Drug Reformulation For A Neglected Disease. The Nanohat Project To Develop A Safer More Effective Sleeping Sickness Drug

Author summarySleeping sickness or human African Trypanosomiasis (HAT) is a disease caused by a parasite, which is transferred to humans by the bite of an infected tsetse fly. There are two disease stages: the first stage is the blood-based stage of the disease and the second stage affects the brain. It is fatal if left untreated. The blood-brain barrier (BBB) makes the brain stage difficult to treat because it prevents 99% of all drugs from entering the brain from the blood. Those anti-HAT drugs that do enter the brain are toxic and have serious side effects. Pentamidine is a less toxic blood stage drug, which our research has shown has a limited ability to cross the BBB due to its removal by proteins called transporters. The objective of this study was to use Pluronic to improve pentamidine delivery to target sites, whilst reducing its side effects. Pluronic is a polymer, which can assemble into micelles and encapsulate the drug. Thus, prolonging its circulation time and protecting it. Our study indicated that the selected Pluronics did not increase the brain delivery of pentamidine. However. Pluronic-pentamidine formulations were identified that harboured trypanocidal activity and did not increase safety concerns compared to unformulated pentamidine.BackgroundHuman African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10-100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations.MethodologyTo do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study.Principal FindingsScreening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline(2) receptor. The reduction in insulin secretion in MIN6 beta-cells by pentamidine may be secondary to pentamidine-mediated activation of beta-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine.SignificanceThese results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.