Nachrichten aus der Brüder-Gemeine, 1828, no. 01

The Moravian Church's monthly journal of its worldwide activities, including in Labrador, published from 1819-94

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 β-cells by pentamidine may be 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. SIGNIFICANCE: These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine

Bondholder Coercion: The Problem of Constrained Choice in Debt Tender Offers and Recapitalizations

<p>The effect MATE1, MATE2 and PMAT inhibitors on the [³H]pentamidine accumulation in hCMEC/D3 (A) and bEnd.3 (B) cells. Cells were incubated with MATE1 inhibitor famotidine (1 μM), MATE2 inhibitor nifekalant (3 μM) or PMAT inhibitor lopinavir (2 μM) and no significant differences were observed compared to control. All data expressed as mean ± SEM, n =.4 passages of cells, with 6 replicates (wells) per timepoint per plate. Data were analysed using two-way ANOVA with SigmaPlot 13.0.</p

Organic cation transporter 1 (OCT1) is involved in pentamidine transport at the human and mouse blood-brain barrier (BBB) - Fig 10

<p>Confocal microscopy image of MRP4 expression in confluent hCMEC/D3 cells (A) and bEnd.3 cells (B). No fluorescent signals were detected when using MRP4 antibody [M4I-10] from Abcam at 1:200 dilution and goat anti-rat Alexa Fluor^® 488 secondary antibody (Abcam, ab181448) at 1:200 dilution.</p

Expression of OCT1 was detected by Transmission Electron Microscopy and immunogold labelling.

<p>hCMEC/D3 (p28) and bEnd.3 cells (p23) were grown to confluency on transwell polycarbonate membrane and stained for OCT1 using the method described. Gold nanoparticles on the membranes (arrows) were counted in the 60 sequential images acquired at 11500 x magnification. Significantly increased expression of OCT1 was found on the luminal membrane compared to the abluminal membrane. ***p<0.001 using student’s t-test.</p

BCRP expression in confluent hCMEC/D3 cells and bEnd.3 cells.

<p>Confocal microscopy image of BCRP expression in confluent hCMEC/D3 cells (A) and bEnd.3 cells (B) was not detected in the plasma membranes when using anti-BCRP antibody (New England Biolabs, 4477S diluted at 1:200)—Western blot band of BCRP was detected at 143 kD which corresponds to the dimer version of BCRP in positive control (C). No band was observed at 70 kD (monomer version of BCRP). Tubulin (55 kD) was used as a loading control. Lane 1 –hCMEC/D3 passage 28, Lane 2- hCMEC/D3 passage 33, Lane 3- bEnd.3 passage 18, Lane 4- bEnd.3 passage 23, Lane 5 –brain endothelial cells isolated from mouse (positive control).</p

The transporter inhibitors used in this study along with [³H]pentamidine.

<p>All inhibitors were used in the presence of 0.05% DMSO and used at the published concentration ranges where they affect transporter activity.</p