IMMOBILIZED PLASMINOGENASE COMPOSITION, PREPARATION PROCESS, USE AND DEVICE COMPRISING SUCH A COMPOSITION

Abstract

Disclosed is an enzymatic composition including:at least one enzyme, termed plasminogenase, for converting, into plasmin, plasminogen from a blood plasma medium including plasminogen;a solid support that is insoluble in aqueous solution, the solid support having dimensions suitable for being able to be retained on a filter having a cut-off threshold of less than or equal to 0.22 m. The plasminogenase is bound to the solid support and remains bound to this support on contact with a blood plasma medium. The composition is in the dry state. Also disclosed is a process for preparing such an enzymatic composition, to the use thereof and to a device (20) for preparing a blood plasma medium ex vivo rich in sterile plasmin and free of enzymatic composition.

Claims

1. Enzymatic composition comprising: at least one enzyme, named plasminogenase, for converting into plasmin at least some of the plasminogen of a blood plasma medium comprising plasminogen; a solid support that is insoluble in aqueous solution, the solid support having dimensions suitable for allowing it to be retained on a filter with a cut-off threshold of less than or equal to 0.22 m; wherein said plasminogenase is linked to the solid support and remains linked to this support on contact with a blood plasma medium, and in that the composition is in dry form.

2. Composition according to claim 1, wherein the solid support is in divided form and formed from particles having three dimensions extending in three mutually orthogonal directions, at least two of the three dimensions being greater than 0.22 m.

3. Composition according to claim 1, wherein the solid support is formed from a material chosen from the group formed from polymethacrylic polymers.

4. Composition according to claim 1, wherein at least one plasminogenase is a serine endopeptidase of the class EC 3.4.21 of the enzyme classification.

5. Composition according to claim 1, wherein at least one plasminogenase is linked to the solid support via at least one covalent bond.

6. Composition according to claim 1, wherein it is sterile.

7. Composition according to claim 1, wherein it is in dehydrated powder form.

8. Composition according to claim 1, wherein at least one plasminogenase is linked to the solid support via a group of atoms comprising a main chain of atoms linearly bonded to each other via covalent bonds, the main chain having a number of atoms at least equal to 4.

9. Composition according to claim 1, wherein each plasminogenase is linked to the solid support so as only to introduce into a blood plasma medium with which the enzymatic composition is placed in contact a mass of free plasminogenase of less than 400 g, said contact being performed according to the process below: a mass of between 0.01 g and 0.5 g of enzymatic composition in dehydrated form is mixed at a temperature of about 37 C. with a volume of between 0.5 mL and 1.0 mL of blood plasma medium; and then the contact is maintained for a time of more than 5 minutes; and then the enzymatic composition and the blood plasma medium are separated by filtration on a filter with a cut-off threshold of less than or equal to 0.22 m; and the mass of plasminogenase released into the blood plasma medium is measured.

10. Composition according to claim 1, wherein it has an activity, named plasminogenase activity, for converting into plasmin the plasminogen of a blood plasma medium with which it is placed in contact under the following conditions: a mass of between 0.01 g and 0.5 g of enzymatic composition in dehydrated form is placed in contact at a temperature of about 37 C. for a time of more than 5 minutes with a volume of between 0.5 mL and 1.0 mL of blood plasma medium, and a plasmin-rich ex-vivo plasma medium is formed, which has, after separation by filtration of the enzymatic composition and of the plasmin-rich ex-vivo plasma medium, an initial enzymatic activity, named plasmin activity, as measured via a para-nitroaniline release test of greater than 0.1 mol of para-nitroaniline released per minute and per millilitre (mL) of plasmin-rich ex-vivo plasma medium, said release test consisting in: mixing in the plasmin-rich ex-vivo plasma medium maintained at a temperature of 37 C. a chromogenic substrate S-2251 of formula (I) below: ##STR00005## at an initial concentration of about 1 mM (i.e. 10.sup.3 mol/L) in the plasmin-rich ex-vivo plasma medium, evaluating, following the mixing, the initial rate of para-nitroaniline release (in mol of para-nitroaniline) per minute and per millilitre (mL) of plasmin-rich ex-vivo plasma medium.

11. Process for preparing an enzymatic composition according to claim 1, in which: at least one enzyme, named plasminogenase, for converting into plasmin at least some of the plasminogen of a blood plasma medium comprising plasminogen is chosen; a solid support that is insoluble in aqueous solution is chosen: which is suitable for forming with each plasminogenase a bond that is stable on contact with a blood plasma medium; and which has dimensions that are suitable for allowing it to be retained on a filter with a cut-off threshold of less than or equal to 0.22 m; and the solid support and each plasminogenase are placed in contact so as to link each plasminogenase to the solid support; and a lyophilization step is performed so as to form the enzymatic composition.

12. Process according to claim 11, wherein a solid support in divided form is chosen, formed from particles having three dimensions extending in three mutually orthogonal directions, at least two of said three dimensions being greater than 0.22 m.

13. Process according to claim 11, wherein at least one step of sterilization of the enzymatic composition is performed.

14. Use of an enzymatic composition according to claim 1 for preparing a sterile plasmin-rich ex-vivo plasma medium free of enzymatic composition.

15. Use according to claim 14, wherein an amount of the enzymatic composition is placed and maintained in contact with an amount of the blood plasma medium comprising plasminogen for a time of between 15 minutes and 60 minutes at 37 C., so as to form the plasmin-rich ex-vivo plasma medium comprising a mass of free plasminogenase of less than 25 g per mL of plasmin-rich ex-vivo plasma medium.

16. Device for preparing a sterile plasmin-rich ex-vivo plasma medium free of enzymatic composition, comprising an amount of enzymatic composition according to claim 1 and a filter with a cut-off threshold of less than or equal to 0.22 m.

17. Device (20) according to claim 16, further comprising: a container (1) containing the amount of enzymatic composition; a device (3) for introducing blood plasma medium into the container (1); a device (11 ) for collecting from the container (1) a plasma medium formed in the container under the effect of the enzymatic composition; the sample collection device (11) and the filter being arranged to allow the filtration of the plasma medium and the production of a filtrate constituting a sterile plasmin-rich ex-vivo plasma medium free of enzymatic composition.

18. Composition according to claim 2, wherein the solid support is formed from a material chosen from the group formed from polymethacrylic polymers.

19. Composition according to claim 2, wherein at least one plasminogenase is a serine endopeptidase of the class EC 3.4.21 of the enzyme classification.

20. Composition according to claim 3, wherein at least one plasminogenase is a serine endopeptidase of the class EC 3.4.21 of the enzyme classification.

Description

EXAMPLE 1

Preparation of an Enzymatic Composition According to the Invention

[0178] 0.2 g of GMP Sepabeads EC-HFA/S material is hydrated in WFI-grade water, and the hydrated material is then rinsed three times successively with 0.7 mL of sterile physiological saline at pH 6.8. The material thus rinsed is placed in contact with 0.7 mL of a solution of plasminogenase (urokinase U0633) in physiological saline having an enzymatic activity of 2 U/mL. Contact is maintained between the support and the enzyme for several hours. The liquid reaction supernatant is removed and three successive rinses are then performed.

[0179] The mass of urokinase present in the ex-vivo plasma medium obtained by placing in contact 0.2 g of the enzymatic composition and 0.7 mL of blood plasma medium for 60 minutes at a temperature of 37 C., measured via the ELISA immunoenzymatic method, is between 2 g and 20 g.

EXAMPLE 2

Effect of Lyophilization on the Activity of the Enzymatic Composition According to the Invention

[0180] Urokinase is immobilized on a GMP Sepabeads EC-HFA/S solid support in divided form via the method described in Example 1. A step of lyophilization of the enzymatic composition obtained is or is not performed. The capacity of the lyophilized or non-lyophilized enzymatic composition is studied by placing the same amount (0.2 g) of enzymatic composition in 0.7 mL of blood plasma medium for a time of 15 minutes or 60 minutes. The plasmin-rich ex-vivo plasma medium and the enzymatic composition are separated by filtration. A solution of the substrate S-2251 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 ex-vivo plasma medium is measured. The results are given in Table 1 below.

TABLE-US-00001 TABLE 1 Lyophilization of the Plasmin activity at Plasmin activity at enzymatic composition 15 minutes, U/mL 60 minutes, U/mL no 0.178-0.193 0.172-0.178 yes 0.174-0.196 0.191-0.194

[0181] Lyophilization of the enzymatic composition has no effect on its activity for converting the plasminogen of a blood plasma medium into plasmin.

[0182] Sterilization of the Enzymatic Composition by Irradiation

[0183] A step of irradiating at 25 kGy the enzymatic composition after lyophilization is performed. The activity of the irradiated enzymatic composition is analysed indirectly by measuring the plasmin activity of a plasmin-rich ex-vivo plasma medium obtained by placing said sterilized enzymatic composition in contact with a blood plasma medium for 15 minutes or 60 minutes. The enzymatic composition is removed by filtration and the plasmin activity of the plasmin-rich ex-vivo plasma media thus obtained is measured in the presence of S-2251. The plasmin activity of the plasmin-rich ex-vivo plasma media obtained after 15 minutes of contact is between 0.116 and 0.148 U/mL and the plasmin activity of the plasmin-rich ex-vivo plasma media obtained after 60 minutes of contact is between 0.200 and 0.218 U/mL. Sterilizing irradiation of the enzymatic composition makes it possible to conserve a plasmin activity of the plasmin-rich ex-vivo plasma medium obtained after 15 minutes of contact which is greater than 0.1 U/mL and a plasmin activity of the plasmin-rich ex-vivo plasma medium obtained after 60 minutes of contact which is greater than 0.2 U/mL.

[0184] Assay of the Free Urokinase in the Plasmin-Rich Ex-Vivo Plasma Medium

[0185] Assay via the ELISA immunoenzymatic technique of the amount of urokinase present in the plasmin-enriched ex-vivo plasma medium obtained by placing a blood plasma medium in contact with the enzymatic composition for 60 minutes at 37 C. is presented in Table 2 below.

TABLE-US-00002 TABLE 2 Enzymatic composition Irradiation, kGy Lyophilization Free urokinase, g 0 no 10.0-13.0 0 yes 7.1-9.1 25 yes 4.3-7.sup.

[0186] Lyophilization coupled with terminal sterilization by irradiation makes it possible to reduce the amount of urokinase released by the enzymatic composition into the plasmin-rich ex-vivo plasma medium.

[0187] The amount of free urokinase present in the plasmin-rich ex-vivo plasma medium obtained by placing a blood plasma medium in contact with an enzymatic composition (comprising a urokinase immobilized on a solid support in divided form) subjected to irradiation is less than the amount of urokinase present in a plasmin-rich ex-vivo plasma medium obtained by placing a blood plasma medium in contact with an enzymatic composition that has not been subjected to irradiation. The combination of treatments by irradiation and lyophilization of the enzymatic composition makes it possible to obtain amounts of urokinase in the plasmin-rich ex-vivo plasma medium that are less than or equal to about 10 g, in particular less than 5 g.

[0188] The enzymatic composition according to the invention makes it possible to convert a blood plasma medium into plasmin-enriched ex-vivo plasma medium with a high plasmin activity. It makes it possible to convert the plasminogen of a blood plasma medium into plasmin without introducing into the ex-vivo plasma medium formed a large amount of immunogenic plasminogenase.

[0189] Preparation of a Blood Plasma Medium and Activation

[0190] An amount of blood is collected in a sterile manner from a patient to be treated, into a sample collection tube (BD Vacutainer, BD Diagnostics, Pont-de-Claix, France) comprising an anticoagulant such as EDTA or sodium citrate. A step of separating the blood cells and the blood plasma medium is performed by centrifugation at 4000 rpm for 15 minutes. The blood plasma medium is placed in contact, in a sterile manner, with the enzymatic composition at a temperature of 37 C. for a time of at least 15 minutes necessary to allow the conversion of plasminogen into plasmin. The enzymatic composition and the plasmin-rich ex-vivo plasma medium free of free urokinase are separated by filtration on a sterilizing filter (cut-off threshold of 0.22 m), for example on a Millex PVDF filter (Millipore) or on an Acrodisk Syringe Filter, PN4602 (Pall). Intraocular injection of a suitable volume of the sterile plasmin-rich ex-vivo plasma medium is then performed.

[0191] Assay of Free Urokinase in the Plasmin-Rich Ex-Vivo Plasma Medium

[0192] Assay via the ELISA immunoenzymatic method of the amount of free urokinase present in the plasmin-rich ex-vivo plasma medium shows a mean amount of free urokinase of less than 20 g.

[0193] A device 20 according to a particular variant of the invention represented in the single figure comprises: [0194] a hermetically closed containerfor example a glass container 1containing an amount of sterile enzymatic composition 2 according to the invention; [0195] means 3 for introducing an amount of blood plasma medium into the container 1 in a sterile manner and for placing said amount of blood plasma medium in contact with the enzymatic composition 2 and comprising: [0196] a sterile syringe 4 for dispensing the amount of blood plasma medium from a blood sample collection tube and for introducing said amount of collected blood plasma medium into the container 1, said sterile dispensing syringe 4 comprising a plunger 6 sliding in a cylinder 7 having an exiting axial end, named dispensing end 8; [0197] a dispensing needle 5 suitable for connecting to the dispensing end 8 of the sterile dispensing syringe 4 and having a pointed end 9 suitable for introduction into the container 1 through a pierceable stopper 10 and for introducing the blood plasma medium into the container 1 under the effect of the translational movement of the plunger 6 sliding in the cylinder 7. The dispensing needle 5 is preferably, for example, a needle of about 20 gauge (outside diameter of 0.9081 mm for a wall thickness of 0.1524 mm); [0198] means 11 for collecting and filtering an amount of plasmin-rich ex-vivo plasma medium under the effect of the enzymatic composition 2, comprising: [0199] a sterile syringe 12 for preparing an amount of plasmin-rich ex-vivo plasma medium from the container 1; [0200] a filtration device 13 having a plasmin-rich ex-vivo plasma medium inlet 14, and an outlet 15 that can be connected to the sterile preparation syringe 12 and which is configured to be able to dispense sterile plasmin-rich ex-vivo plasma medium in the sterile preparation syringe 12, the filtration device 13 comprising a filter 16 that is capable of retaining the enzymatic composition of the plasmin-rich ex-vivo plasma medium flowing between the inlet 14 and the outlet 15 under the effect of the sterile preparation syringe 12, said inlet 14 being suitable to be assembled with and connected hermetically to a needle 17 for collecting plasmin-rich ex-vivo plasma medium from the container 1; [0201] said collection needle 17 being suitable to be placed in blood plasma medium communication with the inlet 14 of the filtration device 13 and having a pointed end 18 suitable for introduction into the container 1 and for collecting plasmin-rich ex-vivo plasma medium from the container 1. The sample collection needle 17 is preferably, for example, a needle of about 20 gauge (outside diameter of 0.9081 mm for a wall thickness of 0.1524 mm).

[0202] The pierceable stopper 10 formed from an elastic polymerfor example chlorobutylwhich is suitable for piercing with the needle 5 and for introducing blood plasma medium into the container 1 and for collecting plasmin-rich ex-vivo plasma medium from the container 1.

[0203] The filtration device 13 is a device for sterilization by filtration on a filter 16 with a cut-off threshold of about 0.22 m, i.e. on a filter that is suitable for retaining particles with a mean diameter of greater than 0.22 m.

[0204] The device 20 comprises a sterile outer packaging envelope 30 for the container 1 comprising the enzyme composition, the means 3 for introducing an amount of blood plasma medium into the container 1 in a sterile manner and the means 11 for collecting and filtering the plasmin-rich ex-vivo plasma medium. The sterile outer packaging envelope 30 containing the means 3 for introducing an amount of blood plasma medium into the container 1 (comprising the sterile dispensing syringe 4 and the dispensing needle 5) in a sterile manner and the means 11 for collecting and filtering plasmin-rich ex-vivo plasma medium (comprising the sterile preparation syringe 12, the filtration device 13 and the sample collection needle 17) may be sterilized after packaging via any suitable sterilization means, the hermetically closed container 1 containing the sterile enzymatic composition 2 according to the invention being sterilized via sterilization means that do not harm the functionality of the enzymatic composition 2.

[0205] In a variant not shown, the device 20 may also comprise an additional sterile needle for injecting into a patient's body a suitable volume of plasmin-rich ex-vivo plasma medium contained in the sterile syringe 12 for preparing the amount of plasmin-rich ex-vivo plasma medium. Such an injection needle is a needle of between 25 and 30 gauge (i.e. the outside diameter of which is between 0.30 mm and 0.50 mm).

[0206] The sterile syringe 12 for preparing the amount of plasmin-rich ex-vivo plasma medium may have an exiting end formed from an adapter of Luer-lock type and the injection needle may have an end complementary to the Luer-lock adapter of the syringe 12.

[0207] A device according to another variant of the invention, not shown, may comprise: [0208] a hermetically closed container 1 containing an amount of sterile enzymatic composition 2 according to the invention; [0209] a sterile syringe 12 for preparing an amount of plasmin-rich ex-vivo plasma medium from the container 1, said sterile syringe 12 being a precision syringe that is capable of containing and delivering a volume of sterile plasmin-rich ex-vivo plasma medium free of enzymatic composition of between 100 L and 250 L; and [0210] a filtration device 13.

[0211] The invention may be the subject of numerous variants without departing from the scope of protection. For example, the constituent components of a device according to the invention may be in disassembled form or in partially assembled form in the outer envelope 30.