Post-exposure treatment with the oxime RS194B rapidly reverses early and advanced symptoms in macaques exposed to sarin vapor

Abstract

Organophosphate (OP) nerve agents and pesticides trigger a common mechanism of neurotoxicity resulting from critical targeting and inhibition of acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Therapeutic countermeasures have thus focused on either administering an oxime post-exposure, that can rapidly reactivate OP-inhibited AChE, or by preventing OP poisoning through administering pre-exposure treatments that scavenge OPs before they inhibit their physiological AChE targets. While several pyridinium aldoxime antidotes are currently approved, their utility is impaired due to their inability to cross the blood-brain barrier (BBB) efficiently. The present study utilized a macaque (Ma) model to demonstrate the efficacy of a novel zwitterionic and centrally acting oxime RS194B to reactivate sarin- and paraoxon-inhibited macaque AChE and butyrylcholinesterase (BChE) in vitro and to further assess the capacity of RS194B to effect a reversal of clinical symptoms following sarin inhalation in vivo. In vitro, oxime reactivation of MaAChE and MaBChE was shown to be comparable to their human orthologs, while the macaque studies indicated that IM administration of 62.5 mg/kg of RS194B and 0.28 mg/kg atropine after continuous exposure to 49.6 μg/kg sarin vapor, rapidly reactivated the inhibited AChE and BChE in blood and reversed both early and advanced clinical symptoms of sarin-induced toxicity following pulmonary exposure within 1 h. The rapid cessation of autonomic and central symptoms, including convulsions, observed in macaques bodes well for the use of RS194B as an intra- or post-exposure human treatment and validates the macaque model in generating efficacy and toxicology data required for approval under the FDA Animal rule.

Fig. 1

RS194B concentration dependence for reactivation at 37°C of macaque RBC-AChE and recombinant CHO-derived (rMaBChE) inhibited by a sarin analogue and paraoxon (POX) in vitro. (A) reactivation of isopropyl methylphosphonyl AChE from macaque blood MaRBC-AChE (B) reactivation of diethylphosphoryl Ma RBC AChE, (C) reactivation of isopropyl methylphosphonyl rMaBChE and (D) reactivation of diethylphosphoryl rMaBChE. Oxime was added at 0.5mM (grey), 1.5 mM (black), 5mM (red) and 15mM (blue).

Figure 2

Reactivation of blood AChE (upper panel) and BChE (lower panel) in three surviving macaques (#42984, 43553 and 43960) following inhalation exposure to sarin. Values are related to pre-exposure levels in the corresponding macaque. The respective macaques received sarin exposure for 10.8, 17.7 and 17.3 min and exhibited the symptoms including tremors following miosis in two of the three animals. Colors from green to red signify the severity of clinical symptoms. Animals were pretreated with aerrHuBChE using a nebulizer and received no oxime.

Figure 3

Post-exposure reactivation by IM RS194B of RBC-AChE and plasma BChE in five macaques (#42840, 43481, 42848, 42029, 44434) exposed to inhaled sarin (49.6 µg/kg). Values are shown as percent of pre-exposure levels in the individual macaque prior to sarin exposure. The time intervals between cessation of sarin administration and injection of RS194B in the surviving animals was 2.75, 0.9, 1.5, 4.2 and 4.5 min respectively. All animals were administered oxime (62.5 mg/kg) IM with atropine (0.28 mg/kg) IM and all survived. Colors from pale pink to red signify the severity of clinical symptoms. The clinical signs indicated were observed at 27.6min (#42840), 33.1min (#43481), 20.3mins (#42840), 19.3 mins (#42029) and 20.5 mins (#44434) following initiation of sarin administration. By contrast, five control sarin-exposed macaques, which did not receive the RS194B, exhibited the typical clinical signs of sarin poisoning including tremors and convulsions and died with AChE levels at 3–4% of baseline in those animals where an initial sample measurement was possible. Animals were pretreated with aer-rHuBChE.

Scheme 1

(A) Ionization equilibria of the hydroxyiminoacetamidoalkyl cyclic amines showing the transition from cations to anions to proceed through zwitterionic and neutral species. The oximate-oxime and amine protonation in the azepine ring have nearly identical pKa’s ~8.8. The neutral species facilitates crossing the blood-brain barrier and oral bioavailability, while the cation will be attracted to the active center gorge and in solution the anion is the nucleophilic, general base catalyst. (B) The lead structure, RS 194B of over 100 congeners synthesized, is shown on the right.