title: In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs).
abstract: BACKGROUND
  Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection.
  
  
  METHODS
  To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-PAM is a substrate for common brain efflux pumps, experiments were performed in the MDCKII-MDR1 cell line, transfected to overexpress the P-gp efflux pump, and the MDCKII-FLuc-ABCG2 cell line, transfected to overexpress the BCRP efflux pump. To determine how transcellular transport influences enzyme reactivation, we developed a modified transwell assay where the inhibited acetylcholinesterase enzyme, substrate, and reporter are introduced into the basolateral chamber. Enzymatic activity was inhibited using paraoxon and parathion.
  
  
  RESULTS
  The permeability of 2-PAM is about 2 × 10(-6) cm s(-1) in MDCK cells and about 1 × 10(-6) cm s(-1) in BC1-hBMECs. Permeability is not influenced by pre-treatment with atropine. In addition, 2-PAM is not a substrate for the P-gp or BCRP efflux pumps.
  
  
  CONCLUSIONS
  The low permeability explains poor brain penetration of 2-PAM and therefore the slow enzyme reactivation. This elucidates one of the reasons for the necessity of sustained intravascular (IV) infusion in response to organophosphate poisoning.
authors.affiliation: Institute for Nanobiotechnology Johns Hopkins University, 3400 North Charles Street, Baltimore, MD, 21218, USA.|Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD, 21218, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Gallagher
authors.affiliation: Department of Radiology and Radiological Science, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD, 21231, USA.
authors.forename: Il
authors.initials: I
authors.lastname: Minn
authors.affiliation: College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 39762-6100, USA.
authors.forename: Janice E
authors.initials: JE
authors.lastname: Chambers
authors.affiliation: Institute for Nanobiotechnology Johns Hopkins University, 3400 North Charles Street, Baltimore, MD, 21218, USA. searson@jhu.edu.|Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD, 21218, USA. searson@jhu.edu.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Searson
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000098:blood supply / Q000187:drug effects / Q000201:enzymology; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002801:Cholinesterase Reactivators / Q000493:pharmacokinetics* / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D055806:Microvessels / Q000187:drug effects / Q000201:enzymology; D010261:Paraoxon / Q000494:pharmacology; D010278:Parathion / Q000494:pharmacology; D011220:Pralidoxime Compounds / Q000493:pharmacokinetics* / Q000494:pharmacology; D020112:Rhodamine 123 / Q000493:pharmacokinetics
chemical_list: D002800:Cholinesterase Inhibitors; D002801:Cholinesterase Reactivators; D005456:Fluorescent Dyes; D011220:Pralidoxime Compounds; D020112:Rhodamine 123; D010278:Parathion; D000110:Acetylcholinesterase; C028797:pralidoxime; D010261:Paraoxon