APPARATUS FOR THE EXTRACORPOREAL TREATMENT OF BLOOD

Abstract

An apparatus for the extracorporeal treatment of blood comprising an extracorporeal blood circuit (2), a pump (6) configured to provide fluid displacement within the extracorporeal blood circuit, and a reaction chamber (8) connected to the extracorporeal blood circuit and configured to receive blood or plasma from the circuit and treat the blood or plasma. The reaction chamber comprises a protease enzyme immobilized to a support, in which the protease enzyme is specific for, and capable of irreversibly cleaving, a human C5a present in the blood or plasma, wherein the abundance of the human C5a in the treated blood or plasma is less than that in the untreated blood or plasma. The apparatus finds utility in the extracorporeal treatment of blood from patients with inflammatory conditions, especially auto-immune disease and sepsis.

Claims

1. A method for the extracorporeal treatment of blood, the method comprising removing blood or a blood fraction from a patient and reacting the blood or blood fraction with a protease enzyme that is specific for and capable of irreversibly cleaving functional C5a, thereby reducing an abundance of functional C5a and increasing an abundance of non-functional C5a in the blood or blood fraction compared to that of untreated blood or blood fraction, and returning the blood or the blood fraction to the patient, wherein the protease enzyme is immobilised to a support.

2. The method of claim 1, wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, SEQ ID NO: 4 or SEQ ID NO: 5.

3. (canceled)

4. The method of claim 1, wherein the reacting step is carried out with an apparatus comprising: an extracorporeal blood circuit; a pump configured to provide fluid displacement with the extracorporeal blood circuit; and a reaction chamber connected to the extracorporeal blood circuit, the reaction chamber configured to receive the blood or blood fraction from the extracorporeal blood circuit and to treat the blood or blood fraction, wherein the reaction chamber contains the protease enzyme immobilised to the support.

5. The method of claim 4, wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, SEQ ID NO: 4 or SEQ ID NO: 5.

6. (canceled)

7. The method of claim 4, wherein the apparatus further comprises separating means adapted to separate the blood or blood fraction into a C5a-containing fraction and a non-C5a containing fraction, wherein the reaction chamber receives the C5a-containing fraction.

8. The method of claim 7, wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, SEQ ID NO: 4 or SEQ ID NO: 5.

9. (canceled)

10. The method of claim 7, wherein the apparatus further comprises means configured to recombine the treated C5a-containing fraction with the non-C5a containing fraction.

11. The method of claim 1, wherein the support includes a coordinated transition metal ion and one or more functional groups; the C-terminus of the protease enzyme comprises a first tag and a second tag, the second tag being separated from the first tag by a spacer; the first tag comprises a motif capable of covalently reacting with the one or more functional groups; and the second tag comprises a motif capable of interacting with the coordinated transition metal ion.

12. The method of claim 11, wherein the first tag is poly-lysine, poly-glutamate, or poly-cysteine, and the second tag is poly-histidine.

13. The method of claim 12, wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, SEQ ID NO: 4, or SEQ ID NO: 5.

14. The method of claim 1, wherein the support includes a silica material, a methacrylate, a polyacrylamide, a polypyrrole, or a polysaccharide.

15. The method of claim 14, wherein the silica material is selected from the group consisting of mesoporous silica, a monodispersed mesoporous silicate, and a Ni2+-modified mesoporous silica.

16. The method of claim 1, wherein the support comprises a multiplicity of beads and the protease enzyme is irreversibly immobilized to a surface of the beads.

17. A method for treating sepsis, the method comprising removing blood or a blood fraction from a patient having sepsis and reacting the blood or blood fraction with a protease enzyme that is specific for and capable of irreversibly cleaving functional C5a, thereby reducing an abundance of functional C5a in the blood or blood fraction, and returning the blood or the blood fraction to the patient, wherein the protease enzyme is immobilised to a support.

18. The method of claim 17, wherein the protease enzyme is a recombinant bacterial C5a protease comprising SEQ ID NO. 3, SEQ ID NO: 4, or SEQ ID NO: 5.

19. (canceled)

20. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The invention description below refers to the accompanying drawings, of which:

[0068] FIG. 1 is a Diagrammatic representation of blood purifying invention.

[0069] The diagram shows the components and blood flow route envisaged for the implementation of the invention. Blood is removed from the patient and fractionated into a high protein plasma fraction and a high blood cell fraction. The former is passed over the active material (immobilized enzyme) in the reaction chamber and then recombined with the latter before return to the patient. Components of the invention are labeled: 1 the overall invention, 2 the extracorporeal blood purification device, 3 blood withdrawal line, 4 patient arm, 5 blood return line, 6 pumping system, 7 blood separator, 8 reaction chamber, 9 cartridge housing blood separation chambers, 10a and 10b blood separation chambers, 11 biocompatible size restrictive semi-permeable membrane, 12 line delivering protein rich plasma to reaction chamber, 13 line delivering blood cell rich fraction to mixing chamber, 14 line delivering treated plasma to mixing chamber, 15 mixing chamber for blood reconstitution, 16 vessel housing active component of reaction chamber, 17 reactive material comprising immobilized enzyme irreversibly coupled to solid support material.

[0070] FIG. 2a shows SDS-PAGE analysis of C5a untreated () and treated (+) with ScpA; and

[0071] FIG. 2b shows the scissile bond in the C5a sequence confirmed by Mass Spec analysis of C5a cleaved with ScpA.

DETAILED DESCRIPTION

[0072] Referring to FIG. 1, there is provided an apparatus for the extracorporeal treatment of blood according to the invention, and indicated generally by the reference numeral 1. The apparatus 1 comprises an extracorporeal blood circuit 2, having a feed line 3 for withdrawing blood from a patients arm 4 for treatment and a return line 5 for returning treated blood to the patient, and an adjustable pump 6 provided in the feed line for providing blood displacement within the blood circuit 2.

[0073] The apparatus also includes a blood separator 7 and a reaction chamber 8 in the circuit 2, the separator 7 being provided upstream of the reaction chamber 8. The separator comprises a cartridge 9 having two chambers 10a and 10b separated by a semi-permeable membrane 11 adapted to allow separation of blood proteins from blood cells. The whole blood passes from the patient to the first chamber 10a, where proteins in blood plasma pass into the second chamber 10b forming a protein rich plasma fraction in the second chamber and leaving blood cells in the first chamber 10a. A tube 12 is provided to transfer the thus-formed protein rich fraction plasma from the second chamber 10b to the reaction chamber 8 where it is treated. A further tube 13 is provided to transfer the cell rich fraction from the first chamber 10a to re-join with treated plasma distally of the reaction chamber 8 at a mixing chamber 15 where the two fractions are mixed prior to being returned to the patient via the whole blood return line 5.

[0074] The reaction chamber comprises a cylindrical vessel 16 filled with functionalized support material 17 containing the immobilized enzyme, thereby providing a large surface area for the treatment of the incoming plasma. The tube 12 feeds into a top of the cylindrical vessel 16, and the plasma filters through the cylinder before exiting the vessel through a tube 14.

[0075] Mesoporous silica (MPS) materials (including but not limited to MCM, SBA, MCF and PMO type materials) are prepared using a templated synthesis method. Ideally these particles will be monodispersed in nature. The particles will have a specific particle size in the range of 0.1-50 m, contain nanopores with a final internal diameter in the range 8-12 nm and have a high surface area 300-800 m.sup.2g.sup.1.

[0076] The surface characteristics of the silica nanocarriers will be modified with a range of functional groups (e.g. NH2, COOH, SH) directly during synthesis of the material, or by post synthesis grafting to facilitate covalent coupling (through the poly-Gluamate or poly-Lysine or Cysteine residues respectively) of the enzyme to the surface after orientation specific adsorption.

[0077] The Ni.sup.2+-modified MPS will be prepared by attachment of 3-iodo-trimethoxypropylsilane to the silicate surface followed by reaction with cyclam and incorporation of the metal ion. This is to generate immobilization of the protease in a controlled orientation.

[0078] In use, the extracorporeal blood circuit is connected to a patient, generally an arm of a patient, and the pump is actuated to withdraw blood from the patient and pump it through the circuit. The whole blood from the patient enters the separator 7 and is separated under pressure into the two fractions. The plasma fraction is pumped from the second chamber 10b to the reaction chamber 8 where the blood percolates through the functionalized cassette bed 17. In the reaction chamber, mediator in the plasma binds to the protease enzyme that is immobilized to the support material, and is cleaved into an inactive form that is released back into the plasma leaving the immobilized enzyme free for another reaction. As a result of the plasma passing through the reaction chamber, the concentration of functional mediator in the plasma is significantly reduced. The thus treated plasma is then pumped to the mixing chamber 15 where it rejoins with the cell rich fraction to form whole blood that is significantly depleted of active mediator protein. The whole blood is returned to the patient via the return line 5.

[0079] It will be appreciated that the use of a separator to filter the blood prior to treatment is optional, and that the treatment of whole blood in the reaction chamber forms part of the invention.

EXPERIMENTAL

Materials and Methods

C5a Peptidase Activity Assays

[0080] Recombinant C5a was produced as an N-term His-tagged fusion (HT-C5a) in accordance with the method of Toth et al., and chemoattractant activity was verified in an under-agarose migration assay (data not shown). The C5a-ase activity of ScpA was demonstrated in reactions consisting of 42 nM ScpA with 37 M HT-C5a, in 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, and 5 mM CaCl.sub.2 for 30 min at 20 C. The observed C5a-ase activity was independent of the presence of Complete Mini EDTAfree inhibitor cocktail (Roche). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of cleaved HT-C5a was performed.

Results

[0081] The activity assay showed that the ScpA cleaved C5a at a single site (FIG. 2a). MS analysis indicated a loss of 830 Da, consistent with the removal of seven residues from the C terminal (FIG. 2b) which removes chemoattractant capabilities.

[0082] The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.

REFERENCES

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