(S)-norketamine and (2S,6S)-hydroxynorketamine exert potent antidepressant-like effects in a chronic corticosterone-induced mouse model of depression

Journal Pre-proof (S)-norketamine and (2S,6S)-hydroxynorketamine exert potent antidepressant-like effects in a chronic corticosterone-induced mouse model of depression

Rei Yokoyama, Momoko Higuchi, Wataru Tanabe, Shinji Tsukada, Megumi Naito, Takumi Yamaguchi, Lu Chen, Atsushi Kasai, Kaoru Seiriki, Takanobu Nakazawa, Shinsaku Nakagawa, Kenji Hashimoto, Hitoshi Hashimoto, Yukio Ago PII:

S0091-3057(20)30055-1

DOI:

https://doi.org/10.1016/j.pbb.2020.172876

Reference:

PBB 172876

To appear in:

Pharmacology, Biochemistry and Behavior

Received date:

28 January 2020

Revised date:

11 February 2020

Accepted date:

19 February 2020

Please cite this article as: R. Yokoyama, M. Higuchi, W. Tanabe, et al., (S)-norketamine and (2S,6S)-hydroxynorketamine exert potent antidepressant-like effects in a chronic corticosterone-induced mouse model of depression, Pharmacology, Biochemistry and Behavior (2020), https://doi.org/10.1016/j.pbb.2020.172876

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© 2020 Published by Elsevier.

Journal Pre-proof Original Investigations

(S)-norketamine

and

(2S,6S)-hydroxynorketamine

exert

potent

antidepressant-like effects in a chronic corticosterone-induced mouse model of depression

Rei Yokoyamaa,†, Momoko Higuchia,†, Wataru Tanabea, Shinji Tsukadaa, Megumi Naitoa, Takumi

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Yamaguchib, Lu Chenb, Atsushi Kasaia, Kaoru Seirikia,c, Takanobu Nakazawaa,d, Shinsaku

Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka

University, Suita, Osaka 565-0871, Japan

Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University,

c

na

Suita, Osaka 565-0871, Japan

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b

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a

-p

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Nakagawab,e,f, Kenji Hashimotog, Hitoshi Hashimotoa,h,i,j,k,*, Yukio Agob,e,f,*

Interdisciplinary Program for Biomedical Sciences, Institute for Transdisciplinary Graduate Degree

d

Department of Pharmacology, Graduate School of Dentistry, Osaka University, Suita, Osaka

565-0871, Japan e

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Programs, Osaka University, Suita, Osaka 565-0871, Japan

Laboratory of Innovative Food Science, Graduate School of Pharmaceutical Sciences, Osaka

University, Suita, Osaka 565-0871, Japan f

Global Center for Medical Engineering and Informatics, Osaka University, Suita, Osaka 565-0871,

Japan g

Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba

260-8670, Japan h

Molecular Research Center for Children's Mental Development, United Graduate School of Child

Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine,

Journal Pre-proof Chiba University and University of Fukui, Suita, Osaka 565-0871, Japan i

Division of Bioscience, Institute for Datability Science, Osaka University, Suita, Osaka 565-0871,

Japan j

Transdimensional Life Imaging Division, Institute for Open and Transdisciplinary Research

Initiatives, Osaka University, Suita, Osaka 565-0871, Japan k

Department of Molecular Pharmaceutical Science, Graduate School of Medicine, Osaka University,

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These authors contributed equally to this work.

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†

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Suita, Osaka 565-0871, Japan

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Running title: Antidepressant effects of (S)-ketamine metabolites

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*Correspondence should be addressed to Yukio Ago or Hitoshi Hashimoto.

Yukio Ago, Ph.D. (Corresponding author for communication with the editorial office)

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Associate Professor; Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University; 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: +81 6 6879 8176; Fax: +81 6 6879 8179; Email: [email protected]

Hitoshi Hashimoto, Ph.D. (Corresponding author) Professor and Chairman; Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University; 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: +81 6 6879 8180; Fax: +81 6 6879 8184; Email: [email protected]

Journal Pre-proof

Abstract

Clinical and preclinical studies have shown that the N-methyl-D-aspartate receptor antagonist ketamine exerts rapid and long-lasting antidepressant effects. Although ketamine metabolites might also have potential antidepressant properties, controversial results have been reported for (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) in particular, and there is little information regarding

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the effects of other ketamine metabolites. Here we aimed to compare the effects of (R)-norketamine ((R)-NK), (S)-NK, (2R,6R)-HNK, and (2S,6S)-HNK in a mouse model of depression induced by

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chronic corticosterone (CORT) injection. None of the ketamine metabolites at doses up to 20 mg/kg

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showed antidepressant-like activity in naïve male C57BL6/J mice. Chronic CORT treatment

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increased immobility in the forced swim test and caused anhedonic-like behaviors in the female encounter test. A single administration of (S)-NK and (2S,6S)-HNK dose-dependently reduced the

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enhanced immobility at 30 min after injection in chronic CORT-treated mice, while (R)-NK or

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(2R,6R)-HNK did not. Additionally, (S)-NK and (2S,6S)-HNK, but not (R)-NK or (2R,6R)-HNK, improved chronic CORT-induced anhedonia at 24 h after the injection. These results suggest that

effects in rodents.

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(S)-ketamine metabolites (S)-NK and (2S,6S)-HNK have potent acute and sustained antidepressant

Keywords ketamine metabolites, depression model, antidepressant, anti-anhedonic, behaviors, C57BL/6J mice

Journal Pre-proof

1. Introduction

Accumulating evidence has indicated that the N-methyl-D-aspartate (NMDA) receptor antagonist ketamine (racemic ketamine; (R,S)-ketamine) produces rapid and potent antidepressant effects in major depressive disorder, including treatment-resistant depression (Berman et al., 2000; Zarate et al., 2006; Murrough et al., 2013; Newport et al., 2015; Su et al., 2017). Intranasal administration of

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(S)-ketamine (esketamine) also showed rapid and sustained (more than two months) antidepressant effects in treatment-resistant depression (Daly et al., 2018) and resulted in rapid improvement in

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depressive symptoms and suicidality in patients at imminent risk for suicide (Canuso et al., 2018).

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An (S)-ketamine nasal spray has been approved as a new antidepressant for treatment-resistant

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depression by the U.S. Food and Drug Administration. Although ketamine is one of the most attractive drugs for the treatment of depression, its adverse effects such as psychotomimetic and

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dissociative effects and abuse potential raise important issues for clinical use of ketamine (Krystal et

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al., 2019). These adverse effects of ketamine might be at least partly accounted for by NMDA receptor inhibition (Ebert et al., 1997; Yang et al., 2019; Zanos et al., 2016). Recently, (R)-ketamine

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(arketamine) and its metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) have attracted much attention, because of their potential as antidepressant drugs without psychotomimetic and reinforcing properties due to a low affinity for NMDA receptors (Lumsden et al., 2019; Yang et al., 2019). Clinical trials of (R)-ketamine and (2R,6R)-HNK in humans are currently underway (Hashimoto, 2019). While there are some preclinical studies that (2R,6R)-HNK exhibits rapid and sustained antidepressant effects (Fukumoto et al., 2019; Lumsden et al., 2019; Pham et al., 2018; Zanos et al., 2016, 2019b), other controversial results have been reported (Shirayama and Hashimoto, 2018; Xiong et al., 2019; Yamaguchi et al., 2018; Yang et al., 2017). The experimental conditions such as mouse strain (CD-1 outbred or BALB/cJ inbred mice vs. C57BL/6J inbred mice), different animal models used (healthy vs. stress-induced or genetic models of depression), and behavioral tests might

Journal Pre-proof account for this discrepancy. More information needs to be presented regarding what conditions are essential for the effects of (2R,6R)-HNK. Additionally, there is little information on the effects of other ketamine metabolites except for (2R,6R)-HNK. Zanos et al. (2016) have shown that (2S,6S)-HNK at 25 mg/kg or more exhibited antidepressant activity in naïve male CD-1 mice. Yang et al. (2018) recently found that (S)-norketamine ((S)-NK) elicits rapid and sustained antidepressant-like effects in inflammation-induced and chronic social defeat stress-induced models of depression, respectively. Interestingly, the antidepressant-like effects of (S)-NK were independent

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of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activation. Additionally,

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while both the rapid and sustained antidepressant-like effects of (2R,6R)-HNK require AMPA

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receptor activation, hippocampal synaptic AMPA receptor protein levels are upregulated 24 h, but not

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1 h, after systemic administration (Zanos et al., 2016). Of note, (2R,6R)-HNK acutely enhances excitatory synaptic transmission in the hippocampus through a concentration-dependent, NMDA

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receptor-independent, and synapse selective increase in glutamate release probability with no direct

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actions on AMPA receptor function (Riggs et al., 2020). Therefore, ketamine metabolites themselves might have potential antidepressant effects, even if they possess no direct activities at NMDA and

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AMPA receptors. In this study, by using a naïve and chronic corticosterone (CORT)-induced depression model in C57BL/6J mice, we aimed to investigate and compare the antidepressant potentials of ketamine metabolites (R)-NK, (S)-NK, (2R,6R)-HNK, and (2S,6S)-HNK on a depression-like state in the forced swim test and anhedonia in the female encounter test (Ago et al., 2015; Hasebe et al., 2017).

2.

Methods

2.1. Animals

All animal studies were approved by the Animal Care and Use Committee of the Graduate School of

Journal Pre-proof Pharmaceutical Sciences, Osaka University. All experimental procedures were conducted under the guidelines of the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Every effort was made to minimize animal suffering and to reduce the number of animals used. Five-week-old male C57BL/6J mice were obtained from SHIMIZU Laboratory Supplies Co., Ltd. (Kyoto, Japan) and housed in cages (28 cm × 17 cm × 12 cm) in groups of five or six animals under controlled environmental conditions (22 ± 1°C; 50 ± 10% relative humidity; a 12-hour

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light-dark cycle, lights on at 8:00 am; food and water ad libitum) for at least 1 week before use in the

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experiments.

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2.2. Drug treatments

Corticosterone was purchased from Sigma-Aldrich (St. Louis, MO, USA). (R)-NK, (S)-NK,

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(2R,6R)-HNK, and (2S,6S)-HNK were purchased from Tocris Bioscience (Bristol, UK).

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Corticosterone was suspended in 0.5% (w/v) carboxymethylcellulose. All other drugs were dissolved in saline (0.9% (w/v) solution of NaCl). Ketamine metabolites were administered intraperitoneally

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(i.p.) at a fixed volume of 10 mL/kg body weight. The doses of the drugs used here were selected according to previous studies (Fukumoto et al., 2019; Lumsden et al., 2019; Yang et al., 2017, 2018; Zanos et al., 2016, 2019a, b). To induce depression-like models, mice were subcutaneously (s.c.) injected once daily (between 10:00 and 12:00 h) with corticosterone (20 mg/10 mL/kg) for 21 consecutive days (Ago et al., 2008, 2013, 2015). Mice treated with a vehicle for the same period were used as the control group.

2.3. Forced swim test

The forced swim test was performed as previously reported (Ago et al., 2008, 2013; Kawasaki et al., 2011). Twenty-four hours after the last injection of corticosterone or vehicle, swimming sessions

Journal Pre-proof were conducted by placing a mouse in the individual acrylic cylinder (25 cm height × 19 cm diameter) containing 25 ± 1 °C water with a depth of 13 cm, so that the mice could not support themselves by touching the bottom with their paws. The performance of the mice for 6 min in the swimming session was videotaped using a digital video camera for subsequent analysis. After the session, the mice were removed from the cylinders, dried with paper towels and placed under a warming lamp until completely dry, and then returned to their home cages. The total immobility time

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was measured during the 6 min swimming session by an observer blind to the treatment.

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2.4. Female encounter test

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The female encounter test was performed as previously reported (Ago et al., 2015; Hasebe et al., 2017). Briefly, a sexually naïve 9-week-old male C57BL/6J mouse (test mouse) was placed in the

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central chamber of an opaque acrylic-modified polyvinyl chloride box (42 cm × 50 cm × 30 cm)

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divided into three interconnected chambers under the illumination of 400 lx (measured in the center zone) (Fig. 3A). The transparent partitions have openings that allow the animal to move freely from

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one chamber to another. After a 90-min habituation period, unfamiliar sexually naïve age- and strain-matched male and female mice were introduced into the intruder boxes (10 cm × 6.5 cm × 20 cm). The resident (test) and intruder mice were allowed to interact through the wire-mesh walls for 10 min, and then the intruder mice were removed. The behaviors of the test mouse were videotaped, and its occupancy in the box and locomotor path were automatically analyzed off-line using ANY-maze video-tracking software (Stoelting Co., Wood Dale, IL, USA). The amount of time spent in each of the three chambers was measured to evaluate the behavioral reactivity of mice to an intruder. Preference for a female encounter was then calculated as a percentage score for each intruder: Preference (%) = (time spent in the female zone/total time spent in the male and female zones) × 100.

Journal Pre-proof 2.5. Measurement of spontaneous locomotor activity

The locomotor activity of mice was measured using a digital counter system with an infrared sensor (Supermex®, Muromachi Kikai Co., Ltd., Tokyo, Japan) (Ago et al., 2008, 2013). Mice were placed individually in a novel clear plastic cage (28 cm × 17 cm × 12 cm). After a 3-h habituation period,

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mice were injected with ketamine metabolites, and then locomotor activity was recorded for 60 min.

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2.6. Statistics

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All results are presented as the mean ± standard error of the mean (SEM). Data were analyzed using

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a one-way analysis of variance (ANOVA) followed by Dunnett’s test for comparing the treated group with a control group. Statistical analyses were performed using the Statview 5.0J software package

Results

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3.

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statistically significant.

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for Apple Macintosh (SAS Institute Inc., Cary, NC, USA). A value of P < 0.05 was considered

Figure 1 shows the acute effects of ketamine metabolites on the immobility times of naïve male C57BL/6J mice in the forced swim test. (R)-NK, (S)-NK, (2R,6R)-HNK, or (2S,6S)-HNK at doses of 20 mg/kg did not affect the immobility times of mice at 30 min after the administration [F4,55 = 1.064, P = 0.3831]. None of ketamine metabolites affected locomotor activity [F4,55 = 0.724, P = 0.5794] (Table 1). Figure 2 shows the acute effects of ketamine metabolites on immobility time in chronic CORT-injected mice in the forced swim test. Chronic CORT increased the immobility times of mice, indicating an enhanced depression-like state. Under this condition, single administration of (S)-NK at 20 mg/kg, but not (R)-NK, significantly attenuated the enhanced immobility 30 min after

Journal Pre-proof administration [F4,55 = 4.039, P = 0.0061]. (2S,6S)-HNK (20 mg/kg), but not (2R,6R)-HNK, also attenuated this enhanced immobility 30 min after their respective administrations [F4,55 = 2.738, P = 0.0377]. Moreover, even higher dose (40 mg/kg) of (2R,6R)-HNK did not cause a significant decrease in immobility time in chronic CORT-injected mice [saline (mean ± SEM; n=12): 197.8 ± 5.8 s, (2R,6R)-HNK (mean ± SEM; n=12): 189.1 ± 8.0 s] (analyzed by Student’s t-test; P = 0.3890). Figure 3 shows the sustained antidepressant-like effects of ketamine metabolites on female

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preference in the female encounter test in chronic CORT- or vehicle-injected mice. Figure 3A shows a representative occupancy profile and the locomotor path of the test mouse (naïve male C57BL/6J

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mouse) during the 10 min female encounter test. Vehicle-injected control mice spent more time in the

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female zone than in the male or center zones, indicating a preference for the female encounter zone

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(Fig. 3B). Chronic CORT injection decreased female preference, indicating an anhedonic state. Single administration of (S)-NK and (2S,6S)-HNK, but not (R)-NK or (2R,6R)-HNK, at a dose of 20

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mg/kg significantly improved the decreased female preference 24 h after the administration [F4,55 =

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3.046, P = 0.0244] (Fig. 3C). In contrast, none of these ketamine metabolites affected female preference in chronic vehicle-injected control mice (data not shown). There is no significant

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difference between saline-treated chronic vehicle- and CORT-injected mice in the locomotor activity, and none of the drugs used here affected the locomotor activity of chronic CORT-injected mice [F5,66 = 0.600, P = 0.7000] (Fig. 3D).

4.

Discussion

In this study, we demonstrated that both (S)-NK and (2S,6S)-HNK exert rapid (30 min) and sustained (24 h) antidepressant-like effects in a chronic CORT-induced model of depression. There is a limited study investigating whether (R)-NK and (S)-NK have antidepressant activity, and an earlier study showed antidepressant-like effects of (R,S)-NK in male CD-1 mice (Sałat et al., 2015). A single administration of (R,S)-NK (50 mg/kg) has been shown to reduce the immobility time in the forced

Journal Pre-proof swim test at 30 min, but not 3 or 7 days after administration. Yang et al. (2018) recently showed that (S)-NK at doses of 5 and 10 mg/kg attenuated the increased immobility in lipopolysaccharide-treated C57BL/6J mice 3 h after injection, while (R)-NK at 20 mg/kg, but not at lower doses, attenuated the immobility. In a chronic social defeat stress-induced model, (S)-NK (10 mg/kg), but not (R)-NK (10 mg/kg), exhibits sustained (24 h) antidepressant-like effects. In this report, the potency of the antidepressant-like effects of (S)-NK was similar to that of its parent compound (S)-ketamine,

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although the antidepressant effects of (S)-NK were less potent than those of (R)-ketamine. Additionally, (R)-NK (20 mg/kg) did not show antidepressant effects in a rat learned helplessness

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model, although (R)-ketamine showed rapid and persistent antidepressant effects in the same model

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(Shirayama and Hashimoto, 2018). Of note, (S)-NK, but not (R)-NK showed anti-anhedonic effects 7

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days after a single dose in the sucrose preference test. We have developed a novel female encounter test to assess anhedonia, a marker of motivation (Ago et al., 2015). A male mouse prefers an

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encounter with a female mouse over a male mouse. The preference for female encounters was absent

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in mouse models of depression such as isolation-reared, chronic CORT-treated, and lipopolysaccharide-treated mice (Ago et al., 2015). The impaired preference in chronic CORT-treated

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mice was improved by a metabotropic glutamate 2/3 receptor antagonist LY341495, but not by a selective serotonin reuptake inhibitor fluvoxamine. Using this test, our study showed that (S)-NK and (2S,6S)-HNK improved the impaired female preference 24 h after injection, suggesting sustained anti-anhedonic effects.

Although ketamine metabolites might have potential antidepressant properties, controversial results have been reported for (2R,6R)-HNK in particular. (2R,6R)-HNK at doses of 5 mg/kg, and more exhibited acute antidepressant-like effects in naïve CD-1 mice in the forced swim test 1 h after injection (Zanos et al., 2016), while in the present study 20 mg/kg of (2R,6R)-HNK did not exhibit anti-depressant-like effects in naïve C57BL/6J mice. This finding suggests that there is a strain difference that accounts for the observed effects. Previous studies also showed that neither systemic (10

mg/kg)

nor

intracerebroventricular

(20

μg)

injection

of

(2R,6R)-HNK

affected

Journal Pre-proof lipopolysaccharide-induced or social defeat stress-induced increases in the immobility of C57BL/6J mice in the forced swim and tail suspension tests at 3–4 h after the injection (Zhang et al., 2018a; Yang et al., 2017). We found that (2R,6R)-HNK at 10, 20 or 40 mg/kg did not affect the enhanced immobility of CORT-injected mice in the forced swim test 30 min after treatment. Regarding the pharmacokinetic profiles of ketamine and its metabolites, concentrations of (R)-ketamine, (R)-NK, and (2R,6R)-HNK in the brain of male adult CD-1 mice at 10 min after i.p. administration of

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(R)-ketamine (10 mg/kg) were about 1.2, 1.1, and 0.4 μg/g, respectively (Zanos et al., 2019a). For male adult C57BL/6J mice, brain levels of (R)-ketamine, (R)-NK, and (2R,6R)-HNK at 10 min after

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i.p. administration of (R)-ketamine (10 mg/kg) were about 3.4, 1.8, and 0.8 μg/g, respectively (Zhang

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et al., 2018b). Additionally, brain (2R,6R)-HNK level in CD-1 mice at 5 min after i.p. administration

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of (2R,6R)-HNK (10 mg/kg) was about 4.5 μg/g (Lumsden et al., 2019). Although the data were obtained from lipopolysaccharide-treated mice, brain (2R,6R)-HNK level in C57BL/6J mice at 5 min

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after i.p. administration of (2R,6R)-HNK (10 mg/kg) was about 10 μg/g (Yamaguchi et al., 2018).

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These findings suggest that the brain concentrations of (2R,6R)-HNK in C57BL/6J mice following the administration of (R)-ketamine or (2R,6R)-HNK (10 mg/kg) are rather higher than those in CD-1

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mice. Therefore, the difference in pharmacokinetic profile between mouse strains cannot simply explain why (2R,6R)-HNK did not exhibit antidepressant-like effects in the present study. Regarding the sustained antidepressant effects, (2R,6R)-HNK has been reported to exert antidepressant-like effects in the forced swim test (naïve CD-1 and BALB/cJ mice) (Pham et al., 2018; Zanos et al., 2016) and the anti-anhedonic effects in the sucrose preference and female urine sniffing tests (chronic CORT-injected CD-1 mice) 24 h after the injection (Zanos et al., 2016). In contrast, the present study showed that (2R,6R)-HNK did not affect the impairment of female preference 24 h after the treatment, suggesting no anti-anhedonic activity. The reason for this discrepancy is currently unknown. Additionally, 10 mg/kg of (2R,6R)-HNK did not exhibit sustained antidepressant-like effects in a social defeat stress model using C57BL/6J mice 24 h after the injection (Yang et al., 2017). These findings suggest that (2R,6R)-HNK might produce acute and sustained

Journal Pre-proof antidepressant-like effects under certain limited conditions or environments. In this study, we unexpectedly observed that (2S,6S)-HNK at 20 mg/kg reduced the enhanced immobility 30 min after injection and improved impaired female preference 24 h after injection in chronic CORT-treated mice, suggesting potent antidepressant-like and anti-anhedonic effects. Few reports are investigating the effects of (2S,6S)-HNK, but Zanos et al. (2016) have demonstrated using naïve male CD-1 mice in the forced swim test that the administration of (2S,6S)-HNK at the dose of

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25 mg/kg induced antidepressant effects at 1 and 24 h after injection. In contrast, in the present study, (2S,6S)-HNK did not affect the immobility times of naïve C57BL/6J mice. A single injection of

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(2S,6S)- HNK also induced antidepressant-like effects in the learned helplessness test at the dose of

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75 mg/kg, but not 25 mg/kg (Zanos et al., 2016). Although the antidepressant potency might differ

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between the mouse strains and animal models used, these findings suggest that (2S,6S)-HNK have rapid and sustained antidepressant properties in depression models. (2S,6S)-HNK had a low affinity

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for the NMDA receptor (Lumsden et al., 2019; Yang et al., 2019) and it did not affect the in vivo

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release of monoamines in mouse prefrontal cortex (Ago et al., 2019). Thus, the mechanism

needed.

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underlying the antidepressant effects of (2S,6S)-HNK remains unclear, and further investigation is

In conclusion, we demonstrated the antidepressant-like and anti-anhedonic actions of (S)-NK and (2S,6S)-HNK in a chronic CORT-induced model of depression. Both (S)-ketamine metabolites have little effects on locomotor activity. A chronic CORT-induced model might have an aspect of treatment-resistant depression, which neither the classical tricyclic antidepressant desipramine nor the selective serotonin reuptake inhibitor fluvoxamine affected the enhanced immobility (Ago et al., 2013, 2015; Koike et al., 2013; Fukumoto et al., 2017). Therefore, (S)-NK and (2S,6S)-HNK might represent safe alternative antidepressants with high potency.

Funding

Journal Pre-proof This study was supported in part by grants from the JSPS KAKENHI, grant numbers JP15H04645 (T.N.), JP18H02574 (T.N.), JP17K19488 (H.H.), JP17H03989 (H.H.), and JP16K08268 (Y.A.); MEXT KAKENHI, grant numbers JP19H04909 (T.N.), JP19H05218 (T.N.), and JP18H05416 (H.H.);

AMED,

grant

numbers

JP19gm1310003

(T.N.),

JP19dm0107122

(H.H.),

and

JP19dm0207061 (H.H.), and JP19dm0107119 (K.H.); grants from the Takeda Science Foundation (H.H. and Y.A.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (Y.A.),

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the Pharmacological Research Foundation, Tokyo (Y.A.).

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Acknowledgments

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This work was also supported in part by AMED under grant number JP19am0101084 (Kazutake Tsujikawa, Ph.D., Graduate School of Pharmaceutical Sciences, Osaka University). We thank Trent

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Conflict of Interest

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this manuscript.

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Rogers, Ph.D. from Edanz Group (https://en-author-services.edanzgroup.com/) for editing a draft of

Dr. Kenji Hashimoto is the inventor of filed patent applications ‘The use of (R)-ketamine in the treatment of psychiatric diseases’ and ‘(S)-norketamine and a salt thereof as pharmaceutical’ by Chiba University. Dr. Kenji Hashimoto also declares that he has received research support and consultant fees from Sumitomo Dainippon Pharma Co., Ltd., Otsuka Pharmaceutical Co., Ltd., and Taisho Pharmaceutical Co., Ltd. The other authors declare no conflicts of interest.

Journal Pre-proof

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Figure legends

Figure 1. The acute effects of ketamine metabolites on the immobility time of naïve male C57BL/6J mice in the forced swim test. (R)-NK (20 mg/kg), (S)-NK (20 mg/kg), (2R,6R)-HNK (20 mg/kg), (2S,6S)-HNK (20 mg/kg), or saline was i.p. injected 30 min before the experiments. Results are expressed as the mean ± SEM of 12 mice per group.

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Figure 2. The acute effects of ketamine metabolites on the increased immobility in chronic corticosterone-treated mice in the forced swim test. Mice were s.c. injected with vehicle or

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corticosterone (CORT, 20 mg/kg) once daily for 21 consecutive days. Twenty-four hours after the

-p

last injection, the forced swim test was performed. (R)-NK (10, 20 mg/kg), (S)-NK (10, 20 mg/kg),

re

(2R,6R)-HNK (10, 20 mg/kg), (2S,6S)-HNK (10, 20 mg/kg), or saline was i.p. injected 30 min before the test session. Results are expressed as the mean ± SEM of 12 mice per group.

P < 0.01,

P < 0.001 compared with the saline-treated chronic vehicle treatment group (Student’s t-test), #P <

lP

***

**

na

0.05, ##P < 0.01 compared with the saline-treated chronic CORT treatment group (Dunnett’s test). Figure 3. The sustained effects of ketamine metabolites on impaired female preference in chronic

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corticosterone-treated mice in the female encounter test. (A) Images of the three-chambered apparatus and interacting mice. Representative occupancy in the apparatus and the locomotor path of the resident mice during a 10-min encounter analyzed by the ANY-maze video tracking system. (B, C) Twenty-four hours after the forced swim test (fig. 2), mice treated with (R)-NK (20 mg/kg), (S)-NK (20 mg/kg), (2R,6R)-HNK (20 mg/kg), (2S,6S)-HNK (20 mg/kg), or saline were subjected to the female encounter test. Time spent in each zone (B) and preference (C) for a female encounter by the resident test mouse are shown. (D) The total distance traveled by the mice during a 10-min encounter was analyzed as locomotor activity. Results are expressed as the mean ± SEM of 12 mice per group. ††P < 0.01,

†††

P < 0.001 compared with the time spent in male zone. *P < 0.05 compared

with saline-treated chronic vehicle treatment group (Student’s t-test), #P < 0.05 compared with saline-treated chronic CORT treatment group (Dunnett’s test).

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Table 1. Effects of ketamine metabolites on the spontaneous locomotor activity of C57BL/6J mice Dose

Locomotor activity (counts)

Saline

–

5,134 ± 1089

(R)-NK

20 mg/kg

6,246 ± 885

(S)-NK

20 mg/kg

7,392 ± 1016

(2R,6R)-HNK

20 mg/kg

6,432 ± 798

(2S,6S)-HNK

20 mg/kg

5,961 ± 982

of

Treatment

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After a 3-h habituation period to a novel environment, mice were intraperitoneally injected with

-p

ketamine metabolites or saline, and then the locomotor activity was measured for 60 min. Results are

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na

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expressed as the mean ± SEM of 12 mice per group.

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Highlights

Chronic corticosterone induced a depression-like state and anhedonia in mice.

•

No ketamine metabolite showed antidepressant effects in naïve C57BL/6J mice.

•

(S)-NK and (2S,6S)-HNK reversed the enhanced immobility in the forced swim test.

•

(S)-NK and (2S,6S)-HNK improved impaired female preferences.

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ro

of

•

Figure 1

Figure 2

Figure 3