Abstract
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 primary 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 (DNDi) recommendations. To do this, we evaluated the physicochemical and structural characteristics of Pluronic-pentamidine formulations, selected appropriate 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 observed reduction in insulin secretion with pentamidine in MIN6 β-cells maybe secondary to pentamidine-mediated activation of β-cell imidazoline receptors and impairment of cell viability. 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. We therefore closed the study before progressing to in vivo efficacy and toxicity studies. Importantly, this MRC DPFS funded study has resulted in the generation of a set of results which are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.
Footnotes
Author Contributions for uploading during submission
LS: PDRA in vivo BBB (PK and In situ brain perfusion technique) and in vitro BBB studies. Data analysis. Result writing.
GNS: PhD Student in vitro BBB studies, haemolysis assay, membrane integrity assay. Data analysis
RCB: in vitro BBB studies.
MF: PhD Student contributed to the initial concept and data sets with P85.
MAS: PDRA insulin secretion and cell viability studies, physical chemistry, data analysis result writing.
SAT: Introduction, method, result and discussion writing, paper collation, co-ordination. CNS drug delivery expert. Obtained funding, concept, project management and experimental design, data analysis and interpretation.
HBS: in vitro anti-trypanosomal assays.
CD, CL, SP, MC, SC, SAT: obtaining funding, concept, project management and experimental design, data analysis and interpretation.
Abbreviations
- BBB
- blood-brain barrier
- CAC
- critical aggregation concentration
- CMC
- critical micellar concentration
- CVO
- circumventricular organs
- DPFS
- developmental pathway funding scheme
- DNDi
- Drugs for Neglected Diseases initiative
- DLS
- dynamic light-scattering
- HAT
- human African trypanosomiasis
- HLB
- hydrophilic-lipophilic balance
- MW
- molecular weight
- MRP
- multi-drug resistance associated protein
- MRC
- Medical Research Council
- PTI
- pentamidine isethionate
- Pgp
- P-glycoprotein
- PFH
- plasma free haemoglobin
- PEO
- poly(ethylene oxide)
- PPO
- poly(propylene oxide)
- SLD
- scattering length density