Accumulation and intracellular distribution of antitrypanosomal diamidine compounds DB75 and DB820 in African trypanosomes

The aromatic diamidine pentamidine has long been used to treat early-stage human African trypanosomiasis (HAT). Two analogs of pentamidine, DB75 and DB820, have been shown to be more potent and less toxic than pentamidine in murine models of trypanosomiasis. The diphenyl furan diamidine, DB75, is the active metabolite of the prodrug DB289, which is currently in phase III clinical trials as a new orally active candidate drug to treat first-stage HAT. The new aza analog, DB820, is the active diamidine of the prodrug DB844, currently undergoing preclinical evaluation as a new candidate to treat HAT of the central nervous system. The exact mechanisms of antitrypanosomal activity of aromatic dications remain poorly understood, with multiple mechanisms hypothesized. Pentamidine is known to be actively transported into trypanosomes and binds to DNA within the nucleus and kinetoplast. A long-hypothesized mechanism of action has been that DNA binding ultimately leads to interference with DNA-associated enzymes. Both DB75 and DB820 are intensely fluorescent, which provides an important tool for determining the kinetics of accumulation and intracellular distribution in trypanosomes. We show in the current study that DB75 and DB820 rapidly accumulate and strongly concentrate within trypanosomes, with intracellular concentrations over 15,000-fold higher than mouse plasma concentrations. Both compounds initially accumulate in the DNA-containing nucleus and kinetoplast, but at later time points, they concentrate in non-DNA-containing cytoplasmic organelles. Analyses of the kinetics of uptake and intracellular distribution are necessary to begin to define antitrypanosomal mechanisms of action of DB75, DB820, and other aromatic diamidines.