DEVICE AND METHOD FOR THE DELIVERY OF A THROMBIN ACTIVATED FIBRIN SEALANT

Abstract

The invention relates to a dispenser of a sealant for hemostasis comprising a container configured to receive a fibrinogen solution and an activation compartment being situated downstream of the container and comprising a functionalized support to which a coagulation factor, preferably thrombin, is immobilized, such that upon dispensing the fibrinogen solution it passes through the activation compartment, thereby generating and dispensing polymerizing fibrin as a sealant. The invention further relates to a support, exchangeable tip and a kit for the dispenser as well as to methods and uses of the dispenser or components thereof for dispensing a sealant to a desired site.

Claims

1. A dispenser of a sealant for hemostasis, comprising: a container configured to receive a fibrinogen solution (8), and an activation compartment, the activation compartment being situated downstream of the container and comprising a functionalized support to which a coagulation factor is electrostatically immobilized, wherein the functionalized support is a functionalized resin comprising particles with a mean size of 150 to 300 μm.

2. The dispenser according to claim 1, wherein the coagulation factor is a fibrinogen-cleaving protease.

3. The dispenser according to claim 1, wherein the functionalized resin is an amino-functionalized resin.

4. The dispenser according to claim 1, wherein the functionalized resin comprises a spacer moiety to which a functionalized group is attached.

5. The dispenser according to claim 1, wherein at least 2000 units (U) of the coagulation factor are immobilized per gram (g) of the support.

6. The dispenser according to claim 1, wherein the resin particles are porous particles with a mean pore diameter of 60 nm to 120 nm.

7. The dispenser according to claim 1, additionally comprising a piston equipped with a plug, said plug being configured to be received in the container and for extruding a fibrinogen solution from the container and through the activation compartment.

8. The dispenser according to claim 1 additionally comprising a filter situated downstream of the activation compartment, wherein said filter is configured to prevent release of the functionalized support.

9. The dispenser according to claim 1, wherein the dispenser comprises a main body comprising the container and an exchangeable tip, wherein the activation compartment is comprised in the exchangeable tip or the main body.

10. The dispenser according to claim 1, wherein the container is filled with a solution comprising fibrinogen.

11. A method for generating and dispensing a sealant to a desired site, comprising the steps of providing a dispenser, the dispenser comprising a container filled with a fibrinogen solution, and an activation compartment, wherein the activation compartment is situated downstream of the container and comprising a functionalized support to which a coagulation factor is electrostatically immobilized, and dispensing the fibrinogen solution to the desired site, wherein upon dispensing, the fibrinogen solution passes through the activation compartment, thereby generating and dispensing polymerizing fibrin as a sealant.

12. A support for use in an activation compartment of a dispenser according to claim 1, producible by a method comprising: providing a functionalized support, which is a functionalized resin comprising particles with a mean size of 150 to 300 μm, providing a solution comprising a coagulation factor, and incubating the solution comprising a coagulation factor with the functionalized support.

13. An exchangeable tip for use in a dispenser according to claim 1, wherein the exchangeable tip comprises means for a releasable attachment to a main body of a dispenser and an activation compartment comprising a functionalized support to which a coagulation factor is electrostatically immobilized, wherein the functionalized support is a functionalized resin comprising particles with a mean size of 150 to 300 μm.

14. (canceled)

15. A kit comprising a dispenser according to claim 1, a solution comprising fibrinogen, and optionally instructions for a method for dispensing a sealant for hemostasis.

16. The dispenser according to claim 2, wherein the fibrinogen-cleaving protease is thrombin.

17. The dispenser according to claim 3, wherein the amino-functionalized resin is an amino methacrylate resin.

18. The dispenser according to claim 4, wherein the spacer moiety comprises or consists of an organic moiety comprising C.sub.2-C.sub.20.

19. The dispenser according to claim 18, wherein the organic moiety is a branched or unbranched alkyl, alkenyl, alkynyl, aryl, aminoalkyl, aminoalkenyl, aminoalkynyl, amide and/or aralkyl group, or combination thereof.

20. The dispenser according to claim 5, wherein at least 5000 units (U) of the coagulation factor are immobilized per gram (g) of the support.

21. The method according to claim 11, wherein the coagulation factor is thrombin.

22. The support according to claim 12, wherein the coagulation factor is thrombin.

23. The exchangeable tip according to claim 13, wherein the coagulation factor is thrombin.

Description

FIGURES

[0162] The present invention is further described by reference to the following figures. The figures exemplify non-limiting and potentially preferred embodiments, presented for further illustration of the invention.

[0163] FIG. 1 Activity of 20 μL of thrombin immobilized resin with epoxy (black) or amino (red and green) functionality, immobilization conditions were 20 mg/mL thrombin and resin:buffer ratio of 1:2. Solid lines represent raw data, while the underlying dashed lines depict corrected values after subtracting fluorescence signal from unmodified resins (bottom dashes lines).

[0164] FIG. 2 Thrombin activity of typical standards used in fluorescence experiments.

[0165] FIG. 3. Fluorescent signal of standards after 60 min, including the regression line used to calculate the apparent activity of immobilized thrombin.

[0166] FIG. 4. Activity of thrombin immobilized on amino-terminated resins with different mean diameters.

[0167] FIG. 5 Schematic representation of a preferred embodiment of a dispenser.

EXAMPLES

[0168] The present invention is further described by reference to the following non-limiting examples. The examples describe non-limiting and practical embodiments, presented for further illustration of the invention.

[0169] Methods of the Examples:

[0170] Thrombin Immobilization.

[0171] Epoxy-(no spacer) and amino-(long C6 spacer) functionalized resins (Purolite GmbH Ratingen, Germany) with a pore size of 600-1200 Å (mean pore size: 879 Å and 805 Å, respectively) and a diameter of 150-300 μm (mean diameter: 222 μm and 198 μm, respectively) were equilibrated by washing with an excess of phosphate buffered saline (PBS) (Lonza Verviers, Belgium,) using a glass filter and moderate negative pressure. After three washing steps, the resins were added to separate 10-50 mg/mL solutions of thrombin (BioPharm Laboratories LLC Bluffdale, USA) in PBS, at a resin:buffer ratio of 1:2-4, and left on a tumbler shaker overnight at RT. After immobilization, the immobilization solution was examined using a Pierce BCA assay kit (Thermo Fisher Scientific GmbH Dreiech, Germany) to determine the immobilization yield. The resin was washed three times using 50 mL PBS, once with 50 mL 0.5 M NaCl and with 50 mL PBS, and stored in PBS at a resin:buffer ratio of 1:2. The collected wash solution was saved and used to determine final immobilization yield.

[0172] Thrombin Activity.

[0173] The activity of the immobilized thrombin was determined using a fluorogenic thrombin substrate (Benzoyl-Phe-Val-Arg-AMC.HCl, Merck GmbH Darmstadt, Germany) and compared to standard amounts of thrombin. Briefly, the tip of a standard 200 μL pipette tip was cut off at approximately 5 mm from the end to enlarge the opening, and 20 μL of resin suspension was pipetted into a black flat well 96-well plate (Nunc Thermo Fisher Scientific GmbH Dreiech, Germany). To the same well, 30 μL of PBS was added, while from each thrombin standard (0-500 U/mL) 50 μL was added to the 96-well plate. As a control, and to determine the background fluorescence signal, 20 μL of unmodified resin together with 30 μL of PBS were added to the plate as well. When each well contained a total of 50 μL liquid (standards), or liquid resin mixture (immobilized thrombin), 100 μL of 1 mM thrombin substrate was added, the plate was immediately transferred to the plate reader (Fluostar Optima, BMG Labtech GmbH Ortenberg, Germany) and the measurement was started. Fluorescence was measured using excitation and emission wavelengths of 350 nm and 430 nm, respectively. The fluorescence signal was measured every 150 s for 1 h.

[0174] Fibrinogen Activation and Gel Formation.

[0175] 200 μl of thrombin immobilized resin was suspended in 200 μl of 0.4 M CaCl.sub.2, after 5 min, the resin was added to a modified syringe tip containing a 1.5 μm pore filter. A 1 mL syringe was used to aspirate 500 μl solution of 90 mg/mL fibrinogen (Merck GmbH, Darmstadt, Germany) in ultrapure H.sub.2O, and the syringe was connected to the tip containing the resin. The fibrinogen solution was then forced through the tip containing the thrombin immobilized resin onto a glass microscope slide and gel formation was observed within 1 min.

Example 1: Immobilization Yield for Thrombin

[0176] Immobilization yield was determined using 500 mg of amino-terminated resin in 2 mL of PBS with varying thrombin concentrations. Initial yield was calculated by determining the protein content of immobilization solutions, while the final yield included the amount of thrombin lost after washing.

[0177] Table 1 exhibits the yield and final yield of thrombin immobilization on amino-terminated resins and demonstrate that for these resin high yields can be obtained for different thrombin concentrations.

Example 2: Determination of Thrombin Activity

[0178] Activity of thrombin was determined for epoxy- and amino-terminated resins (FIG. 1), while in the case of amino-terminated resin both low (technical grade) and high purity thrombin (technical: 90-250 U/mg, high purity: >1500 U/mg) were used. Activity of thrombin immobilized on epoxy-terminated resins was lowest, and resulted in an increasing fluorescence signal during the time of the measurements, reaching a maximum of approximately 10000 au. When immobilized on amino-terminated resins, thrombin activity was considerably higher and increased exponentially during the first minutes of the measurement, before continuing linearly after 30 min. In the case of technical grade thrombin, the maximum values obtained were around 20000 au, while the use of high purity thrombin resulted in values exceeding 25000 au.

[0179] To compare the activity of immobilized thrombin to the activity of free thrombin, several standards were included in each experiment (FIG. 2).

[0180] Apparent thrombin activity of immobilized thrombin was calculated using thrombin standards and the obtained fluorescent signal after 60 min.

[0181] Table 2 shows the results for apparent activity of immobilized thrombin for different resin types. Particular good results were obtained for amino-terminated resins, in particular in case of high thrombin grade.

[0182] Activity of thrombin was also determined for amino-terminated resins with different mean diameter of the resin particles (FIG. 4). Activity of thrombin immobilized on a resin with a mean diameter of 388 μm was lowest, and resulted in slow linear fluorescence signal increase during the time of the measurements, reaching a maximum of approximately 7000 au. When using resins with mean particles sizes of 59 μm or 116 μm thrombin activity was higher with a slow approximately linear increase reaching 15000 au at 60 min.

[0183] In contrast thrombin activity on resins with a mean particle size of 198 μm exhibit a fast exponential increase, showing a more than 4 fold increase within the first 10 min up to approximately 18000 au and a continuing linear increase over the duration of observation reaching at 60 min more than 25 000 au.

[0184] The fast exponential increase of fluorescent signal upon addition of the substrate to resins containing thrombin may serves as a reliable indicator for preservation of thrombin activity after immobilization.

[0185] Standards indicate that free thrombin results in a fast increase in fluorescence signal as large amounts of the substrate are converted to its fluorescent product. Inhibition of the free movement of thrombin by covalent immobilization onto epoxy-terminated resins resulted in a linear increase in signal over time and/or resins with suboptimal particles sizes. While immobilization by electrostatic interactions to the amino groups of the amino-terminated resins, did not limit the free movement of thrombin and resulted in signal profiles similar to that of free thrombin, in particular when using particles with mean diameters of approximately 200 μm.

[0186] Results for the thrombin activity for different resin particle sizes are summarized in Table 3, indicating a substantial increase for preferred resin with particles sizes ranging from 150 to 300 μm.

Example 3: Dispenser Design

[0187] FIG. 5 depicts a schematic representation of a preferred embodiment of a dispenser 1, which takes the form of a single-barrel syringe. The dispenser 1 comprises a container 3 filled with a fibrinogen solution 8 situated upstream of an activation compartment 5. Within the activation compartment 5 a functionalized support 7 is located to which a coagulation factor, preferably thrombin is immobilized. A piston 11 equipped with a plug 13 allows for dispensing the sealant. By pushing the piston 11 the fibrinogen solution 8 is extruded from the container 3 and forced to pass the activation compartment 5. During this process fibrinogen-cleavage activity of the immobilized coagulation factor, preferably thrombin, generates polymerizing fibrin. A tip 9 allows for precise deposition of the resulting fibrin sealant at the desired site. Downstream of the activation compartment 5 the dispenser 1 preferably comprises a filter 15 to prevent the release of any part of the functionalized support 7 into the tip 9 and thus the site of deposition.

TABLE-US-00001 TABLE 1 Immobilization yield for thrombin Table 1. Yield and final yield of thrombin immobilization on amino-terminated resins. Thrombin Immobilization Wash concentration yield out Final immobilization yield (mg/mL) (mg) (mg) (mg/g) μmol/g 5 9.6 5.8 8.2 0.23 10 18.6 6.7 23.8 0.66 25 45.0 12.7 64.5 1.79 50 94.7 31.2 124.4 3.46

TABLE-US-00002 TABLE 2 Apparent activity of thrombin for different resin types Immobilization Fluorescence Apparent Resin concentration Thrombin after 60 min activity type (mg/mL) grade (au) (U/g resin) Amino 20 90-250 U/mg 21397 6785 Amino 20  >1500 U/mg 28116 51226 Epoxy 20 90-250 U/mg 10683 2920

TABLE-US-00003 TABLE 3 Comparison of thrombin activity for different sizes of resin particles Size range Mean size Porosity Acitivity* Resin name (μm) (μm) (nm) (%) LifeTech ECR8409F 150-300 198 60-120 100 LifeTech ECR8415M 300-710 413 120-180  57.5 LifeTech ECR8409F 150-300 198 60-120 100 D6159  50-150 116 60-120 68.4 D6161 30-80 59 120-180  64.2 *Max fluorescent signal of thrombin substrate as compared to ECR8409F in paired experiments

LIST OF REFERENCE SIGNS

[0188] 1 dispenser [0189] 3 container [0190] 5 activation compartment [0191] 7 functionalized support [0192] 8 fibrinogen solution [0193] 9 tip [0194] 11 piston [0195] 13 plug [0196] 15 filter