Lack of Antidepressant Effects of (2R,6R)-Hydroxynorketamine in a Rat Learned Helplessness Model: Comparison with (R)-Ketamine

Lack of Antidepressant Effects of (2R,6R)-Hydroxynorketamine in a Rat Learned Helplessness Model:... Background: (R)-Ketamine exhibits rapid and sustained antidepressant effects in animal models of depression. It is stereoselectively metabolized to (R)-norketamine and subsequently to (2R,6R)-hydroxynorketamine in the liver. The metabolism of ketamine to hydroxynorketamine was recently demonstrated to be essential for ketamine’s antidepressant actions. However, no study has compared the antidepressant effects of these 3 compounds in animal models of depression. Methods: The effects of a single i.p. injection of (R)-ketamine, (R)-norketamine, and (2R,6R)-hydroxynorketamine in a rat learned helplessness model were examined. Results: A single dose of (R)-ketamine (20 mg/kg) showed an antidepressant effect in the rat learned helplessness model. In contrast, neither (R)-norketamine (20 mg/kg) nor (2R,6R)-hydroxynorketamine (20 and 40 mg/kg) did so. Conclusions: Unlike (R)-ketamine, its metabolite (2R,6R)-hydroxynorketamine did not show antidepressant actions in the rat learned helplessness model. Therefore, it is unlikely that the metabolism of ketamine to hydroxynorketamine is essential for ketamine’s antidepressant actions. Keywords: metabolism, (R)-ketamine, (R)-norketamine (2R,6R)-hydroxynorketamine, learned helplessness Introduction Recently conducted meta-analyses revealed that the -meth N yl- approximately 3- to 4-fold greater anesthetic potency and D-aspartate receptor antagonist ketamine exhibits rapid and sus- greater undesirable psychotomimetic side effects than ( )- R tained antidepressant effects in patients with treatment-resistant ketamine (Domino et al., 2010). Several groups including our depression (Newport et al, 2015; Kishimoto et al, 2016). Thus, keta- own have demonstrated that ()-ketamine sho R wed greater mine is the most attractive antidepressant for the treatment of potency and longer-lasting antidepressant effects than ( )-S treatment-resistant depression (Monteggia and Zarate, 2015; ketamine in animal models of depression (Zhang et al., 2014; Duman et al., 2016; Hashimoto, 2016b), although the precise mech- Yang et al., 2015, 2017, 2018; Zanos et al., 2016; Fukumoto anisms underlying its antidepressant actions remain unknown. et al., 2017). Unlike (S)-ketamine, (R)-ketamine does not induce (R,S)-Ketamine is a racemic mixture containing equal psychotomimetic side effects or exhibit abuse potential in parts of (R)-ketamine and (S)-ketamine. (S)-Ketamine shows rodents (Yang et al., 2015). Furthermore, single or repeated Received: August 8, 2017; Revised: October 24, 2017; Accepted: November 14, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 84 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/1/84/4633900 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Shirayama and Hashimoto | 85 Significance Statement The rapid and sustained antidepressant effects of ketamine in patients with treatment-resistant depression are the most important discovery in the field of depression research in a half-century. However, the precise mechanisms underlying the antidepressant effects of ketamine remain unknown. A recent study (Zanos et al., 2016) reported that the metabolism of ketamine to hydroxynorketamine (HNK) is essential for ketamine’s antidepressant effects. In particular, (2R,6R)-HNK, a metabolite of (R)-ketamine, plays a key role in the antidepressant actions. However, here we report that, unlike (R)-ketamine, its metabolites (R)-norketamine and (2R,6R)-HNK did not elicit antidepressant effects in a rat learned helplessness model. It is, therefore, unlikely that the metabolism of ketamine to HNK is necessary for ketamine’s antidepressant actions. intermittent administration of ()-ketamine S , but not of (R )- Methods and Materials ketamine, resulted in the loss of parvalbumin immunoreactiv- ity in the prefrontal cortex and hippocampus (Yang et al., 2015, Animals 2016). Moreover, with the results using [C]raclopride and posi- Male Sprague-Dawley rats (200–230 g, 7 weeks old; Charles-River tron emission tomography, we reported a marked reduction Japan) were used. The animals were housed under a 12-h-light/- of dopamine D receptor binding in the conscious monkey 2/3 dark cycle with free access to food and water. The protocol was striatum after a single infusion of ()-ketamine S , but not of (R )- approved by the Chiba University Institutional Animal Care and ketamine (Hashimoto et al., 2017). These findings suggest that Use Committee (permission no: 28–394 and 29–328). All efforts (S)-ketamine, but not (R )-ketamine, can cause a marked release were made to minimize suffering. of dopamine from presynaptic terminals, which is associated with acute psychotomimetic effects (Hashimoto et al., 2017). Taking these findings together, (R)-ketamine could be a poten- Drugs tially safer antidepressant without detrimental side effects in humans than (S)-ketamine (Hashimoto, 2016a, 2016b, 2017). (R)-Ketamine hydrochloride was prepared by recrystallization of It is well known that ketamine is rapidly metabolized into nor - (R,S)-ketamine (Ketalar, ketamine hydrochloride, Daiichi Sankyo ketamine and subsequently into hydroxynorketamine (HNK) by Pharmaceutical Ltd) and D-(-)-tartaric acid, as described previ- microsomal cytochrome P450 enzymes (through N-demethylation ously (Zhang et al., 2014). (R)-Norketamine hydrochloride was and hydroxylation) in the liver (Figure 1) (Turfus et al., 2009 Zhao ; prepared as described previously (Zanos et al., 2016). The purity et  al., 2012; Zanos et  al., 2016; Hashimoto, 2017). The metabo- of these stereoisomers was determined by a high-performance lism of ketamine to HNK was also recently demonstrated to be liquid chromatography (CHIRALPAK IA, column size: 250 x 4.6 essential for the antidepressant actions of ketamine (Zanos et al., mm, mobile phase: n-hexane/dichloromethane/diethylamine 2016). In particular, (2R ,6R)-HNK, a metabolite from (R)-ketamine, (75/25/0.1), Daicel Corporation). (2R,6R)-HNK hydrochloride was plays a key role in the antidepressant actions (Zanos et al., 2016). provided from Taisho Pharmaceutical Ltd as reported previ- However, increasing attention has been drawn to the antide- ously (Zanos et al., 2016). ( R)-Ketamine, (R)-norketamine, and pressant actions of (2R ,6R)-HNK (Abdallah, 2017). In the present (2R,6R)-HNK were dissolved in 0.9% NaCl. Other compounds study, we examined the effects of a single systemic injection of were purchased commercially. The doses of (R)-ketamine and (R)-ketamine and its two major metabolites, (R )-norketamine and its metabolites were selected as previously reported (Yang et al., (2R,6R)-HNK, in a rat learned helplessness (LH) model. 2015, 2017; Zanos et al., 2016). Figure 1. Metabolism of (R)-ketamine in the liver. In the liver, (R)-ketamine is metabolized to (R)-norketamine (major pathway) and (2R,6R)-hydroxyketamine (minor pathway), subsequently (2R,6R)-hydroxynorketamine (HNK). Downloaded from https://academic.oup.com/ijnp/article-abstract/21/1/84/4633900 by Ed 'DeepDyve' Gillespie user on 16 March 2018 86 | International Journal of Neuropsychopharmacology, 2018 2, the rats were subjected to 30 inescapable electric foot-shocks Stress Paradigm (LH Model) (0.65 mA, 30-second duration, administered at random intervals To create an LH paradigm, the animals are initially exposed to averaging 18–42 seconds) (Figure 2A, C). On day 3, a 2-way con- ditioned avoidance test was performed as a post-shock test to uncontrollable stress. When the animal is later placed in a situ- ation where the shock is controllable (escapable), the animal not determine whether the rats would exhibit the predicted escape deficits (Figure 2A, C). This screening session consisted of 30 tri- only fails to acquire the escape response but also often makes no efforts to escape the shock at all. The LH behavioral tests als in which electric foot-shocks (0.65 mA, 6-second duration, administered at random intervals with a mean of 30 seconds) were performed using the Gemini Avoidance System (San Diego Instruments) (Shirayama et  al., 2015, 2017). This apparatus is were preceded by a 3-second conditioned stimulus tone that remained on until the shock was terminated. Rats with more divided into 2 compartments by a retractable door. On days 1 and Figure 2. Effects of a single injection of (R)-ketamine, (R)-norketamine, and (2R,6R)- HNK in a rat LH model. (A) Rats received inescapable electric shock (IES) treatments on 2 days (days 1 and 2), passed a post-shock test (PS) on day 3, and were designated as learned helplessness (LH) rats with depression-like phenotype. On day 3, vehicle (saline: 2 mL/kg), (R)-ketamine (20 mg/kg), (R)-norketamine (20 mg/kg), or (2R,6R)- HNK (20 and 40 mg/kg) was administered i.p. into LH rats. On day 8 (5 days after a single injection), conditioned avoidance (CA) test to study the antidepressant effect was performed. (B) The failure number of LH (1-way ANOVA: F  = 3.755, P = .0167). 4,24 The escape latency of LH (1-way ANOVA: F  = 3.973, P = .013). Data are shown as mean ± SEM (n = 5–8). The number in the parenthesis is the dose (mg/kg). *P < .05 4,24 compared with vehicle-treated group. (C) Rats received IES treatments on 2 days (days 1 and 2), passed a PS on day 3, and were designated as LH rats with depression- like phenotype. On day 3, either vehicle (saline: 2 mL/kg), (R)-ketamine (20 mg/kg), or (2R,6R)- HNK (20 mg/kg) was administered i.p. into LH rats. CA test was performed on day 4 (24 hours after a single injection). (D) The failure number of LH (1-way ANOVA: F  = 13.52, P < .0001). The escape latency of LH (1-way ANOVA: F  = 14.73, 2,14 2,14 P = .0004). Data are shown as mean ± SEM (n = 5 or 6). The number in the parenthesis is the dose (mg/kg). **P < .01, ***P < .001 compared with vehicle-treated group. R-KT: (R)-ketamine, R-NKT: (R)-norketamine, R-HNK: (2R,6R)-hydroxynorketamine. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/1/84/4633900 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Shirayama and Hashimoto | 87 than 25 escape failures among the 30 trials were regarded as model of depression, although (2R,6R)-HNK did not have anti- having reached the LH criterion and were used in further experi- depressant effects (Yang et al., 2017). Collectively, it seems that ments. Approximately 65% of the rats met this criterion. unlike (R)-ketamine, (2R,6R)-HNK does not have an antidepres- In the experiment 1, on day 3, the rats received i.p. injection sant effect in rodent models of depression, inconsistent with the of saline (2  mL/kg), (R)-ketamine (20  mg/kg), (R)-norketamine findings by Zanos et al. (2016). (20 mg/kg), or (2R,6R)-HNK (20 and 40 mg/kg) (Figure 2A). On day Zanos et al. (2016) reported more potent antidepressant 8 (5 days after a single injection), a 2-way conditioned avoidance effects of (2R,6R)-HNK, which is exclusively derived from (R)- test was performed (Figure  2A). In the experiment 2, on day 3, ketamine. A single injection of (2R,6R)-HNK (10 or 20 mg/kg) the rats received i.p. injection of saline (2 mL/kg), (R)-ketamine reversed chronic corticosterone-induced anhedonia assessed (20  mg/kg), or (2R,6R)-HNK (20mg/kg) (Figure  2C). On day 4 (24 with the sucrose preference and female urine sniffing behav- hours after a single injection), a 2-way conditioned avoidance ioral tasks as well as social avoidance induced by chronic social test was performed (Figure  2C). This test session consisted of defeat stress (Zanos et al., 2016). They reported sustained (24 30 trials in which electric foot-shocks (0.65 mA, 30-second dur - hours) antidepressant effects of (2R,6R)-HNK in LH model (Zanos ation, administered at random intervals with a mean of 30 sec- et al, 2016). However, we could not find sustained (24 hours) anti- onds) were preceded by a 3-second conditioned stimulus tone depressant effects of (2R,6R)-HNK (20 mg/kg) in the LH model, that remained on until the shock was terminated. The number although (R)-ketamine (20 mg/kg) showed sustained (24 hours) of escape failures and the latency until escape for each of the 30 antidepressant effects in the same model (Figure 2D). Thus, we trials were recorded by the Gemini Avoidance System. were unable to detect antidepressant activity induced by (2R,6R)- HNK in any of our 3 models (inflammation, social defeat stress, and LH), although rapid and sustained antidepressant effects Statistical Analysis were detected for (R)-ketamine (Yang et al., 2017; this study). The The data are shown as the mean ± SEM. The analyses were per - reasons for this discrepancy (Zanos et al., 2016, vs Yang et al., formed using GraphPad Prism 5 (GraphPad Software Inc). The 2017, and this study) remain unknown. Nonetheless, our nega- data were analyzed using 1-way ANOVA, followed by posthoc tive findings regarding the lack of an antidepressant effect of Tukey test. The criterion for significance was P < .05. (2R,6R)-HNK in rodents with depression-like phenotype need to be replicated by other groups in future studies. It is well known that (R)-ketamine is stereoselectively Results N-demethylated by liver microsomal cytochrome P450 into (R)-norketamine (Hijazi and Boulieu, 2002; Desta et  al., 2012; Effects of a Single Intraperitoneal Injection of (R)- Zanos et  al., 2016) (Figure  1). ( R)-Norketamine is further Ketamine, (R)-Norketamine, and (2R,6R)-HNK in metabolized to (2R,6R)-HNK arising from hydroxylation of the LH Rats cyclohexanone ring (Figure 1). In addition to -demeth N ylation, (R)-ketamine is also metabolized by the hydroxylation of the cyclohexanone ring to produce (2R ,6R)-hydroxyketamine. Experiment 1 (2R,6R)-HNK is also prepared by the N-demethylation of To examine the antidepressant effects of (R)-ketamine and its (2R,6R)-hydroxyketamine (Desta et  al., 2012; Zanos et  al., 2 metabolites in a LH model, saline (2  mL/kg), (R)-ketamine 2016) (Figure  1). A  study showed that the plasma levels of (20 mg/kg), (R)-norketamine (20 mg/kg), or (2R,6R)-HNK (20 and norketamine and HNK were higher after a single injection of 40  mg/kg) was administered i.p. into the LH rats. The LH rats ketamine, although plasma levels of hydroxyketamine were that received a single injection of (R)-ketamine (20 mg/kg, 5 days very low (Zanos et al., 2016), suggesting that the metabolism after a single injection) exhibited significant improvements in of (R)-ketamine to (2R,6R)-HNK via (R)-norketamine is the their conditioned avoidance test results, relative to vehicle- major pathway of (R)-ketamine in mice (Figure  1). Therefore, treated LH rats (Figure 2A,B). In contrast, a single administration it is noteworthy that ()-norketamine and (2 R R,6R)-HNK, the of (R)-norketamine (20 mg/kg), or (2R,6R)-HNK (20 and 40 mg/kg) two major metabolites from (R)-ketamine, did not exert anti- did not improve their conditioned avoidance test results in LH depressant effects, although antidepressant effects of ( )-R rats (Figure 2A,B). ketamine were detected in the same model. We have recently reported that a single bilateral infu- Experiment 2 sion of (R)-ketamine into the infralimbic (IL) area of the The LH rats that received a single injection of (R)-ketamine medial prefrontal cortex (mPFC) and the DG and CA3 of (20 mg/kg, 24 hours after a single injection) exhibited significant the hippocampus exerted antidepressant effects in LH rats improvements in their conditioned avoidance test results rela- (Shirayama and Hashimoto, 2017). Furthermore, a study tive to vehicle-treated LH rats (Figure 2C,D). In contrast, (2R,6R)- showed that neuronal inactivation of the IL of mPFC com- HNK (20 mg/kg, 24 hours after a single injection) did not improve pletely blocked the antidepressant effects of (R,S)-ketamine, their conditioned avoidance test results in LH rats (Figure 2C,D). and that microinfusion of (R,S)-ketamine into IL of mPFC produced an antidepressant effect in control unstressed rats Discussion (Fuchikami et al., 2015). These findings suggest a crucial role for the IL area of the mPFC, DG, and CA3 in the antidepres- In the present study, we established that a single systemic (R)- sant action of (R )-ketamine itself (NOT metabolite) in a rat ketamine injection (24 hours and 5 days after a single injection) LH model, since (R )-ketamine (or (R,S)-ketamine) might not showed antidepressant effects in a rat LH model of depres- be metabolized in the brain. Given the key role of hepatic sion, whereas a single systemic injection of (R)-norketamine cytochrome P450 enzymes in ketamine metabolism (Turfus or (2R,6R)-HNK did not. It should be noted that a higher dose et al., 2009; Zhao et al., 2012; Zanos et al., 2016), it is unlikely (40 mg/kg) of (2R,6R)-HNK did not have antidepressant effects. that the metabolism of (R)-ketamine in the liver plays a role We have also recently reported the potent and longer-lasting in the antidepressant actions of ()-ketamine R . antidepressant effects of (R)-ketamine in a social defeat stress Downloaded from https://academic.oup.com/ijnp/article-abstract/21/1/84/4633900 by Ed 'DeepDyve' Gillespie user on 16 March 2018 88 | International Journal of Neuropsychopharmacology, 2018 In conclusion, unlike (R)-ketamine, neither (R)-norketamine Kishimoto T, Chawla JM, Hagi K, Zarate CA, Kane JM, Bauer nor (2R,6R)-HNK elicited antidepressant effects in LH model, M, Correll CU (2016) Single-dose infusion ketamine and suggesting that the metabolism of (R)-ketamine might not play non-ketamine N-methyl-D-aspartate receptor antago- a key role in its robust antidepressant action. nists for unipolar and bipolar depression: a meta-analy- sis of efficacy, safety and time trajectories. Psychol Med 46:1459–1472. Acknowledgments Monteggia LM, Zarate C Jr (2015) Antidepressant actions of keta- mine: from molecular mechanisms to clinical practice. Curr We thank Dr. Shigeyuki Chaki (Taisho Pharmaceutical Co, Ltd) for providing (2R,6R)-HNK. We also thank Yuko Fujita (Chiba Opin Neurobiol 30:139–143. Newport DJ, Carpenter LL, McDonald WM, Potash JB, Tohen M, University) for her technical assistance. This study was supported by Strategic Research Program for Nemeroff CB, APA Council of Research Task Force on Novel Biomarkers and Treatments (2015) Ketamine and other Brain Sciences, AMED, Japan (to K.H.). NMDA antagonists: early clinical trials and possible mecha- nisms in depression. Am J Psychiatry 172:950–966. Statement of Interest Shirayama Y, Hashimoto K (2017) Effects of a single bilateral infusion of R-ketamine in the brain regions of a learned Dr. Shirayama has received research support from Eli Lilly, Eisai, helplessness model of depression. Eur Arch Psychiatry Clin MSD, Pfizer, and Mitsubishi-Tanabe. Dr. Hashimoto has received Neurosci 267:177–182. research support from Dainippon-Sumitomo, Mochida, Otsuka, Shirayama Y, Yang C, Zhang JC, Ren Q, Yao W, Hashimoto K (2015) and Taisho. Dr. Hashimoto is an inventor on a filed patent appli- Alterations in brain-derived neurotrophic factor (BDNF) cation on “The use of (R)-ketamine in the treatment of psychi- and its precursor proBDNF in the brain regions of a learned atric diseases” by Chiba University. helplessness rat model and the antidepressant effects of a TrkB agonist and antagonist. Eur Neuropsychopharmacol References 25:2449–2458. Abdallah CG (2017) What’s the buzz about hydroxynorketamine? Turfus SC, Parkin MC, Cowan DA, Halket JM, Smith NW, Is it the history, the story, the debate, or the promise? Biol Braithwaite RA, Elliot SP, Steventon GB, Kicman AT (2009) Psychiatry 81:61–63. Use of human microsomes and deuterated substrates: an Desta Z, Moaddel R, Ogburn ET, Xu C, Ramamoorthy A, Venkata alternative approach for the identification of novel metabo- SL, Sanghvi M, Goldberg ME, Torjman MC, Wainer IW (2012) lites of ketamine by mass spectrometry. Drug Metab Dispos Stereoselective and regiospecific hydroxylation of ketamine 37:1769–1778. and norketamine. Xenobiotica 42:1076–1087. Yang C, Shirayama Y, Zhang JC, Ren Q, Yao W, Ma M, Dong C, Domino EF (2010) Taming the ketamine tiger. 1965. Hashimoto K (2015) R-ketamine: a rapid-onset and sustained Anesthesiology 113:678–684 antidepressant without psychotomimetic side effects. Transl Duman RS, Aghajanian GK, Sanacora G, Krystal JH (2016) Psychiatry 5:632. Synaptic plasticity and depression: new insights from stress Yang C, Han M, Zhang JC, Ren Q, Hashimoto K (2016) Loss of and rapid-acting antidepressants. Nat Med 22:238–249. parvalbumin-immunoreactivity in mouse brain regions after Fuchikami M, Thomas A, Liu R, Wohleb ES, Land BB, DiLeone RJ, repeated intermittent administration of esketamine, but not Aghajanian GK, Duman RS (2015) Optogenetic stimulation of R-ketamine. Psychiatric Res 239:281–283. infralimbic PFC reproduces ketamine’s rapid and sustained Yang C, Qu Y, Abe M, Nozawa D, Chaki S, Hashimoto K (2017) antidepressant actions. Proc Natl Acad Sci USA 112:8106–8111. (R)-ketamine shows greater potency and longer last- Fukumoto K, Toki H, Iijima M, Hashihayata T, Yamaguchi JI, ing antidepressant effects than its metabolite (2R,6R)- Hashimoto K, Chaki S (2017) Antidepressant potential of (R)- hydroxynorketamine. Biol Psychiatry 82:43–44. ketamine in rodent models: comparison with (S)-ketamine. J Yang C, Ren Q, Qu Y, Zhang JC, Ma M, Dong C, Hashimoto K (2018) Pharmacol Exp Ther 361:9–16. Mechanistic target of rapamycin-independent antidepres- Hashimoto K (2016a) R-ketamine: a rapid-onset and sustained sant effects of (R)-ketamine in a social defeat stress model. antidepressant without risk of brain toxicity. Psychol Med Biol Psychiatry 83:18–28. 46:2449–2451. Zanos P, Moaddel R, Morris PJ, Georgiou P, Fischell J, Elmer GI, Hashimoto K (2016b) Ketamine’s antidepressant action: Alkondon M, Yuan P, Pribut HJ, Singh NS, Dossou KS, Fang Y, beyond NMDA receptor inhibition. Expert Opin Ther Targets Huang XP, Mayo CL, Wainer IW, Albuquerque EX, Thompson 20:1389–1392. SM, Thomas CJ, Zarate CA Jr, Gould TD (2016) NMDAR- Hashimoto K (2017) Rapid antidepressant activity of ketamine independent antidepressant actions of ketamine metabo- beyond NMDA receptor. In: The NMDA Receptors (Hashimoto lites. Nature 533:481–486. K, ed), pp 69–81. New York: Humana Press. Zhao X, Venkata SL, Moaddel R, Luckenbaugh DA, Brutsche Hashimoto K, Kakiuchi T, Ohba H, Nishiyama S, Tsukada H NE, Ibrahim L, Zarate CA Jr, Mager DE, Wainer IW (2012) (2017) Reduction of dopamine D receptor binding in the Simultaneous population pharmacokinetic modelling of 2/3 striatum after a single administration of esketamine, but ketamine and three major metabolites in patients with not R-ketamine: a PET study in conscious monkeys. Eur Arch treatment-resistant bipolar depression. Br J Clin Pharmacol Psychiatry Clin Neurosci 267:173–176. 74:304–314. Hijazi Y, Boulieu R (2002) Contribution of CYP3A4, CYP2B6, and Zhang JC, Li SX, Hashimoto K (2014) R(-)-ketamine shows greater CYP2C9 isoforms to N-demethylation of ketamine in human potency and longer lasting antidepressant effects than S(+)- liver microsomes. Drug Metab Dispos 30:853–858. ketamine. Pharmacol Biochem Behav 116:137–141. 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Lack of Antidepressant Effects of (2R,6R)-Hydroxynorketamine in a Rat Learned Helplessness Model: Comparison with (R)-Ketamine

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Abstract

Background: (R)-Ketamine exhibits rapid and sustained antidepressant effects in animal models of depression. It is stereoselectively metabolized to (R)-norketamine and subsequently to (2R,6R)-hydroxynorketamine in the liver. The metabolism of ketamine to hydroxynorketamine was recently demonstrated to be essential for ketamine’s antidepressant actions. However, no study has compared the antidepressant effects of these 3 compounds in animal models of depression. Methods: The effects of a single i.p. injection of (R)-ketamine, (R)-norketamine, and (2R,6R)-hydroxynorketamine in a rat learned helplessness model were examined. Results: A single dose of (R)-ketamine (20 mg/kg) showed an antidepressant effect in the rat learned helplessness model. In contrast, neither (R)-norketamine (20 mg/kg) nor (2R,6R)-hydroxynorketamine (20 and 40 mg/kg) did so. Conclusions: Unlike (R)-ketamine, its metabolite (2R,6R)-hydroxynorketamine did not show antidepressant actions in the rat learned helplessness model. Therefore, it is unlikely that the metabolism of ketamine to hydroxynorketamine is essential for ketamine’s antidepressant actions. Keywords: metabolism, (R)-ketamine, (R)-norketamine (2R,6R)-hydroxynorketamine, learned helplessness Introduction Recently conducted meta-analyses revealed that the -meth N yl- approximately 3- to 4-fold greater anesthetic potency and D-aspartate receptor antagonist ketamine exhibits rapid and sus- greater undesirable psychotomimetic side effects than ( )- R tained antidepressant effects in patients with treatment-resistant ketamine (Domino et al., 2010). Several groups including our depression (Newport et al, 2015; Kishimoto et al, 2016). Thus, keta- own have demonstrated that ()-ketamine sho R wed greater mine is the most attractive antidepressant for the treatment of potency and longer-lasting antidepressant effects than ( )-S treatment-resistant depression (Monteggia and Zarate, 2015; ketamine in animal models of depression (Zhang et al., 2014; Duman et al., 2016; Hashimoto, 2016b), although the precise mech- Yang et al., 2015, 2017, 2018; Zanos et al., 2016; Fukumoto anisms underlying its antidepressant actions remain unknown. et al., 2017). Unlike (S)-ketamine, (R)-ketamine does not induce (R,S)-Ketamine is a racemic mixture containing equal psychotomimetic side effects or exhibit abuse potential in parts of (R)-ketamine and (S)-ketamine. (S)-Ketamine shows rodents (Yang et al., 2015). Furthermore, single or repeated Received: August 8, 2017; Revised: October 24, 2017; Accepted: November 14, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 84 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/1/84/4633900 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Shirayama and Hashimoto | 85 Significance Statement The rapid and sustained antidepressant effects of ketamine in patients with treatment-resistant depression are the most important discovery in the field of depression research in a half-century. However, the precise mechanisms underlying the antidepressant effects of ketamine remain unknown. A recent study (Zanos et al., 2016) reported that the metabolism of ketamine to hydroxynorketamine (HNK) is essential for ketamine’s antidepressant effects. In particular, (2R,6R)-HNK, a metabolite of (R)-ketamine, plays a key role in the antidepressant actions. However, here we report that, unlike (R)-ketamine, its metabolites (R)-norketamine and (2R,6R)-HNK did not elicit antidepressant effects in a rat learned helplessness model. It is, therefore, unlikely that the metabolism of ketamine to HNK is necessary for ketamine’s antidepressant actions. intermittent administration of ()-ketamine S , but not of (R )- Methods and Materials ketamine, resulted in the loss of parvalbumin immunoreactiv- ity in the prefrontal cortex and hippocampus (Yang et al., 2015, Animals 2016). Moreover, with the results using [C]raclopride and posi- Male Sprague-Dawley rats (200–230 g, 7 weeks old; Charles-River tron emission tomography, we reported a marked reduction Japan) were used. The animals were housed under a 12-h-light/- of dopamine D receptor binding in the conscious monkey 2/3 dark cycle with free access to food and water. The protocol was striatum after a single infusion of ()-ketamine S , but not of (R )- approved by the Chiba University Institutional Animal Care and ketamine (Hashimoto et al., 2017). These findings suggest that Use Committee (permission no: 28–394 and 29–328). All efforts (S)-ketamine, but not (R )-ketamine, can cause a marked release were made to minimize suffering. of dopamine from presynaptic terminals, which is associated with acute psychotomimetic effects (Hashimoto et al., 2017). Taking these findings together, (R)-ketamine could be a poten- Drugs tially safer antidepressant without detrimental side effects in humans than (S)-ketamine (Hashimoto, 2016a, 2016b, 2017). (R)-Ketamine hydrochloride was prepared by recrystallization of It is well known that ketamine is rapidly metabolized into nor - (R,S)-ketamine (Ketalar, ketamine hydrochloride, Daiichi Sankyo ketamine and subsequently into hydroxynorketamine (HNK) by Pharmaceutical Ltd) and D-(-)-tartaric acid, as described previ- microsomal cytochrome P450 enzymes (through N-demethylation ously (Zhang et al., 2014). (R)-Norketamine hydrochloride was and hydroxylation) in the liver (Figure 1) (Turfus et al., 2009 Zhao ; prepared as described previously (Zanos et al., 2016). The purity et  al., 2012; Zanos et  al., 2016; Hashimoto, 2017). The metabo- of these stereoisomers was determined by a high-performance lism of ketamine to HNK was also recently demonstrated to be liquid chromatography (CHIRALPAK IA, column size: 250 x 4.6 essential for the antidepressant actions of ketamine (Zanos et al., mm, mobile phase: n-hexane/dichloromethane/diethylamine 2016). In particular, (2R ,6R)-HNK, a metabolite from (R)-ketamine, (75/25/0.1), Daicel Corporation). (2R,6R)-HNK hydrochloride was plays a key role in the antidepressant actions (Zanos et al., 2016). provided from Taisho Pharmaceutical Ltd as reported previ- However, increasing attention has been drawn to the antide- ously (Zanos et al., 2016). ( R)-Ketamine, (R)-norketamine, and pressant actions of (2R ,6R)-HNK (Abdallah, 2017). In the present (2R,6R)-HNK were dissolved in 0.9% NaCl. Other compounds study, we examined the effects of a single systemic injection of were purchased commercially. The doses of (R)-ketamine and (R)-ketamine and its two major metabolites, (R )-norketamine and its metabolites were selected as previously reported (Yang et al., (2R,6R)-HNK, in a rat learned helplessness (LH) model. 2015, 2017; Zanos et al., 2016). Figure 1. Metabolism of (R)-ketamine in the liver. In the liver, (R)-ketamine is metabolized to (R)-norketamine (major pathway) and (2R,6R)-hydroxyketamine (minor pathway), subsequently (2R,6R)-hydroxynorketamine (HNK). Downloaded from https://academic.oup.com/ijnp/article-abstract/21/1/84/4633900 by Ed 'DeepDyve' Gillespie user on 16 March 2018 86 | International Journal of Neuropsychopharmacology, 2018 2, the rats were subjected to 30 inescapable electric foot-shocks Stress Paradigm (LH Model) (0.65 mA, 30-second duration, administered at random intervals To create an LH paradigm, the animals are initially exposed to averaging 18–42 seconds) (Figure 2A, C). On day 3, a 2-way con- ditioned avoidance test was performed as a post-shock test to uncontrollable stress. When the animal is later placed in a situ- ation where the shock is controllable (escapable), the animal not determine whether the rats would exhibit the predicted escape deficits (Figure 2A, C). This screening session consisted of 30 tri- only fails to acquire the escape response but also often makes no efforts to escape the shock at all. The LH behavioral tests als in which electric foot-shocks (0.65 mA, 6-second duration, administered at random intervals with a mean of 30 seconds) were performed using the Gemini Avoidance System (San Diego Instruments) (Shirayama et  al., 2015, 2017). This apparatus is were preceded by a 3-second conditioned stimulus tone that remained on until the shock was terminated. Rats with more divided into 2 compartments by a retractable door. On days 1 and Figure 2. Effects of a single injection of (R)-ketamine, (R)-norketamine, and (2R,6R)- HNK in a rat LH model. (A) Rats received inescapable electric shock (IES) treatments on 2 days (days 1 and 2), passed a post-shock test (PS) on day 3, and were designated as learned helplessness (LH) rats with depression-like phenotype. On day 3, vehicle (saline: 2 mL/kg), (R)-ketamine (20 mg/kg), (R)-norketamine (20 mg/kg), or (2R,6R)- HNK (20 and 40 mg/kg) was administered i.p. into LH rats. On day 8 (5 days after a single injection), conditioned avoidance (CA) test to study the antidepressant effect was performed. (B) The failure number of LH (1-way ANOVA: F  = 3.755, P = .0167). 4,24 The escape latency of LH (1-way ANOVA: F  = 3.973, P = .013). Data are shown as mean ± SEM (n = 5–8). The number in the parenthesis is the dose (mg/kg). *P < .05 4,24 compared with vehicle-treated group. (C) Rats received IES treatments on 2 days (days 1 and 2), passed a PS on day 3, and were designated as LH rats with depression- like phenotype. On day 3, either vehicle (saline: 2 mL/kg), (R)-ketamine (20 mg/kg), or (2R,6R)- HNK (20 mg/kg) was administered i.p. into LH rats. CA test was performed on day 4 (24 hours after a single injection). (D) The failure number of LH (1-way ANOVA: F  = 13.52, P < .0001). The escape latency of LH (1-way ANOVA: F  = 14.73, 2,14 2,14 P = .0004). Data are shown as mean ± SEM (n = 5 or 6). The number in the parenthesis is the dose (mg/kg). **P < .01, ***P < .001 compared with vehicle-treated group. R-KT: (R)-ketamine, R-NKT: (R)-norketamine, R-HNK: (2R,6R)-hydroxynorketamine. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/1/84/4633900 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Shirayama and Hashimoto | 87 than 25 escape failures among the 30 trials were regarded as model of depression, although (2R,6R)-HNK did not have anti- having reached the LH criterion and were used in further experi- depressant effects (Yang et al., 2017). Collectively, it seems that ments. Approximately 65% of the rats met this criterion. unlike (R)-ketamine, (2R,6R)-HNK does not have an antidepres- In the experiment 1, on day 3, the rats received i.p. injection sant effect in rodent models of depression, inconsistent with the of saline (2  mL/kg), (R)-ketamine (20  mg/kg), (R)-norketamine findings by Zanos et al. (2016). (20 mg/kg), or (2R,6R)-HNK (20 and 40 mg/kg) (Figure 2A). On day Zanos et al. (2016) reported more potent antidepressant 8 (5 days after a single injection), a 2-way conditioned avoidance effects of (2R,6R)-HNK, which is exclusively derived from (R)- test was performed (Figure  2A). In the experiment 2, on day 3, ketamine. A single injection of (2R,6R)-HNK (10 or 20 mg/kg) the rats received i.p. injection of saline (2 mL/kg), (R)-ketamine reversed chronic corticosterone-induced anhedonia assessed (20  mg/kg), or (2R,6R)-HNK (20mg/kg) (Figure  2C). On day 4 (24 with the sucrose preference and female urine sniffing behav- hours after a single injection), a 2-way conditioned avoidance ioral tasks as well as social avoidance induced by chronic social test was performed (Figure  2C). This test session consisted of defeat stress (Zanos et al., 2016). They reported sustained (24 30 trials in which electric foot-shocks (0.65 mA, 30-second dur - hours) antidepressant effects of (2R,6R)-HNK in LH model (Zanos ation, administered at random intervals with a mean of 30 sec- et al, 2016). However, we could not find sustained (24 hours) anti- onds) were preceded by a 3-second conditioned stimulus tone depressant effects of (2R,6R)-HNK (20 mg/kg) in the LH model, that remained on until the shock was terminated. The number although (R)-ketamine (20 mg/kg) showed sustained (24 hours) of escape failures and the latency until escape for each of the 30 antidepressant effects in the same model (Figure 2D). Thus, we trials were recorded by the Gemini Avoidance System. were unable to detect antidepressant activity induced by (2R,6R)- HNK in any of our 3 models (inflammation, social defeat stress, and LH), although rapid and sustained antidepressant effects Statistical Analysis were detected for (R)-ketamine (Yang et al., 2017; this study). The The data are shown as the mean ± SEM. The analyses were per - reasons for this discrepancy (Zanos et al., 2016, vs Yang et al., formed using GraphPad Prism 5 (GraphPad Software Inc). The 2017, and this study) remain unknown. Nonetheless, our nega- data were analyzed using 1-way ANOVA, followed by posthoc tive findings regarding the lack of an antidepressant effect of Tukey test. The criterion for significance was P < .05. (2R,6R)-HNK in rodents with depression-like phenotype need to be replicated by other groups in future studies. It is well known that (R)-ketamine is stereoselectively Results N-demethylated by liver microsomal cytochrome P450 into (R)-norketamine (Hijazi and Boulieu, 2002; Desta et  al., 2012; Effects of a Single Intraperitoneal Injection of (R)- Zanos et  al., 2016) (Figure  1). ( R)-Norketamine is further Ketamine, (R)-Norketamine, and (2R,6R)-HNK in metabolized to (2R,6R)-HNK arising from hydroxylation of the LH Rats cyclohexanone ring (Figure 1). In addition to -demeth N ylation, (R)-ketamine is also metabolized by the hydroxylation of the cyclohexanone ring to produce (2R ,6R)-hydroxyketamine. Experiment 1 (2R,6R)-HNK is also prepared by the N-demethylation of To examine the antidepressant effects of (R)-ketamine and its (2R,6R)-hydroxyketamine (Desta et  al., 2012; Zanos et  al., 2 metabolites in a LH model, saline (2  mL/kg), (R)-ketamine 2016) (Figure  1). A  study showed that the plasma levels of (20 mg/kg), (R)-norketamine (20 mg/kg), or (2R,6R)-HNK (20 and norketamine and HNK were higher after a single injection of 40  mg/kg) was administered i.p. into the LH rats. The LH rats ketamine, although plasma levels of hydroxyketamine were that received a single injection of (R)-ketamine (20 mg/kg, 5 days very low (Zanos et al., 2016), suggesting that the metabolism after a single injection) exhibited significant improvements in of (R)-ketamine to (2R,6R)-HNK via (R)-norketamine is the their conditioned avoidance test results, relative to vehicle- major pathway of (R)-ketamine in mice (Figure  1). Therefore, treated LH rats (Figure 2A,B). In contrast, a single administration it is noteworthy that ()-norketamine and (2 R R,6R)-HNK, the of (R)-norketamine (20 mg/kg), or (2R,6R)-HNK (20 and 40 mg/kg) two major metabolites from (R)-ketamine, did not exert anti- did not improve their conditioned avoidance test results in LH depressant effects, although antidepressant effects of ( )-R rats (Figure 2A,B). ketamine were detected in the same model. We have recently reported that a single bilateral infu- Experiment 2 sion of (R)-ketamine into the infralimbic (IL) area of the The LH rats that received a single injection of (R)-ketamine medial prefrontal cortex (mPFC) and the DG and CA3 of (20 mg/kg, 24 hours after a single injection) exhibited significant the hippocampus exerted antidepressant effects in LH rats improvements in their conditioned avoidance test results rela- (Shirayama and Hashimoto, 2017). Furthermore, a study tive to vehicle-treated LH rats (Figure 2C,D). In contrast, (2R,6R)- showed that neuronal inactivation of the IL of mPFC com- HNK (20 mg/kg, 24 hours after a single injection) did not improve pletely blocked the antidepressant effects of (R,S)-ketamine, their conditioned avoidance test results in LH rats (Figure 2C,D). and that microinfusion of (R,S)-ketamine into IL of mPFC produced an antidepressant effect in control unstressed rats Discussion (Fuchikami et al., 2015). These findings suggest a crucial role for the IL area of the mPFC, DG, and CA3 in the antidepres- In the present study, we established that a single systemic (R)- sant action of (R )-ketamine itself (NOT metabolite) in a rat ketamine injection (24 hours and 5 days after a single injection) LH model, since (R )-ketamine (or (R,S)-ketamine) might not showed antidepressant effects in a rat LH model of depres- be metabolized in the brain. Given the key role of hepatic sion, whereas a single systemic injection of (R)-norketamine cytochrome P450 enzymes in ketamine metabolism (Turfus or (2R,6R)-HNK did not. It should be noted that a higher dose et al., 2009; Zhao et al., 2012; Zanos et al., 2016), it is unlikely (40 mg/kg) of (2R,6R)-HNK did not have antidepressant effects. that the metabolism of (R)-ketamine in the liver plays a role We have also recently reported the potent and longer-lasting in the antidepressant actions of ()-ketamine R . antidepressant effects of (R)-ketamine in a social defeat stress Downloaded from https://academic.oup.com/ijnp/article-abstract/21/1/84/4633900 by Ed 'DeepDyve' Gillespie user on 16 March 2018 88 | International Journal of Neuropsychopharmacology, 2018 In conclusion, unlike (R)-ketamine, neither (R)-norketamine Kishimoto T, Chawla JM, Hagi K, Zarate CA, Kane JM, Bauer nor (2R,6R)-HNK elicited antidepressant effects in LH model, M, Correll CU (2016) Single-dose infusion ketamine and suggesting that the metabolism of (R)-ketamine might not play non-ketamine N-methyl-D-aspartate receptor antago- a key role in its robust antidepressant action. nists for unipolar and bipolar depression: a meta-analy- sis of efficacy, safety and time trajectories. Psychol Med 46:1459–1472. Acknowledgments Monteggia LM, Zarate C Jr (2015) Antidepressant actions of keta- mine: from molecular mechanisms to clinical practice. Curr We thank Dr. Shigeyuki Chaki (Taisho Pharmaceutical Co, Ltd) for providing (2R,6R)-HNK. We also thank Yuko Fujita (Chiba Opin Neurobiol 30:139–143. Newport DJ, Carpenter LL, McDonald WM, Potash JB, Tohen M, University) for her technical assistance. This study was supported by Strategic Research Program for Nemeroff CB, APA Council of Research Task Force on Novel Biomarkers and Treatments (2015) Ketamine and other Brain Sciences, AMED, Japan (to K.H.). NMDA antagonists: early clinical trials and possible mecha- nisms in depression. Am J Psychiatry 172:950–966. Statement of Interest Shirayama Y, Hashimoto K (2017) Effects of a single bilateral infusion of R-ketamine in the brain regions of a learned Dr. Shirayama has received research support from Eli Lilly, Eisai, helplessness model of depression. Eur Arch Psychiatry Clin MSD, Pfizer, and Mitsubishi-Tanabe. Dr. Hashimoto has received Neurosci 267:177–182. research support from Dainippon-Sumitomo, Mochida, Otsuka, Shirayama Y, Yang C, Zhang JC, Ren Q, Yao W, Hashimoto K (2015) and Taisho. Dr. Hashimoto is an inventor on a filed patent appli- Alterations in brain-derived neurotrophic factor (BDNF) cation on “The use of (R)-ketamine in the treatment of psychi- and its precursor proBDNF in the brain regions of a learned atric diseases” by Chiba University. helplessness rat model and the antidepressant effects of a TrkB agonist and antagonist. Eur Neuropsychopharmacol References 25:2449–2458. Abdallah CG (2017) What’s the buzz about hydroxynorketamine? Turfus SC, Parkin MC, Cowan DA, Halket JM, Smith NW, Is it the history, the story, the debate, or the promise? Biol Braithwaite RA, Elliot SP, Steventon GB, Kicman AT (2009) Psychiatry 81:61–63. Use of human microsomes and deuterated substrates: an Desta Z, Moaddel R, Ogburn ET, Xu C, Ramamoorthy A, Venkata alternative approach for the identification of novel metabo- SL, Sanghvi M, Goldberg ME, Torjman MC, Wainer IW (2012) lites of ketamine by mass spectrometry. Drug Metab Dispos Stereoselective and regiospecific hydroxylation of ketamine 37:1769–1778. and norketamine. Xenobiotica 42:1076–1087. Yang C, Shirayama Y, Zhang JC, Ren Q, Yao W, Ma M, Dong C, Domino EF (2010) Taming the ketamine tiger. 1965. Hashimoto K (2015) R-ketamine: a rapid-onset and sustained Anesthesiology 113:678–684 antidepressant without psychotomimetic side effects. Transl Duman RS, Aghajanian GK, Sanacora G, Krystal JH (2016) Psychiatry 5:632. Synaptic plasticity and depression: new insights from stress Yang C, Han M, Zhang JC, Ren Q, Hashimoto K (2016) Loss of and rapid-acting antidepressants. Nat Med 22:238–249. parvalbumin-immunoreactivity in mouse brain regions after Fuchikami M, Thomas A, Liu R, Wohleb ES, Land BB, DiLeone RJ, repeated intermittent administration of esketamine, but not Aghajanian GK, Duman RS (2015) Optogenetic stimulation of R-ketamine. Psychiatric Res 239:281–283. infralimbic PFC reproduces ketamine’s rapid and sustained Yang C, Qu Y, Abe M, Nozawa D, Chaki S, Hashimoto K (2017) antidepressant actions. Proc Natl Acad Sci USA 112:8106–8111. (R)-ketamine shows greater potency and longer last- Fukumoto K, Toki H, Iijima M, Hashihayata T, Yamaguchi JI, ing antidepressant effects than its metabolite (2R,6R)- Hashimoto K, Chaki S (2017) Antidepressant potential of (R)- hydroxynorketamine. Biol Psychiatry 82:43–44. ketamine in rodent models: comparison with (S)-ketamine. J Yang C, Ren Q, Qu Y, Zhang JC, Ma M, Dong C, Hashimoto K (2018) Pharmacol Exp Ther 361:9–16. 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International Journal of NeuropsychopharmacologyOxford University Press

Published: Jan 1, 2018

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