EP3283626A1 - Immobilized plasminogenase composition, preparation process, use and device comprising such a composition - Google Patents
Immobilized plasminogenase composition, preparation process, use and device comprising such a compositionInfo
- Publication number
- EP3283626A1 EP3283626A1 EP16729958.5A EP16729958A EP3283626A1 EP 3283626 A1 EP3283626 A1 EP 3283626A1 EP 16729958 A EP16729958 A EP 16729958A EP 3283626 A1 EP3283626 A1 EP 3283626A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- medium
- plasmin
- plasminogenase
- rich
- plasma
- 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- Withdrawn
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Classifications
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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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
- C12N11/087—Acrylic polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
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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
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/484—Plasmin (3.4.21.7)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
-
- 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
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
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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
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6456—Plasminogen activators
- C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
Definitions
- the invention relates to an enzymatic composition comprising a plasminogenase immobilized on a solid support, a process for preparing such an enzymatic composition, the use of such an enzymatic composition for the preparation of an autologous plasmatic medium ex vivo and rich in plasmin and a device comprising such an enzymatic composition.
- the invention particularly relates to an enzyme composition and a device comprising such an enzyme composition which are suitable for use in the treatment of a pathology in a patient in which an autologous composition enriched with autologous plasmin is required.
- an enzyme composition enriched with autologous plasmin is required.
- a device comprising such an enzyme composition which are suitable for use in the treatment of a pathology in a patient in which an autologous composition enriched with autologous plasmin is required.
- Certain pathological indications in ophthalmology such as vitreo-macular tractions (TVM) generate macular holes created by differential tangential tensile stresses occurring between the vitreous body and the retina.
- the treatment of these pathologies requires releasing the retina from these constraints.
- the known treatments are mainly of a surgical nature aimed at mechanically separating the retina and the vitreous body.
- WO2010 / 125148 discloses a treatment of a detachment of the retina in traction (in English "tractional retinal detachment") in which a sterile composition obtained by addition of heterologous urokinase is administered into the vitreous humor of a patient. to the patient's plasma.
- This sterile composition comprises heterologous urokinase and is likely to pose a problem of immunological and inflammatory reaction in the patient.
- an injectable composition comprising a recombinant protein formed from the catalytic domain of plasmin and the effect of the injection of such an injectable composition at the vitreo-retinal interface of ex vivo human eyes or eyes of in vivo cat.
- Such a recombinant protein is however complex in its manufacture.
- the presence of heterologous recombinant protein in such an injectable composition leads to a problem of immune reaction in the patient receiving this injectable composition.
- the cost of this injectable composition is high, so that alternative solutions to the use of recombinant proteins are sought, with which the risks of immune reaction would also be minimized.
- JP2007 / 068497 a plasminogen plasmin conversion process previously extracted from plasma. JP2007 / 068497 does not teach a method for converting plasma plasminogen directly into a plasma into plasmin and preparing an autologous plasma enriched with plasmin.
- the invention therefore aims to solve all of these problems.
- the invention aims at providing an enzymatic composition, a method of preparation and the use of such an enzymatic composition, and a device comprising such an enzymatic composition which makes it possible to convert plasminogen ex vivo from a plasma blood medium. in plasmin under the effect of the enzymatic composition.
- the invention makes it possible to prepare an ex vivo plasmatic medium rich in plasmin free of heterologous enzyme or having only a residual amount of minimal free heterologous enzyme and in particular insufficient to provoke an immune reaction during the bringing into contact-in particular injection- of said ex vivo plasmin-rich plasma medium into contact with tissue (s) of a patient.
- the aim of the invention is to propose an enzymatic composition, a method of preparation and the use of such an enzymatic composition, and a device comprising such an enzymatic composition adapted to allow rapid conversion - especially in a period of between 15 min and 60 min at 37 ° C - of plasminogen from a plasmatic blood plasma medium.
- the invention relates to an enzymatic composition
- an enzymatic composition comprising:
- plasminogenase at least one enzyme, referred to as plasminogenase, for conversion to plasmin of plasminogen of a plasmatic blood medium comprising plasminogen;
- the solid support having dimensions adapted to be retained on a filter having a cutoff threshold less than or equal to 0.22 ⁇ ;
- said plasminogenase is bound to the solid support and remains bound to this support in contact with a blood plasma medium and in that the composition is in a dry state.
- blood plasma medium means any liquid medium free of viable blood cells and resulting directly from a fractionation treatment of blood-in particular human blood-liquid not coagulated, under conditions adapted to allow separation of blood cells (red blood cells, leukocytes, platelets) and blood plasma medium free of viable blood cells. Such separation can be achieved for example by centrifugation or cell sorting in a microfluidic process;
- ex vivo plasma medium denotes any plasma plasmatic medium extracted from the human or animal body
- plasminogenase denotes any enzyme exhibiting plasminogen conversion activity to plasmin by cleavage of a peptide bond of plasminogen
- stable bond refers to all the forces ensuring, under predetermined conditions, the cohesion between groups of atoms, in particular between the solid support and at least one plasminogenase when the enzymatic composition is contacted by immersion at a temperature of the order of 37 ° C in a blood plasma medium;
- the invention relates to an enzymatic composition
- an enzymatic composition comprising at least one immobilized plasminogenase on a solid support in the divided state, said enzymatic composition being adapted to be able to:
- the plasminogenase, the solid support and the bond formed between the plasminogenase and the solid support are selected so that the plasminogenase remains bound to the solid support when the enzyme composition is brought into contact with a blood plasma medium.
- the enzymatic composition according to the invention is in the dry state.
- dry state translates, in particular, the fact that the enzymatic composition according to the invention does not bring to the blood plasma medium with which it is brought into contact any liquid, in particular any aqueous liquid, capable of modifying the ionic composition and / or the ionic concentrations of the blood plasma medium in which the enzyme composition is contacted. It does not therefore substantially modify the osmotic properties of the plasma blood medium with which it is put in contact. It also does not modify, by its only addition in the blood plasma medium, the concentration of the plasminogen present in the blood plasma medium in which it is added.
- the enzymatic composition according to the invention does not release into the blood plasma medium brought into contact with the enzymatic composition of plasminogenase in the free state or releases into the blood plasma medium brought into contact with the enzyme composition a small amount of plasminogenase in the free state.
- the enzymatic composition according to the invention is therefore likely to be used in a process for the preparation of a plasmin-rich ex vivo plasma medium substantially free of heterologous plasminogenase, from a blood plasma medium formed from blood taken from a patient.
- Such an autologous ex vivo plasma plasmin-rich medium substantially free of heterologous plasminogenase is intended for use in the same patient.
- the inventor has observed that it is possible to immobilize at least one plasminogenase on a solid support in the dissolved state which is insoluble in aqueous solution while retaining a plasminogenase activity.
- Such an enzymatic composition makes it possible, on the one hand, to effectively convert plasminogen from a plasma blood medium to plasmin and, on the other hand, facilitates the separation of the enzyme composition from an ex vivo plasmatic medium rich in plasmin obtained by placing in contact with a blood plasma medium and the enzymatic composition, by limiting - in particular by completely avoiding - a significant untimely release of plasminogenase in this ex vivo plasma rich plasmin medium.
- the plasmin rich ex vivo plasma medium is therefore substantially free of exogenous and heterologous plasminogenase.
- the solid support is in the divided state and formed of particles having three dimensions extending in three orthogonal directions to each other, at least two of the three dimensions being greater than 0.22 ⁇ .
- each particle size is greater than 0.22 ⁇ .
- At least two of the dimensions of the solid support particles in the divided state are between 1 ⁇ and 500 ⁇ , in particular between 10 ⁇ and 500 ⁇ , preferably between 100 ⁇ and 500 ⁇ .
- each particle size of the solid support in the divided state is between 1 ⁇ and 500 ⁇ , especially between 10 ⁇ and 500 ⁇ , preferably between 100 ⁇ and 500 ⁇ .
- the dimensions of the particles of the solid support in the divided state are chosen so that the enzymatic composition is retained on a sterilization filter, that is to say on a filter having a cut-off threshold less than or equal to 0.22. ⁇ .
- the solid support is formed of a porous material.
- the porous material has pores with an average diameter of between 5 nm and 50 nm, preferably between 10 nm and 20 nm.
- the porous material is chosen from the group formed of rigid materials.
- the solid support is formed of a material chosen from the group formed of high specific surface materials.
- the solid support of the enzymatic composition is a solid support excluding the solid supports cited in JP2007 / 068497.
- the solid support of the enzymatic composition according to the invention is a solid support excluding the Sepharose 4FF / BB-CNBr support, the Sepharose 4FF-NHS support, the Sepharose 4B-ECH support and the Affi-Prep 10 support.
- the solid support in particular the solid support in the divided state, is formed of a material chosen from the group consisting of polyglucoside polymers and poly-methacrylic polymers.
- the solid support is selected from the group consisting of SEPABEADS ® EC-HFA / S and GE Healthcare Epoxy-activated Sepharose TM supports.
- the solid support in the divided state is formed of spherical particles of average diameter between 100 ⁇ and 300 ⁇ .
- the solid support-in particular the solid support in the divided state- is formed of a material chosen from the group formed of poly-methacrylic polymers.
- the solid support is formed of a material selected from the group formed in particular poly-methacrylic copolymers, for example poly (glycidyl methacrylic (GMA) / ethylene dimethacrylic copolymers (EDMA).
- each plasminogenase - is a serine endopeptidase - in particular an antithrombotic activity endopeptidase - of class EC 3.4.21 of the classification of enzymes.
- At least one plasminogenase - in particular each plasminogenase - is selected from the group consisting of a urokinase, a streptokinase, a nattokinase and a tissue plasminogen activator (t-PA).
- at least one plasminogenase is urokinase U0633 (Sigma-Aldrich, Lyon, France).
- the enzymatic composition comprises a single plasminogenase.
- the enzyme composition may comprise a plurality of different plasminogenases in admixture.
- At least one plasminogenase is bound to the solid support by at least one stable bond selected from the group consisting of a covalent bond, an ionic bond, a covalent bond coordination (or dative linkage) and a van der Waals-type hydrophobic interaction.
- At least one stable bond is chosen to resist contacting with an aqueous solution of NaCl at a concentration of 0.5 M.
- At least one plasminogenase - in particular each plasminogenase - is bound to the solid support by at least one covalent bond.
- at least one such covalent bond is formed by chemical reaction between a free amine group of said plasminogenase and an epoxy group of the solid support.
- the enzymatic composition comprises a single plasminogenase or a mixture of a plurality of plasminogenases.
- the enzymatic composition is adapted to form, by placing in contact with an enzymatic composition according to the invention and with a plasma blood medium, an ex vivo plasmin-rich plasma medium which is substantially pyrogen-free.
- An enzymatic composition according to the invention is suitable for not releasing a heterologous compound-in particular heterologous plasminogenase-in a plasmin-rich ex vivo plasma medium obtained by bringing the enzymatic composition into contact with a plasma blood medium-in particular with a temperature of the order of 37 ° C-, said heterologous compound being capable of inducing an immune reaction in a subject treated with an amount of plasmin-rich ex vivo plasma medium.
- the pyrogenic / pyrogen-free character of the plasmin-rich ex vivo plasmatic medium obtained by contacting a blood plasma medium and an enzymatic composition according to the invention by measuring the body temperature of rabbits injected with Autologous ex vivo plasma medium rich in plasmin.
- the lack of temperature increase rabbit body following injection reflects the pyrogen-free character of plasmin-rich plasma ex vivo medium.
- the enzymatic composition is free of any microbial germ, in particular of any pathogenic microbial germ.
- the enzymatic composition is sterile.
- the enzymatic composition is in the dry state. It may be in the form of a dry powder adapted to be put directly in contact with a plasma blood medium, in particular without the addition of water or aqueous solution (such as water for solution for injection or saline) and to allow a plasmin conversion of at least a portion of the plasminogen of the blood plasma medium under the action of said plasminogenase in contact with the blood plasma medium.
- aqueous solution such as water for solution for injection or saline
- the enzymatic composition is in the dry state and dehydrated; it can therefore be kept in a dry and dehydrated state; it is adapted to be put directly in contact with a plasma blood medium, in particular without any addition of water or aqueous solution (such as water for solution for injection or saline) and to allow conversion to plasmin at least a part of the plasminogen of the plasma blood medium under the action of said plasminogenase in contact with the blood plasma medium.
- aqueous solution such as water for solution for injection or saline
- the enzymatic composition is in the form of dehydrated powder.
- each plasminogenase is bound to the solid support so as to introduce, into a plasma blood medium in contact with which the enzyme composition is placed, a free plasminogenase mass of less than 400 ⁇ g, said contact being carried out according to the method below:
- a mass of between 0.01 g and 0.5 g, in particular of the order of 0.1 g of enzyme composition in the state, is mixed at a temperature of about 37.degree. dehydrated with a volume of between 0.5 mL and 1.0 mL - in particular of the order of 0.7 mL of blood plasma medium, then;
- the contact is maintained for a period greater than 5 minutes, in particular between 5 minutes and 60 minutes, then;
- the enzymatic composition and the blood plasma medium are separated by filtration on a filter having a cutoff threshold less than or equal to 0.22 ⁇ , and;
- the mass of plasminogenase released in the plasma blood medium is measured.
- the mass of free plasminogenase in the blood plasma medium in contact with which the enzyme composition is placed is less than 200 ⁇ g, especially less than 100 ⁇ g, preferably less than 50 ⁇ g.
- the mass of free plasminogenase in the blood plasma medium in contact with which the enzyme composition is placed is less than 40 ⁇ g, especially less than 30 ⁇ g, in particular less than 25 ⁇ g, preferably less than 20 ⁇ g. more preferably less than 10 ⁇ g.
- the mass of free plasminogenase in the blood plasma medium in contact with which the enzyme composition is placed is less than 20 ⁇ g, especially less than 15.0 ⁇ g, in particular less than 8.0 ⁇ g, preferably less than 3.0 ⁇ g, more preferably less than 1.5 ⁇ g.
- the mass of free plasminogenase in the blood plasma medium in contact with which the enzyme composition is placed is less than 10 ⁇ g, especially less than 8.0 ⁇ g, in particular less than 6.0 ⁇ g, preferably less than 5.0 ⁇ g, more preferably less than 2 ⁇ g.
- the mass of free plasminogenase in the blood plasma medium in contact with which the enzyme composition is placed is less than 0.5 ⁇ g.
- the assay of the plasminogenase released by any suitable method for example by quantitative enzyme immunoenzymatic assay of "ELISA" type using a primary antibody specific for plasminogenase and a quantifiable secondary antibody and carrying out a calibration curve to from solutions containing known amounts of plasminogenase in blood plasma medium.
- plasminogenase activity released in plasmin-rich ex vivo plasma medium by an indirect method by analyzing the temporal variation of the plasmin activity of the plasmin-rich ex vivo plasmatic medium obtained after removal by filtration of the enzyme composition. .
- the quasi-stability of the plasmin activity reflects the absence of free plasminogenase in the ex vivo plasmin-rich plasma medium, whereas a significant increase in plasmin activity can reflect the presence of free plasminogenase in plasmin-rich ex vivo plasma medium.
- the enzymatic composition according to the invention makes it possible to form an ex vivo plasmin-rich plasma medium which is weakly immunogenic.
- At least one plasminogenase - in particular each plasminogenase - is bound to the solid support by a group of atoms comprising a main chain of atoms linearly linked to each other by covalent bonds (ie say the chain of greater number of atoms), the main chain having a number of atoms at least equal to 4, in particular between 10 and 15 atoms.
- the enzymatic composition has an activity, called plasminogenase activity, of plasminogen plasmin conversion of a blood plasma medium in contact with which it is placed under the following conditions:
- a mass of between 0.01 g and 0.5 g, in particular of the order of 0.1 g, of said enzymatic composition in the dehydrated state in contact with temperature is placed of the order of 37 ° C. for a period of greater than 5 minutes, in particular between 5 minutes and 60 minutes, with a volume of between 0.5 ml and 1.0 ml, in particular of the order of 0.7 ml. blood plasma medium, and
- plasmin activity an initial enzymatic activity, called plasmin activity, as measured by a test for the release of ara.
- plasmin activity an initial enzymatic activity, as measured by a test for the release of ara.
- -nitro-aniline greater than 0.1 ⁇ -notably between 0.1 and 0.3 ⁇ , preferably of the order of 0.2 ⁇ - ara-nitro-aniline released per minute and per milliliter (mL) plasmin rich plasma medium ex vivo
- said release test consisting of:
- Plasmin activity is measured in a plasmin-rich ex vivo plasma medium obtained by contacting a blood plasma medium and an enzymatic composition according to the invention by an indirect method in which an amount of enzyme composition is placed in in contact with an amount of blood plasma medium comprising plasminogen at 37 ° C for a period of time between 5 min and 60 min and under conditions adapted to allow a plasminogen conversion of the plasma plasmatic medium to plasmin and the formation of a plasmin-rich ex vivo plasma medium.
- a separation step is then carried out, in particular by sterile filtration, of the enzymatic composition and plasmin-rich ex vivo plasma medium formed by conversion of plasminogen from the blood plasma medium to plasmin.
- this sterilizing filtration step in a single filtration stage on a filter having a cut-off threshold of less than or equal to 0.22 ⁇ .
- this sterilizing filtration step in several stages comprising a first filtration of separation of the enzyme composition and plasmin-rich ex vivo plasma medium, the first filtration being non-sterilizing, and then a second filtration of the medium.
- ex vivo plasmatic rich in plasmin free of enzymatic composition said second filtration being a filter sterilizing filtration having a cut-off threshold less than or equal to 0.22 ⁇ .
- the plasmin activity of the ex vivo plasmatic medium rich in plasmin is determined.
- a measuring medium of the plasmin activity is prepared in a spectrophotometric measuring tank by mixing, at 37 ° C., a volume of plasma-rich ex vivo plasma medium and the same volume of an aqueous solution of plasmin.
- a chromogenic substrate S-2251 HD-Val-Leu-Lys-pNA "2HC £, Chromogenix, Le Pre Saint Gervais, FRANCE
- Vm is the total volume (in mL) of the measuring medium
- ⁇ is the molecular extinction coefficient (in M -1 cm -1 ) of para-xixaniline at 405 nm;
- L is the length (in cm) of the optical path of the spectrophotometric measurement vessel, and;
- Vp is the volume (in mL) of plasmin-rich ex vivo plasma medium introduced into the spectrophotometric measuring cell.
- the basal plasmin activity of a non-plasmin enriched blood plasma medium is measured as a control by replacing, in the above protocol, the plasmin-rich ex vivo plasmatic medium with the same volume of plasma plasmatic medium from which the ex vivo plasmatic medium rich in plasmin is derived. From the plasmin activity value of the ex vivo plasmin-rich plasma medium is deduced the basal plasmin activity value thus calculated.
- the invention also extends to a method for preparing an enzymatic composition according to the invention.
- the invention also relates to a method for preparing an enzymatic composition according to the invention, in which:
- At least one enzyme called plasminogenase, is chosen for converting plasminogen plasmin to a plasmatic blood medium comprising plasminogen;
- an insoluble solid support is chosen in aqueous solution
- the solid support is brought into contact with each plasminogenase so as to bind each plasminogenase to the solid support, and;
- a lyophilization step is carried out so as to form the enzymatic composition.
- each plasminogenase is immobilized on the solid support simply by contacting each plasminogenase in liquid solution with the solid support.
- the solid support is immersed in a liquid solution - in particular aqueous - of each plasminogenase, which is kept under stirring for a time sufficient to allow coupling between the solid support and each plasminogenase.
- a lyophilization step is then carried out so as to form the enzyme composition in the dehydrated state and retain the activity of the plasminogenase (s).
- conditions are selected-in particular temperature conditions-which are adapted to form the enzyme composition.
- At least one plasminogenase in particular each plasminogenase, is selected from the group consisting of serine endopeptidases, in particular endopeptidases with antithrombotic activity, of class EC 3.4.21 of the classification of enzymes.
- a solid support is chosen in the divided state and formed of particles having three dimensions extending in three directions orthogonal to each other, at least two of said three dimensions being greater than 0 , 22 ⁇ .
- each particle size is greater than 0.22 ⁇ .
- the solid support in the group of solid supports in the divided state is chosen having particles of substantially spherical shape and of diameter greater than 0.22 ⁇ . Such a solid support is therefore likely to be able to be retained on filtration devices having a maximum permeation size of 0.22 ⁇ .
- At least two of the three dimensions of the solid support particles in the divided state are between 1 ⁇ and 500 ⁇ , in particular between 10 ⁇ and 500 ⁇ , preferably between 100 ⁇ and 500 ⁇ .
- each particle size of the solid support in the divided state is between 1 ⁇ and 500 ⁇ , especially between 10 ⁇ and 500 ⁇ , preferably between 100 ⁇ and 500 ⁇ .
- a solid support is chosen in the divided state which can be retained by a filtration separation device having a cut-off point of the order of 0.22 ⁇ , for example a separation device.
- filtration comprising a filter of polytetrafluoroethylene (PTFE) or polyvinylidene fluoride (PVDF).
- PTFE polytetrafluoroethylene
- PVDF polyvinylidene fluoride
- the solid support in the divided state is formed of a material comprising at least one surface ligand capable of forming at least one stable bond-in particular at least one covalent bond-with at least one plasminogenase.
- the surface ligand is formed of a group of atoms comprising a main chain of atoms linearly linked to each other by covalent bonds (that is to say the chain of greatest number of atoms), the main chain having a number of atoms at least equal to 4-in particular between 10 and 15-.
- At least one surface ligand comprises an epoxy group.
- at least one surface ligand is an amino-epoxide group bonded to the solid support and of formula (III) below:
- the material of the solid support-in particular of the solid support in the divided state- is chosen from the group formed of functionalized poly-glycosides and functionalized methacrylate polymers.
- the solid support material in the divided state is selected from the group consisting of poly-glycosides functionalized at the surface by at least one epoxy group and methacrylate polymers functionalized at the surface by at least one epoxy group.
- the solid support in the divided state is chosen from the group formed by SEPABEADS ® EC-HFA / S supports, the average particle diameter of which is between 100 ⁇ and 300 ⁇ , and GE Healthcare Epoxy-activated Sepharose TM.
- At least one sterilization stage is carried out.
- At least one sterilization step is carried out by any known sterilization method to at least partially preserve the activity of the plasminogenase.
- at least one sterilization step is carried out by irradiation.
- At least one step of sterilization of the enzyme composition is carried out.
- at least one sterilization step of the enzyme composition is carried out by irradiation.
- at least one irradiation sterilization step is carried out at a predetermined temperature. We can perform such a sterilization step by irradiation at a temperature below 0 ° C.
- at least one irradiation sterilization step is carried out at a temperature above 0 ° C.
- At least one sterilization step is carried out by successive irradiations, said successive irradiations being interspersed with at least one cooling phase.
- At least one irradiation sterilization step is carried out, releasing an amount of energy of between 5 ⁇ 10 3 J / Kg (5 kGy) and 5 ⁇ 10 4 J / Kg (50 kGy).
- at least one sterilization step is carried out by irradiation with a radiation selected from the group formed by ⁇ -radiation and ⁇ -radiation.
- a lyophilization step of the enzyme composition is carried out.
- An enzymatic composition is formed in the form of dehydrated powder.
- At least one sterilization step is carried out by irradiation of the enzyme composition after lyophilization.
- a method according to the invention makes it possible to obtain a sterile enzymatic composition.
- a sterile and pyrogen-free enzymatic composition is obtained.
- the invention also extends to an enzymatic composition that can be obtained by a process according to the invention.
- the invention also extends to any use of an enzymatic composition according to the invention for the preparation of an ex vivo plasmatic medium rich in sterile plasmin and free from enzymatic composition.
- the invention also extends to any use of an enzymatic composition according to the invention for the preparation of a plasmin-rich ex vivo plasmatic medium, in particular for the preparation of a plasmoly-rich sterile ex vivo plasma medium.
- an enzymatic composition according to the invention for converting at least a part of the plasminogen from a plasma plasmatic medium to plasmin, which is the active form of plasminogen, and to form a plasma ex vivo medium which is rich in plasmin without significant release of free plasminogenase into plasmin-rich ex vivo plasma medium and by limiting the risks of inducing an immune reaction in a patient who has been administered ex plasmin rich plasma medium.
- ex vivo plasmatic medium rich in plasmin is thus obtained which can be used in the context of any preventive or curative treatment of a pathology in which a transformation of plasminogenase into autologous plasmin is required, for example in the context of the treatment. of a cardiovascular pathology or in the context of a surgical intervention, in particular during an intravitreal intervention of a treatment - in particular during a surgical intervention - of vitreo-macular disorders in ophthalmology.
- Such plasmin-rich ex vivo plasma medium can be obtained in a sterile, solid support-free and substantially enzyme-free form by separation / filtration on a sterilization filter.
- an amount of the enzymatic composition is placed in contact with an amount of plasma plasma medium comprising plasminogen for a period of between 15 min and 60 min, so as to form the ex vivo plasma medium.
- the enzymatic composition is maintained in contact with the blood plasma medium at a temperature close to the optimum temperature of the plasminogenase, especially at a temperature of the order of 37.degree.
- Such a method is simple in its implementation in that it requires only contacting the enzyme composition and the amount of blood plasma medium and maintaining the mixture at a predetermined temperature for a period of time suitable to allow a Plasminogenase conversion to plasmin under the effect of plasminogenase, followed by a plasma medium sample ex vivo rich in plasmin formed under the effect of plasminogenase, said sample comprising a filtration -partement a sterilizing filtration-.
- the plasmin-rich ex vivo plasmatic medium is adapted to be used directly, for example by intravitreal injection, during an intravitreal intervention of a treatment - especially during a surgical procedure - of vitreo-macular disorders in ophthalmology.
- the plasmin rich ex vivo plasma medium is autologous.
- an enzymatic composition according to the invention is used in a method implemented in a room dedicated to performing an intravitreal injection (IVT) and in which an authorized person, in particular a surgeon, an ophthalmologist , a caregiver, proceeds:
- the plasmin rich ex vivo plasma medium is separated from the enzyme composition by filtration by means of a filter capable of retaining the enzyme composition.
- separation of the plasma-rich ex vivo plasma medium and the enzyme composition is carried out by filtration using a filter having a cut-off point of the order of 0.22 ⁇ .
- the separation of plasmin-rich ex vivo plasma medium and of the enzyme composition is a sterilizing separation of the ex vivo plasma rich plasmin medium.
- An enzymatic composition according to the invention is used during a treatment in which an plasmin-rich ex vivo plasma medium is injected into the vitreous body of a patient in order to release the vitreomacular stresses by hydrolysis of protein fibers.
- An enzymatic composition according to the invention is used for the preparation of an ex vivo plasmin-rich plasma medium which is autologous-that is to say which is obtained from a blood plasma medium derived from the blood of a patient. patient and prepared for injection into this patient alone and which lacks a heterologous protein-that is, lacking a sufficient amount of heterologous protein capable of inducing an immune response in the patient.
- the enzymatic composition is used for the preparation of an ex vivo plasmin-rich plasma medium which is pyrogen-free.
- the conditions for obtaining a pyrogen-free plasmin-rich ex vivo plasma medium are validated by measuring the body temperature of a rabbit which has been injected with plasmin-rich ex vivo plasma medium obtained from a quantity of medium. blood plasma of said rabbit.
- the absence of an increase in body temperature of the rabbit following the injection reflects the pyrogen-free character of the ex vivo plasmin-rich plasma medium and validates the conditions for obtaining it.
- the enzymatic composition is used for the preparation of an ex vivo plasmin-rich plasma medium which is sterile.
- the invention also extends to a device for the preparation of an ex vivo blood plasma medium rich in sterile plasmin and free from enzymatic composition, comprising a quantity of enzymatic composition according to the invention and a filter having a lower cutoff threshold. or equal to 0.22 ⁇ .
- the invention also extends to a device comprising:
- a container-in particular a hermetically sealed container-containing the amount of enzyme composition a device for introducing blood plasma medium into the container;
- the sampling device (11) and the filter being arranged to allow the filtration of the plasma medium and obtaining a filtrate constituting a sterile plasmin-rich ex vivo plasmatic medium free of enzyme composition.
- a device according to the invention is advantageously in the form of a kit, that is to say a kit comprising several elements in the separated state, of which at least:
- the container containing an amount of a sterile enzymatic composition according to the invention, said container being hermetically sealed and adapted to preserve the sterility of the enzymatic composition,
- a plasmin rich ex vivo plasma medium sampling device comprising the enzymatic composition
- the filter is a sterilization filter of plasmin-rich ex vivo plasmatic medium and of forming an ex vivo plasmatic medium rich in sterile plasmin and free from enzymatic composition.
- each of the constituent elements of the kit is packaged separately sterilely in an individual package.
- kits are sterilely packaged together in a sterile state in a common package. They may be in the assembled state or in the disassembled state or in a partially assembled state and in a sterile state.
- a device in a third embodiment of a device according to the invention, some of the constituent elements of the kit excluding the container
- the enzymatic composition containing the enzyme composition is packaged together in the assembled state or in the dissociated state or in a partially assembled state in a non-sterile state in a common package and then sterilized by any known and adapted sterilization process.
- the container containing the enzyme composition can be sterilized by any sterilization process respecting the enzymatic activity of the immobilized enzyme.
- the device for introducing the quantity of plasma plasmatic medium into the container comprises:
- a syringe comprising a piston sliding in a cylinder provided with a dispensing outlet end;
- a needle adapted to be connected with the open end of the syringe
- the syringe and the needle being adapted to cooperate and to allow:
- the blood plasma preparation of the middle tube is a blood collection tube, such as a Vacutainer ® tube (BD Diagnostics, Le Pont de Claix, France), adapted to enable said removal and, if appropriate, to oppose the coagulation of said blood plasma medium removed.
- a blood collection tube such as a Vacutainer ® tube (BD Diagnostics, Le Pont de Claix, France)
- BD Diagnostics Le Pont de Claix, France
- the needle of the introduction device has a length adapted to allow the removal of blood plasma medium in the sampling tube and blood plasma medium preparation.
- the sampling device comprises:
- a sterile syringe for taking a quantity of plasma-rich ex vivo plasma medium in the container; the filter adapted to be inserted between the sterile syringe and a sampling needle in the plasmin-rich ex vivo plasma medium container comprising the enzymatic composition, said filter being capable of receiving the plasma-rich ex vivo plasma medium and the composition enzymatic, to retain the enzymatic composition and to deliver into the syringe plasmatic environment ex vivo rich in plasmin and free of enzymatic composition.
- the device may also comprise an additional sterile dispensing needle of the plasma plasmin-rich plasma medium in the body of a patient, as part of the treatment of a cardiovascular pathology or in the context of a surgical intervention, in particular during an intravitreal intervention of a treatment - especially during a surgical intervention - in ophthalmology.
- the container is a bottle equipped with a stopper formed of polymer adapted to be pierced by a needle and to allow introduction of the plasma blood medium into the container and a sample of the plasma plasmin rich blood plasma medium. from the container.
- the filter is a sterilizing filter having a cut-off threshold less than or equal to 0.22 ⁇ , that is to say on filter adapted to be able to retain particles whose average diameter is greater than 0 , 22 ⁇ -including bacteria, yeasts, fungi-.
- the device comprises an outer sterile packaging envelope enclosing at least the sterile container comprising the sterile enzyme composition, at least one sterile syringe, at least one sterile needle and the sterile filtration device.
- the assembly formed of the syringe, the needle, the filtration device and the sterile packaging outer casing can be sterilized by a sterilization treatment (irradiation, ethylene oxide, or other ... ) then associated with the sterile container comprising the enzyme composition.
- the invention also relates to an enzymatic composition, a method of preparation, the use of such an enzymatic composition and a device or kit for treating a blood plasma medium, characterized in combination by all or some of the characteristics mentioned above. or below.
- the enzymatic activity of a solution comprising a plasminogenase was determined by measuring the initial rate of the predetermined temperature hydrolysis reaction of substrate S-2251 (HD-Val-Leu-Lys-pNA ). 2HC £, Chromogenix, Werfen France, Pre Saint Gervais, France) introduced into the solution.
- a measuring medium at 37 ° C. is formed in a spectrophotometric measuring tank by mixing a volume of a solution of plasminogenase in physiological saline and of the same volume of an aqueous solution of S-2251 at a concentration of the order of 2 mM (so that the concentration of S-2251 is of the order of 1 mM in the measuring medium) and likely to release para-mimaniline ( ⁇ ⁇ 10 000 M _1 .cm _1 ) under the action of plasminogenase / urokinase. From the mixture, the evolution of the absorbance (optical density, OD 40 5 nm ) at 405 nm of the measuring medium at 37 ° C.
- Plasminogenase activity (I) in which:
- £ is the molecular extinction coefficient (in M -1 cm -1 ) of para-mlmaniline at 405 nm;
- L is the length (in cm) of the optical path of the spectrophotometric measurement vessel, and;
- Vs is the volume (in mL) of plasminogenase solution introduced into the spectrophotometric measuring tank.
- plasmin activity The enzymatic activity of plasmin (called plasmin activity) of an ex vivo plasmatic medium rich in plasmin devoid of enzymatic composition and obtained by contacting at 37 ° C. with a quantity of enzymatic composition according to the invention with an amount of plasma plasma medium comprising plasminogen for a period of time ranging from 5 min to 60 min by spectrophotometric measurement of the formation of ara-nitroaniline from S-2251.
- plasmin activity The enzymatic activity of plasmin (called plasmin activity) of an ex vivo plasmatic medium rich in plasmin devoid of enzymatic composition and obtained by contacting at 37 ° C. with a quantity of enzymatic composition according to the invention with an amount of plasma plasma medium comprising plasminogen for a period of time ranging from 5 min to 60 min by spectrophotometric measurement of the formation of ara-nitroaniline from S-2251.
- a measuring medium is prepared in a spectrophotometric measuring tank by mixing at 37 ° C. a volume of plasmin-rich ex vivo plasma medium and the same volume of an aqueous solution of S-2251 (HD-Val-Leu). - Lys-pNA " 2HC £, Chromogenix, Werfen France, Pre Saint Gervais, FRANCE) at a concentration of the order of 2 mM (so that the concentration of S-2251 is of the order of 1 mM in the measuring medium) and capable of releasing para-mimaniline under the action of plasmin from the plasma-rich ex vivo plasma medium.
- the evolution of the absorbance (optical density, OD 40 5 nm ) at 405 nm of the measuring medium is measured at 37 ° C. for 5 minutes.
- the slope at the origin of the evolution curve of the absorbance at 405 nm is evaluated, that is to say the initial speed Vi of the reaction expressed in A abs / min.
- the plasmin activity of the plasmin-rich ex vivo plasmatic medium (expressed in U / ml plasmin-rich ex vivo plasma medium) is given by the following formula (II): Vi.Vm.lO 3
- Vm is the volume (in mL) of the measuring medium
- L is the length (in cm) of the optical path of the spectrophotometric measurement vessel, and;
- Vp is the volume (in mL) of plasmin-rich ex vivo plasma medium introduced into the spectrophotometric measuring cell.
- the amount of plasminogenase of a plasmin-rich ex vivo plasma medium is determined by a fluorescence measurement according to any method known per se, for example by assaying by the enzyme-linked immunosorbent assay using a primary antibody.
- specificity of the plasminogenase in particular an antibody directed against human urokinase-for example, ABcam rabbit antibody ab24121-
- a quantifiable secondary antibody for example, a goat antibody directed against rabbit IgG and conjugated to the HRP ("Horse Raddish Peroxidase") in the Presence of Amplex ® UltraRed
- HRP Hexe Raddish Peroxidase
- a solid support is chosen in the divided state in the group of supports formed of a porous hydrophilic material, in particular in the group of supports formed of particles of hydrophilic material. porous and having surface grafting groups of epoxy nature. It is possible to choose solid carrier is Epoxy-GE Healthcare (GE Healthcare Epoxy- activated Sepharose TM). It is also possible to choose the SEPABEADS ® EC-EP / S support as a solid support (Resindion, Binasca, Italy).
- SEPABEADS ® EC-HFA / S split solid support Resindion, Binasca, Italy
- the particles of material SEPABEADS ® EC-HFA / S are formed of polymethacrylate and functionalized on the surface by minoepoxide groups of formula (III) below:
- the average porosity of the support is between 10 nm and 20 nm.
- an immobilization of a plasminogenase - for example a urokinase, or a streptokinase, or a nattokinase, or a tissue plasminogen activator (t-PA) - is carried out. on a solid material in the divided state.
- an amount of dry solid material is hydrated in an aqueous hydration composition.
- the aqueous hydration composition may be, for example, osmosis water, water "for injection” (so-called PPI) or sterile physiological saline.
- 0.2 g of solid material is placed dried in -for example divided state 0.2g SEPABEADS ® EC-HFA / O status sec in 40 ml of water for injection for 1 hour at room temperature, then three successive rinses of the solid material hydrated with 0.7 ml of sterile physiological saline pH 6.8.
- the solid was then rinsed material into contact with 0.7 ml of an aqueous solution in particular of sterile pyrogen-free saline or sterile intraocular irrigating solution (BSS Bioaqua ®, "Balanced Salt Solution”) - of human urokinase (U0633, Sigma-Aldrich, Lyon, France) comprising on the order of 2 units (2 U / mL) of plasminogenase activity per mL.
- BSS Bioaqua ® sterile intraocular irrigating solution
- U0633 human urokinase activity per mL
- the contact between the solid support and the enzyme is maintained for several hours.
- the liquid reaction supernatant is removed and the enzymatic composition thus obtained is then rinsed 3 times with 0.7 ml of a 1M NaCl solution in PPI water, or preferably sterile physiological saline.
- the synthesis of the enzyme composition is carried out under suitable conditions to form an enzymatic composition having a content of pyrogenic compounds lower than the upper acceptable limit value for an injectable composition - in particular less than 0 , 5 units of EU endotoxin / mL-.
- the solid support in the divided state is a SEPABEADS ® EC-HFA / S support obtained according to a Good Manufacturing Practices (GMP) process and having a reduced content of pyrogenic endotoxins.
- the consumables used are certified sterile and apyrogenous consumables.
- Glassware and laboratory equipment (bottle for hydration of the support, freeze-drying bottles, stoppers and spatulas) are treated before use with an alkaline detergent solution (E-toxa Clean, 1%) for 16 hours, rinsed with water and sterilized. All manipulations are performed under laminar flow hood. Starting reagents and solvents are chosen which are pyrogen-free and the steps for preparing the enzyme composition are carried out under optimum conditions of sterility.
- the solid support is then rinsed three times with 15.6 ml of serum physiological, then is brought into contact for several hours with 15.6 ml of urokinase (urokinase solution U0633 in sterile physiological saline, sterilized by filtration on a filter having a cut-off threshold of 0.22 ⁇ or less) at a concentration of of 2 U / mL of sterile saline so as to form the enzymatic composition.
- Three rinses of the enzyme composition are then carried out with 15.6 ml of physiological saline.
- the rinse liquid is removed and 0.2 g aliquots of wet enzyme composition are sampled in sterile lyophilization flasks.
- the aliquots of wet resin are lyophilized and then stored at 4 ° C.
- 0.7 ml of blood plasma medium is placed in contact with an aliquot of enzymatic composition obtained above at 37 ° C. for a period of between 5 min and 60 min.
- Plasmid rich ex vivo plasma medium is separated from the enzymatic composition by filtration.
- Plasmin activity (U / mL ex plasmin rich plasma medium) is measured in the presence of S-2251 substrate, the medium being placed at a temperature of 37 ° C.
- An enzymatic activity of the order of 0.2 ⁇ 0.1 U / ml is observed for a contact time of the blood plasma medium and of the enzymatic composition of between 5 min and 60 min, in particular of the order of 15 minutes. .
- SEPABEADS ® EC-HFA / S "GMP” material is hydrated in PPI water, and then three successive rinses of the hydrated material are made with 0.7 ml of sterile physiological saline at pH 6.8.
- the thus rinsed material is brought into contact with 0.7 ml of a solution of plasminogenase (urokinase U0633) in physiological saline having an enzymatic activity of 2 U / ml.
- plasminogenase urokinase U0633
- the liquid reaction supernatant is removed, and then three successive rinses are carried out
- the mass of urokinase present in the ex vivo plasma medium obtained by contacting 0.2 g of the enzyme composition and 0.7 ml of blood plasma medium for 60 minutes at a temperature of 37 ° C., measured by the immunoenzymatic method "ELISA" is between 2 ⁇ g and 20 ⁇ g.
- Urokinase is immobilized on a solid support in the SEPABEADS® EC-HFA / S "GMP" divided state by the method described in Example 1.
- a lyophilization step of the enzymatic composition obtained is carried out or not.
- the capacity of the lyophilized or non-lyophilized enzyme composition is studied by placing the same amount (0.2 g) of enzyme composition in 0.7 ml of blood plasma medium for a period of 15 minutes or 60 minutes. Plasmid-rich ex vivo plasma medium and enzymatic composition are separated by filtration.
- a solution of S-2251 substrate at a final concentration of 1 mM in the measuring medium is added to the plasmin-rich ex vivo plasma medium at 37 ° C. and the plasmin activity of the plasma medium is measured ex vivo.
- Table 1 The results are given in Table 1 below.
- Lyophilization of the enzyme composition has no effect on its plasminogen conversion activity from a plasma plasmatic medium to plasmin.
- An irradiation step is carried out at 25 kGy of the enzyme composition after lyophilization.
- the activity of the irradiated enzymatic composition is indirectly analyzed by measuring the plasmin activity of a medium. plasmin rich plasma ex vivo obtained by contacting said sterilized enzymatic composition and a blood plasma medium for 15 min or 60 min.
- the enzymatic composition is removed by filtration and the plasmin activity of ex vivo plasmin-rich plasma media thus obtained is measured in the presence of S-2251.
- Plasmin activity of plasmin-rich ex vivo plasmatic medium obtained after 15 min of contact is between 0.116 and 0.148 U / mL and plasmin activity of plasmin-rich ex vivo plasma medium obtained after 60 min of contact is between 0.200. and 0.218 U / mL.
- the sterilizing irradiation of the enzymatic composition makes it possible to maintain plasmin activity of the plasmin-rich ex vivo plasmatic medium obtained after 15 min of contact which is greater than 0.1 U / ml and a plasmin activity of the ex vivo plasma medium rich in plasma.
- plasmin obtained after 60 min of contact which is greater than 0.2 U / mL.
- Lyophilization coupled with terminal sterilization by irradiation makes it possible to reduce the amount of urokinase released by the enzyme composition in the ex vivo plasma medium rich in plasmin.
- the amount of free urokinase present in plasmin-rich ex vivo plasma medium obtained by contacting a blood plasma medium and an enzymatic composition (comprising immobilized urokinase on a solid support in the divided state) subjected to Irradiation is less than the amount of urokinase present in plasmin-rich ex vivo plasma medium obtained by contacting a blood plasma medium with a non-irradiated enzyme composition.
- the combination of irradiation and lyophilization treatments of the enzyme composition makes it possible to obtain amounts of urokinase in the ex vivo plasmin-rich plasma medium that are less than or equal to about 10 ⁇ g, in particular less than 5 ⁇ g.
- the enzymatic composition according to the invention makes it possible to convert a plasmatic blood medium into plasmin-enriched plasma ex vivo plasma having a high plasmin activity. It makes it possible to convert plasminogen from a plasma blood medium to plasmin without introducing into the ex vivo plasma medium formed a large quantity of immunogenic plasminogenase.
- Sterile is removed a quantity of blood of a patient to treatment in a sampling tube (BD Vacutainer ®, BD Diagnostics, Le Pont de Claix, France) comprising an anticoagulant EDTA type or sodium citrate.
- a step of separation of the blood cells and the blood plasma medium is carried out by centrifugation at 4000 rpm for 15 min.
- the blood plasma medium is intimately contacted with the enzyme composition at a temperature of 37 ° C for a period of at least 15 minutes necessary to allow the conversion of plasminogen to plasmin.
- a sterilization filter (0.22 ⁇ cut-off point), for example on a Millex PVDF filter (Millipore) or on an Acrodisk Syringe Filter filter, PN4602 (Pall), is used to separate the enzymatic composition and the rich ex vivo plasma medium.
- plasmin free of free urokinase We then proceed to an intraocular injection of a volume adapted from the plasmatic ex vivo medium rich in sterile plasmin.
- the assay by the enzyme immunoassay method "ELISA" of the amount of free urokinase present in the plasmin-rich ex vivo plasma medium shows an average amount of free urokinase of less than 20 ⁇ g.
- a device 20 according to a particular variant of the invention shown in the single figure comprises:
- means 3 for introducing a quantity of blood plasma medium sterilely into the container 1 and for bringing said quantity of plasma blood medium into contact with the enzymatic composition 2 and comprising;
- a sterile syringe 4 for dispensing the quantity of blood plasma medium from a blood collection tube and introducing said quantity of blood plasma medium taken from the container 1, said sterile dispensing syringe 4 comprising a piston 6 sliding in a cylinder 7 provided with an end, said end 8 distribution, axial opening;
- a dispensing needle 5 adapted to be connected with the dispensing end 8 of the sterile dispensing syringe 4 and having a pointed end 9 adapted to be introduced into the container 1 through a pierceable cap 10 and allow the introduction of the blood plasma medium into the container 1 under the effect of the displacement in translation of the piston 6 sliding in the cylinder 7.
- the dispensing needle 5 is for example preferably a needle of the order of 20 gauge (outer diameter of 0.9081 mm for a wall thickness of 0.1524 mm); means 11 for sampling and filtering a quantity of plasma-rich ex vivo plasma medium under the effect of the enzymatic composition 2, comprising;
- a sterile syringe 12 for the preparation of a quantity of plasma-rich ex vivo plasma medium from the container 1; a filtration device 13 equipped with an input 14 of plasmin-rich ex vivo plasma medium, and an outlet 15 which can be connected to the sterile preparation syringe 12 and shaped in order to be able to deliver rich ex vivo plasma medium.
- the filtration device 13 comprising a filter 16 capable of retaining the enzymatic composition of the plasma-rich ex vivo plasmatic medium flowing between the inlet 14 and the outlet 15 under the effect of the sterile preparation syringe 12, said inlet 14 being adapted to be assembled and hermetically connected to a plasmin-rich ex vivo plasma medium collection needle 17 of the container 1;
- sampling needle 17 being adapted to be placed in blood plasma medium communication with the inlet 14 of the filtration device 13 and having a pointed end 18 adapted to be introduced into the container 1 and allow a sampling of the plasma medium ex vivo rich in plasmin from the container 1.
- the sampling needle 17 is for example preferably a needle of the order of 20 gauge (Outer diameter of 0.9081 mm for a wall thickness of 0.1524 mm) .
- the pierceable cap formed of an elastic polymer-for example, chlorobutyl- adapted to be pierced by the needle 5 and allowing the introduction of blood plasma medium into the vessel 1 and the removal of the ex vivo rich plasma medium. plasmin from the container 1.
- the filtration device 13 is a filter filtration sterilization device 16 having a cut-off point of the order of 0.22 ⁇ , that is to say on a filter adapted to be able to retain particles whose average diameter is greater than 0.22 ⁇ .
- the device 20 comprises an outer sterile packaging envelope 30 of the container 1 comprising the enzyme composition, the means 3 for introducing a quantity of blood plasma medium sterile into the container 1 and the means 11 for sampling and filtration ex vivo plasmatic medium rich in plasmin.
- the sterile outer packaging envelope 30 enclosing the means 3 for introducing a sterile amount of blood plasma medium into the container 1 (including the sterile dispensing syringe 4 and dispensing needle 5) and the dispensing means 11.
- sampling and filtration ex vivo plasmin-rich plasma medium can be sterilized after packaging by any suitable sterilization means, the container 1 hermetically closed and containing the sterile enzymatic composition 2 according to the invention being sterilized by sterilization means respecting the functionality of the enzymatic composition 2.
- the device 20 may also comprise an additional sterile injection needle in the body of a patient of a suitable volume of plasmin-rich plasma ex vivo medium contained in the sterile syringe 12 for the preparation of the quantity ex vivo plasmatic medium rich in plasmin.
- an injection needle is a needle of between 25 and 30 gauge (that is to say, whose outer diameter is between 0.30 mm and 0.50 mm).
- the sterile syringe 12 for preparing the amount of plasmin-rich ex vivo plasma medium may have an open end formed of a "luer-lock” type adapter and the injection needle may have a complementary end of the adapter. "Luer-lock" of the syringe 12.
- a device may comprise: a hermetically sealed container 1 containing an amount of sterile enzymatic composition 2 according to the invention;
- a sterile syringe 12 for the preparation of a quantity of plasmin-rich ex vivo plasmatic medium from the container 1, said sterile syringe being a precision syringe capable of being able to contain and deliver a volume of ex vivo plasmatic medium rich in plasmin sterile and free of enzymatic composition between 100 ⁇ . and 250 ⁇ ., and;
- the constituent elements of a device according to the invention may be in the disassembled state or in the partially mounted state in the outer envelope.
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Abstract
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FR1553347A FR3035120B1 (en) | 2015-04-15 | 2015-04-15 | IMMOBILIZED PLASMINOGENASE COMPOSITION, PREPARATION METHOD, USE AND DEVICE COMPRISING SUCH COMPOSITION |
PCT/FR2016/050869 WO2016166484A1 (en) | 2015-04-15 | 2016-04-14 | Immobilized plasminogenase composition, preparation process, use and device comprising such a composition |
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US (1) | US20180135038A1 (en) |
EP (1) | EP3283626A1 (en) |
JP (1) | JP2018515131A (en) |
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US20100028904A1 (en) * | 2007-03-30 | 2010-02-04 | Centocor, Inc. | Way to obtain high expression clones of mammalian cells using a methylcellulose with fluorescent protein a or g and fluorescent screening method |
US20100272740A1 (en) * | 2007-10-17 | 2010-10-28 | Alexey Vertegel | Micro- and nanoscale devices for delivery of active fibronolytic agents |
AU2008331545B2 (en) * | 2007-11-29 | 2014-01-16 | Grifols Therapeutics Inc. | Recombinantly modified plasmin |
CN201351160Y (en) * | 2008-05-20 | 2009-11-25 | 博傲西腾医疗科技(上海)有限公司 | Device for extracting autologous blood plasminogen |
EP2297317A4 (en) * | 2008-06-04 | 2011-12-07 | Talecris Biotherapeutics Inc | Composition, method and kit for preparing plasmin |
US20110020370A1 (en) * | 2008-12-11 | 2011-01-27 | Elias Georges | Slc7a5 directed diagnostics and therapeutics for neoplastic disease |
FR2941371B1 (en) * | 2009-01-28 | 2011-01-21 | Alaxia | DEVICE FOR THE EXTENDED PREPARATION OF MEDICINAL PREPARATIONS |
WO2010125148A2 (en) | 2009-04-30 | 2010-11-04 | Catherine Blondel | Methods for treating ocular conditions |
-
2015
- 2015-04-15 FR FR1553347A patent/FR3035120B1/en not_active Expired - Fee Related
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2016
- 2016-04-14 US US15/566,642 patent/US20180135038A1/en not_active Abandoned
- 2016-04-14 EP EP16729958.5A patent/EP3283626A1/en not_active Withdrawn
- 2016-04-14 CN CN201680034582.2A patent/CN107787366A/en active Pending
- 2016-04-14 JP JP2018505541A patent/JP2018515131A/en active Pending
- 2016-04-14 WO PCT/FR2016/050869 patent/WO2016166484A1/en active Application Filing
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WO2016166484A1 (en) | 2016-10-20 |
CN107787366A (en) | 2018-03-09 |
FR3035120A1 (en) | 2016-10-21 |
FR3035120B1 (en) | 2020-02-07 |
US20180135038A1 (en) | 2018-05-17 |
JP2018515131A (en) | 2018-06-14 |
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