Development of a sensitive HPLC method to measure in vitro permeability of E- and Z-isomeric forms of thiosemicarbazones in Caco-2 monolayers

Abstract

In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (<22%) and accuracy (85-115% of true values) were obtained over a range of 1.0-100μM for Bp4eT and 1.5-300μM for Bp4aT. Limits of detection were 0.3μM and 1μM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1μM and 5μM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100μM and 300μM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery.

Figure 1. Structures of the chelators discussed in this article

General structure of the DpT analogs; general structure of the BpT analogs; 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311); 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670); di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT); 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT); 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT); desferrioxamine (DFO); 2-benzoylpyridine thiosemicarbazone (BpT); and 2-benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone (Bp44mT).

Figure 2. Representative chromatograms showing Z-to-E isomer conversion over time for Bp4eT and Bp4aT in aqueous solution

(A) Blank HBSS buffer; (B) 50 µM Bp4eT and 10 µM Dp44mT in HBSS buffer (20 µL) injected immediately after preparation of the solution from the solid; (C) Under the same conditions as (B), but after a 24 h incubation at 4°C; (D) 50 µM Bp4aT and 10 µM Dp44mT in HBSS buffer (20 µL) injected immediately after preparation from the solid; and (E) under the same conditions as (C), but after a 24 h incubation at 4°C. IS, internal standard, Dp44mT. Results are mean ± SD (3 determinations) in a typical experiment of three performed.

Figure 3

Cellular iron chelation by Bp4eT and Bp4aT using THP-1 cells in culture. THP-1 cells were treated with iron(II) sulfate (10 µM) for 3 h at 37°C. After this, the cells were loaded with calcein-AM (0.2 µM) for 10 min at 37°C, and then 10 µM of the indicated iron chelator was added. Fluorescence intensity of calcein was measured in real time starting just after the addition of chelator. Here, the size of the labile iron pool (LIP) in the cells is represented as fractional fluorescence (F−F_i)/F_i, where F_i = fluorescence in the presence of quencher at time 0, and F = fluorescence at a given time. LIP is plotted against time of incubation with chelator (in min). Results are means of 3 determinations in a typical experiment of three performed.

Figure 4. Effect of the chelators Bp4eT, Bp4aT and BpT on the growth of Caco-2 cells

Caco-2 cells were grown on 96 well plates (2 × 10⁵ cells/well) and treated either of the chelators for 24 h/37°C. Then the cells were loaded with 0.2 µM calcein-AM for 10 min/37°C and fluorescence was measured. Fluorescence intensity of the treated cells was compared to the untreated (control) cells and expressed as % of Control Fluorescence Intensity for the chelators, Bp4eT, Bp4aT, and also BpT, a chelator with low anti-proliferative activity. Fluorescence intensity (% control) was plotted against concentration (in µM). Results are mean ± SD (3 determinations) in a typical experiment of three experiments performed.

Figure 5. Transport of Bp4eT and Bp4aT across Caco-2 Monolayers

Bp4eT or Bp4aT was loaded onto either the apical (AP) or the basolateral (BL) side of Caco-2 monolayers at the indicated concentrations and incubated at 37°C for the indicated times (30, 60 and 120 min). Samples were collected from the receiving side and the total amount of chelator present was measured by HPLC. Total amount measured (in nmol) is plotted against time of incubation. A, B: Bp4eT; C, D: Bp4aT. Solid triangles: 100 µM of chelator; solid squares, 300 µM of chelator. Results are mean ± SD (3 determinations) in a typical experiment of three experiments performed.