In vitro characterization of pralidoxime transport and acetylcholinesterase reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs)

Erin Gallagher, Il Minn, Janice E. Chambers, Peter C. Searson

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article number10
JournalFluids and barriers of the CNS
Volume13
Issue number1
DOIs
StatePublished - Jul 11 2016

ASJC Scopus subject areas

  • Neurology
  • Developmental Neuroscience
  • Cellular and Molecular Neuroscience

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