Electrophysiological evaluation of pentamidine and 17-AAG in human stem cell-derived cardiomyocytes for safety assessment.

Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs' actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. Cav1.2 or Nav1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.

Affinity capillary electrophoresis for identification of active drug candidates in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is an autosomal dominantly inherited degenerative disease with a slow progression. At the present, there is no commercially available treatment, but sustained effort is currently undertaken for the development of a promising lead compound. In the present paper we report the development of a fast, versatile, and cost-effective affinity capillary electrophoresis (ACE) method for the screening and identification of potential drug candidates targeting pathological ARN probes relevant for DM1. The affinity studies were conducted in physiologically relevant conditions using 50 mM HEPES buffer (pH 7.4) in a fused silica capillary dynamically coated with poly(ethylene oxide), by testing a library of potential ligands against (CUG)50 RNA as target probe with a total run time of 4-5 h/ligand. For the most promising ligands, their affinity parameters were assessed and some results formerly reported on the affinity of pentamidine (PTMD) and neomycin against CUG repeats were confirmed. To the best of the authors' knowledge, the estimated binding stoichiometry for some of the tested compounds (i.e., ~ 121:1 for PTMD against the tested RNA probe) is reported for the first time. Additionally, the potential of a novel pentamidine like compound, namely 1,2-ethane bis-1-amino-4-benzamidine (EBAB) with much lower in vivo toxicity than its parent compound has also been confirmed studying its effect on a live cell model by fluorescence microscopy. Further tests, such as the evaluation of the rescue in the mis-splicing of the involved genes, can be performed to corroborate the potential therapeutic value of EBAB in DM1 treatment. Graphical abstract ᅟ.

Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents.

To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications.

Comparative efficacy assessment of pentamidine isethionate and diminazene aceturate in the chemotherapy of Trypanosoma brucei brucei infection in dogs.

The chemotherapeutic efficacy of diminazene aceturate (Berenil)--a standard veterinary trypanocide and pentamidine isethionate (PMI)--a human trypanocide was compared in dogs experimentally infected with Trypanosoma brucei brucei. Also, the activities of the drugs on some serum liver enzymes were evaluated before and after treatment to ascertain the relative safety of the drugs. Fifteen local dogs (mongrels) were used for the study. Three of the dogs were uninfected controls, and twelve were infected with a stock of T. brucei brucei. Three of the infected dogs were untreated controls, three were given diminazene aceturate (DA) at 7 mg/kg body weight intramuscularly (i/m), another three received pentamidine isethionate (PMI) at 4 mg/kg i/m on days 14, 17, 19, 27, 29, and 31 post infection (PI) and the remaining three dogs were also given same dose of PMI on days 14, 16, 18, 20, 22, 24 and 26 PI. Both trypanocides effectively cleared the parasites from the blood of the infected treated dogs. However, the infection subsequently relapsed at day 42 PI in one of the dogs in the DA treated group which later died at day 70 PI. Relapse infection was not recorded with the PMI treated groups although two dogs died in the PMI treated group II (treatment at days 14, 17, 19, 27, 29, and 31 PI) without showing relapsed parasitaemia. The packed cell volume (PCV), red blood cell (RBC) count, and haemoglobin (Hb) level which decreased significantly following infection, were reversed by the trypanocidal treatment. The reversal in the red cell values was faster in the PMI treated groups than in the DA treated group. The serum alkaline phosphate (SAP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels increased following infection and drug administration. The increase in the enzyme levels was greater in the DA treated groups than PMI treated groups. It was thus concluded that PMI given at 4 mg/kg i/m at days 14, 16, 18, 20, 22, 24, and 26 PI constituted a safe and efficient trypanocide and exhibited a superior trypanocidal action than DA in T. brucei brucei infected dogs.

In vitro assessment of susceptibility of Acanthamoeba polyphaga to drugs using combined methods of dye-binding assay and uptake of radiolabelled adenosine.

A technique based on binding of eosin dye to cell was applied to quantitate Acanthamoeba trophozoites in culture. Using this technique in combination with the uptake of radiolabelled adenosine, we assessed the activities of triazole (saperconazole), imidazole (ketoconazole, miconazole) and diamidine (berenil, pentamidine, dibromopropamidine) compounds against A. polyphaga trophozoites. The quantity of dye bound to the trophozoites correlated (r = 0.99) with the number of organisms per well. When inhibition of the growth of Acanthamoeba was the only parameter used to measure the effectiveness of these drugs, saperconazole was found to be most active with IC50 (concentration of drug that inhibited by 50%, the growth of A. polyphaga trophozoites incubated at 28 degrees C for 48 h) of 0.95 microM. The IC50s of the other drugs ranged from > or = 1500 microM for micronazole, the least active, to 3.0 microM for berenil. However, when efficacy was assessed by combining inhibition of growth with the uptake of [14C-8]adenosine by the drug treated organism, the diamidines, particularly berenil and pentamidine were considered most potent.

Semiautomated assessment of in vitro activity of potential antileishmanial drugs.

We have compared the in vitro activity of six agents against macrophage-contained Leishmania tropica amastigotes determined by the conventional Giemsa staining procedure, with the activity determined by the semiautomated assessment of incorporation of radiolabeled uracil into the nucleic acid of the organisms. Although the mean 50% effective dose of Pentostam by Giemsa staining (4.1 micrograms/ml) was somewhat higher than that by uracil incorporation (2.8 micrograms/ml), the ED50S for the other two clinical agents (pentamidine, 0.035 versus 0.037 micrograms/ml; amphotericin B, 0.67 versus 0.70 micrograms/ml) and for three promising experimental agents (ketoconazole, 11.3 versus 11.3 micrograms/ml; the 8-aminoquinoline WR 6026, 1.6 versus 1.5 micrograms/ml formycin B, 0.018 versus 0.017 micrograms/ml) were virtually identical. The radiolabeling technique has several advantages over the Giemsa staining procedure. These include the need for relatively few macrophages, rapid and objective data generation, and viability of the test organism being measured. The successful application of the radiolabeling technique to at least six different chemical classes of compounds suggests that it would be useful for the routine assessment of antileishmanial activity in vitro.