title: 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.affiliation: Center for Sickle Cell Disease, Department of Medicine, Howard University College of Medicine, Washington, DC, United States.
authors.forename: Zufan
authors.initials: Z
authors.lastname: Debebe
authors.forename: Sergei
authors.initials: S
authors.lastname: Nekhai
authors.forename: Meseret
authors.initials: M
authors.lastname: Ashenafi
authors.forename: David B
authors.initials: DB
authors.lastname: Lovejoy
authors.forename: Danuta S
authors.initials: DS
authors.lastname: Kalinowski
authors.forename: Victor R
authors.initials: VR
authors.lastname: Gordeuk
authors.forename: W Malcolm
authors.initials: WM
authors.lastname: Byrnes
authors.forename: Des R
authors.initials: DR
authors.lastname: Richardson
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Karla
mesh_terms: D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007501:Iron / Q000378:metabolism; D007502:Iron Chelating Agents / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology; D007536:Isomerism; D007546:Isoquinolines / Q000378:metabolism; D057230:Limit of Detection; D008954:Models, Biological; D015203:Reproducibility of Results; D013882:Thiosemicarbazones / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics* / Q000494:pharmacology
chemical_list: D007502:Iron Chelating Agents; D007546:Isoquinolines; D013882:Thiosemicarbazones; C017475:lucifer yellow; D007501:Iron