In vitro evaluation, biodistribution and scintigraphic imaging in mice of radiolabeled anthrax toxins.
BACKGROUND
There is a lot of interest towards creating therapies and vaccines for Bacillus anthracis, a bacterium which causes anthrax in humans and which spores can be made into potent biological weapons. Systemic injection of lethal factor (LF), edema factor (EF) and protective antigen (PA) in mice produces toxicity, and this protocol is commonly used to investigate the efficacy of specific antibodies in passive protection and vaccine studies. Availability of toxins labeled with imageable radioisotopes would allow to demonstrate their tissue distribution after intravenous injection at toxin concentration that are below pharmacologically significant to avoid masking by toxic effects.


METHODS
LF, EF and PA were radiolabeled with (188)Re and (99m)Tc, and their performance in vitro was evaluated by macrophages and Chinese hamster ovary cells toxicity assays and by binding to macrophages. Scintigraphic imaging and biodistribution of intravenously (IV) injected (99m)Tc-and (123)I-labeled toxins was performed in BALB/c mice.


RESULTS
Radiolabeled toxins preserved their biological activity. Scatchard-type analysis of the binding of radiolabeled PA to the J774.16 macrophage-like cells revealed 6.6 x 10(4) binding sites per cell with a dissociation constant of 6.7 nM. Comparative scintigraphic imaging of mice injected intravenously with either (99m)Tc-or (123)I-labeled PA, EF and LF toxins demonstrated similar biodistribution patterns with early localization of radioactivity in the liver, spleen, intestines and excretion through kidneys. The finding of renal excretion shortly after IV injection strongly suggests that toxins are rapidly degraded which could contribute to the variability of mouse toxigenic assays. Biodistribution studies confirmed that all three toxins concentrated in the liver and the presence of high levels of radioactivity again implied rapid degradation in vivo.


CONCLUSIONS
The availability of (188)Re and (99m)Tc-labeled PA, LF and EF toxins allowed us to confirm the number of PA binding sites per cell, to provide an estimate of the dissociation constant of PA for its receptor and to demonstrate tissue distribution of toxins in mice after intravenous injection.
Dadachova
Ekaterina
E
Department of Nuclear Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA. edadacho@aecom.yu.edu
Rivera
Johanna
J
Revskaya
Ekaterina
E
Nakouzi
Antonio
A
Cahill
Sean M
SM
Blumenstein
Michael
M
Xiao
Hui
H
Rykunov
Dmitry
D
Casadevall
Arturo
A
D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000493:pharmacokinetics*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D007457:Iodine Radioisotopes; D051379:Mice; D008807:Mice, Inbred BALB C; D011868:Radioisotopes; D012211:Rhenium; D013667:Technetium; D014018:Tissue Distribution
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D007457:Iodine Radioisotopes; D011868:Radioisotopes; C030325:anthrax toxin; D012211:Rhenium; D013667:Technetium