3-[5-{2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-1-yl]
thymidine (N5-2OH) is a first generation 3-carboranyl
thymidine analog (3CTA) that has been intensively studied as a
boron-10 ((10)B) delivery agent for
neutron capture therapy (NCT). N5-2OH is an excellent substrate of
thymidine kinase 1 and its favorable biodistribution profile in rodents led to successful preclinical NCT of rats bearing intracerebral RG2
glioma. The present study explored cellular influx and efflux mechanisms of N5-2OH, as well as its intracellular anabolism beyond the monophosphate level. N5-2OH entered cultured human CCRF-CEM cells via passive diffusion, whereas the
multidrug resistance-associated protein 4 appeared to be a major mediator of N5-2OH monophosphate efflux. N5-2OH was effectively monophosphorylated in cultured murine L929 [
thymidine kinase 1 (TK1(+))] cells whereas formation of N5-2OH monophosphate was markedly lower in L929 (TK1(-)) cell variants. Further metabolism to the di- and
triphosphate forms was not observed in any of the cell lines. Regardless of monophosphorylation, parental N5-2OH was the major intracellular component in both TK1(+) and TK1(-) cells.
Phosphate transfer experiments with
enzyme preparations showed that N5-2OH monophosphate, as well as the monophosphate of a second 3-carboranyl
thymidine analog [3-[5-(o-carboran-1-yl)pentan-1-yl]
thymidine (N5)], were not substrates of
thymidine monophosphate kinase. Surprisingly, N5-diphosphate was phosphorylated by
nucleoside diphosphate kinase although N5-triphosphate apparently was not a substrate of
DNA polymerase. Our results provide valuable information on the cellular metabolism and pharmacokinetic profile of 3-carboranyl
thymidine analogs.