Transport Inhibitors to enhance central and peripheral nervous system antidote activity to organophosphate (OP) exposure

Abstract ID 127388 Poster Board 137 Background and Aims: RS194B is an ionizable, zwitterionic oxime with potent OP antidotal activity. Its cation and zwitterionic species enter the active center gorge and reactivate the OP-inhibited acetylcholinesterase (AChE); the neutral species crosses blood-brain barrier, but its ionized species are extruded by P-glycoprotein (P-gp) transporters in the central nervous system (CNS). The P-gp transporters are also responsible for eliminating RS194B unchanged by renal mechanism. Thus, administering P-gp inhibitors such as tariquidar (TQD) to compete with RS194B decreases RS194B extrusion to sustain its CNS concentrations and diminish renal clearance. Alternative P-gp inhibitors inhibiting brain extrusion of RS194B remain unknown. We describe exploratory data among different RS194B combinations and to find the optimal range of P-gp inhibitors that can enhance the RS194B concentration in mouse whole brain to relative plasma concentration. Methods: This is an ongoing exploratory in-vivo study of antidote combinations reactivating OP-inhibited AChE. Sampling of both whole brain and plasma in various combinations of RS194B was conducted at 15 and 30 minutes post-administration of the antidote. RS194B was dissolved by incremental dropwise addition of HCl lowering the pH to 6.5, resulting in clear solutions. P-gp transport inhibitors included zwitterionic ciprofloxacin (20-60 mg/kg) and levofloxacin (40-60 mg/kg), cobicistat (30 mg/kg), diltiazem (DIL, 3-60 mg/kg), metformin (50 mg/kg), TQD (7.5 mg/kg). They were dissolved in 5% DMSO and sterile water, resulting in clear solutions. A total dose of P-gp inhibitor in approximately 0.5 - 1 mL volumes was injected intraperitoneally. Ten minutes after P-gp inhibitor administration, a total dose of 70 mg/kg RS194B in approximately 50 - 100 μL volumes was injected intramuscularly. Total RS194B concentrations were measured by a validated LC-MS/MS assay with a detection limit of approximately 20 picograms. Areas under the curve (AUC) ratios at 30 minutes were calculated between RS194B alone and RS194B in combination with prospective transport inhibitors. Results: Initial analysis included 24 BALB/c male mice at 8 weeks of age and weighing 20-30 grams. Whole brain and plasma data of the RS194B antidote and antidote-transporter inhibitor combinations were measured at 15 minutes and 30 minutes. Overall, the combination of RS194B+TQD shows the highest RS194B AUC 30 minutes post-administration, indicating a 3.6-fold and 4.4-fold greater increase than with RS194B alone in whole brain and plasma, respectively (Table 1). Addition of DIL (60 mg/kg) yielded an increase in RS194B brain concentration, second only to the combination of RS194B+TQD. However, mice experienced limping on the injection sites. We reduced DIL dosing and found that 10 mg/kg DIL seemed to be the optimal dose for mice, showing a 2.9-fold and 3.9-fold increase than RS194B alone in mouse whole brain and plasma, respectively (Table 1). Conclusions: Of all six candidate transport inhibitors, the most promising P-gp inhibitors are TQD and DIL for maintaining high RS194B concentrations in mouse whole brain and plasma. We are collecting more data with these two leads to confirm our exploratory findings. Supported by an American Foundation for Pharmaceutical Education Fellowship to Xiaoying Huang and conducted at UCSD