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.

• Authors: Zufan Debebe, Sergei Nekhai, Meseret Ashenafi, David B Lovejoy, Danuta S Kalinowski, Victor R Gordeuk, W Malcolm Byrnes, Des R Richardson, Pradeep K Karla
• Journal: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (J Chromatogr B Analyt Technol Biomed Life Sci) Vol. 906 Pg. 25-32 (Oct 01 2012) ISSN: 1873-376X [Electronic] Netherlands
• PMID: 22954966 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier B.V. All rights reserved.

Chemical References

• Iron Chelating Agents
• Isoquinolines
• Thiosemicarbazones
• lucifer yellow
• Iron

Topics

• Caco-2 Cells
• Cell Line, Tumor
• Cell Membrane Permeability
• Cell Survival (drug effects)
• Chromatography, High Pressure Liquid (methods)
• Humans
• Iron (metabolism)
• Iron Chelating Agents (analysis, chemistry, pharmacokinetics, pharmacology)
• Isomerism
• Isoquinolines (metabolism)
• Limit of Detection
• Models, Biological
• Reproducibility of Results
• Thiosemicarbazones (analysis, chemistry, pharmacokinetics, pharmacology)