Abstract:
A drug targeting both the inflammatory initiators (lipopolysaccharide; LPS) and mediators [tumor necrosis factor- (TNF- )]
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 2-macroglobulin (A2M). The conjugate binds TNF- 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- 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
g/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.
