Article Figures & Data
Figures
- Fig. 1.
Metabolic formation of hydroxynorketamines from ketamine. (R,S)-ketamine (KET) is N-demethylated to form (R,S)-norketamine (norKET), which is then further metabolized to form the HNKs and dehydronorketamine (DHNK). Via this pathway, (R,S)-norKET is hydroxylated to form the HNKs as shown [see Portmann et al. (2010); Desta et al. (2012)]. Ketamine additionally undergoes direct hydroxylation to form the 6-hydroxyketamines (HKs), which are then N-demethylated to form the (2,6)-HNKs (Portmann et al., 2010; Desta et al., 2012).
- Fig. 2.
Putative synaptic mechanisms of (2R,6R)- and (2S,6S)-hydroxynorketamine. (2R,6R)- HNK acts on the presynaptic terminal to increase glutamate release, possibly via signaling mechanisms convergent with mGlu2, whereby (2R,6R)-HNK disinhibits the mGlu2-induced cAMP release, or via another glutamate release mechanism. Subsequent to enhanced glutamate release, AMPAR activation leads to enhanced BDNF release, TrkB activation, and subsequent activation of plasticity-relevant signal cascades, including an increase in protein kinase B (AKT), extracellular signal–related kinases (ERK)/mitogen-activated proteain kinases (MAPK), and mTORC1 pathway activity. These signaling cascades result in protein synthesis, including increased AMPAR expression, and synaptogenesis, ultimately promoting enhanced synaptic strength. (2R,6R)-HNK may also disrupt TrkB/AP-2 interactions, thereby inhibiting TrkB endocytosis and enhancing TrkB stability at the synapse. Additionally, (2S,6S)-HNK has moderate affinity to inhibit NMDARs and may act to increase intracellular signal cascades via an NMDAR inhibition–dependent pathway, including inhibition of eEF2 signaling in addition to increased AKT, ERK/MAPK, and mTORC1 signaling.
Tables
Species Experimental Details Concentration After ketamine dosing Humans Estimated peak unbound brain concentration after a typical antidepressant treatment (0.5 mg/kg, i.v., over 40 min) ≤37.8 ± 14.3 nMa,b,c Mice Peak total (bound and unbound) brain concentrations after a dose (10 mg/kg, i.p.) frequently reported to induce relevant behavioral actions 1.54–2.46 µmol/kgb,d,e After (2R,6R)-HNK dosing Mice Peak total concentrations after a dose (10 mg/kg, i.p.) frequently reported to induce relevant behavioral actions measured in: a) Whole brain 10.66–17.80 µmol/kgd,f,g,h b) CSF 18.40 µMg c) Extracellular hippocampal space 7.57 ± 2.13 µMh ↵a Peak unbound brain concentrations were estimated based upon peak plasma levels 157 ± 59.2 nM.
↵b Concentrations correspond to racemic mixture of (2R,6R;2S,6S)-HNK.
↵c Shaffer et al. (2019).
↵d Zanos et al. (2016).
↵e Zanos et al. (2019a).
↵f Pham et al. (2018).
↵g Yamaguchi et al. (2018).
↵h Lumsden et al. (2019).
- TABLE 2
NMDAR ligand binding and functional inhibition
Ketamine Ki and IC50 values are only reported from studies that compared the effects of ketamine to HNKs under similar experimental conditions.
Compound Affinity/Potency References NMDAR binding affinity (MK-801 displacement) (R,S)-ketamine Ki = 0.25–1.06 µM Moaddel et al. (2013); Morris et al. (2017) (2S,6S)-HNK Ki = 7.34–21.19 µM (2R,6R)-HNK Ki > 100 µM (2R,5R)-HNK Ki > 100 µM (2S,5S)-HNK Ki > 100 µM (2R,5S)-HNK Ki > 100 µM (2S,5R)-HNK Ki > 100 µM (2R,4R)-HNK Ki > 100 µM (2S,4S)-HNK Ki > 100 µM (2R,4S)-HNK Ki > 100 µM (2S,4R)-HNK Ki > 100 µM (2R,6R)-HNK Ki > 10 µM Zanos et al. (2016) (2S,6S)-HNK Ki > 10 µM Inhibition of NMDAR-mediated current amplitude, X. laevis oocytes GluN subunits 1A/2A 1A/2B 1A/2C 1A/2D (R,S)-ketamine IC50 = 3.3 µM IC50 = 0.9 µM IC50 = 1.7 µM IC50 = 2.4 µM Dravid et al. (2007); Lumsden et al. (2019) (2R,6R)-HNK IC50 = 498 µM IC50 = 258 µM IC50 = 202 µM IC50 = 287 µM (2S,6S)-HNK IC50 = 43 µM IC50 = 21 µM IC50 = 15 µM 13 µM Inhibition of NMDAR-mediated current amplitude, HEK-293 cells GluN subunits 1A/2A 1A/2B (R,S)-ketamine IC50 = 0.8 µM at pH 6.8 ND Abbott and Popescu (2020) IC50 = 0.5 µM at pH 7.2 (2R,6R)-HNK IC50 = 46 µM at pH 6.8 IC50 = 39 µM at pH 6.8 IC50 = 46 µM at pH 7.2 IC50 = 69 µM at pH 7.2 Inhibition of NMDAR-mediated fEPSP slope, SC-CA1 mouse hippocampal slices (R,S)-ketamine IC50 = 4.5 µM Lumsden et al. (2019) (2R,6R)-HNK IC50 = 211.9 µM (2S,6S)-HNK IC50 = 47.2 µM Inhibition of NMDAR-mediated mEPSC amplitude, CA1 neurons in rat hippocampal slices (R,S)-ketamine IC50 = 6.4 µM Lumsden et al. (2019) (2R,6R)-HNK IC50 = 63.7 µM Inhibition of NMDAR-mediated mEPSCs, primary mouse hippocampal neurons (R,S)-ketamine 50 µM (∼90% inhibition) Suzuki et al. (2017) (2R,6R)-HNK 50 µM (∼40% inhibition) 10 µM (no inhibition) Inhibition of NMDA-induced whole-cell current charge (R,S)-ketamine IC50 = 45.9 µM Lumsden et al. (2019) (2R,6R)-HNK IC50 > 1000 µM (2S,6S)-HNK IC50 > 1000 µM Attenuation of NMDA-induced lethality in mice (R,S)-ketamine ED50 = 6.4 mg/kg, i.p. Lumsden et al. (2019) (2R,6R)-HNK ED50 = 227.8 mg/kg, i.p. (2S,6S)-HNK ED50 = 18.6 mg/kg, i.p. ND, not determined; SC-CA1, Schaffer collateral-CA1 hippocampal synapse.
- TABLE 3
Effects on synaptic glutamatergic transmission
Contained in this table are synaptic effects other than direct effects on NMDAR function, which are shown in Table 2. Studies and experiments that did not identify an effect are not summarized in this table; please refer to the text for these details.
Concentration/Dose Timing Effect System References In vitro or ex vivo application (2R,6R)-HNK 0.3–30 µM (EC50 = 3.3 µM) 1-h exposure Increased fEPSP slopea SC-CA1, rat hippocampal slices Riggs et al. (2019) 10 µM 1-h exposure Zanos et al. (2016) 0.3–30 µM (IC50 = 3.8 µM) 1-h exposure Decreased paired-pulse ratio Riggs et al. (2019) 10 µM 20-min exposure Increased sEPSC frequency and amplitude Rat CA1 stratum radiatum interneurons Zanos et al. (2016) 10 µM 15- to 25-min exposure Increased mEPSC frequency (no change in amplitude) Rat CA1 pyramidal neurons Riggs et al. (2019) 10 µM 15-min exposure Increased mEPSC frequency and amplitude Rat ventrolateral periaqueductal gray neurons Chou et al. (2018); Ye et al. (2019) 10 µM 1- to 17-min exposure No change in AMPA-evoked currents Rat primary cortical neurons Shaffer et al. (2019) 10 µM 20-min exposure No change in sEPSC frequency or amplitude Mouse CA1 interneurons 3–30 µM (EC50 = 7.8 µM) 2-h exposure No change in baseline fEPSP Slope SC-CA1, hippocampal slices from 14-day-old mouse Kang et al. (2020) Decreased LTP (2S,6S)-HNK 10 µM 20-min exposure No change in sEPSC frequency or amplitude Rat CA1 interneurons Zanos et al. (2016) 10 µM 1- to 17-min exposure No change in AMPA-evoked currents Rat primary cortical neurons Shaffer et al. (2019) 1–10 µM (EC50 = 1.0 µM) 2-h exposure No change in baseline fEPSP Slope SC-CA1, hippocampal slices from 14-day-old mouse Kang et al. (2020) Decreased LTP Racemic (2R,6R;2S,6S)-HNK 20 µM 30-min exposure No change in fEPSP slope SC-CA1, rat hippocampal slices Michaelsson et al. (2019) In vivo administration 10 mg/kg, i.p. 24 h post-treatment Increased glutamate release Mouse prefrontal cortex, microdialysis Pham et al. (2018) 1 nmol/side bilateral intracortical infusion 24 h post-treatment 0.075 mg/kg, i.p. 1 wk post-treatment Attenuation of AMPAR-mediated EPSC bursts Mouse hippocampal pyramidal neurons Chen et al. (2020) 5 mg/kg, i.p. 3.5 h or 1 day post-treatment Increased magnitude of and slowed decay of LTP SC-CA1, Wistar-Kyoto stress-susceptible rats, in vivo electrophysiology Aleksandrova et al. (2020) 10 mg/kg, i.p. 24 h post-treatment Reversed stress-induced increases in paired-pulse ratios Rat ventrolateral periaqueductal gray neurons Chou et al. (2018) Reversed stress-induced decreased in inward AMPAR-mediated currents Reversed stress-induced decreases in AMPA-evoked currents Reversed stress-induced decreases in mEPSC frequency and amplitude 10 mg/kg, i.p. 24 h post-treatment Increased mEPSC frequency and amplitude Rat ventrolateral periaqueductal gray neurons Ye et al. (2019) Decreased paired-pulse ratio Increased stimulus response relationship Increased AMPA-evoked currents 10 mg/kg, i.p. 24 h post-treatment Decreased mEPSC frequency and amplitude Mouse dopaminergic ventral tegmental neurons Yao et al. (2018) Decreased AMPAR:NDMAR ratio No change in firing frequency 10 mg/kg, i.p. 24 h post-treatment Decreased LTP Mouse nucleus accumbens core Yao et al. (2018) No change in stimulus response relation or paired-pulse ratio 30 mg/kg, i.p. 24 h post-treatment Increased serotonin and hypocretin-evoked sEPSC frequency and amplitude Mouse prefrontal cortex neurons Fukumoto et al. (2019) SC-CA1, Schaffer collateral-CA1 hippocampal synapse; sEPSC, spontaneous excitatory postsynaptic current.
↵a Shaffer et al. (2019) reported no change in SC-CA1 fEPSP slope after a 1-h exposure to 10 µM (2R,6R)-HNK.
- TABLE 4
Biochemical effects implicating glutamatergic neurotransmission
Studies and experiments that did not identify an effect are not summarized in this table; please refer to the text for these details.
Compound Concentration/Dose Timing Effect System References BDNF (2R,6R)-HNK 10 mg/kg, i.p. 24 h post-treatment Increased BDNF protein levels Mouse hippocampus Zanos et al. (2016) 10 mg/kg, i.p. 30 min post-treatment Increased BDNF protein levels Mouse hippocampus Lumsden et al. (2019) 10 and 50 nM 1-h exposure Increased BDNF release Rat primary neurons Fukumoto et al. (2019) 10–100 µM 15-min exposure Decreased TrkB/AP-2 interactions MG87:TRKB cells Fred et al. (2019) (2S,6S)-HNK 10 mg/kg, i.p. 30–60 min post-treatment Increased extracellular BDNF levels Mouse prefrontal cortex Anderzhanova et al. (2020) cAMP (2R,6R)-HNK 10 µM 15-min exposure Increased cAMP accumulation C6 cells Wray et al. (2019) eEF2 (2R,6R)-HNK 10 mg/kg, i.p. 1 h post-treatment Decreased eEF2 phosphorylation Mouse hippocampus Zanos et al. (2016) 24 h post-treatment 50 µM 30-min exposure Decreased eEF2 phosphorylation Mouse primary neurons Suzuki et al. (2017) mTOR and downstream pathways (2R,6R)-HNK 10 mg/kg, i.p. 30 min post-treatment Increased p-mTOR Mouse hippocampus Lumsden et al. (2019) 30 mg/kg, i.p. 30 min post-treatment Increased p-mTOR Mouse prefrontal cortex Fukumoto et al. (2019) 1–50 nM 1-h exposure Increased pERK Rat primary neurons Fukumoto et al. (2019) (2S,6S)-HNK 20 mg/kg, i.v. 20 min post-treatment Increased p-mTOR Rat prefrontal cortex Paul et al. (2014) 0.01–1 nM 1-h exposure PC-12 cells 20 mg/kg, i.v. 20–60 min post-treatment Increased p4E-BP1 Rat prefrontal cortex Paul et al. (2014) Increased pp70S6K 0.5 nM 1-h exposure Increased p4E-BP1 PC-12 cells Paul et al. (2014) 0.5–1 nM 1-h exposure Increased pERK 0.1–1 nM 1-h exposure Increased pAkt pAkt, phosphorylated protein kinase B; p-MTOR, phosphorylated mTOR; pp70S6K, phosphorylated p70S6 kinase; p4E-BP1, phosphorylated eukaryotic initiation factor 4E-binding protein 1.
- TABLE 5
Changes in AMPAR expression
Studies and experiments that did not identify an effect are not summarized in this table; please refer to the text for these details.
Compound Effective Dose/Concentration Timing Effect System References (2R,6R)-HNK 10 mg/kg, i.p. 24 h post-treatment Increased GluA1 protein expression Mouse hippocampus Zanos et al. (2016) Increased GluA2 protein expression 1–10 µM 90-min exposure Increased GluA1 surface protein expression Rat primary neurons Shaffer et al., (2019) 0.1–10 µM 180-min exposure 200–400 nM 24-h exposure Increased GluA1 mRNA expression U251-MG human glioblastoma cells Ho et al. (2018) 400 nM Increased GluA2 mRNA expression 400 nM Increased GluA4 mRNA expression 400 nM 24-h exposure Increased GluA4 mRNA expression Human iPSC-derived astrocytes Ho et al. (2018) (2S,6S)-HNK 200–400 nM 24-h exposure Increased GluA1 mRNA expression U251-MG human glioblastoma cells Ho et al. (2018) 400 nM Increased GluA2 mRNA expression 400 nM Increased GluA4 mRNA expression 400 nM 24-h exposure Increased GluA1 mRNA expression Human iPSC-derived astrocytes Ho et al. (2018) 400 nM Increased GluA2 mRNA expression 400 nM Increased GluA4 mRNA expression - TABLE 6
Effects of (2R,6R)-HNK on cellular morphology
Subeffective or ineffective concentrations or doses are not included in this summary table. Please refer to the text for full details.
Concentration Timing Effect System References 0.5 µM 3 days after 1- to 6-h drug exposure Increased dendrite length Mouse primary mesencephalic dopaminergic neurons Cavalleri et al. (2018) Increased dendrite number Increased soma area 0.5 µM 3 days after 1- to 6-h drug exposure Increased dendrite length Human iPSC-derived dopaminergic neurons Cavalleri et al. (2018); Collo et al., (2018) Increased dendrite number Increased size of dopaminergic bodies Behavioral Test Outcome Predictive of Antidepressant Effectiveness Forced swim Decreased immobility time (increased swimming) Learned helplessness Decreased escape failures/reversal of escape deficits Novelty-suppressed feeding Reduced latency to eat Sucrose preference Reversal of sucrose consumption preference deficits, or increased preference (vs. water) Female urine sniffing preference Reversal of preference deficits, or increased preference (vs. water or male urine) Social preference Reversal of social preference deficits - TABLE 8
Preclinical behavioral effects
Subeffective doses and studies that did not identify an effect are not summarized in this table; please refer to the text (section IV.A.1.b. Direct antidepressant-relevant effects of hydroxynorketamines) for these details.
Behavior or Test Time Post-Treatment Effective Doses Species References Antidepressant-relevant effects (2R,6R)-HNK Forced swim test 1 h 5–125 mg/kg, i.p.; 15–150 mg/kg, by mouth Mouse Zanos et al. (2016, 2019b); Highland et al. (2019); Lumsden et al., (2019); Aguilar-Valles et al. (2020) 3.62–36.2 pmol/side, intra-vlPAG Rat Chou et al. (2018) 24 h 5–125 mg/kg, i.p.; 15–150 mg/kg, by mouth; 0.036–1 nmol/side, intra-PFC Mouse Zanos et al. (2016, 2019b); Highland et al. (2019); Pham et al. (2018); Fukumoto et al. (2019); Lumsden et al. (2019); Chen et al. (2020) 10 mg/kg, i.p.; 3.62 pmol/side, intra-vlPAG Rat Chou et al. (2018) 3 days 10 mg/kg, i.p. Mouse Zanos et al. (2016) 10 mg/kg, i.p.; 3.62 pmol/side, intra-vlPAG Rat Chou et al. (2018) 5 days 30 mg/kg, i.p. Mouse Fukumoto et al. (2019) 7 days 10 mg/kg, i.p.; 3.62 pmol/side, intra-vlPAG Rat Chou et al. (2018) 14 days 0.025–0.075 mg/kg, i.p. Mouse Chen et al., (2020) 21 days 10 mg/kg, i.p.; 3.62 pmol/side, intra-vlPAG Rat Chou et al., (2018) Learned helplessness test 24 h 5–75 mg/kg, i.p.; 50–150 mg/kg, by mouth; 3–10 nmol/side, i.c.v. Mouse Zanos et al. (2016); Highland et al. (2019); Zanos et al. (2019a) 5 days 10 nmol/side, i.c.v. Mouse Zanos et al. (2019a) Adolescent exposure to live predator prior to learned helplessness test 24 h 20 mg/kg, i.p. Mouse Elmer et al. (2020) Novelty-suppressed feeding test 30 min 10 mg/kg, i.p. Mouse Lumsden et al. (2019) 1 h 10–20 mg/kg, i.p. Mouse Zanos et al. (2016); Aguilar-Valles et al. (2020) 3 days 30 mg/kg, i.p.; 3.62 pmol/side, intra-PFC Mouse Fukumoto et al. (2019) Chronic CORT–induced sucrose preference deficits 24 h 10 mg/kg, i.p. Mouse Zanos et al. (2016) Chronic CORT–induced female urine preference deficits 24 h 10 mg/kg, i.p. Mouse Zanos et al. (2016) Female urine preference 24 h 30 mg/kg, i.p. Mouse Fukumoto et al. (2019) CSDS-induced sucrose preference deficits 24 h 10 mg/kg, i.p. Mouse Zanos et al. (2019b) CSDS-induced social interaction deficits 24 h 20 mg/kg, i.p. Mouse Zanos et al. (2016) Footshock stress–induced sucrose preference 1 h 10 mg/kg, i.p.; 3.62 pmol/side, intra-vlPAG Rat Chou et al. (2018) 24 h 3 days 7 days 21 days 2 days 3–30 nmol/side, i.c.v. Mouse Zanos et al. (2019a) IBD-induced sucrose preference deficits 1 h 1 pg/side, intra-vlPAG Mouse Ko et al. (2020) Tail suspension test (in IBD model) 1 h 1 pg/side, intra-vlPAG Mouse Ko et al. (2020) (2S,6S)-HNK Forced swim test 1 h 25 mg/kg, i.p. Mouse Zanos et al. (2016) 24 h 25 mg/kg, i.p. Mouse Zanos et al. (2016) Learned helplessness test 24 h 75 mg/kg, i.p. Mouse Zanos et al. (2016) Forced swim test (after chronic CORT) 30 min 20 mg/kg, i.p. Mouse Yokoyama et al. (2020) Chronic CORT–induced sucrose preference deficits 30 min 20 mg/kg, i.p Mouse Yokoyama et al. (2020) Attenuation of learned fear 14 days 0.025–30 mg/kg, i.p. Mousea Chen et al. (2020) Analgesic effects (2R,6R)-HNK Elevated von Frey threshold 24 h 10 mg/kg, i.p. Mouse Kroin et al. (2019) Reduced mechanical allodynia in CRPS1 model 0–4 days 10 mg/kg/day × 10 3 days, i.p. Mouse Kroin et al. (2019) Reduced mechanical allodynia in postoperative pain model 0–5 days 10 mg/kg/day × 10 3 days, i.p. Mouse Kroin et al. (2019) Aggression and social dominance (2R,6R)-HNK Enhanced aggression 1 h 10 mg/kg, i.p.; 1–10 pg/side, intra-vlPAG Rat Chou (2020) 24 h 10 mg/kg, i.p.; 1–10 pg/side, intra-vlPAG Rat Ye et al. (2019); Chou (2020) 3 days 10 mg/kg, i.p.; 10 pg/side, intra-vlPAG Rat Ye et al. (2019) 7 days 14 days 21 days 28 days Enhanced social dominance 24 h 10 mg/kg, i.p. Rat Ye et al. (2019) CORT, corticosterone; CSDS, chronic social defeat stress; IBD, inflammatory bowel disease; vlPAG, ventrolateral periaqueductal gray.
↵a Effect observed only in female, but not male, mice.
