liposome pour la préparation d'un médicament présentant une activité anti-coagulante et immuno-modulatrice.The invention also relates to the liposome destabilase complex as a medicament, and the use of a liposome monomeric or polymeric destabilase complex for the preparation of a medicament having anti-coagulant and immunomodulatory activity.
L'invention concerne également un dispositif de purification d'un complexe de destabilase à partir de sangsues médicinales, comprenant une colonne d'affinité chargée avec des anticorps anti 6-keto-prostaglandine.The invention also relates to a device for purifying a destabilase complex from medicinal leeches, comprising an affinity column loaded with anti-6-keto-prostaglandin antibodies.
L'invention concerne également une prothèse médicale implantable, dans laquelle au moins une partie de la prothèse est couverte avec un habillage, ledit habillage comprenant un complexe destabilase liposome selon l'invention.The invention also relates to an implantable medical prosthesis, in which at least part of the prosthesis is covered with a covering, said covering comprising a destabilase liposome complex according to the invention.
Dans un mode de réalisation préféré, la prothèse implantable est un support stent. D'autres objets et avantages de l'invention apparaîtront dans la description détaillée qui suit.In a preferred embodiment, the implantable prosthesis is a stent support. Other objects and advantages of the invention will appear in the detailed description which follows.
Le procédé de purification du complexe de destabilase stable selon l'invention comprend une étape de chromatographie par affinité à l'aide d'anticorps de 6-keto- PGFlα (6-keto-prostaglandine Flα), immobilisés sur un support insoluble dans l'eau. Le principe de la chromatographie par immuno-affinité est connu de l'homme du métier et repose sur la spécificité d'anticorps mono ou polyclonaux pour capturer des antigènes protéiques spécifiques à partir d'extraits naturels complexes. Par contre les inventeurs ont réussi à obtenir de façon tout à fait surprenante un complexe destabilase aux activités biologiques conservées à la fois anti-coagulantes et immuno- modulatrices, et capable de se structurer en liposomes.The method for purifying the stable destabilase complex according to the invention comprises a step of affinity chromatography using antibodies of 6-keto-PGFlα (6-keto-prostaglandin F lα ), immobilized on a support insoluble in l 'water. The principle of immunoaffinity chromatography is known to those skilled in the art and is based on the specificity of mono or polyclonal antibodies to capture specific protein antigens from complex natural extracts. On the other hand, the inventors have succeeded in obtaining, quite surprisingly, a destabilase complex with preserved biological activities which is both anti-coagulant and immunomodulatory, and capable of being structured into liposomes.
Les anticorps sont couplés à une phase solide chromatographique, typiquement d' agarose, par des liaisons covalentes (bromure de cyanogène par exemple CNBR), ou
d'autres couplages chimiques ciblant les groupes amino, hydroxyle, carboxyle ou sulphidryle des immunoglobulines de manière à former une matrice solide. La phase solide couplée aux anticorps est alors placée dans une colonne de chromatographie par affinité. Le mélange de l'antigène cible et des contaminants est dilué dans un tampon de liaison puis appliqué sur la colonne. Les contaminants non adsorbes sont éliminés par le lavage avec différents tampons. L'élution de l'antigène protéique cible est ensuite obtenue en utilisant par exemple des conditions de pH extrêmes, des changements de forces ioniques.The antibodies are coupled to a solid chromatographic phase, typically of agarose, by covalent bonds (cyanogen bromide, for example CNBR), or other chemical couplings targeting the amino, hydroxyl, carboxyl or sulphidryl groups of the immunoglobulins so as to form a solid matrix. The solid phase coupled to the antibodies is then placed in an affinity chromatography column. The mixture of target antigen and contaminants is diluted in a binding buffer and then applied to the column. Non-adsorbed contaminants are removed by washing with different buffers. The elution of the target protein antigen is then obtained using for example extreme pH conditions, changes in ionic strengths.
L'hirudine et les inhibiteurs de kallikréine ont une activité seulement dans la phase aqueuse. La destabilase a une activité seulement dans la phase non aqueuse.Hirudin and kallikrein inhibitors have activity only in the aqueous phase. Destabilase has activity only in the nonaqueous phase.
Le complexe de destabilase stable obtenu par les inventeurs présente des propriétés hydrophobes conditionnées par le composant prostaglandine, et des propriétés hydrophiles par les polypeptides du liposome.The stable destabilase complex obtained by the inventors exhibits hydrophobic properties conditioned by the prostaglandin component, and hydrophilic properties by the liposome polypeptides.
La forme monomère du complexe destabilase a un poids moléculaire de 25 kDa, est stable, présente une capacité d'agrégation qui se traduit par la formation de liposomes. Elle est obtenue à partir d'extraits totaux de sangsues, ou d'une fraction en particulier des sécrétions de glandes salivaires ou du sang du tube digestif de la sangsue. A l'issue de la purification par chromatographie par affinité, le complexe destabilase obtenu comprend les composants suivants : hirudine, prostaglandine (substance analogue à la prostaciclyne), inhibiteur de kallikréine, destabilase. Le complexe destabilase présente les propriétés suivantes : activité antithrombotique par l'hirudine, augmentation du temps de recalcification du plasma sanguin par les inhibiteurs de kallikréine, blocage de l'adhésion et de l'agrégation plaquettaire par les substances analogues aux prostacyclines, dissolution de fibrines stabilisés par la destabilase.The monomeric form of the destabilase complex has a molecular weight of 25 kDa, is stable, has an aggregation capacity which results in the formation of liposomes. It is obtained from total extracts of leeches, or from a fraction in particular of secretions from the salivary glands or from the blood of the digestive tract of the leech. After purification by affinity chromatography, the destabilase complex obtained comprises the following components: hirudin, prostaglandin (substance similar to prostaciclyne), kallikrein inhibitor, destabilase. The destabilase complex has the following properties: antithrombotic activity by hirudin, increased time for recalcification of blood plasma by kallikrein inhibitors, blocking of adhesion and platelet aggregation by prostacyclin-like substances, dissolution of fibrins stabilized by destabilase.
Ces propriétés du complexe destabilase déterminent son efficacité antithrombotique, thrombolytique (2,5 fois supérieures à celles du prototype), immuno-modulatrice, et son activité hypotensive complètement absente chez le prototype. On présente ci-après les méthodes de mesure de l'activité biologique utilisées pour l'extrait de sangsues purifié et sous forme de liposome, puis quatre exemples de
réalisation démontrant l'activité à la fois anti-coagulante et immuno-modulatrice du complexe destabilase selon l'invention.These properties of the destabilase complex determine its antithrombotic, thrombolytic (2.5 times greater than those of the prototype), immunomodulatory, and hypotensive activity completely absent in the prototype. The following are the methods for measuring the biological activity used for the purified leech extract and in the form of a liposome, then four examples of realization demonstrating both the anti-coagulant and immunomodulatory activity of the destabilase complex according to the invention.
1/ L'activité antithrombine est déterminée par l'allongement de la durée de précipitation du fibrinogène par la thrombine. On détermine la durée de formation d'un précipité dans un système comprenant 0,2 ml d'une solution de fibrinogène à 0,3%, et 0,1 ml du complexe destabilase après fixation 0,1 ml d'une solution de thrombine comprenant une unité d'activité thrombine. L'activité de l'hirudine est exprimée en unités internationales antithrombines (ATU NIH). 2/ Le temps de recalcification du plasma sanguin est déterminé dans un système comprenant 0,1 ml de plasma sanguin citrique et 0,1 ml de complexe destabilase après fixation de 0,1 ml d'une solution 0,025 M de CaCl2. L'augmentation de ce paramètre par deux correspond à une unité APC.1 / The antithrombin activity is determined by the lengthening of the duration of fibrinogen precipitation by thrombin. The duration of the formation of a precipitate is determined in a system comprising 0.2 ml of a 0.3% fibrinogen solution, and 0.1 ml of the destabilase complex after fixing 0.1 ml of a thrombin solution. comprising a thrombin activity unit. Hirudin activity is expressed in international antithrombin units (ATU NIH). 2 / The time for recalcification of the blood plasma is determined in a system comprising 0.1 ml of citric blood plasma and 0.1 ml of destabilase complex after fixing 0.1 ml of a 0.025 M solution of CaCl 2 . The increase of this parameter by two corresponds to one APC unit.
3/ Le contenu en prostaglandines est déterminé en utilisant un radio immuno-essai pour le 6-keto-PGFlα obtenu auprès de la Compagnie « Amersham ». 4/ L'action antithrombotique du complexe destabilase est déterminée sur des rats en utilisant la méthode de thromboformation de Wessler (5). Le niveau de blocage de la thromboformation est évalué par rapport au contrôle. Pour cela on étudie les résultats suite à une séparation de 4 heures entre l'injection du complexe destabilase et une injection de sérum sanguin humain activé par le froid. Ce degré de blocage est exprimé en % par rapport au contrôle (volume égal d'une solution saline normale).3 / The prostaglandin content is determined using a radio immunoassay for 6-keto-PGFl α obtained from the "Amersham" Company. 4 / The antithrombotic action of the destabilase complex is determined on rats using the thromboformation method of Wessler (5). The level of thromboformation blockage is evaluated in relation to the control. For this, we study the results following a 4-hour separation between the injection of the destabilase complex and an injection of cold-activated human blood serum. This degree of blockage is expressed in% relative to the control (equal volume of normal saline solution).
5/ L'action hypotensive est déterminée suite à l'administration orale de 0,2 ml d'un complexe destabilase chez des rats d'une lignée SHR (spontanément hypertensive). Le niveau initial de pression était de 165±5 mm. Après 10 heures d'analyse, le niveau de pression chez le rat est mesuré dans la veine tail. Le niveau d'efficacité hypotensive est exprimé en pourcentage par rapport au contrôle (volume égal administré d'une solution saline normale).5 / The hypotensive action is determined following the oral administration of 0.2 ml of a destabilase complex in rats of a SHR line (spontaneously hypertensive). The initial pressure level was 165 ± 5 mm. After 10 hours of analysis, the pressure level in the rat is measured in the tail vein. The level of hypotensive efficiency is expressed as a percentage relative to the control (equal volume administered with normal saline).
6/ L'activité cytophage de neutrophiles est déterminée par la méthode connue de Chernushenko (6). Les recherches ont été effectuées sur vingt rats pubères. Une solution aqueuse de complexe destabilase a été injectée en intraveineuse quotidiennement pendant dix jours à une dose de 0,5 ml (n = 10). Un volume égal d'une solution 0,85% NaCl a été injecté chez des animaux contrôle (n = 10). A l'issue du délai approprié, du sang a été collecté chez les animaux, et l'activité cytophage des neutrophiles a été étudiée. Un index de phagocytose a été défini : un index cytophage (CI) et un pourcentage de phagocytose (Ph).
7/ L'influence sur l'activité cellulaire a été étudiée en mesurant l'inhibition de l'activité de composants du système du complément. On a utilisé également une méthode de définition de l'activité hémolytique du sérum humain dilué (7). Les résultats ont été les suivants. Exemple 1.6 / The cytophagous activity of neutrophils is determined by the known method of Chernushenko (6). The research was carried out on twenty pubescent rats. An aqueous solution of destabilase complex was injected intravenously daily for ten days at a dose of 0.5 ml (n = 10). An equal volume of 0.85% NaCl solution was injected into control animals (n = 10). At the end of the appropriate period, blood was collected from the animals, and the cytophagous activity of the neutrophils was studied. A phagocytosis index has been defined: a cytophagous index (CI) and a percentage of phagocytosis (Ph). 7 / The influence on cellular activity has been studied by measuring the inhibition of the activity of components of the complement system. A method of defining the hemolytic activity of diluted human serum was also used (7). The results were as follows. Example 1.
Le protocole est le suivant :The protocol is as follows:
- Prendre dix sangsues médicinales (au total 12 g), homogénéiser, mélanger à de l'eau distillée jusqu'à obtenir un volume total d'homogénéisat de 24 ml (ratio en volumes- Take ten medicinal leeches (in total 12 g), homogenize, mix with distilled water until a total volume of homogenate of 24 ml is obtained (volume ratio
1 :1), broyer, collecter le surnageant. - Mettre le surnageant de l'homogénéisât sur une colonne d' agarose comportant des anticorps immobilisés anti 6-keto-PGFlα.1: 1), grind, collect the supernatant. - Put the supernatant of the homogenate on an agarose column comprising immobilized anti-6-keto-PGFl α antibodies.
L' elution est obtenue par un tampon comprenant 0,2 M de glycine avec 0,15 M HC1 etThe elution is obtained by a buffer comprising 0.2 M glycine with 0.15 M HC1 and
0,5 M NaCl. Le volume de l'éluat est de 35 ml.0.5 M NaCl. The volume of the eluate is 35 ml.
Comme cela est visible sur le tableau 1, le produit final a une activité fibrinolytique, antithrombine, augmente le temps de recalcification, contient de la prostaglandine, et en plus du fort potentiel antithrombolytique, possède une action hypotensive réduisant la pression artérielle de 25% (la ramenant pratiquement à la normale).As can be seen in Table 1, the final product has fibrinolytic, antithrombin activity, increases recalcification time, contains prostaglandin, and in addition to the high antithrombolytic potential, has a hypotensive action reducing blood pressure by 25% ( bringing it back practically to normal).
Exemple 2. 5 ml d'une sécrétion de glandes salivaires de sangsues ont été introduits dans une colonne d' agarose avec des anticorps immobilisés anti 6-keto-PGFlα,. L'élution est obtenue à l'aide d'un tampon phosphate 0,5 M, pH 6,4. Le volume de l'éluat est de 10 ml. Comme indiqué sur le tableau 1, le produit final a une activité fibrinolytique antithrombine, augmente le temps de recalcification, contient de la prostaglandine, et en plus d'un fort potentiel antithrombique et thrombolitique, possède une action hypotensive, réduisant la pression artérielle de 25% (la ramenant pratiquement à la normale). Le complexe destabilase augmente l'index cytophage et le pourcentage de phagocytose (tableau 1), démontrant l'action immuno-stimulative.Example 2. 5 ml of a secretion from the salivary glands of leeches were introduced into an agarose column with immobilized anti-6-keto-PGF1 α antibodies. The elution is obtained using a 0.5 M phosphate buffer, pH 6.4. The volume of the eluate is 10 ml. As shown in Table 1, the final product has fibrinolytic antithrombin activity, increases recalcification time, contains prostaglandin, and in addition to a high antithrombic and thrombolitic potential, has a hypotensive action, reducing blood pressure by 25 % (bringing it back to practically normal). The destabilase complex increases the cytophagous index and the percentage of phagocytosis (Table 1), demonstrating the immunostimulatory action.
Exemple 3.Example 3.
10 ml de sang du tube digestif de sangsues médicinales (trois mois après leur dernier repas) ont été placés dans une colonne de L-Glutamine-Sépharose. L'élution est obtenue avec une solution 1 M KC1. Le volume de l'éluat est de 20 ml. Comme indiqué sur le tableau 1, le produit final a une activité fibrinolytique antithrombine,
augmente le temps de recalcification, contient de la prostaglandine, et en plus d'un fort potentiel antithrombique et thrombolytique, possède une action hypotensive, réduisant la pression artérielle de 25% (la ramenant pratiquement à la normale). Le complexe destabilase augmente l'index cytophage et le pourcentage de phagocytose (tableau 1), démontrant l'action immuno-stimulative.10 ml of blood from the digestive tract of medicinal leeches (three months after their last meal) were placed in an L-Glutamine-Sepharose column. The elution is obtained with a 1 M KC1 solution. The volume of the eluate is 20 ml. As indicated in table 1, the final product has fibrinolytic antithrombin activity, increases the recalcification time, contains prostaglandin, and in addition to a high antithrombic and thrombolytic potential, has a hypotensive action, reducing blood pressure by 25% (bringing it back to practically normal). The destabilase complex increases the cytophagous index and the percentage of phagocytosis (Table 1), demonstrating the immunostimulatory action.
Exemple 4.Example 4.
Le protocole est le suivant : prendre vingt sangsues médicinales (au total 21 g), extraire la partie avant de l'animal, homogénéiser, et recueillir l'extrait aqueux. 10 ml de l'extrait sont placés dans une colonne de L-Lysine-Sépharose. L'élution est obtenue avec une solution 1 M KC1. Le volume de l'éluat est de 20 ml.The protocol is as follows: take twenty medicinal leeches (total 21 g), extract the front part of the animal, homogenize, and collect the aqueous extract. 10 ml of the extract are placed in an L-Lysine-Sepharose column. The elution is obtained with a 1 M KC1 solution. The volume of the eluate is 20 ml.
Comme indiqué sur le tableau 1, le produit final a une activité fibrinolytique antithrombine, augmente le temps de recalcification, contient de la prostaglandine, et en plus d'un fort potentiel antithrombique et thrombolytique, possède une action hypotensive, réduisant la pression artérielle de 25%) (la ramenant pratiquement à la normale). Le complexe destabilase augmente l'index cytophage et le pourcentage de phagocytose (tableau 1), démontrant l'action immuno-stimulative.
As shown in Table 1, the final product has fibrinolytic antithrombin activity, increases recalcification time, contains prostaglandin, and in addition to a high antithrombic and thrombolytic potential, has a hypotensive action, reducing blood pressure by 25 %) (bringing it back to almost normal). The destabilase complex increases the cytophagous index and the percentage of phagocytosis (Table 1), demonstrating the immunostimulatory action.
Tableau 1 : définition activités d'extraits de sangsues.Table 1: definition activities of leech extracts.
En ce qui concerne l'application de l'extrait purifié sur les stents, l'homme du métier dispose d'un grand nombre de méthodes possibles. Ainsi les stents supports du complexe destabilase obtenu par les inventeurs peuvent être de types très variés. Par exemple un stent présentera une surface polymérique externe sur laquelle sera placée une matrice gélatineuse, la matrice incluant le complexe de destabilase sous forme de liposomes. Selon une réalisation, la surface polymérique sera liée par des liaisons
covalentes avec la matrice gélatineuse. Le gel polymérique peut avoir par exemple une épaisseur de 10 à 50μm à l'état non comprimé. Ce gel peut être choisi par exemple dans le groupe constitué par des acides polycarboxyliques, des polymères cellulosiques, de la gélatine, de la polyvinylpyrrolidone, des polymères anhydrides maléiques, des polyamides, des alcools polyvinyliques, des oxydes de polyéthylène, de l'acide polyacrylique.
With regard to the application of the purified extract to the stents, a person skilled in the art has a large number of possible methods. Thus the support stents of the destabilase complex obtained by the inventors can be of very varied types. For example, a stent will have an external polymeric surface on which a gelatinous matrix will be placed, the matrix including the destabilase complex in the form of liposomes. According to one embodiment, the polymer surface will be linked by bonds covalent with the gelatinous matrix. The polymer gel can, for example, have a thickness of 10 to 50 μm in the uncompressed state. This gel can be chosen, for example, from the group consisting of polycarboxylic acids, cellulosic polymers, gelatin, polyvinylpyrrolidone, maleic anhydride polymers, polyamides, polyvinyl alcohols, polyethylene oxides, polyacrylic acid. .
Claims
1. Complexe de destabilase stable, sous forme de monomère, capable de s'agréger en complexe destabilase polymère formant un liposome, susceptible d'être obtenu à partir de sangsues médicinales par un procédé comprenant :1. A stable destabilase complex, in the form of a monomer, capable of aggregating into a polymeric destabilase complex forming a liposome, capable of being obtained from medicinal leeches by a process comprising:
- une chromatographie d'affinité avec lesdits anticorps de la 6-keto- prostaglandine immobilisés sur une colonne appropriée ;- affinity chromatography with said antibodies to 6-ketoprostaglandin immobilized on an appropriate column;
- l'élution à force ionique élevée du complexe destabilase, caractérisé en ce qu'il présente une activité antithrombine d'au moins 700 ATU/mg, une durée de recalcification du plasma d'au moins 800 APC/mg, une activité fibrinolytique d'au moins 40mm2/mg, et une activité immunomodulatrice.- the elution at high ionic strength of the destabilase complex, characterized in that it has an antithrombin activity of at least 700 ATU / mg, a duration of plasma recalcification of at least 800 APC / mg, a fibrinolytic activity d '' at least 40mm 2 / mg, and immunomodulatory activity.
2. Composition pharmaceutique comprenant un complexe destabilase selon la revendication 1 et un véhicule pharmaceutiquement acceptable.2. Pharmaceutical composition comprising a destabilase complex according to claim 1 and a pharmaceutically acceptable vehicle.
3. Composition cosmétique comprenant un complexe de destabilase selon la revendication 1.3. Cosmetic composition comprising a destabilase complex according to claim 1.
4. Complexe de destabilase selon la revendication 1 en tant que médicament.4. A destabilase complex according to claim 1 as a medicament.
5. Utilisation d'un complexe de destabilase selon la revendication 1 pour la préparation d'un médicament présentant une activité anti-coagulante et immunomodulatrice.5. Use of a destabilase complex according to claim 1 for the preparation of a medicament having an anti-coagulant and immunomodulatory activity.
6. Prothèse médicale implantable, dans laquelle au moins une partie de la prothèse est couverte avec un habillage, ledit habillage comprenant un complexe de destabilase selon la revendication 1.6. An implantable medical prosthesis, in which at least part of the prosthesis is covered with a covering, said covering comprising a destabilase complex according to claim 1.
7. Prothèse implantable selon la revendication 6, caractérisé en ce qu'il s'agit d'un support stent. 7. Implantable prosthesis according to claim 6, characterized in that it is a stent support.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0210042 | 2002-08-07 | ||
FR0210042A FR2843304B1 (en) | 2002-08-07 | 2002-08-07 | EXTRACTS OF BLOOD FOR STENTS |
PCT/FR2003/002473 WO2004015096A2 (en) | 2002-08-07 | 2003-08-06 | Leech extracts for stents |
Publications (1)
Publication Number | Publication Date |
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EP1527169A2 true EP1527169A2 (en) | 2005-05-04 |
Family
ID=30470988
Family Applications (1)
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EP03756529A Withdrawn EP1527169A2 (en) | 2002-08-07 | 2003-08-06 | Leech extracts for stents |
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US (1) | US20060110425A1 (en) |
EP (1) | EP1527169A2 (en) |
JP (1) | JP2005534331A (en) |
AU (1) | AU2003282192A1 (en) |
FR (1) | FR2843304B1 (en) |
WO (1) | WO2004015096A2 (en) |
Cited By (1)
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CN105307634A (en) * | 2013-03-15 | 2016-02-03 | 沃纳奇尔科特有限责任公司 | Pharmaceutical soft gelatin capsule dosage form with modified guar gum |
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FR2884829B1 (en) * | 2005-04-22 | 2008-01-11 | Ricarimpex | PROCESS FOR OBTAINING A BACTERIAL EXTRACT OF AEROMONAS HYDROPHILA FROM BLOOD, AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAME |
US8709473B1 (en) * | 2009-01-28 | 2014-04-29 | Abbott Cardiovascular Systems Inc. | Method of targeting hydrophobic drugs to vascular lesions |
RU2560839C1 (en) * | 2014-03-20 | 2015-08-20 | Общество с ограниченной ответственностью "Малое инновационное предприятие Центр экологических и оздоровительных технологий" | Composition of cosmetic product of hero-protective action |
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IT1243196B (en) * | 1990-08-03 | 1994-05-24 | Arval Spa | LYOPHILIZED NATIVE COLLAGEN SHEETS CONTAINING COSMETIC FORMULAS FOR THE TREATMENT OF COUPEROSE |
RU2112528C1 (en) * | 1994-12-23 | 1998-06-10 | Институт биоорганической химии им.М.М.Шемякина и Ю.А.Овчинникова РАН | Method of preparing enzyme destabilase showing fibrinolytic, thrombolytic, endo- and exo-gamma)-lys-isopeptidase and amidolytic activity |
RU2129429C1 (en) * | 1995-09-05 | 1999-04-27 | Московское акционерное общество "Биокон" | Method of isolating complex destabilase-prostaglandin from medicinal leech |
CA2268009A1 (en) * | 1996-10-15 | 1998-04-23 | The Procter & Gamble Company | Detergent compositions containing isopeptidase |
WO2001047572A2 (en) * | 1999-12-29 | 2001-07-05 | Advanced Cardiovascular Systems, Inc. | Device and active component for inhibiting formation of thrombus-inflammatory cell matrix |
-
2002
- 2002-08-07 FR FR0210042A patent/FR2843304B1/en not_active Expired - Fee Related
-
2003
- 2003-08-06 AU AU2003282192A patent/AU2003282192A1/en not_active Abandoned
- 2003-08-06 WO PCT/FR2003/002473 patent/WO2004015096A2/en active Application Filing
- 2003-08-06 US US10/523,937 patent/US20060110425A1/en not_active Abandoned
- 2003-08-06 EP EP03756529A patent/EP1527169A2/en not_active Withdrawn
- 2003-08-06 JP JP2004526981A patent/JP2005534331A/en active Pending
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Cited By (2)
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CN105307634A (en) * | 2013-03-15 | 2016-02-03 | 沃纳奇尔科特有限责任公司 | Pharmaceutical soft gelatin capsule dosage form with modified guar gum |
CN105307634B (en) * | 2013-03-15 | 2020-03-10 | 沃纳奇尔科特有限责任公司 | Pharmaceutical soft gelatin capsule dosage form containing modified guar gum |
Also Published As
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US20060110425A1 (en) | 2006-05-25 |
FR2843304A1 (en) | 2004-02-13 |
WO2004015096A8 (en) | 2005-05-06 |
JP2005534331A (en) | 2005-11-17 |
WO2004015096A3 (en) | 2004-04-08 |
FR2843304B1 (en) | 2005-12-16 |
AU2003282192A1 (en) | 2004-02-25 |
WO2004015096A2 (en) | 2004-02-19 |
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