title: Polymyxin B-conjugated alpha 2-macroglobulin as an adjunctive therapy to sepsis: Modes of action and impact on lethality.
abstract: A drug targeting both the inflammatory initiators (lipopolysaccharide; LPS) and mediators [tumor necrosis factor-alpha (TNF-alpha)] should have advantage over a "single-factor targeting strategy" in sepsis prevention trials. We have prepared conjugates of polymyxin B (PMB) and the cytokine binding protein alpha2-macroglobulin (A2M). The conjugate binds TNF-alpha as well as LPS as studied by electrophoresis and phase partitioning. Compared with free PMB, the conjugate is nontoxic to cells and does not affect the viability of human monocytes. The A2M-PMB conjugate binds to the A2M receptor (CD91/low-density lipoprotein receptor-related protein 1) with affinity similar to that of the nonmodified protein. Fluorescein isothiocyanate-labeled LPS in the presence of A2M-PMB is rapidly transported into fibroblasts for degradation via receptor-mediated endocytosis. In vitro, A2M-PMB demonstrated inhibition of LPS-induced secretion of TNF-alpha from isolated monocytes as well as in the whole blood assay. The efficacy of the drug was tested in mice after induction of acute inflammation (LPS model) and after induction of a polymicrobial sepsis by cecal ligation and puncture (CLP) model. Treatment of mice with A2M-PMB up to 250 microg/g body weight was not toxic to the animal. When the drug was administered 30 min before or 30 min after the LPS challenge, a survival rate of 90 and 70%, respectively, was obtained compared with the placebo control group (5%). A2M-PMB also protected mice after induction of polymicrobial sepsis when administered 30 min before CLP. These results support our hypothesis that A2M-PMB acts as a polyvalent drug to target different host mediators as well as sepsis inducer at the same time.
authors.affiliation: Institute of Biochemistry, University of Leipzig, Leipzig, Germany. birg@medizin.unileipzig.de
authors.forename: Gerd
authors.initials: G
authors.lastname: Birkenmeier
authors.forename: Silke
authors.initials: S
authors.lastname: Nicklisch
authors.forename: Christiane
authors.initials: C
authors.lastname: Pockelt
authors.forename: Andualem
authors.initials: A
authors.lastname: Mossie
authors.forename: Volker
authors.initials: V
authors.lastname: Steger
authors.forename: Christiane
authors.initials: C
authors.lastname: Gläser
authors.forename: Sunna
authors.initials: S
authors.lastname: Hauschildt
authors.forename: Elke
authors.initials: E
authors.lastname: Usbeck
authors.forename: Klaus
authors.initials: K
authors.lastname: Huse
authors.forename: Ulli
authors.initials: U
authors.lastname: Sack
authors.forename: Michael
authors.initials: M
authors.lastname: Bauer
authors.forename: Angelika
authors.initials: A
authors.lastname: Schäfer
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000627:therapeutic use* / Q000633:toxicity; D001424:Bacterial Infections / Q000401:mortality / Q000517:prevention & control; D015153:Blotting, Western; D002469:Cell Separation; D002908:Chronic Disease; D016207:Cytokines / Q000378:metabolism; D003911:Dextrans / Q000627:therapeutic use / Q000633:toxicity; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007249:Inflammation / Q000139:chemically induced / Q000188:drug therapy; D008033:Limulus Test; D008070:Lipopolysaccharides; D051379:Mice; D008807:Mice, Inbred BALB C; D011112:Polymyxin B / Q000627:therapeutic use* / Q000633:toxicity; D018121:Receptors, Cytokine / Q000378:metabolism; D011955:Receptors, Drug / Q000378:metabolism; D018805:Sepsis / Q000139:chemically induced / Q000188:drug therapy* / Q000401:mortality; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000627:therapeutic use* / Q000633:toxicity
chemical_list: D000900:Anti-Bacterial Agents; D016207:Cytokines; D003911:Dextrans; D008070:Lipopolysaccharides; D018121:Receptors, Cytokine; D011955:Receptors, Drug; D014409:Tumor Necrosis Factor-alpha; D000511:alpha-Macroglobulins; D011112:Polymyxin B