Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-09-15
2004-08-10
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S334000, C604S005010, C604S005020, C604S005030, C604S005040
Reexamination Certificate
active
06774102
ABSTRACT:
ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT
Not applicable.
BACKGROUND OF THE INVENTION
The invention relates to blood treating material having the capability of selectively removing endotoxin and cytokine inducing substances from blood or plasma by extracorporeal adsorption for therapeutic septic shock treatment.
Endotoxins are lipopolysaccharides from gram-negative bacteria and are the leading cause of sepsis and septic shock, having mortality rates of more than 50%. Endotoxins can persist in blood subsequent to infection even in the absence of live bacteria. Endotoxin molecules have a highly conserved region, which consists of Lipid-A moiety comprising several long fatty acid chains and sugar rings with at least two negatively charged phosphate groups. The Lipid-A moiety is connected to polysaccharide chains which vary greatly depending on bacteria type. The pathological effect is mainly derived from the Lipid-A moiety of the molecule. The accessibility of the Lipid-A moiety is largely modulated by the nature of the polysaccharide chains and the surrounding media, including such factors as salinity, water, sugar molecules, plasma, blood, pH, detergents, and the like. For example, high salt concentration leads to micellar and other supermolecular structures of endotoxins, resulting in different activities.
Endotoxin is assayed by measuring its cytokine-inducing effect on CD14-positive leucocytes, to produce, e.g. TNF-&agr;, which can be analyzed by an ELISA technique using a commercially available kit, e.g. R&D Systems, Bad Homburg, Germany or by an LAL induced chromogenic substrate reaction (Chromogenics, Moeldwagen, Sweden). The molecular weight of endotoxin ranges from 5000 Da to some millions Da depending on the polysaccharide chain length and its supermolecular structure.
Most extracorporeal removal strategies have exploited the negatively charged phosphate groups of endotoxin using positively charged adsorbent materials immobilized on a variety of substrates. Kodama, et al. (EP 0107119, EP 0129786) disclosed polycationic Polymyxin B covalently immobilized on polystyrene fibers and described adsorbing endotoxins from blood in a device filled with woven fibers of such material. Otto, et al. (EP 424698) also disclosed immobilized polycationic Polymyxin B on poly(comethacrylate) beads for adsorbing endotoxin from blood. Falkenhagen, et al. [
Artificial Organs
(1996) 20:420] described adsorbing endotoxin from plasma on polycationic polyethyleneimine-coated cellulosic beads. Mitzner, et al. [
Artificial Organs
(1993) 17(9):775] described polyethyleneimine and Polymyxin B immobilized on macroporous cellulose beads for endotoxin removal from plasma. A product incorporating the Kodama technique has been marketed in Japan; however, the fact that Polymyxin B is strongly nephrotoxic has been a drawback preventing registration in other countries due to the risk of Polymyxin B leaching into blood. Polyethyleneimine as an endotoxin ligand has the twin disadvantages that it strongly adsorbs heparin and also interacts with platelets, leading to coagulation problems in an in vivo application. The potential for adsorbing plasma proteins poses a significant problem for developing an endotoxin adsorbent that is both specific and selective.
European applications (EP 0494848, EP 0129786, Pharmacia Upjohn) disclosed endotoxin removal using an arginine ligand on sepharose. Whereas in vitro trials appeared promising, no further development appears to have been made. Anspach (WO97/33683, DE 19609479) described the immobilization of cationic ligands such as polylysine, N,N-diethylaminoethane, lysine, arginine, histidine or histamine onto polyamide microfiltration membranes and disclosed data on removal of up to about 50% endotoxin from protein solutions. However, applicability was restricted to solutions having a lower protein content than blood or plasma. Hoess, et al. (WO 95/05393) described a peptide having endotoxin adsorbent property. The peptide was composed of hydrophilic, positively charged aminoacids alternated with hydrophobic aminoacids. No data was reported on endotoxin adsorption from blood or plasma. Evans, et al. (WO 96/41185) described immobilizing amidine groups on macroporous beads such that the groups had a specific spacing between the positively charged centers. The material was reportedly suitable for endotoxin removal from plasma and other fluids; however, the material does not appear to be commercially available, Otto, et al. (EP application 0858831) disclosed endotoxin removal from whole blood using albumin as a ligand covalently immobilized onto macroporous polymethyl methacrylate beads. Although in vitro data under static conditions in plasma showed excellent endotoxin adsorption capacity, when applied to whole blood under flowing conditions as in a therapeutic application, the endotoxin removal behavior was very restricted, perhaps due to the weak binding of endotoxin onto immobilized albumin.
Other workers have explored the use of non-selective surfaces for removing endotoxin removal from plasma. Ash, et al. (Biologic DTPF-system TM, ISFA-congress, Saarbrüken/Germany, Apr. 15-19, 1999) treated endotoxin containing plasma in vitro with fine powdered charcoal, having no ligand, with a surface area of approximately 1000 m
2
/g charcoal/10 ml plasma. Although high endotoxin removal was reported, the report did not give more details as to what other components had been removed, including beneficial substances. Tetta, et al. (EP 0787500) described the use of positively charged ion-exchange beads for endotoxin removal from plasmas and reported 90% removal in animal trials.
In summary one approach is to remove as much endotoxin as possible simply by using very large adsorbent areas. However, non-specific binding can result in collateral removal of normal blood components such as certain antibodies and coagulation factors. The collateral removal is undesirable. Also, the use of non-specific binding materials is restricted to treatment of plasma, in order to avoid cell activation. Non-specific binding materials are considered impractical for a whole-blood application due to the potential risk of unexpected side reactions.
A different approach is represented by the disclosures of Kodama et al. supra or Otto et al supra based on the use of specific-binding ligands such as polymyxin B or histidine. While such ligands demonstrate sufficient specificity to avoid collateral removal of blood components, the binding capacity is variable across the range of endotoxins likely to be encountered. In order to compensate for low binding capacity a large adsorption chamber might be required, necessitating an unacceptably large extracorporeal blood volume to achieve rapid endotoxin clearance. The lack of binding capacity for a polymyxin B ligand adsorbent was revealed in animal studies in which the adsorbent was only able to clear the endotoxin for a limited time [Otto et al (1997)
Therapeutic Apheresis
1:67]. Although the animals lived somewhat longer than untreated controls, the survival rate was not affected.
The use of serum albumin as an adsorbent has been reported. Non-covalent attachment of albumin to bead-type support materials has been reported by Hirae et al. EP 800862, and Suzuki et al EP 028937. Covalently attached albumin has also been disclosed (Otto, EP 848831). The rationale for using albumin is that it already fictions as a binding and transport protein in the bloodstream. The practical use of immobilized albumin is limited by the fact that albumin does not bind endotoxin with sufficient avidity.
Hemodialysis membranes with higher protein adsorption as e.g. AN69 (Gambro-Hospal) are considered to be good surfaces for very low incidence of sepsis related reactions, due to their endotoxin and cytokine adsorption capability.
SUMMARY OF THE INVENTION
The problem solved by the present invention is to devise endotoxin adsorption ligands which, on the one hand, have sufficient heterogeneity to effectively adsorb a large variety of endotoxins, while at
Beck Werner
Bell Carl-Martin
Storr Markus
Gambro Dialysatoren GmbH & Co. KG
Greenlee Winner and Sullivan P.C.
Low Christopher S. F.
Lukton David
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