Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function Eric W. Lumsdena,1, Timothy A. Troppolib,1, Scott J. Myersc, Panos Zanosd, Yasco Aracavaa, Jan Kehre,f, Jacqueline Lovettg, Sukhan Kimc, Fu-Hua Wange,f, Staffan Schmidte,f, Carleigh E. Jenned, Peixiong Yuanh, Patrick J. Morrisi, Craig J. Thomasi, Carlos A. Zarate Jr.h, Ruin Moaddelg, Stephen F. Traynelisc,2, Edna F. R. Pereiraa,j,2, Scott M. Thompsonb,d,2, Edson X. Albuquerquea,j,k,2, and Todd D. Gouldd,j,l,m,2,3 aDepartment of Epidemiology and Public Health, Division of Translational Toxicology, University of Maryland School of Medicine, Baltimore, MD 21201; bDepartment of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201; cDepartment of Pharmacology, Emory University, Atlanta, GA 30329; dDepartment of Psychiatry, University of Maryland School of Medicine, Baltimore, MD 21201; eDepartment of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm, Sweden; fPronexus Analytical AB, SE-167 33 Bromma, Sweden; gBiomedical Research Center, National Institute on Aging, Intramural Research Program, National Institutes of Health, Baltimore, MD 21224; hSection on the Neurobiology and Treatment of Mood Disorders, Intramural Research Program, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892; iDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, Intramural Research Program, National Institutes of Health, Bethesda, MD 20892; jDepartment of Pharmacology, University of Maryland School of Medicine, Baltimore, MD 21201; kDepartment of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201; lDepartment of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, MD 21201; and mVeterans Affairs Maryland Health Care System, Baltimore, MD 21201 Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved January 24, 2019 (received for review September 19, 2018) Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) Significance is a putative fast-acting antidepressant candidate. Although inhibi- tion of NMDA-type glutamate receptors (NMDARs) is one mecha- Standard antidepressant treatments require weeks to show effec- ’ nism proposed to underlie ketamine s antidepressant and adverse tiveness. A single subanesthetic dose of ketamine rapidly attenu- R R PHARMACOLOGY effects, the potency of (2 ,6 )-HNK to inhibit NMDARs has not been ates many clinical signs and symptoms of depression; however, established. We used a multidisciplinary approach to determine the ketamine treatment also has many adverse effects, including dis- R R effects of (2 ,6 )-HNK on NMDAR function. Antidepressant-relevant sociation and potential for abuse, which are mediated by NMDA R R behavioral responses and (2 ,6 )-HNK levels in the extracellular glutamate receptor (NMDAR) inhibition. Previous work has revealed compartment of the hippocampus were measured following sys- that the ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) R R temic (2 ,6 )-HNK administration in mice. The effects of ketamine, induces antidepressant-like responses in rodents while minimizing R R S S (2 ,6 )-HNK, and, in some cases, the (2 ,6 )-HNK stereoisomer were the adverse effects observed with ketamine. The results of this i ii evaluated on the following: ( ) NMDA-induced lethality in mice, ( ) study, using a multitude of experimental approaches, confirm that NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) antidepressant-relevant concentrations of (2R,6R)-HNK are not suf- iii in the CA1 field of mouse hippocampal slices, ( )NMDAR- ficient to block NMDARs. This provides a basis for work directed at mediated miniature excitatory postsynaptic currents (mEPSCs) and alternative molecular targets and toward novel drugs that exert NMDA-evoked currents in CA1 pyramidal neurons of rat hippocam- rapid antidepressant effects independent of NMDAR inhibition and iv Xenopus pal slices, and ( ) recombinant NMDARs expressed in oo- NMDAR-mediated adverse effects. cytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, Author contributions: E.W.L., T.A.T., S.J.M., P.Z., Y.A., J.K., S.F.T., E.F.R.P., S.M.T., E.X.A., the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was and T.D.G. designed research; E.W.L., T.A.T., S.J.M., P.Z., Y.A., J.K., J.L., S.K., F.-H.W., S.S., found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. C.E.J., P.Y., P.J.M., C.J.T., C.A.Z., and R.M. performed research; E.W.L., T.A.T., S.J.M., P.Z., The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal Y.A., J.K., C.E.J., and S.F.T. analyzed data; and E.W.L., T.A.T., P.Z., E.F.R.P., S.M.T., E.X.A., and T.D.G. wrote the paper. extracellular concentrations of ∼8 μM, which were well below con- Conflict of interest statement: T.D.G. has received research funding from Janssen, Roche, and centrations required to inhibit synaptic and extrasynaptic NMDARs Allergan Pharmaceuticals, and served as a consultant for FSV7 LLC during the preceding in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less 3 years. The authors declare competing financial interests: T.D.G., P.Z., R.M., P.J.M., C.J.T., potent than ketamine at inhibiting NMDARs. These data demon- and C.A.Z. are listed as co-inventors on a patent application for the use of (2R,6R)-hydroxy- strate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)- norketamine and (2S,6S)-hydroxynorketamine in the treatment of depression, anxiety, anhe- donia, suicidal ideation, and post-traumatic stress disorders. C.A.Z. and R.M. are listed as co- HNK and support the conclusion that direct NMDAR inhibition does inventors on a patent for the use of (2R,6R)-hydroxynorketamine, (S)-dehydronorketamine, not contribute to antidepressant-relevant effects of (2R,6R)-HNK. and other stereoisomeric dehydro- and hydroxylated metabolites of ketamine metabolites in the treatment of depression and neuropathic pain. R.M., P.J.M., C.J.T., and C.A.Z. have as- depression | ketamine | hydroxynorketamine | antidepressant | signed patent rights to the US Government but will share a percentage of any royalties that may be received by the Government. P.Z. and T.D.G. have assigned their patent rights to the NMDA receptor University of Maryland, Baltimore, but will share a percentage of any royalties that may be received by the University of Maryland, Baltimore. S.F.T. received research support from ajor depressive disorder (MDD) occurs in about 16% of Janssen, is a consultant for Janssen, is a member of the Scientific Advisory Board for Sage Therapeutics, is a co-founder of NeurOp, Inc., and co-inventor on Emory-owned IP. S.J.M. owns Mthe population over the course of a lifetime (1). It is esti- stock in NeurOp, Inc., which is developing NMDAR inhibitors for use in treating neurological mated that MDD affected nearly 7% of all US adults in 2016, disease and disorders. All other authors declare no competing interests. and that one-half of those individuals were prescribed typical This article is a PNAS Direct Submission. antidepressant medications as part of their treatment regimen Published under the PNAS license. (2). Although such typical antidepressants, including selective 1E.W.L. and T.A.T. contributed equally to this work. serotonin and norepinephrine reuptake inhibitors, tricyclic an- 2S.F.T., E.F.R.P., S.M.T., E.X.A., and T.D.G. contributed equally to this work. tidepressants, and monoamine oxidase inhibitors, can sometimes 3To whom correspondence should be addressed. Email: [email protected]. mitigate clinical symptoms of MDD, the onset of action of these This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. drugs is very slow, requiring daily administration over weeks or 1073/pnas.1816071116/-/DCSupplemental. months for clinical improvement (3). In addition, ∼30% of pa- www.pnas.org/cgi/doi/10.1073/pnas.1816071116 PNAS Latest Articles | 1of10 Downloaded by guest on September 30, 2021 tients suffering from MDD fail to respond after attempting (14). Nevertheless, reports that at high concentrations (2R,6R)-HNK multiple treatments (3). The use of (R,S)-ketamine (ketamine) inhibits NMDAR function led to the suggestion that NMDAR in- for the treatment of MDD has generated much excitement be- hibition accounts for (2R,6R)-HNK’s antidepressant-relevant effects cause it reduces, and in some patients eliminates, many core (31, 32). symptoms of depression, including depressed mood, anhedonia, The present study systematically assessed the effects of (2R,6R)- and suicidal ideation, within hours following i.v. administration of HNK on NMDAR function. Tests exploring behavioral despair and ’ a single subanesthetic dose. Furthermore, ketamine is effective in hyponeophagia in mice were employed to confirm the metabolite s – antidepressant-relevant effects. Analytical assays were used to quantify patients who are refractory to typical antidepressants (4 9). R R Although ketamine is a promising alternative to standard clinically plasma, whole-brain, and extracellular hippocampal levels of (2 ,6 )- used antidepressants, it induces adverse effects at antidepressant doses, HNK following systemic treatment of mice with a dose that produces particularly dissociation (10). Furthermore, ketamine, a derivative of the antidepressant-relevant effects. A series of functional tests including in illicit drug phencyclidine, is widely abused (11). Ketamine is rapidly and vivo NMDA-induced lethality and ex vivo electrophysiological mea- stereoselectively metabolized in the liver to a number of metabolites, surements of NMDAR activity in hippocampal neurons and in oocytes including the norketamines, hydroxyketamines, dehydronorketamines, expressing distinct NMDAR subtypes (GluN1/GluN2A, GluN2B, and the hydroxynorketamines (HNKs) (12). Demethylation of the GluN2C, or GluN2D) were used to determine the potency for R R methyl amine on ketamine’s central cyclohexyl ring generates the nor- (2 ,6 )-HNK to inhibit NMDARs.