Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant α7 and α4β2 neuronal nicotinic acetylcholine receptors in Xenopus oocytes

Ketamine is a dissociative anaesthetic that is formulated as Ketalar, which contains the preservative benzethonium chloride (BCl). We have studied the effects of pure racemic ketamine, the preservative BCl and the Ketalar mixture on human neuronal nicotinic acetylcholine receptors (nAChRs) composed of the α7 subunit or α4 and β2 subunits expressed in Xenopus laevis oocytes. Ketamine inhibited responses to 1 mM acetylcholine (ACh) in both the human α7 and α4β2 nAChRs, with IC50 values of 20 and 50 μM respectively. Inhibition of the α7 nAChRs occurred within a clinically relevant concentration range, while inhibition of the α4β2 nAChR was observed only at higher concentrations. The Ketalar formulation inhibited nAChR function more effectively than was expected given its ketamine concentration. The surprising increased inhibitory potency of Ketalar compared with pure ketamine appeared to be due to the activity of BCl, which inhibited both α7 (IC50 value of 122 nM) and α4β2 (IC50 value of 49 nM) nAChRs at concentrations present in the clinical formulation of Ketalar. Ketamine is a noncompetitive inhibitor at both the α7 and α4β2 nAChR. In contrast, BCl causes a parallel shift in the ACh dose-response curve at the α7 nAChR suggesting competitive inhibition. Ketamine causes both voltage-dependent and use-dependent inhibition, only in the α4β2 nAChR. Since α7 nAChRs are likely to be inhibited during clinical use of Ketalar, the actions of ketamine and BCl on this receptor subtype may play a role in the profound analgesia, amnesia, immobility and/or autonomic modulation produced by this anaesthetic. Keywords: Neuronal nicotinic acetylcholine receptors, benzethonium chloride, ketamine, anaesthetic mechanism Introduction Ketamine is a dissociative anaesthetic, that is an effective analgesic, amnestic and hypnotic, but causes hallucinations that limit its usefulness as a clinical anaesthetic agent. Due to its potency as an analgesic and its sympathomimetic properties, ketamine finds extensive use in the treatment of burn patients in intensive care settings. In the early 1980's Lodge described ketamine as a selective inhibitor of NMDA receptor activity and neuronal nicotinic receptors (nAChRs) on Renshaw cells of the cat spinal cord (Anis et al., 1983). Ketamine is also an antagonist of NMDA receptors in rat cortex (Harrison & Simmonds, 1985) and blocks these receptors at sub-clinical concentrations, by acting both as a use-dependent open channel blocker (Honey et al., 1985) and through allosteric inhibition (Orser et al., 1997). Ketamine inhibits the response to acetylcholine or nicotine in both muscle and neuronal type nAChRs (Sumikawa et al., 1983; Wachtel & Wegrzynowicz, 1992; Scheller et al., 1996; Durieux, 1995 Furuya et al., 1999; Flood & Krasowski, 2000; Friederich et al., 2000; Sasaki et al., 2000; Yamakura et al., 2000). In the case of nAChRs containing the α4 subunit, inhibition occurs at concentrations similar to those effective in inhibiting NMDA responses (Flood & Krasowski, 2000; Friederich et al., 2000; Sasaki et al., 2000). Ketamine is marketed in the United States as Ketalar, a racemic mixture of ketamine containing benzethonium chloride (BCl). BCl is included in Ketalar as a preservative, at a concentration of 0.1 mg with 10 mg of ketamine. BCl is also used as a topical antiseptic alone. BCl is an inhibitor of acetylcholine esterase (AChE) and choline esterase (Zaman et al., 1997) as well as muscarinic receptors for acetylcholine (ACh) (Durieux & Nietgen, 1997). The secondary structure of BCl consists of a quaternary ammonium atom as does acetylcholine, though it also contains 2 benzyl groups. (Figure 1). Figure 1 Chemical structures of acetylcholine, the native nicotinic agonist and benzethonium chloride, a nicotinic and muscarinic antagonist. In the present study we investigated the mechanism of action of ketamine and BCl at both human heteromeric α4β2 and homomeric α7 type nAChRs that are found in the CNS. We compared the activity of each drug alone with the activity of the clinical formulation, Ketalar, for evidence of potentially clinically significant interaction.