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

BACKGROUND: 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. METHODOLOGY: 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. PRINCIPAL FINDINGS: 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 ß-cells by pentamidine may be secondary to pentamidine-mediated activation of ß-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. SIGNIFICANCE: These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.

Organic cation transporter 1 (OCT1) is involved in pentamidine transport at the human and mouse blood-brain barrier (BBB).

Pentamidine is an effective trypanocidal drug used against stage 1 Human African Trypanosomiasis (HAT). At the blood-brain barrier (BBB), it accumulates inside the endothelial cells but has limited entry into the brain. This study examined transporters involved in pentamidine transport at the human and mouse BBB using hCMEC/D3 and bEnd.3 cell lines, respectively. Results revealed that both cell lines expressed the organic cation transporters (OCT1, OCT2 and OCT3), however, P-gp was only expressed in hCMEC/D3 cells. Polarised expression of OCT1 was also observed. Functional assays found that ATP depletion significantly increased [3H]pentamidine accumulation in hCMEC/D3 cells (***p<0.001) but not in bEnd.3 cells. Incubation with unlabelled pentamidine significantly decreased accumulation in hCMEC/D3 and bEnd.3 cells after 120 minutes (***p<0.001). Treating both cell lines with haloperidol and amantadine also decreased [3H]pentamidine accumulation significantly (***p<0.001 and **p<0.01 respectively). However, prazosin treatment decreased [3H]pentamidine accumulation only in hCMEC/D3 cells (*p<0.05), and not bEnd.3 cells. Furthermore, the presence of OCTN, MATE, PMAT, ENT or CNT inhibitors/substrates had no significant effect on the accumulation of [3H]pentamidine in both cell lines. From the data, we conclude that pentamidine interacts with multiple transporters, is taken into brain endothelial cells by OCT1 transporter and is extruded into the blood by ATP-dependent mechanisms. These interactions along with the predominant presence of OCT1 in the luminal membrane of the BBB contribute to the limited entry of pentamidine into the brain. This information is of key importance to the development of pentamidine based combination therapies which could be used to treat CNS stage HAT by improving CNS delivery, efficacy against trypanosomes and safety profile of pentamidine.

Delivery of antihuman African trypanosomiasis drugs across the blood-brain and blood-CSF barriers.

Human African trypanosomiasis (HAT or sleeping sickness) is a potentially fatal disease caused by the parasite, Trypanosoma brucei sp. The parasites are transmitted by the bite of insect vectors belonging to the genus Glossina (tsetse flies) and display a life cycle strategy that is equally spread between human and insect hosts. T.b. gambiense is found in western and central Africa whereas, T.b. rhodesiense is found in eastern and southern Africa. The disease has two clinical stages: a blood stage after the bite of an infected tsetse fly, followed by a central nervous system (CNS) stage where the parasite penetrates the brain; causing death if left untreated. The blood-brain barrier (BBB) makes the CNS stage difficult to treat because it prevents 98% of all known compounds from entering the brain, including some anti-HAT drugs. Those that do enter the brain are toxic compounds in their own right and have serious side effects. There are only a few drugs available to treat HAT and those that do are stage specific. This review summarizes the incidence, diagnosis, and treatment of HAT and provides a close examination of the BBB transport of anti-HAT drugs and an overview of the latest drugs in development.