Elsevier

Acta Tropica

Volume 91, Issue 2, July 2004, Pages 131-143
Acta Tropica

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

https://doi.org/10.1016/j.actatropica.2004.03.010Get rights and content

Abstract

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.

Introduction

African trypanosomes cause a variety of diseases in humans and animals. Trypanosoma brucei subspecies brucei is not infective to humans, but causes a wasting disease known as ngana in African livestock. T. b. rhodesiense is morphologically indistinguishable from the ngana parasite, but causes an acute form of sleeping sickness in humans. T. b. gambiense disease produces chronic trypanosomiasis in humans. Individuals suffering from both forms of sleeping sickness will die without effective treatment. In chronic sleeping sickness (T. b. gambiense), there is evidence that trypanosomes are sequestered from most therapeutic agents when present within the central nervous system (CNS), thus providing a source of relapse following clearance of bloodstream forms (Raseroka and Ormerod, 1985).

Several drugs are currently used to treat sleeping sickness. Suramin must be administered intravenously and is not effective against the CNS stage of the disease. Melarsoprol is an arsenical compound that is effective against secondary-stage disease, but can cause fatal drug reactions and reports of clinical treatment failure are increasing (Barrett and Fairlamb, 1999). Difluoromethylornithine (DFMO), the most recent development in sleeping sickness therapy, reverses CNS symptoms, but must be administered intravenously for a minimum of 7 days and resistance has been documented (Iten et al., 1995). Pentamidine, introduced over 50 years ago, is still used to treat primary-stage disease, but does not target parasites within the central nervous system because it is unable to penetrate the blood–brain barrier (BBB).

Structure–activity experiments resulted in a new class of pentamidine derivatives retaining the terminal amidines, but possessing varied linker moieties. These compounds exhibited potent and broad spectrum anti-microbial activity against such varied organisms as Pneumocystis carinii (Tidwell and Bell, 1993), Cryptosporidium parvum (Blagburn et al., 1998), Leishmania sp. (Bell et al., 1990, Das and Boykin, 1977), Plasmodium falciparum (Bell et al., 1990), Crithidia fasiculata (Gutteridge, 1969), Giardia lambia (Bell et al., 1993), Candida albicans (Del Poeta et al., 1998), Cryptococcus neoformans (Del Poeta et al., 1998), and Trypanosoma spp. (Das and Boykin, 1977). Although the mechanism of antimicrobial activity has not been determined, numerous possibilities including binding the minor groove of DNA have appeared in the literature (Tidwell and Bell, 1993).

Increased interest in the diamidine compounds as new agents to treat AIDS-associated opportunistic infections led to the synthesis of 2,5-bis(4-amidinophenyl)furan (DB75), a compound with anti-trypanosomal and anti-P. carinii activity (Tidwell et al., 1990). DB75 is highly active against trypanosomes when injected, but has poor oral activity. Permeability experiments using cultured intestinal epithelial cells suggest that the absence of oral activity is due to poor oral absorption (Zhou et al., 2002). DB75 is highly hydrophilic due to the dicationic diamidine functional groups; both cationic moieties are presumed necessary for anti-trypanosomal activity (Steck et al., 1982). In 1996, the first reported bis-O-methylamidoxime prodrug 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289) was developed (Boykin et al., 1996). This prodrug DB289 is rapidly metabolized to DB75 in the liver by cytochrome P450 and cytochrome b5 enzymes. However, the potential of either DB75 or DB289 to target parasites within brain tissue has not been determined. DB289 has completed preclinical toxicity and Phase II(a) clinical trials for early-stage sleeping sickness, and is currently under Phase II(b) investigation in central Africa. Although the antimicrobial potency of DB289 has been established, treatment of late-stage African trypanosomiasis demands the development of drugs that can cross the BBB. Single-pass carotid artery experiments with DB289 suggest that it is rapidly and significantly cleared by the brain (Zong et al., 2000). Chronic mouse models using T. b. gambiense infected mice were not cleared of bloodstream parasites using oral DB289 even at toxic dosage regimes, indicating that some parasites may be sequestered in sites unavailable to the compound. Similar tests have not been carried out with intravenous DB289.

A useful characteristic of DB75 and structurally related diamidines is the innate fluorescence, likely due to structural resonance across the entire molecule, whereby excitation with ultraviolet light produces an intense blue fluorescent signal. The direct visualization of DB75 via microscopy and quantitation by HPLC presented in this report were possible due to this quality. These fluorescent properties preclude the use of secondary tags or antibodies, which might influence or alter the behavior of DB75 during in vitro or in vivo experiments. Tracking temporal and spatial distribution of DB75 and DB289 within brain tissue will provide an empirical foundation for further experiments and a greater understanding of trypanosome targeting during cerebral sleeping sickness.

Section snippets

Animal experiments

Adult male Swiss–Webster mice (Charles River Labs, Cambridge, MA, USA) were used in all experiments. Animal use adhered to the University of North Carolina–Chapel Hill Institutional Animal Care and Use Committee (IACUC) guidelines for humane treatment of laboratory animals. Systemic dosing of DB75 was performed on normal male mice (12–18 weeks) by caudal vein injection of DB75 in sterile 0.9% NaCl in water. The final dose for intravenous DB75 was 7.5 μmol/kg. This dose was chosen because it was

Fluorescence microscopy of murine brain tissue after intravenous DB75

Fluorescence was observed in frozen mouse brain tissue sections 24 h after tail-vein injection with 7.5 μmol/kg DB75 (Fig. 1), an effective, non-toxic dose as demonstrated in early-stage mouse models. Fluorescence was not pervasive throughout the brain, however, but instead was localized in cells adjacent to vascular spaces, including cells in the choroid plexus, meninges, and parenchymal endothelial cells (Fig. 1). DB75-associated fluorescence was not observed in cells of the brain parenchyma

Discussion

Results presented here demonstrate that the anti-microbially active dicationic compound DB75 does not effectively cross the blood–brain barrier following intravenous administration to mice. DB75 was detected by HPLC analysis in brain homogenates of treated mice within 15 min post-injection and remained detectable in homogenates for long periods. However, fluorescence microscopy revealed that DB75-associated fluorescence was sequestered within specific regions, namely the choroid plexus,

Acknowledgements

This work was supported by grants from the Bill and Melinda Gates Foundation.

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