Pharmacological evaluation of clinically relevant concentrations of (2R,6R)-hydroxynorketamine

Ketamine is a rapid-onset antidepressant whose efficacy long outlasts its pharmacokinetics. Multiple studies suggest ketamine's antidepressant effects require increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-dependent currents, which have recently been exclusively attributed to its N-methyl-d-aspartate receptor-inactive metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK). To investigate this AMPAR-activation claim further, we estimated and evaluated preclinically and clinically relevant unbound brain HNK concentrations (C_b,u). (2S,6S)-HNK and (2R,6R)-HNK were novelly synthesized, and their neuropharmacokinetic profiles were determined to project relevant C_b,u. Using concentrations (0.01-10 μM) bracketing the pertinent cross-species C_b,u, both compounds' AMPAR modulation was assessed in vitro by electrophysiological recordings and GluA1 surface expression. Neither (2S,6S)-HNK nor (2R,6R)-HNK bound orthosterically to or directly functionally activated AMPARs. (2R,6R)-HNK failed to evoke AMPAR-centric changes in any electrophysiological endpoint from adult rodent hippocampal slices. Conversely, time- and concentration-dependent increases in GluA1 expression occurred only with (2R,6R)-HNK (≥0.1 μM at ≥90 min). The (2R,6R)-HNK concentrations that increased GluA1 expression are consistent with its maximal C_b,u (0.92-4.84 μM) at reportedly efficacious doses of ketamine or (2R,6R)-HNK in mouse depression models, but ≥3-fold above its projected maximal human C_b,u (≤37.8 ± 14.3 nM) following ketamine's clinically antidepressant infusion. These findings provide insight into the observed AMPAR-affecting (2R,6R)-HNK concentrations versus its exposures attained clinically at an antidepressant ketamine dose. To optimize any clinical study with (2R,6R)-HNK to fully assess its translational pharmacology, future preclinical work should test (2R,6R)-HNK concentrations and/or C_b,u of 0.01-0.1 μM to parallel its projected human C_b,u at a clinically antidepressant ketamine dose.