Hydroxynorketamine pharmacokinetics and antidepressant behavioral effects of the (2,6)- and (5R)-methyl-(2R,6R)-hydroxynorketamines

Jaclyn N Highland; Patrick J Morris; Kylie M Konrath; Lace M Riggs; Natalie R Hagen; Panos Zanos; Chris F Powels; Ruin Moaddel; Craig J Thomas; Amy Q Wang; Todd D Gould

doi:10.1021/acschemneuro.1c00761

. Author manuscript; available in PMC: 2023 Feb 16.

Published in final edited form as: ACS Chem Neurosci. 2022 Feb 3;13(4):510–523. doi: 10.1021/acschemneuro.1c00761

Hydroxynorketamine pharmacokinetics and antidepressant behavioral effects of the (2,6)- and (5R)-methyl-(2R,6R)-hydroxynorketamines

Jaclyn N Highland ^a,^b, Patrick J Morris ^c, Kylie M Konrath ^d, Lace M Riggs ^a,^e, Natalie R Hagen ^d, Panos Zanos ^a,^f,^g,^k, Chris F Powels ^a, Ruin Moaddel ^h, Craig J Thomas ^c, Amy Q Wang ^d, Todd D Gould ^a,^f,^i,^j,^*

^aDepartment of Psychiatry, University of Maryland School of Medicine, Baltimore MD 21201, USA.

^bProgram in Toxicology, University of Maryland School of Medicine, Baltimore MD 21201, USA.

^cDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, Intramural Research Program, National Institutes of Health, Rockville MD 20850, USA.

^dTherapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville MD 20850, USA.

^eProgram in Neuroscience, University of Maryland School of Medicine, Baltimore MD 21201, USA.

^fDepartment of Pharmacology, University of Maryland School of Medicine, Baltimore MD 21201, USA.

^gDepartment of Physiology, University of Maryland School of Medicine, Baltimore MD 21201, USA.

^hBiomedical Research Center, National Institute on Aging, Intramural Research Program, National Institutes of Health, Baltimore MD 21224, USA.

ⁱDepartment of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore MD 21201, USA.

^jVeterans Affairs Maryland Health Care System, Baltimore MD 21201, USA.

^kCurrent address: Department of Psychology, University of Cyprus, Nicosia 1678, Cyprus

Correspondence: Todd D. Gould, MD, Department of Psychiatry, University of Maryland School of Medicine, Rm. 936 MSTF, 685 W. Baltimore St., Baltimore, MD 21201, USA, Phone: 410-706-5585, gouldlab@me.com

Author Contributions

JNH and TDG were responsible for the overall experimental design. PJM and CJT were responsible for the synthesis and chemical characterization of the HNKs. JNH and LMR performed the tissue collections for which the bioanalytical assessments were performed by KMK and NRH, and pharmacokinetics analyzed by AQW with support from RM. The behavioral assessments were performed by JNH, PZ, and CFP. JNH conducted the analysis and presentation of data unless otherwise noted. JNH outlined and wrote the first draft of the paper, which was edited by TDG and reviewed by all authors.

PMCID: PMC9926475 NIHMSID: NIHMS1867317 PMID: 35113535

Abstract

(R,S)-ketamine is rapidly metabolized to form a range of metabolites in vivo, including 12 unique hydroxynorketamines (HNKs) that are distinguished by a cyclohexyl ring hydroxylation at the 4, 5, or 6 position. While both (2R,6R)- and (2S,6S)-HNK readily penetrate the brain and exert rapid antidepressant-like actions in preclinical tests following peripheral administration, the pharmacokinetic profiles and pharmacodynamic actions of the ten other HNKs have not been examined. We assessed the pharmacokinetic profiles of all 12 HNKs in the plasma and brains of male and female mice and compared the relative potencies of the four (2,6)-HNKs to induce antidepressant-relevant behavioral effects in the forced swim test in male mice. While all HNKs were readily brain-penetrable following intraperitoneal injection, there were robust differences in peak plasma and brain concentrations and exposures. Forced swim test immobility rank-order of potency, from most to least potent, was (2R,6S)-, (2S,6R)-, (2R,6R)-, and (2S,6S)-HNK. We hypothesized that distinct structure-activity relationships and the resulting potency of each metabolite is linked to the unique substitution patterns and resultant conformation of the 6-membered cyclohexanone ring system. To explore this, we synthesized (5R)-methyl-(2R,6R)-HNK, which incorporates a methyl substitution on the cyclohexanone ring. (5R)-methyl-(2R,6R)-HNK exhibited similar antidepressant-like potency to (2R,6S)-HNK. These results suggest that conformation of the cyclohexanone ring system in the (2,6)-HNKs is an important factor underlying potency and that additional engineering of this structural feature may improve the development of a new generation of HNKs. Such HNKs may represent novel drug candidates for the treatment of depression.

Keywords: pharmacokinetics, antidepressant, hydroxynorketamine, structure-activity relationship, depression

INTRODUCTION

Ketamine undergoes rapid and stereoselective metabolism forming a number of metabolites that include twelve distinct hydroxynorketamines (HNKs; Figure 1A). A total of twelve HNK metabolites (distinguished by the site of cyclohexyl ring hydroxylation at the 4, 5, or 6 position and unique stereochemistry at two stereocenters) are formed from the metabolism of ketamine in vivo ^1-7. Of these, the (2,6)-HNKs are the most prevalent HNK metabolites identified after ketamine administration ^1,2,4,5,8,9.

(A) Structures of the 12 hydroxynorketamines (HNKs) formed *in vivo* following ketamine administration: (*2R,6R*)-, (*2S,6S*)-, (*2R,6S*)-, (*2S,6R*)-, (*2R,5R*)-, (*2S,5S*)-, (*2R,5S*)-, (*2S,5R*)-HNK, (*2R,4R*)-HNK, (*2S,4S*)-, (*2R,4S*)-, and (*2S,4R*)-HNK. (B) Structure of (5R)-methyl (Me)-(*2R,6R*)-HNK.

Ketamine rapidly (often within hours) exerts rapid antidepressant effects in patients suffering from depression following a single administration, which lasts for days and can be sustained with repeated administration ^10-14. There is preclinical evidence to suggest that the (2,6)-HNKs play a critical role in mediating the sustained antidepressant-relevant actions of the parent compound ketamine ^4,15. Additionally, independent of their role in ketamine’s antidepressant actions, a growing number of studies have demonstrated that direct administration of (2R,6R)-HNK and, to a lesser extent, (2S,6S)-HNK rapidly induces antidepressant-relevant effects in rodent studies, including behavioral effects used to predict antidepressant efficacy in a number of rodent tests ^4,9,15-25. When compared under similar experimental conditions, (2R,6R)-HNK has demonstrated greater behavioral potency than (2S,6S)-HNK in rodent assays used to predict antidepressant efficacy⁴. Consistent with this, it has been reported that (2R,6R)-, but not (2S,6S)-HNK, exerts behavioral effects when tested at equivalent doses ^17,26.

It is possible that additional HNK metabolites exert antidepressant-relevant behavioral actions with even greater potency compared to (2R,6R)-HNK or (2S,6S)-HNK. Furthermore, as preclinical studies have shown that (2R,6R)-HNK, but not (2S,6S)-HNK lacks the dissociative properties and abuse potential of ketamine ^4,9,27, identifying the structural characteristics that confer enhanced antidepressant-like effects may inform the development of novel drug candidates that not only demonstrate enhanced efficacy, but also have an improved safety profile. Finally, while earlier studies demonstrated that (2R,6R)- and (2S,6S)-HNK readily penetrate the brain within minutes of systemic administration in rodents ^{4,6,9,15,19,28-30} and have similar elimination profiles ^4,29, little is known about the pharmacokinetic profile of the other 10 HNKs or whether they exert antidepressant-like actions.

Here, we examined the pharmacokinetic profiles of the 12 HNK stereoisomers in the plasma and brains of male and female CD-1 mice. Based upon the previously observed antidepressant-like behavioral effects of (2R,6R)- and (2S,6S)-HNK ^4,9,15-25, we evaluated the antidepressant-like behavioral effects of the four (2,6)-HNKs: (2R,6R), (2S,6S), (2R,6S), and (2S,6R)-HNK in the mouse forced swim test and compared their relative potencies. Finally, we assessed a novel drug candidate, (5R)-methyl-(2R,6R)-HNK, as a means to evaluate the structure activity relationships around the cyclohexanone ring system to potentially manipulate the three-dimensional molecular confirmation in a manner that confers favorable antidepressant-like behavioral potency.

RESULTS AND DISCUSSION

Pharmacokinetic profiles of the 12 HNKs in mouse plasma and brain

Plasma concentrations of HNKs

All HNKs were detected in the plasma of male and female mice between 10 min (earliest time point tested) and 4 h (latest time point tested) after i.p. injection. Peak plasma levels (C_max) for all HNKs were observed in both sexes at the earliest time point, i.e., 10 min post-treatment (Figure 2 and Table 1). Robust differences for effects of sex and compound were observed in both plasma C_max and AUC (Table 1, 2). Approximately 6-fold differences were observed between the dose-normalized plasma C_max of HNKs in both sexes, with the highest levels observed for (2S,6S)-HNK in both sexes, and the lowest observed for (2S,6R)-HNK in males and (2S,5S)-HNK in females (Table 1). Approximately 11- and 12-fold differences were observed in the dose-normalized HNK AUC among male and female mice, respectively (Table 1). Consistent with the trends noted for C_max levels, the highest AUC was observed following (2S,6S)-HNK injection in male and female mice, while the lowest AUC was observed for (2S,6R)-HNK in males and (2S,5S)-HNK in females (Table 1). Plasma levels decreased rapidly after dosing and plasma elimination half-lives ranged from 28-47 min in females and from 27-55 min in males (Table 1).

Figure 2. — Plasma concentrations following intraperitoneal administration of (A) (*2R,6R*)-hydroxynorketamine (HNK; 4.3 mg/kg free base dose), (B) (*2S,6S*)-HNK (4.3 mg/kg free base dose), (C) (*2R,6S*)-HNK (5 mg/kg), (D) (*2S,6R*)-HNK (5 mg/kg), (E) (*2R,5R*)-HNK (5 mg/kg), (F) (*2S,5S*)-HNK (5 mg/kg), (G) (*2R,5S*)-HNK (5 mg/kg), (H) (*2S,5R*)-HNK (5mg/kg), (I) (*2R,5R*)-HNK (5 mg/kg), (J) (*2S,4S*)-HNK (5 mg/kg), (K) (*2R,4S*)-HNK (5 mg/kg), and (L) (*2S,4R*)-HNK (4.3 mg/kg free base dose) to male (M; solid lines) and female (F; dashed lines) mice. Inset area-under-the-curve (AUC) of the plots of concentration vs. time. (M) Dose normalized plasma AUC and (N) dose normalized peak plasma concentrations (C_max) for the 12 HNKs. Data points and error bars represent mean and SEM, respectively, of results obtained from 3-4 mice/group. *p<0.05, **p<0.01, ***p<0.001.

Table 1.

Hydroxynorketamine pharmacokinetics in male and female mice^a.

HNK	Sex	C_max^b (mean ± SEM)		AUC^b (mean ± SEM)		B:P ratio	t_1/2
HNK	Sex	Plasma (ng/ml•mg/kg)	Brain (ng/g•mg/kg)	Plasma (ng/ml•hr•mg/kg)	Brain (ng/g•hr•mg/kg)	B:P ratio	Plasma (min(h))	Brain (min(h))
*(2R,6R)-* ^c	Male	310 ± 26.1	380 ± 19.3	125 ± 8.6	165 ± 13.9	1.3	38 (0.6)	34 (0.6)
*(2R,6R)-* ^c	Female	401 ± 18.3	530 ± 17.2	163 ± 7.9	220 ± 8.8	1.3	36 (0.6)	34 (0.6)
*(2S,6S)-* ^c	Male	663 ± 36.4	712 ± 47.3	485 ± 15.5	462 ± 20.6	1.0	44 (0.7)	41 (0.7)
*(2S,6S)-* ^c	Female	760 ± 17.0	993 ± 27.1	555 ± 24.1	689 ± 30.9	1.2	46 (0.8)	44 (0.7)
*(2R,6S)-*	Male	189 ± 23.0	173 ± 5.4	119 ± 7.9	67.8 ± 2.9	0.6	55 (0.9)	43 (0.7)
*(2R,6S)-*	Female	169 ± 8.2	166 ± 12.3	76.9 ± 4.8	55.7 ± 3.5	0.7	47 (0.8)	23 (0.4)
*(2S,6R)-*	Male	104 ± 23.9	117 ± 30.6	44.9 ± 6.1	53.4 ± 8.0	1.2	45 (0.7)	45 (0.8)
*(2S,6R)-*	Female	142 ± 9.4	177 ± 19.8	61.0 ± 5.2	85.6 ± 8.9	1.4	47 (0.8)	45 (0.8)
*(2R,5R)-*	Male	155 ± 12.6	142 ± 5.6	61.8 ± 5.1	73.9 ± 4.5	1.2	30 (0.5)	27 (0.4)
*(2R,5R)-*	Female	133 ± 7.8	152 ± 8.8	48.2 ± 2.2	69.5 ± 2.8	1.4	31 (0.5)	28 (0.5)
*(2S,5S)-*	Male	133 ± 4.2	138 ± 8.3	47.2 ± 1.8	57.1 ± 2.4	1.2	27 (0.5)	16 (0.3)
*(2S,5S)-*	Female	128 ± 9.1	129 ± 2.8	43.8 ± 2.7	55.0 ± 2.0	1.3	34 (0.6)	17 (0.3)
*(2R,5S)-*	Male	180 ± 10.6	106 ± 6.8	82.2 ± 4.8	70.1 ± 5.1	0.9	28 (0.5)	27 (0.5)
*(2R,5S)-*	Female	209 ± 13.4	154 ± 3.9	113 ± 3.8	115 ± 4.0	1.0	29 (0.5)	27 (0.5)
*(2S,5R)-*	Male	443 ± 20.1	242 ± 9.8	454 ± 15.4	309 ± 14.0	0.7	37 (0.6)	40 (0.7)
*(2S,5R)-*	Female	405 ± 10.6	258 ± 15.4	271 ± 14.4	259 ± 16.2	1.0	28 (0.5)	31 (0.5)
*(2R,4R)-*	Male	367 ± 92.1	431 ± 43.5	369 ± 28.2	496 ± 38.0	1.3	35 (0.6)	38 (0.6)
*(2R,4R)-*	Female	505 ± 19.0	490 ± 26.4	289 ± 11.2	358 ± 16.4	1.2	29 (0.5)	29 (0.5)
*(2S,4S)-*	Male	184 ± 1.8	127 ± 9.5	80.4 ± 3.1	57.8 ± 3.8	0.7	39 (0.7)	34 (0.6)
*(2S,4S)-*	Female	267 ± 8.5	234 ± 10.9	115 ± 4.8	101 ± 4.5	0.9	42 (0.7)	35 (0.6)
*(2R,4S)-*	Male	640 ± 24.7	758 ± 21.4	382 ±24.6	422 ± 34.6	1.1	39 (0.6)	32 (0.5)
*(2R,4S)-*	Female	406 ± 25.9	495 ± 28.7	198 ± 8.0	194 ± 9.6	1.0	44 (0.7)	15 (0.3)
*(2S,4R)-* ^c	Male	112 ± 11.3	160 ± 10.2	67.4 ±3.3	62.6 ± 3.4	0.9	48 (0.5)	23 (0.4)
*(2S,4R)-* ^c	Female	133 ± 8.9	190± 13.7	63.3 ± 3.7	64.3 ± 3.5	1.0	47 (0.4)	21 (0.3)
*(5R)-Me-(2R,6R)-*	Male	565 ± 30.2	324 ± 13.0	544 ± 51.9	191 ± 14.4	0.4	49 (0.8)	48 (0.8)
*(5R)-Me-(2R,6R)-*	Female	427 ± 31.8	352 ± 22.4	371 ± 51.7	149 ± 10.1	0.4	49 (0.8)	41 (0.7)

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In the plasma, T_max was 10 min for all HNKs in both sexes; in the brain, T_max was 10 min for all HNKs in both sexes except for (2R,5S)-HNK (T_max = 10 min in males and 30 min in females) and (2R,4R)-HNK (T_max = 30 min in males and 10 min in females).

For each C_max or AUC, respectively, the dose is adjusted to account for the free base content of the compound.

(2R,6R)-, (2S,6S)-, (2S,4R)-HNK, and (5R)-methyl (Me)-(2R,6R)-HNK were administered as a HCl salt (equivalent to free base dose 4.31 mg/kg for (2R,6R)-, (2S,6S)-, (2S,4R)-HNK and 4.37 mg/kg for (5R)-Me-(2R,6R)-HNK); all other HNKs were administered as the free base (5 mg/kg). Abbreviations: AUC, area under the concentration vs. time curve normalized to free base dose; B:P ratio; brain to plasma AUC ratio; C_max, maximum observed concentration normalized to free base dose; F, female; hr, hours; HNK, hydroxynorketamine; T_max, time of observed maximal concentrations; M, male; t_1/2, terminal half-life.

Table 2.

Sex-dependent differences in concentrations of HNKs.

HNK	Plasma		Brain
HNK	Concentration over time	Total exposure (AUC)	Concentration over time	Total exposure (AUC)
*(2R,6R)-*	F > M at 10, 30 min Two-way ANOVA: effect of sex, F(1,30)=375.4, p=0.0013 effect of time, F(4,30)<0.0001, p<0.0001 interaction, F(4,30)=4.958, p=0.0034 post-hoc comparison, effect of sex at: 10 min, p=0.0004 30 min, p=0.0086 1 hr, p>0.9999 2 hr, p>0.9999 4 hr, p>0.9999	F > M	F > M at 10, 30 min Two-way ANOVA: effect of sex, F(1,30)=35.06, p<0.0001 effect of time, F(4,30)=746.7, p<0.0001 interaction, F(4,30)=17.99, p<0.0001 post-hoc comparison, effect of sex at: 10 min, p<0.0001 30 min, p<0.0001 1 hr, p=0.7458 2 hr, p=0.9984 4 hr, p=0.9984	F > M
*(2S,6S)-*	main effect of sex, F > M Two-way ANOVA: effect of sex, F(1,28)=7.018, p=0.0131 effect of time, F(4,28)=503.6, p<0.0001 interaction, F(4,28)=2.173, p=0.0980	F > M	F > M at 10, 30 min Two-way ANOVA: effect of sex, F(1,29)=41.43, p<0.0001 effect of time, F(4,29)=370.7, p<0.0001 interaction, F(4,29)=9.878, p<0.0001 post-hoc comparison, effect of sex at: 10 min, p<0.0001 30 min, p=0.0005 1 hr, p=0.1921 2 hr, p=0.9494 4 hr, p=0.9494	F > M
*(2R,6S)-*	main effect of sex, M > F Two-way ANOVA: effect of sex, F(1,30)=5.136, p=0.0308 effect of time, F(4,30)=160.4, p<0.0001 interaction, F(4,30)=0.3086, p=0.0308	M > F	NS Two-way ANOVA: effect of sex, F(1,30)=0.7799, p=0.3842 effect of time, F(4,30)=516.9, p<0.0001 interaction, F(4,30)=0.1980, p=0.9375	M > F
*(2S,6R)-*	NS Two-way ANOVA: effect of sex, F(1,29)=2.413, p=0.1312 effect of time, F(4,29)=77.73, p<0.0001 interaction, F(4,29)=2.034, p=0.1157	F > M	main effect of sex, F > M Two-way ANOVA: effect of sex, F(1,30)=5.109, p=0.0312 effect of time, F(4,30)=27.44, p<0.0001 interaction, F(4,30)=1.153, p=0.3512	F > M
*(2R,5R)-*	main effect of sex, M > F Two-way ANOVA: effect of sex, F(1,30)=5.404, p=0.0270 effect of time, F(4,30)=239.4, p<0.0001 interaction, F(4,29)=1.912, p=0.1342	M > F	NS Two-way ANOVA: effect of sex, F(1,30)=0.2341, p=0.6320 effect of time, F(4,30)=359.1, p<0.0001 interaction, F(4,30)=2.149, p=0.0991	NS
*(2S,5S)-*	NS Two-way ANOVA: effect of sex, F(1,30)=0.9101, p=0.3477 effect of time, F(4,30)=516.8, p<0.0001 interaction, F(4,30)=0.2707, p=0.8945	NS	NS Two-way ANOVA: effect of sex, F(1,30)=0.5869, p=0.4496 effect of time, F(4,30)=613.1, p<0.0001 interaction, F(4,30)=0.8868, p=0.4838	NS
*(2R,5S)-*	F > M at 10, 30 min Two-way ANOVA: effect of sex, F(1,30)=19.25, p=0.0001 effect of time, F(4,30)=370.6, p<0.0001 interaction, F(4,30)=9.187, p<0.0001 post-hoc comparison, effect of sex at: 10 min, p=0.0090 30 min, p<0.0001 1 hr, p=0.9781 2 hr, p=0.9907 4 hr, p=0.9907	F > M	F > M at 10, 30 min Two-way ANOVA: effect of sex, F(1,30)=42.93, p<0.0001 effect of time, F(4,30)=229.2, p<0.0001 interaction, F(4,30)=20.08, p<0.0001 post-hoc comparison, effect of sex at: 10 min, p=0.0003 30 min, p<0.0001 1 hr, p=0.9923 2 hr, p=0.9923 4 hr, p=0.9923	F > M
*(2S,5R)-*	M > F at 30, 60 min Two-way ANOVA: effect of sex, F(1,30)=49.49, p<0.0001 effect of time, F(4,30)=377.0, p<0.0001 interaction, F(4,30)=7.273, p=0.0003 post-hoc comparison, effect of sex at: 10 min, p=0.1424 30 min, p<0.0001 1 hr, p<0.0001 2 hr, p=0.2335 4 hr, p=0.7899	M > F	NS Two-way ANOVA: effect of sex, F(1,30)=0.2874, p=0.5959 effect of time, F(4,30)=149.5, p<0.0001 interaction, F(4,30)=2.214, p=0.0913	M > F
*(2R,4R)-*	F > M at 10 min Two-way ANOVA: effect of sex, F(1,30)=0.02068, p=0.8866 effect of time, F(4,30)=67.30, p<0.0001 interaction, F(4,30)=3.643, p=0.0156 post-hoc comparison, effect of sex at: 10 min, p=0.0239 30 min, p=0.1483 1 hr, p=0.9036 2 hr, p=0.9036 4 hr, p=0.9670	M > F	F > M at 10 min Two-way ANOVA: effect of sex, F(1,30)=0.2419, p=0.6264 effect of time, F(4,30)=52.31, p<0.0001 interaction, F(4,30)=4.156, p=0.0085 post-hoc comparison, effect of sex at: 10 min, p=0.0214 30 min, p=0.0835 1 hr, p=0.7408 2 hr, p=0.7408 4 hr, p=0.9480	M > F
*(2S,4S)-*	F > M at 10, 30 min Two-way ANOVA: effect of sex, F(1,30)=75.48, p<0.0001 effect of time, F(4,30)=1196, p<0.0001 interaction, F(4,30)=41.32, p<0.0001 post-hoc comparison, effect of sex at: 10 min, p<0.0001 30 min, p=0.0035 1 hr, p=0.9900 2 hr, p=0.9900 4 hr, p=0.9900	F > M	F > M at 10, 30 min Two-way ANOVA: effect of sex, F(1,30)=66.74, p<0.0001 effect of time, F(4,30)=393.2, p<0.0001 interaction, F(4,30)=34.58, p<0.0001 post-hoc comparison, effect of sex at: 10 min, p<0.0001 30 min, p=0.0016 1 hr, p=0.9785 2 hr, p=0.9891 4 hr, p=0.9891	F > M
*(2R,4S)-*	M > F at 10, 30 min Two-way ANOVA: effect of sex, F(1,30)=79.77, p<0.0001 effect of time, F(4,30)=399.2, p<0.0001 interaction, F(4,30)=18.95, p<0.0001 post-hoc comparison, effect of sex at: 10 min, p<0.0001 30 min, p<0.0001 1 hr, p=0.0531 2 hr, p=0.4818 4 hr, p=0.9671	M > F	M > F at 10, 30, 60 min Two-way ANOVA: effect of sex, F(1,30)=64.71, p<0.0001 effect of time, F(4,30)=356.8, p<0.0001 interaction, F(4,30)=13.86, p<0.0001 post-hoc comparison, effect of sex at: 10 min, p<0.0001 30 min, p=0.0004 1 hr, p=0.0325 2 hr, p=0.4716 4 hr, p=0.9214	M > F
*(2S,4R)-*	F > M at 10 min Two-way ANOVA: effect of sex, F(1,30)=0.3430, p=0.5625 effect of time, F(4,30)=227.3, p<0.0001 interaction, F(4,30)=2.883, p=0.0393 post-hoc comparison, effect of sex at: 10 min, p=0.0152 30 min, p=0.8735 1 hr, p=0.7932 2 hr, p=0.9813 4 hr, p=0.9813	NS	F > M at 10 min Two-way ANOVA: effect of sex, F(1,29)=1.203, p=0.2811 effect of time, F(4,29)=352.0, p<0.0001 interaction, F(4,29)=3.218, p=0.0265 post-hoc comparison, effect of sex at: 10 min, p=0.0044 30 min, p=0.9715 1 hr, p=0.9611 2 hr, p=0.9829 4 hr, p>0.9999	NS
(5R)-Me-(2R,6R)-	main effect of sex, M > F Two-way ANOVA: effect of sex, F(1,30)=4.726, p=0.0377 effect of time, F(4,30)=36.20, p<0.0001 interaction, F(4,30)=1.424, p=0.2502	M > F	NS Two-way ANOVA: effect of sex, F(1,30)=0.3025, p=0.5864 effect of time, F(4,30)=196.1, p<0.0001 interaction, F(4,30)=1.337, p=0.2790	M > F

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Note: When a significant interaction (effect of sex x time) was detected in the two-way ANOVA, Holm- Šidák post-hoc comparison test was performed to compare the effect of sex at each time point. AUCs were compared using a standard t-test. Abbreviations: AUC, area under the concentration vs. time curve; F, female; HNK, hydroxynorketamine; M, male, NS, not statistically different. Abbreviations: AUC, area under the curve; F, female; HNK, hydroxynorketamine; M, male; NS, no statistical difference.

For most HNKs, C_max and AUC were sex-dependent (Figure 2 and Table 2). Female mice had higher C_max and AUC of (2R,6R)-HNK (Figure 2A and Table 2), (2S,6S)-HNK (Figure 2B and Table 2), (2R,5S)-HNK (Figure 2G and Table 2), and (2S,4S)-HNK (Figure 2J and Table 2), compared to males that received the same compound at the same dose. Following administration of (2S,6R)-HNK, AUCs were greater in females compared to males, although differences in plasma concentrations at fixed time points did not reach statistical significance (Figure 2D; see Table 2 for statistical results). Following administration of (2S,4R)-HNK, plasma concentrations were greater in females than males at 10 min post-injection, but total exposures were not different between the sexes (Figure 2L; see Table 2 for statistical results). While plasma concentrations were higher during the first 10 min following (2R,4R)-HNK treatment in females than males, AUC were greater in males (Figure 2I and Table 2). Compared to females, male mice had higher C_max and AUC of (2R,6S)-HNK (Figure 2C), (2R,5R)-HNK (Figure 2E and Table 2), (2S,5R)-HNK (Figure 2H and Table 2), and (2R,4S)-HNK (Figure 2K and Table 2). No sex-dependent differences in C_max and AUC were detected following treatment with (2S,5S)-HNK (Figure 2F and Table 2).

Brain concentrations of HNKs

All 12 HNKs readily penetrated the brains of mice, where they were detected at the earliest sampling timepoint (10 min post-injection; Figure 3). Peak brain levels were at 10 min timepoint for all HNKs, with the exception of (2R,5S)-HNK in females and (2S,5R)-HNK, (2R,4R)-HNK in males, where peak levels were observed at 30 min post-injection (Figure 3). Similar to the plasma, robust differences as a function of sex and compound were observed in the C_max and AUC (see Table 1, 2). Namely, approximately 7- and 8-fold differences were observed in the dose-normalized peak brain concentrations in male and female mice, respectively (Table 1). The highest brain levels (normalized to dose) were observed following (2S,6S)-HNK dosing in females and (2R,4S)-HNK in males, while the lowest were observed following (2R,4S)-HNK in males and (2S,5S)-HNK in females (Table 1). Approximately 9- and 12-fold differences were observed in the dose-normalized AUC of HNKs among male and female mice, respectively (Table 1). Males and females exhibited the highest AUC of (2R,4R)-HNK and (2S,6S)-HNK, respectively, and the lowest AUC of (2S,6R)-HNK and (2S,5S)-HNK, respectively. The AUC ratios of brain to plasma AUC of HNKs ranged between 0.6-1.3 for males and 0.7-1.4 for females (Table 1). Brain levels decreased rapidly, with elimination half-lives ranging from 15-45 min in females and 16-45 min in males (Table 1). Most HNKs could be detected in the brain up to 4 h post-treatment, with the following exceptions: (2S,5S)- and (2S,4R)-HNK were below LLOQs in both sexes, while (2R,6S)- and (2R,4S)-HNK were below LLOQs in females, but not males, at 4 h post-treatment.

Peak concentrations and AUCs of HNKs in the brain were also sex dependent (Figure 3 and summarized in Table 2). Female mice had greater C_max and AUC of (2R,6R)-HNK (Figure 3A and Table 2), (2S,6S)-HNK (Figure 3B and Table 2), (2S,6R)-HNK (Figure 3D and Table 2), (2R,5S)-HNK (Figure 3G and Table 2), and (2S,4S)-HNK (Figure 3J and Table 2), compared to males. Although (2S,4R)-HNK resulted in higher brain concentrations in female mice during the first 10 min post-administration, no differences were observed in its AUC in between male and female mice (Figure 3L and Table 2). Consistent with its plasma profile (Figure 2I and Table 2), (2R,4R)-HNK dosing resulted in higher brain levels in female than male mice during the first 10 min post-treatment, but AUC were higher in male mice (Figure 3I and Table 2). By contrast, male mice had greater C_max and AUC of (2R,4S)-HNK than females (Figure 3K and Table 2). Following systemic administration, AUC of (2R,6S)-HNK (Figure 3C and Table 2) and (2S,5R)-HNK (Figure 3H and Table 2) were greater in male mice relative to females, although no statistically significant sex-dependent differences in brain concentrations over time were observed. No sex-dependent differences were observed in C_max or AUC of (2R,5R)- and (2S,5S)-HNK (Figure 3E and F, respectively, and Table 2).

Effects of the (2,6)-HNKs on forced swim test immobility time

Based upon previous studies demonstrating that (2R,6R)- and (2S,6S)-HNK exert antidepressant-relevant behavioral effects in mice with differing potencies^4,9,15-25 we focused our investigation on the behavioral antidepressant actions of the (2,6)-HNKs. The effects of (2R,6R)-HNK (Figure 4A), (2S,6S)-HNK (Figure 4B), (2R,6S)-HNK (Figure 4C), and (2S,6R)-HNK (Figure 4D) were evaluated in male mice in the forced swim test, 24 h after treatment.

Relative to vehicle, (2R,6R)-HNK reduced immobility time at 10, 30, and 100 mg/kg (Figure 4A), whereas (2S,6S)-HNK reduced immobility at 30 and 100 mg/kg (Figure 4B), consistent with a previous report of their relative potencies in increasing escape-directed behavior ⁴. For each subsequent experiment, 30 mg (2R,6R)-HNK served as an internal experimental control and each HNK was also compared to an internal vehicle control. (2R,6S)-HNK (Figure 4C) and (2S,6R)-HNK (Figure 4D) reduced immobility time at the doses of 1, 3, and 10 mg/kg and at 3 and 10 mg/kg, respectively. This demonstrates, for the first time, that (2R,6S)- and (2S,6R)-HNK are capable of exerting antidepressant-relevant behavioral effects, both with enhanced potency compared to (2R,6R)-HNK. Of note, both (2R,6S)- and (2S,6R)-HNK exhibited U-shaped dose response curves, reducing immobility time at doses ≤10 mg/kg, but not at doses ≥30 mg/kg (Figure 4C-D), in contrast with (2R,6R)- and (2S,6S)-HNK, which did not demonstrate U-shaped dose responses within the tested range (up to 100 mg/kg; Figure 4A-B).

Pharmacokinetic profile of (5R)-methyl-(2R,6R)-HNK in mouse plasma and brain

Based upon the finding that (2R,6S)-HNK reduced forced swim test immobility with greater potency compared to (2R,6R)-HNK (Figure 4), we hypothesized that differences in the equilibria between the three-dimensional conformation of these compounds (Figure 5A) likely contributes to their behavioral potencies.

Figure 5. — (A) Three-dimensional structures of (*2R,6R*)-hydroxynorketamine (HNK), (*2R,6S*)-HNK, and (5R)-methyl (Me)-(*2R,6R*)-HNK. (B) Plasma and (C) brain concentrations of (5R)-Me-(*2R,6R*)-HNK following intraperitoneal administration (5 mg/kg HCl salt; equivalent to 4.37 mg/kg free base dose). Inset area-under-the-curve (AUC) of the plots of concentration vs. time. (D) (5R)-Me-(*2R,6R*)-HNK (dosed as HCl salt) reduces immobility time in male mice in the forced swim test 24 hours after treatment, with minimal effective dose of 1 mg/kg (i.p.). Data are the mean ± SEM. n=13-15/group. *p<0.05, **p<0.01. Individual n values and statistical results are summarized in Supplemental Table S1.(*2R,6R*)-hydroxynorketamine and (*2S,6S*)-hydroxynorketamine chemical structures reproduced with permission from ³¹. Copyright 2017 American Chemical Society.

Single-crystal analysis of (2R,6R)-HNK and (2R,6S)-HNK revealed highly divergent orientations of the amine and aryl substitutions on the cyclohexanone ring (Figure 5A), with the (2R,6S)-HNK orienting the aryl group in an equatorial confirmation and the (2R,6R)-HNK placing this group in an axial position Figure 5B; see ³¹. We hypothesized that additional modifications could drive the confirmational equilibrium into a more biologically favorable composition. Thus, the novel compound (5R)-methyl-(2R,6R)-HNK was designed and synthesized (see Supplemental Information) whereby the methyl group addition on the cyclohexanone ring system was intended to drive the overall confirmation of the molecule toward a three-dimensional confirmation with improved biological activity (see Supplemental Information).

The pharmacokinetic profile of (5R)-methyl-(2R,6R)-HNK was obtained revealing that, following intraperitoneal dosing, (5R)-methyl-(2R,6R)-HNK is detected in the plasma of mice between 10 min and 4 hr, with peak plasma levels observed at the earliest sampling time point, 10 min post-injection (Figure 5B). The plasma elimination t_1/2 was 49 min in both sexes (Table 1). (5R)-methyl-(2R,6R)-HNK penetrated the brain, with peak brain levels observed at the earliest sampling timepoint, 10 min after administration (Figure 5C). The brain to plasma AUC ratio was 0.4 and the brain elimination t_1/2 was 48 min in males and 41 min in females (Table 1). Sex-dependent differences were observed in the plasma levels of (5R)-methyl-(2R,6R)-HNK, with an overall effect of sex detected in the plasma concentrations over time (Figure 5B and Table 2), while no statistical difference was observed in brain concentrations over time (Figure 5C and Table 2). The plasma and brain AUCs of (5R)-methyl-(2R,6R)-HNK were increased in male mice, relative to females (Figure 5B-C and Table 2).

Effects of (5R)-methyl-(2R,6R)-HNK on forced swim test immobility time

(5R)-methyl-(2R,6R)-HNK was tested in male mice within the range of 0.3-10 mg/kg and compared to a vehicle. 30 mg/kg (2R,6R)-hydroxynorketamine was included as an internal experimental control. Similar to (2R,6S)-HNK, (5R)-methyl-(2R,6R)-HNK reduced immobility time in the forced swim test with a minimum effective dose of 1 mg/kg and was effective within the range of 1-10 mg/kg (with a trend to reduce immobility, p=0.06, observed at the dose of 3 mg/kg; Figure 5D). (5R)-methyl-(2R,6R)-HNK exerted antidepressant-relevant behavioral effects in the forced swim test (Figure 5D), with similar potency compared to (2R,6S)-HNK (Figure 4C), suggesting that the three-dimensional confirmation of the cyclohexanone ring system may be a manipulatable feature of the HNKs.

There are 12 unique HNKs that are formed in vivo following ketamine administration in mice ⁴ and humans ^1-3,7. The current study provides the first in-depth characterization of these 12 HNKs’ pharmacokinetics following their direct administration to male and female mice. The pharmacokinetic parameters established here are critical in guiding future studies of these compounds in mice. Overall, the data demonstrate that although HNKs exhibit robust differences in C_max and AUC in the plasma (C_max, ~6-fold differences; AUC, ~11-12-fold differences) and brains (C_max, 7-8-fold differences; AUC, 9-12-fold differences; Table 1) of mice, both rapid elimination profiles (half-lives ranging from 27-55 min (0.4-0.9 h) in the plasma and 15-45 min (0.3-0.8 h) in the brain; Table 1) and capacity to penetrate the brain (brain to plasma AUC ratios between 0.6-1.4, see Table 1) are conserved among the HNK stereoisomers. Notably, the ability of the 12 HNKs to rapidly penetrate the brain following systemic (i.p.) dosing suggests that these compounds would be capable of exerting pharmacodynamic effects in the brain, including antidepressant-relevant behavioral effects. Considering that C_max was observed at the earliest time point studied in most instances, we note that the true peak plasma and brain concentrations likely occur earlier than 10 min after dosing, and the t_1/2 and other outcomes could change modestly from those estimates provided here. Indeed, in an earlier study where plasma was collected at 2.5- and 5-min post-injection following i.p. administration to mice, peak plasma levels of (2R,6R)-HNK were observed at 2.5 min, and peak brain levels at 5 min ¹⁹.

This study is also the first to investigate the antidepressant-relevant behavioral effects of the four (2,6)-HNKs in the mouse forced swim test. Our results demonstrate that (2R,6R)-HNK has greater behavioral potency (minimum effective dose 10 mg/kg, i.p.; Figure 4A) compared to (2S,6S)-HNK (minimum effective dose 30 mg/kg, i.p.; Figure 4B). This is consistent with an earlier study,⁴ which reported that (2R,6R)-HNK reduced forced swim test immobility time at doses as low as 5 mg/kg, i.p. and reduced escape failures in the learned helplessness test at doses as low as 3 mg/kg, i.p., whereas (2S,6S)-HNK required doses of 25 and 75 mg/kg, i.p., respectively, to exert effects in the same behavioral tests. This is particularly striking, considering that following systemic treatment with equivalent doses, C_max and AUC of (2S,6S)-HNK are approximately 2-3-fold greater than those of (2R,6R)-HNK in male mice (see Table 1). Considering these pharmacokinetic differences, it is likely that (2R,6R)-HNK is substantially more potent than (2S,6S)-HNK with respect to engaging its targets in the brain.

The relatively greater behavioral potency of (2R,6R)-HNK compared to that of (2S,6S)-HNK is also consistent with earlier studies demonstrating that, when tested at equivalent doses (10 mg/kg, i.p.), (2R,6R)-, but not (2S,6S)-HNK, reduced forced swim test immobility time in mice ¹⁷ and reversed learned helplessness in rats ²⁶. However, these findings are in contrast to one study that reported (2S,6S)-, but not (2R,6R)-HNK, reduced forced swim test immobility time and measures of anhedonia following chronic stress in mice when each was tested at the same dose 20 mg/kg, i.p.; ²³. It is unclear whether differences in experimental design, species, or strain may underlie these apparent discrepancies. Nevertheless, by testing across a 100-fold range of doses and comparing compounds under the same experimental conditions, the present data support that (2R,6R)-HNK exhibits greater behavioral potency, relative to (2S,6S)-HNK, in the forced swim test.

Both (2R,6S)-HNK (effective at 1 mg/kg, i.p.; Figure 4C) and (2S,6R)-HNK (minimum effective dose 3 mg/kg, i.p.; Figure 4D) exerted behavioral effects in the forced swim test at lower doses compared to either (2R,6R)- or (2S,6S)-HNK. (2R,6S)- and (2S,6R)-HNK may be even more potent compared to (2R,6R)-HNK as suggested by the data presented in Figure 4, since both compounds exhibit greater behavioral potency(effective at 1 mg/kg, i.p. and 3 mg/kg, i.p., respectively), despite reaching lower brain concentrations. Namely, C_max and AUC were approximately two-fold lower following (2R,6S)-HNK dosing and approximately three-fold lower following (2S,6R)-HNK dosing, compared to equivalent dosing with (2R,6R)-HNK (see Table 1).

Altogether, the rank-order of potencies in the forced swim test was determined to be (2R,6S)-, (2S,6R)-, (2R,6R)-, and (2S,6S)-HNK, from most to least effective. We note that the range of doses tested did not allow the determination of a true minimally effective dose of (2R,6S)-HNK, which reduced immobility at the lowest dose tested, 1 mg/kg. Nonetheless, the data led to synthesis and characterization of the novel compound (5R)-methyl-(2R,6R)-HNK, as an approach to manipulate the confirmational equilibrium of the cyclohexanone ring system to produce superior antidepressant-like effects (Figure 5). In particular, the methyl group substitution at the C5 position causes (5R)-methyl-(2R,6R)-HNK to localize the aryl group equatorial to the cyclohexyl ring while the amine and hydroxyl groups are localized axial, similar to (2R,6S)-HNK (Figure 5A) and in contrast with (2R,6R)-HNK Figure 5A; see ³¹. Similar to the HNKs formed in vivo from ketamine, (5R)-methyl-(2R,6R)-HNK rapidly penetrated the brain of mice following an intraperitoneal injection, where it was detected at the earliest sampling timepoint, 10 min post-injection. In the forced swim test, (5R)-methyl-(2R,6R)-HNK reduced immobility time (minimum effective dose 1 mg/kg, i.p.; Figure 5D) with similar potency as (2R,6S)-HNK. These data suggest that the three-dimensional conformation of (5R)-methyl-(2R,6R)-HNK may recapitulate that of (2R,6S)-HNK. Future studies should evaluate the effects of (2R,6S)-, (2S,6R)-, and (5R)-methyl-(2R,6R)-HNK as well as structurally similar compounds on biochemical and synaptic outcomes, and, importantly, compare their relative potencies to induce such effects, in order to understand how the potency to engage putative targets is related to the observed rank-order of behavioral potency. In this study, we focused on assessing and comparing the behavioral actions of the (2,6)-HNKs, based upon the demonstrated antidepressant-relevant behavioral actions of (2R,6R)- and (2S,6S)-HNK ^4,9,15-25. However, additional studies are needed to characterize the behavioral, biochemical, and synaptic actions of the (2,4)- and (2,5)-HNKs in order to fully understand the structure-function relationship underlying the antidepressant-relevant behavioral effects of the HNKs, which may inform the development of HNKs, or structurally similar novel compounds, as novel antidepressant drugs.

We note the present data are consistent with an N-methyl-D-aspartate receptor (NMDAR) inhibition-independent mechanism contributing to the actions of the (2,6)-HNKs. In particular, (2S,6S)-HNK has moderate inhibitory actions on NMDARs (K_i = 7.34-1.19 μM) compared to ketamine Ki = 0.25-1.06 μM; ^19,31,32. By contrast, compared to ketamine (2R,6R)-HNK has markedly lower potency to bind or inhibit NMDARs (K_i > 100 μM) ^4,19,31-35, and does not meaningfully inhibit NMDARs at concentrations relevant to exposures identified in the current study ¹⁹. The relatively low binding affinities of (2R,6S)- and (2S,6R)-HNK are similar to that of (2R,6R)-HNK all were reported to be >100 μM; ³¹. Both compounds reduced immobility time in the forced swim test at doses ≤10 mg/kg, i.p., despite producing lower brain concentrations than similar doses of (2R,6R)-HNK, suggesting that the brain concentrations of (2R,6S)- and (2S,6R)-HNK necessary to induce antidepressant-like actions are even lower than those required for (2R,6R)-HNK, and, therefore, also below the threshold for NMDAR inhibition.

While this study focused on evaluating the behavioral actions of the (2,6)-HNKs following their direct administration to mice, the enhanced potency of (2R,6S)-HNK relative to the other (2,6)-HNKs tested may suggest that the (2R,6S)-HNK metabolite contributes to ketamine’s antidepressant actions. However, future studies are needed to test this possibility directly and to establish the full range of (2R,6S)-HNK’s behavioral actions.

There are several important limitations to the present study. First, only one behavioral outcome (immobility time in the forced swim test) was evaluated and only at a single time point (24 h post-injection). It is possible that the relative potencies of the different (2,6)-HNKs may differ in additional behavioral tests thought to be predictive of antidepressant efficacy or at different times relative to testing (e.g., it is possible that some HNKs may have longer-lasting actions or exert more robust effects when evaluated earlier or later relative to dosing). It is important for future studies to evaluate the effects of the (2,6)-HNKs as well as the other HNKs that have not yet been tested, in a variety of behavioral tests, including those that predict antidepressant potency and in those that aim to characterize adverse behavioral effects. Second, most HNKs demonstrated a sex-dependent difference in their pharmacokinetic profiles, an important consideration for future behavioral studies. To control for these differences, we included only male mice in this initial behavioral characterization of the (2,6)-HNKs. Future studies should examine the effects of the HNKs in females to fully understand how biological sex may alter the behavioral potencies of these compounds. Finally, the effects of the HNKs on biochemical and/or synaptic outcomes thought to underlie antidepressant-relevant behavioral effects are yet to be evaluated.

Altogether, the data presented here demonstrate for the first time that the relative rank-order of potency of the four (2,6)-HNKs to reduce forced swim test immobility time, from most to least potent, is (2R,6R)-, (2S,6R)-, (2R,6R)-, and (2S,6S)-HNK. This is the first indication that (2R,6S)-HNK has greater potency compared to (2R,6R)-HNK, exerting antidepressant-relevant behavioral effects at 10-fold lower doses, despite its lower relative brain concentrations. Utilizing a novel compound (5R)-methyl-(2R,6R)-HNK we also highlight the potential of further modifications to the cyclohexanone ring system as a means to drive the HNK into biologically advantageous three-dimensional confirmations. While these compounds require further study, these data suggest that (2R,6S)-HNK, (5R)-methyl-(2R,6R)-HNK, or additional HNKs yet to be tested, may represent novel, potent drug candidates for the treatment of depression.

MATERIALS AND METHODS

Animals

Male and female CD-1 mice (Charles Rivers Laboratories, Raleigh, NC, USA), 8-11 weeks old at the time of testing, were habituated to the University of Maryland (Baltimore, MD, USA) animal facility for at least one week prior to testing. CD-1 mice were selected based on previous studies establishing the antidepressant-relevant behavioral actions of ketamine and (2R,6R)-HNK in this strain ^{4,15,19,21,27}. Mice were group housed 4-5 per cage with a constant 12-hour light cycle (lights on/off at 07:00/19:00). Food and water were available ad libitum. All experiments were performed during the light phase. All animal studies were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Drugs

(2R,6R)-HNK hydrochloride, (2S,6S)-HNK hydrochloride, (2R,6S)-HNK, (2S,6R)-HNK, (2R,5R)-HNK, (2S,5S)-HNK, (2R,5S)-HNK, (2S,5R)-HNK, (2R,4R)-HNK, (2S,4S)-HNK, (2R,4S)-HNK, and (2S,4R)-HNK hydrochloride (Figure 1A) were synthesized at the National Center for Advancing Translation Sciences (National Institutes of Health, Rockville, MD, USA) as previously described ³¹. (5R)-methyl-(2R,6R)-HNK (Figure 1B) was obtained from Organix Inc. (Woburn, MA, USA) or synthesized internally at the National Center for Advancing Translational Sciences (Rockville, MD, USA); synthesis and characterization, and CIF file are found in the Supplemental Information. All compounds were dissolved in 20% (w/v) cyclodextrin (Sigma Aldrich, USA) in saline and administered intraperitoneally (i.p.) at a volume of 7.5 ml/kg.

Pharmacokinetic studies

Dosing and sample collection

HNKs were administered via i.p. injection; this route of administration was selected for the pharmacokinetic studies because i.p. administration has most frequently been used in studies demonstrating the behavioral actions of HNKs in the mouse forced swim test ^4,18-21,27. HNKs were administered at a dose of 5 mg/kg (the free base dose for compounds provided as a hydrochloride salt—(2R,6R)-HNK, (2S,6S)-HNK, and (2S,4R)-HNK—is equivalent to 4.31 mg/kg; (5R)-methyl-(2R,6R)-HNK is equivalent to 4.37 mg/kg) and a volume of 7.5 ml/kg, in separate cohorts of male and female mice (n=4 per sex, compound, and collection time point). At 10 min (0.167 h), 30 min (0.5 h), 1 h, 2 h, and 4 h post-treatment, mice were deeply anesthetized (3.5% isoflurane for approximately 2 min). Sampling time points were based on the established pharmacokinetic profiles of (2R,6R)-HNK and (2S,6S)-HNK in mice ⁴. Trunk blood was collected into 1.5-ml polypropylene tubes containing 30 μl of disodium EDTA (0.5 M, pH 8.0) and kept on ice until plasma collection (<30 min). Brains were then removed, flash-frozen in isopentane, and stored at −80°C until processing and analysis. Blood was centrifuged at 8000 × g for 6 min at 4°C to obtain plasma. Plasma was collected into clean microcentrifuge tubes and stored at −80°C until processing and analysis.

UPLC-MS/MS analysis of plasma and brain samples

Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods were developed and optimized for each HNK to determine the concentration of each HNK in mouse plasma and brain samples following systemic administration. Mass spectrometric analysis was performed on a Waters Xevo TQ-S triple quadrupole instrument using electrospray ionization in positive mode with the selected reaction monitoring. The separation of HNKs from endogenous components was performed on a Waters Acquity UPLC with 0.5 ml/min flow rate and gradient elution. The mobile phase A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Plasma and brain concentrations of (2R,6R)-, (2S,6S)-, (2R,4S)-, and (2S,4R)-HNK were determined by hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC UPLC-MS/MS) using a BEH amide column (1.7 μ, 2.1 x 50 mm). Plasma and brain concentrations of (2R,6S)-, (2S,6R)-, (2R,5R)- (2S,5S)-, (2R,5S)-, (2S,5R)-, (2R,4R)-, and (2S,4S)-HNK; and (5R)-methyl-(2R,6R)-HNK were determined by reversed phase UPLC-MS/MS methods based upon a previously described protocol with several modifications ^1,4 using a BEH C₁₈ column (1.7 μM, 2.1 x 50 mm). The gradient for the HILIC UPLC method was: 0-0.2 min 98% B; 0.2-1.4 min 98% to 35% B; 1.4-1.8 min 35% to 10% B, 1.9-2.4 min 98% B. The gradient for the reversed phase UPLC method was: 0-0.2 min 1% B; 0.2-1.4 min 1% to 35% B; 1.4-1.8 min 35% to 98% B, 1.8-2.2 min 98% B, 2.3 min 1% B. The analytical run time of the UPLC methods was 2.5 min. The calibration standards and quality control samples of each HNK were prepared in blank mouse plasma and brain homogenate. Calibration standards ranged from 5.0-2500 ng/ml, or 5.0-5000 ng/mL; depending on the concentration ranges of HNKs. A linear regression with 1/x² weighting was used to construct the calibration curve. Quality control samples of each HNK were prepared at 10.0 ng/ml, 100 ng/ml, and 2500 ng/ml (or 5000 ng/mL) in the corresponding matrix. Brains were weighed and homogenized in 3 volumes of water. (2R,6R)-d4-HNK was used as internal standard. 200 μL of 50 ng/ml (2R,6R)-d4-HNK solution in acetonitrile was used to precipitate proteins in 10 μL plasma and 40 μL brain homogenate samples. The supernatant was transferred to a 96-well plate and 1.0 μL supernatant was injected for UPLC-MS/MS analysis. The data were acquired using MassLynx and analyzed using TargetLynx version 4.1. The lower limit of quantification (LLOQ) was 5.0 ng/ml.

Pharmacokinetic analysis

The pharmacokinetic parameters of (2R,6R)- (2S,6S)-, (2R,6S)-, (2S,6R)-, (2R,5R)- (2S,5S)-, (2R,5S)-, (2S,5R)-, (2R,4R)- (2S,4S)-, (2R,4S)-, (2S,4R), and (5R)-methyl-(2R,6R)-HNK were calculated using non-compartmental analysis (Model 200) in the pharmacokinetic software Phoenix WinNonlin (version 7.0, Certara, St. Louis, MO). The area under the plasma and tissue concentration versus time curve (AUC) was calculated using the linear trapezoidal method. The slope of the elimination phase was estimated by log linear regression using at least 3 data points and the terminal rate constant (λ) was derived from the slope. AUC_0→∞ was calculated as the sum of the AUC_0→t (where t is the time of the last measurable concentration) and C_t/λ. The apparent elimination half-life (t½) was calculated as 41.58/λ (min) or 0.693/λ (hr).

Forced-swim test

The 2,6-HNKs were tested in separate cohorts of male mice (n=10-18/group; only male mice were tested to control for sex-dependent differences in pharmacokinetics and brain levels) and all injections were performed by a male experimenter. (2R,6R)-HNK, (2S,6S)-HNK, (2R,6S)-HNK, (2S,6R)-HNK were administered at the doses of 1, 3, 10, 30, and 100 mg/kg. (5R)-methyl-(2R,6R)-HNK was administered at the doses of 0.3, 1, 3, and 10 mg/kg. (2R,6S)-HNK and (2S,6R)-HNK were dosed as a free base whereas (2R,6R)-HNK and (2S,6S)-HNK, and (5R)-methyl-(2R,6R)-HNK were dosed as a hydrochloride salt. Vehicle and (2R,6R)-HNK (30 mg/kg) were included as controls in each cohort. Twenty-four h after treatment, mice were tested in the forced swim test, according to previously described methods ^4,27. Briefly, mice were placed into a clear Plexiglass cylinder (20 cm height × 15 cm diameter) filled to a depth of 15 cm with water (23 ± 1°C) and subjected to a 6 min forced swim session. Swim sessions were recorded using a digital video camera. The time spent immobile (defined as passive floating with no movements other than those necessary to keep the head above water) was scored during the final 4 min of the session by a trained experimenter blind to the treatment groups.

Statistical analyses

Statistical analyses were performed using GraphPad Prism software (v8; GraphPad Software, Inc.). In cases of two-group comparisons, all statistical tests were two-tailed, and significance was assigned as p<0.05. Significant results are indicated with asterisks in the figures (*p<0.05, **p<0.01, ***p<0.001). Data are presented as the mean ± standard error of the mean (SEM). Mice were randomized to treatment groups and experiments were performed in a blinded manner.

For the pharmacokinetic analysis of HNK, sex-dependent differences in concentrations over time were analyzed using a two-way ANOVA with sex and time as factors, followed by Holm-Šidák post-hoc comparisons when significant main effects were observed. The experimental design (in which plasma and brain for a single experimental time point came from independent groups of mice, representing a between-group comparison as a function of time) resulted in a single experimentally derived AUC per compound and sex. Thus, to enable the comparison of AUCs between sexes, a previously described bootstrapping method ³⁶ was utilized to estimate the mean and SEM for each AUC. Briefly, to calculate the mean and SEM of the AUC, N=1000 of 1024 possible combinations were randomly selected to form N time series, and then 1000 AUCs and the corresponding mean and SEM determined for each sex. Sex-dependent differences in AUCs were determined using a standard t-test.

For the behavioral testing of HNKs in the forced-swim test, differences in immobility time were analyzed via one-way ANOVA followed by Holm-Šidák post-hoc comparisons (with all groups compared to the saline control group) when a significant main effect of the model was observed.

Supplementary Material

NIHMS1867317-supplement-SI.pdf^{(560.5KB, pdf)}

Funding Sources

This work was supported by NIH R01-MH107615 and VA Merit Awards 1I01BX004062 and 101BX003631-01A1 to TDG, and by the NIA (RM), NIMH (CAZ), and NCATS (CJT & AQW) NIH intramural research programs.

ABBREVIATIONS

AUC: area under the curve
C_max: maximum concentration
HNK: hydroxynorketamine
NMDAR: N-methyl-D-aspartate receptor
t_1/2: half-life
UPLC-MS/MS: ultra-performance liquid chromatography-tandem mass spectrometry

Footnotes

Conflict of Interest

RM is listed as a co-inventor on a patent for the use of (2R,6R)-hydroxynorketamine, (S)-dehydronorketamine and other stereoisomeric dehydro- and hydroxylated metabolites of (R,S)-ketamine in the treatment of depression and neuropathic pain. PJM, PZ, RM, CJT, and TG are listed as co-inventors on a patents for the synthesis and structure or use of (2R,6R)-hydroxynorketamine and (2S,6S)-hydroxynorketamine. JNH, PJM, PZ, RM, and TG are co-inventor on a patent application for (5R)-methyl-(2R,6R)-HNK and related structure relevant to the treatment of psychiatric disorders. RM, PM, and CJT have assigned their patent rights to the U.S. government but will share a percentage of any royalties that may be received by the government. JNH, PZ and TG have assigned their patent rights to the University of Maryland Baltimore but will share a percentage of any royalties that may be received by the University of Maryland Baltimore. TDG has received research funding from Allergan and Roche Pharmaceuticals and has served as a consultant for FSV7 LLC, during the preceding three years. All other authors declare no competing interests.

Disclosure

Experimental data was previously included in a doctoral dissertation (Highland, J.N., “Characterization of ketamine’s (2,6)-hydroxynorketamine metabolites: pharmacokinetic and behavioral considerations for antidepressant applications. 2020.)

SUPPORTING INFORMATION

(5R)-methyl-(2R,6R)-HNK synthesis and characterization, including X-ray crystallography and CIF file; Individual n values and statistical results are summarized for Figure 4 and Figure 5 data.

REFERENCES

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1867317-supplement-SI.pdf^{(560.5KB, pdf)}

[R1] 1.Moaddel R, Venkata SL, Tanga MJ, Bupp JE, Green CE, Iyer L, Furimsky A, Goldberg ME, Torjman MC & Wainer IW A parallel chiral-achiral liquid chromatographic method for the determination of the stereoisomers of ketamine and ketamine metabolites in the plasma and urine of patients with complex regional pain syndrome. Talanta 82, 1892–1904 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] 2.Zarate CA Jr., Brutsche N, Laje G, Luckenbaugh DA, Venkata SL, Ramamoorthy A, Moaddel R & Wainer IW Relationship of ketamine's plasma metabolites with response, diagnosis, and side effects in major depression. Biol Psychiatry 72, 331–338 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Zhao X, Venkata SL, Moaddel R, Luckenbaugh DA, Brutsche NE, Ibrahim L, Zarate CA Jr., Mager DE & Wainer IW Simultaneous population pharmacokinetic modelling of ketamine and three major metabolites in patients with treatment-resistant bipolar depression. Br J Clin Pharmacol 74, 304–314 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Zanos P, Moaddel R, Morris PJ, Georgiou P, Fischell J, Elmer GI, Alkondon M, Yuan P, Pribut HJ, Singh NS, Dossou KS, Fang Y, Huang XP, Mayo CL, Wainer IW, Albuquerque EX, Thompson SM, Thomas CJ, Zarate CA Jr. & Gould TD NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature 533, 481–486 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Moaddel R, Sanghvi M, Ramamoorthy A, Jozwiak K, Singh N, Green C, O'Loughlin K, Torjman M & Wainer IW Subchronic administration of (R,S)-ketamine induces ketamine ring hydroxylation in Wistar rats. J Pharm Biomed Anal 127, 3–8 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] 6.Leung LY & Baillie TA Comparative pharmacology in the rat of ketamine and its two principal metabolites, norketamine and (Z)-6-hydroxynorketamine. J Med Chem 29, 2396–2399 (1986). [DOI] [PubMed] [Google Scholar]

[R7] 7.Farmer CA, Gilbert JR, Moaddel R, George J, Adeojo L, Lovett J, Nugent AC, Kadriu B, Yuan P, Gould TD, Park LT & Zarate CA Jr. Ketamine metabolites, clinical response, and gamma power in a randomized, placebo-controlled, crossover trial for treatment-resistant major depression. Neuropsychopharmacology 45, 1398–1404 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.Sandbaumhuter FA, Theurillat R & Thormann W Separation of hydroxynorketamine stereoisomers using capillary electrophoresis with sulfated beta-cyclodextrin and highly sulfated gamma-cyclodextrin. Electrophoresis 38, 1878–1885 (2017). [DOI] [PubMed] [Google Scholar]

[R9] 9.Highland JN, Zanos P, Riggs LM, Georgiou P, Clark SM, Morris PJ, Moaddel R, Thomas CJ, Zarate CA Jr., Pereira EFR & Gould TD Hydroxynorketamines: pharmacology and potential therapeutic applications. Pharmacological Reviews 73, 763–791 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR, Charney DS & Krystal JH Antidepressant effects of ketamine in depressed patients. Biol Psychiatry 47, 351–354 (2000). [DOI] [PubMed] [Google Scholar]

[R11] 11.Fava M, Freeman MP, Flynn M, Judge H, Hoeppner BB, Cusin C, Ionescu DF, Mathew SJ, Chang LC, Iosifescu DV, Murrough J, Debattista C, Schatzberg AF, Trivedi MH, Jha MK, Sanacora G, Wilkinson ST & Papakostas GI Correction: Double-blind, placebo-controlled, dose-ranging trial of intravenous ketamine as adjunctive therapy in treatment-resistant depression (TRD). Mol Psychiatry 25, 1604 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Zarate CA Jr., Singh JB, Carlson PJ, Brutsche NE, Ameli R, Luckenbaugh DA, Charney DS & Manji HK A randomized trial of an N-methyl-D-aspartate antagonist in treatment-resistant major depression. Arch Gen Psychiatry 63, 856–864 (2006). [DOI] [PubMed] [Google Scholar]

[R13] 13.Murrough JW, Perez AM, Pillemer S, Stern J, Parides MK, aan het Rot M, Collins KA, Mathew SJ, Charney DS & Iosifescu DV Rapid and longer-term antidepressant effects of repeated ketamine infusions in treatment-resistant major depression. Biol Psychiatry 74, 250–256 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] 14.Riggs LM & Gould TD Ketamine and the Future of Rapid-Acting Antidepressants. Annu Rev Clin Psychol 17, 207–231 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Zanos P, Highland JN, Liu X, Troppoli TA, Georgiou P, Lovett J, Morris PJ, Stewart BW, Thomas CJ, Thompson SM, Moaddel R & Gould TD (R)-Ketamine exerts antidepressant actions partly via conversion to (2R,6R)-hydroxynorketamine, while causing adverse effects at sub-anaesthetic doses. Br J Pharmacol 176, 2573–2592 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Chen BK, Luna VM, LaGamma CT, Xu X, Deng SX, Suckow RF, Cooper TB, Shah A, Brachman RA, Mendez-David I, David DJ, Gardier AM, Landry DW & Denny CA Sex-specific neurobiological actions of prophylactic (R,S)-ketamine, (2R,6R)-hydroxynorketamine, and (2S,6S)-hydroxynorketamine. Neuropsychopharmacology 45, 1545–1556 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Chou D, Peng HY, Lin TB, Lai CY, Hsieh MC, Wen YC, Lee AS, Wang HH, Yang PS, Chen GD & Ho YC (2R,6R)-hydroxynorketamine rescues chronic stress-induced depression-like behavior through its actions in the midbrain periaqueductal gray. Neuropharmacology 139, 1–12 (2018). [DOI] [PubMed] [Google Scholar]

[R18] 18.Fukumoto K, Fogaca MV, Liu RJ, Duman C, Kato T, Li XY & Duman RS Activity-dependent brain-derived neurotrophic factor signaling is required for the antidepressant actions of (2R,6R)-hydroxynorketamine. Proc Natl Acad Sci U S A 116, 297–302 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Lumsden EW, Troppoli TA, Myers SJ, Zanos P, Aracava Y, Kehr J, Lovett J, Kim S, Wang FH, Schmidt S, Jenne CE, Yuan P, Morris PJ, Thomas CJ, Zarate CA Jr., Moaddel R, Traynelis SF, Pereira EFR, Thompson SM, Albuquerque EX & Gould TD Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function. Proc Natl Acad Sci U S A 116, 5160–5169 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] 20.Pham TH, Defaix C, Xu X, Deng SX, Fabresse N, Alvarez JC, Landry DW, Brachman RA, Denny CA & Gardier AM Common Neurotransmission Recruited in (R,S)-Ketamine and (2R,6R)-Hydroxynorketamine-Induced Sustained Antidepressant-like Effects. Biol Psychiatry 84, e3–e6 (2018). [DOI] [PubMed] [Google Scholar]

[R21] 21.Zanos P, Highland JN, Stewart BW, Georgiou P, Jenne CE, Lovett J, Morris PJ, Thomas CJ, Moaddel R, Zarate CA Jr. & Gould TD (2R,6R)-hydroxynorketamine exerts mGlu2 receptor-dependent antidepressant actions. Proc Natl Acad Sci U S A 116, 6441–6450 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Elmer GI, Tapocik JD, Mayo CL, Zanos P & Gould TD Ketamine metabolite (2R,6R)-hydroxynorketamine reverses behavioral despair produced by adolescent trauma. Pharmacol Biochem Behav, 172973 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] 23.Yokoyama R, Higuchi M, Tanabe W, Tsukada S, Naito M, Yamaguchi T, Chen L, Kasai A, Seiriki K, Nakazawa T, Nakagawa S, Hashimoto K, Hashimoto H & Ago Y (S)-norketamine and (2S,6S)-hydroxynorketamine exert potent antidepressant-like effects in a chronic corticosterone-induced mouse model of depression. Pharmacol Biochem Behav 191, 172876 (2020). [DOI] [PubMed] [Google Scholar]

[R24] 24.Casarotto PC, Girych M, Fred SM, Kovaleva V, Moliner R, Enkavi G, Biojone C, Cannarozzo C, Sahu MP, Kaurinkoski K, Brunello CA, Steinzeig A, Winkel F, Patil S, Vestring S, Serchov T, Diniz C, Laukkanen L, Cardon I, Antila H, Rog T, Piepponen TP, Bramham CR, Normann C, Lauri SE, Saarma M, Vattulainen I & Castren E Antidepressant drugs act by directly binding to TRKB neurotrophin receptors. Cell (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R25] 25.Aguilar-Valles A, De Gregorio D, Matta-Camacho E, Eslamizade MJ, Khlaifia A, Skaleka A, Lopez-Canul M, Torres-Berrio A, Bermudez S, Rurak GM, Simard S, Salmaso N, Gobbi G, Lacaille JC & Sonenberg N Antidepressant actions of ketamine engage cell-specific translation via eIF4E. Nature 590, 315–319 (2021). [DOI] [PubMed] [Google Scholar]

[R26] 26.Chou D. Brain-derived neurotrophic factor in the ventrolateral periaqueductal gray contributes to (2R,6R)-hydroxynorketamine-mediated actions. Neuropharmacology 170, 108068 (2020). [DOI] [PubMed] [Google Scholar]

[R27] 27.Highland JN, Morris PJ, Zanos P, Lovett J, Ghosh S, Wang AQ, Zarate CA Jr., Thomas CJ, Moaddel R & Gould TD Mouse, rat, and dog bioavailability and mouse oral antidepressant efficacy of ( 2R,6R)-hydroxynorketamine. J Psychopharmacol, 269881118812095 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Yamaguchi JI, Toki H, Qu Y, Yang C, Koike H, Hashimoto K, Mizuno-Yasuhira A & Chaki S (2R,6R)-Hydroxynorketamine is not essential for the antidepressant actions of (R)-ketamine in mice. Neuropsychopharmacology 43, 1900–1907 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] 29.Moaddel R, Sanghvi M, Dossou KS, Ramamoorthy A, Green C, Bupp J, Swezey R, O'Loughlin K & Wainer IW The distribution and clearance of (2S,6S)-hydroxynorketamine, an active ketamine metabolite, in Wistar rats. Pharmacol Res Perspect 3, e00157 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.Lilius TO, Viisanen H, Jokinen V, Niemi M, Kalso EA & Rauhala PV Interactions of (2S,6S;2R,6R)-Hydroxynorketamine, a Secondary Metabolite of (R,S)-Ketamine, with Morphine. Basic Clin Pharmacol Toxicol 122, 481–488 (2018). [DOI] [PubMed] [Google Scholar]

[R31] 31.Morris PJ, Moaddel R, Zanos P, Moore CE, Gould TD, Zarate CA Jr. & Thomas CJ Synthesis and N-Methyl-d-aspartate (NMDA) Receptor Activity of Ketamine Metabolites. Org Lett 19, 4572–4575 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Hydroxynorketamine pharmacokinetics and antidepressant behavioral effects of the (2,6)- and (5R)-methyl-(2R,6R)-hydroxynorketamines

Jaclyn N Highland

Patrick J Morris

Kylie M Konrath

Lace M Riggs

Natalie R Hagen

Panos Zanos

Chris F Powels

Ruin Moaddel

Craig J Thomas

Amy Q Wang

Todd D Gould

Abstract

INTRODUCTION

Figure 1. Hydroxynorketamine structures.

RESULTS AND DISCUSSION

Pharmacokinetic profiles of the 12 HNKs in mouse plasma and brain

Plasma concentrations of HNKs

Figure 2. Plasma levels of hydroxynorketamines in mice.

Table 1.

Table 2.

Brain concentrations of HNKs

Figure 3. Brain levels of hydroxynorketamines in mice.

Effects of the (2,6)-HNKs on forced swim test immobility time

Figure 4. (2,6)-hydroxynorketamines reduce immobility time in the mouse forced swim test.

Pharmacokinetic profile of (5R)-methyl-(2R,6R)-HNK in mouse plasma and brain

Figure 5. (5R)-methyl-(2R,6R)-hydroxynorketamine reduces forced swim test immobility time.

Effects of (5R)-methyl-(2R,6R)-HNK on forced swim test immobility time

MATERIALS AND METHODS

Animals

Drugs

Pharmacokinetic studies

Dosing and sample collection

UPLC-MS/MS analysis of plasma and brain samples

Pharmacokinetic analysis

Forced-swim test

Statistical analyses

Supplementary Material

Funding Sources

ABBREVIATIONS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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