Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug

Human 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. To 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. Screening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline2 receptor. The reduction in insulin secretion in MIN6 β-cells by pentamidine maybe secondary to pentamidine-mediated activation of β-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. These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine. * aq : aqueous BBB : blood-brain barrier CAC : critical aggregation concentration CMC : critical micellar concentration CVO : circumventricular organs CMH : Cyanmethaemoglobin DPFS : developmental pathway funding scheme DPD : dissipative particle dynamics DNDi : Drugs for Neglected Diseases initiative DLS : dynamic light-scattering HAT : human African trypanosomiasis HLB : hydrophilic-lipophilic balance iv : intravenous IR : inward rectifying MW : molecular weight MRP : multi-drug resistance associated protein MRC : Medical Research Council na : not available ppm : parts per million PTI : pentamidine isethionate Pgp : P-glycoprotein PFH : plasma free haemoglobin PEO : poly(ethylene oxide) PPO : poly(propylene oxide) sal : saline SLD : scattering length density