e Human African trypanosomiasis (HAT), a neglected tropical disease, is fatal without treatment. Pentamidine, a cationic diamidine, has been used to treat first-stage (hemolymphatic) HAT since the 1940s, but it is ineffective against second-stage (meningoencephalitic, or central nervous system [CNS]) infection. Novel diamidines (DB75, DB820, and DB829) have shown promising efficacy in both mouse and monkey models of first-stage HAT. However, only DB829 cured animals with second-stage infection. In this study, we aimed to determine the mechanisms underlying the differential efficacies of these diamidines against HAT by conducting a comprehensive pharmacokinetic characterization. This included the determination of metabolic stability in liver microsomes, permeability across MDCK and MDR1-MDCK cell monolayers, interaction with the efflux transporter MDR1 (P-glycoprotein 1 or P-gp), drug binding in plasma and brain, and plasma and brain concentration-time profiles after a single dose in mice. The results showed that DB829, an azadiamidine, had the highest systemic exposure and brain-to-plasma ratio, whereas pentamidine and DB75 had the lowest. None of these diamidines was a P-gp substrate, and the binding of each to plasma proteins and brain differed greatly. The brain-to-plasma ratio best predicted the relative efficacies of these diamidines in mice with second-stage infection. In conclusion, pharmacokinetics and CNS penetration influenced the in vivo efficacies of cationic diamidines against first-and second-stage HAT and should be considered when developing CNS-active antitrypanosomal diamidines. Human African trypanosomiasis (HAT; sleeping sickness) is a neglected tropical disease caused by subspecies of Trypanosoma brucei and is endemic to sub-Saharan Africa (1). HAT appears in two stages, the hemolymphatic stage (first stage), when parasites are confined to the blood and lymph, and the meningoencephalitic stage (second stage), when parasites infect the central nervous system (CNS), which accounts for the majority of diagnosed cases (2, 3). Without treatment, HAT leads to coma and, eventually, death. Current treatments for second-stage HAT include melarsoprol, eflornithine monotherapy, and nifurtimox-eflornithine combination therapy (NECT). However, these treatments are limited by inconvenient parenteral injections, complex infusion schedules, increasing treatment failure, and/or severe toxicity (1, 3). Hence, new or improved chemotherapeutic agents are needed for second-stage HAT.Pentamidine, an aromatic diamidine (Fig. 1), has been used to treat first-stage HAT since the 1940s, typically via intramuscular injection (4). However, it is ineffective against second-stage HAT due to low blood-brain barrier (BBB) permeability (5). DB75 (furamidine) is a structural analogue of pentamidine (Fig. 1) and possesses potent in vitro antitrypanosomal activities (50% inhibitory concentrations [IC 50 s] of Ͻ10 ng/ml against wild-type T. brucei strains) that are similar to those of pentamidine (6). Pentamidine and DB75 parti...

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