Abstract
Background: Nerve agents (NAs) can irreversibly inhibit acetylcholinesterase (AChE). An effective NA antidote should permeate the blood–brain barrier (BBB) to reactivate the inhibited AChE in brain. There is an urgent requirement for the large-scale evaluation and screening of antidotes. Existing methods for evaluating reactivators in vitro can only examine the reactivation effect of drugs and not brain-target properties. The current Transwell BBB model can only evaluate the drug penetration performance for crossing the barrier, but not the pharmacodynamics. Methods: Highly purified rat brain microvascular endothelial cells (RBMECs) from 2-week-old Sprague Dawley rats were inoculated into the upper chamber of Transwell plates to establish a BBB model. Three key parameters of AChE reactivation were determined by the Ellman method: the minimum detection limit of AChE, the effective dosage of NAs (70% enzyme inhibition rate), and the optimal dosage of reactivators. AChE and NAs were added to the lower pool of Transwell plates to simulate central poisoning, and antidotes of reactivators were added to the upper pool to simulate drug administration. The AChE activity of samples, collected from the lower pool, was measured. A liposomal nanomedicine loaded with the reactivator asoxime chloride (HI-6) was prepared using the extraction method and tested by the model.Results: The obtained RBMECs exhibited a typical monolayer “paving stone” morphology, and tight junctions were expressed among the RBMECs. The concentrations of AChE, sarin, and the reactivator were 0.07 mg/mL, 10–6 v/v, and 0.03 mg/mL, respectively. The reaction rate of the reactivators obtained from the model was significantly lower than that obtained from the non-model group. Furthermore, a nanomedicine loaded with HI-6 was synthesized. The final results and rules obtained from the model were in accordance with those evaluated in vivo. Conclusion: The therapeutic effect of antidotes can be rapidly and accurately evaluated using this model. In addition to small-molecule drugs, nanomedicines can also be evaluated by this method. A liposomal nanomedicine with a high reactivation rate against the nerve agent sarin was discovered.
Evaluation of Potency of Known Oximes (Pralidoxime, Trimedoxime, HI-6, Methoxime, Obidoxime) to in vitro Reactivate Acetylcholinesterase Inhibited by Pesticides (Chlorpyrifos and Methylchlorpyrifos and Nerve Agent (Russian VX)
