Distribution and quantitation of the anti-trypanosomal diamidine 2,5-bis(4-amidinophenyl)furan (DB75) and its N-methoxy prodrug DB289 in murine brain tissue.

The current epidemic of sleeping sickness, also known as human African trypanosomiasis in sub-Saharan Africa places nearly 60 million people at risk for developing this life threatening infection. Although effective treatments for early-stage sleeping sickness exist, these drugs usually require extended dosing schedules and intravenous administration. New treatments are also needed for cerebral (late) stage trypanosomiasis. 2,5-Bis(4-amidinophenyl)furan (DB75), a pentamidine analog, has potent in vitro and in vivo anti-trypanosomal activity. However, DB75 does not exhibit significant oral bioavailability and has proved to be ineffective against mouse models of late-stage sleeping sickness regardless of administration route. To circumvent the limited oral bioavailability of DB75, an N-methoxy prodrug 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289) was designed and developed initially as a compound to treat AIDS-related Pneumocystis carinii pneumonia (PCP). Despite excellent oral activity against early-stage sleeping sickness, oral administration of DB289 exhibited limited efficacy in mouse models of late-stage disease. DB289 has recently entered Phase II(b) clinical trials to treat primary-stage sleeping sickness in Central Africa. The current study takes advantage of the innate fluorescence of DB75 and DB289 along with specific and sensitive quantitative analyses to examine plasma and brain distribution of these compounds. Animals were dosed with intravenous DB75, oral DB289, and intravenous DB289. Following intravenous administration, DB75 was readily detectable in whole brain extracts and persisted for long periods. Fluorescence microscopy revealed that DB75 did not penetrate into brain parenchyma, however, but was sequestered within cells lining the blood-brain and blood-cerebrospinal fluid barriers. In contrast, brain tissue of mice treated with oral DB289 exhibited diffuse fluorescence within the brain parenchyma, suggesting that the prodrug was not trapped within blood-brain barrier cells (BBB). However, maximal brain concentrations of the active compound DB75 were very low (13 nmol/mg of tissue at 24 h). Intravenous administration of DB289 resulted in a qualitatively similar fluorescence pattern to oral DB289, indicating again that DB289 and DB75 were present within brain parenchyma, not only in barrier regions. Furthermore, peak DB75 tissue levels were higher (61 nmol/mg of tissue at 24 h) than with oral prodrug. The near five-fold increase in brain levels of DB289 combined with parenchymal localization of compound fluorescence after intravenous administration suggest that the unaltered prodrug penetrates the blood-brain barrier, and may be subject to in situ biotransformation. Intravenous administration of DB289 should be evaluated in mouse models of late-stage sleeping sickness.