Covalent polymyxin B conjugate with human immunoglobulin G as an antiendotoxin reagent.
Polymyxin B (PMB) is a cyclic decapeptide antibiotic which also binds and neutralizes endotoxin. Unfortunately, PMB can be considerably nephrotoxic at clinically utilized doses, thereby limiting its utility as a therapeutic antiendotoxin reagent. We sought to change the pharmacokinetics and toxicity profile of PMB by covalently linking it to a human immunoglobulin G (IgG) carrier. Conjugates of PMB with IgG were prepared by EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]-mediated amide formation. Analysis by dot enzyme-linked immunosorbent assay with an anti-PMB monoclonal antibody showed that the purified conjugate contained bound PMB. The IgG-PMB conjugate reacted with lipid A and J5 lipopolysaccharide in Western blot assays in a manner comparable to that of whole antiserum with anti-lipid A reactivity; unconjugated IgG had no reactivity. The PMB bound in the conjugate retained its endotoxin-neutralizing activity compared to that of unbound PMB as evidenced by its dose-dependent inhibition of tumor necrosis factor release by endotoxin-stimulated human monocytes in vitro; unconjugated IgG had no activity. By this assay, the PMB-IgG conjugate was determined to have approximately 3.0 microg of bound functional PMB per 100 microg of total protein of conjugate (five molecules of PMB per IgG molecule). The PMB-IgG conjugate was also bactericidal against clinical strains of Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to unconjugated IgG with MBCs of <4 microg of conjugate per ml for each of the tested strains. The conjugate appeared to be nontoxic at the highest doses deliverable and provided statistically significant protection from death to galactosamine-sensitized, lipopolysaccharide-challenged mice in a dose-dependent fashion when administered prophylactically 2 h before challenge. However, neither free PMB nor the PMB-IgG conjugate could protect mice challenged with endotoxin 2 h after administration. This suggests that these reagents can play a role in prophylaxis but not in therapy of sepsis. These experiments demonstrated that the PMB-IgG conjugate retains bound yet functional PMB as evidenced by its endotoxin-neutralizing activity both in vitro and in vivo. Further work is required to define the role that this or related conjugate compounds may play in the prophylaxis of endotoxin-mediated disease.
Drabick
J J
JJ
Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. jdrabino@aol.com
Bhattacharjee
A K
AK
Hoover
D L
DL
Siber
G E
GE
Morales
V E
VE
Young
L D
LD
Brown
S L
SL
Cross
A S
AS
D000900:Anti-Bacterial Agents / Q000276:immunology / Q000494:pharmacology*; D004337:Drug Carriers; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D007074:Immunoglobulin G; D007136:Immunoglobulins / Q000494:pharmacology*; D007711:Klebsiella pneumoniae / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D009000:Monocytes / Q000187:drug effects* / Q000378:metabolism; D011112:Polymyxin B / Q000276:immunology / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000187:drug effects / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004337:Drug Carriers; D007074:Immunoglobulin G; D007136:Immunoglobulins; D014409:Tumor Necrosis Factor-alpha; C042866:antilipopolysaccharide antibodies; D011112:Polymyxin B