Further Characterization of 4-Bromomisonidazole as a Potential Detector of Hypoxic Cells

$[{}^{14}{\rm C}]\text{Bromomisonidazole}$ was prepared by direct bromination of [ring-2] $[{}^{14}{\rm C}]\text{misonidazole}$ in dioxane. The uptake and binding of the two labeled sensitizers were compared in vitro in 1-mm EMT-6 spheroids which contain a necrotic core. Using liquid scintillation counting it was shown that spheroids incubated with 50 μM $[{}^{14}{\rm C}]\text{bromomisonidazole}$ concentrated drug above levels in the medium by 1 1/2 hr and achieved maximum concentration by 10 hr with no further increase at 23 hr. Spheroids incubated with 50 μM $[{}^{14}{\rm C}]\text{misonidazole}$ may concentrate the sensitizer more slowly but ultimately reached the same fivefold increase over levels in the medium by 23 hr as was observed for bromomisonidazole. Autoradiographs prepared from spheroids after incubation with $[{}^{14}{\rm C}]\text{misonidazole}$ or $[{}^{14}{\rm C}]\text{bromomisonidazole}$ showed silver grains preferentially located over viable hypoxic cells in the inner half of the spheroid rim adjacent to the necrotic center, with lower grain density over nonviable necrotic areas and many fewer grains over oxic cells at the periphery of the spheroid. The results indicate that both severely and moderately hypoxic cells may preferentially bind $[{}^{14}{\rm C}]\text{bromomisonidazole}$. The data support the potential of radiolabeled bromomisonidazole for in vivo imaging pending additional studies of the metabolism of this agent.