Recently, (2 R,6 R)-HNK, which is derived from the metabolism of (R)-ketamine, has been proposed to mediate the antidepressant effects of (R,S)-ketamine [7]. In the present study, we investigated whether the generation of (2 R,6 R)-HNK is necessary for (R)-ketamine to exert its antidepressant effects by examining the pharmacokinetic/pharmacodynamic relationship and by blocking metabolism to (2 R,6 R)-HNK using CYP inhibitors. In addition, we investigated the role of (R)-norketamine, another major metabolite of (R)-ketamine, in the antidepressant actions.

In the LPS-induced inflammation model of mice, (R)-ketamine attenuated the increase in the immobility time of LPS-treated mice in the FST without affecting locomotor activity, indicating antidepressant effects. In contrast, both (R)-norketamine and (2 R,6 R)-HNK did not show significant antidepressant effects at the same dose. This result is consistent with previous findings that (2 R,6 R)-HNK (10 mg/kg) did not exhibit significant antidepressant effects in the LPS-induced inflammation model and a chronic social defeat stress model, while both (R)-ketamine (10 mg/kg) and (S)-ketamine (10 mg/kg) showed antidepressant activity [14]. Moreover, very recently, Shirayama and Hashimoto [13] have reported that both (R)-norketamine (20 mg/kg) and (2 R,6 R)-HNK (20 and 40 mg/kg) had no effect in a rat learned helplessness model, while (R)-ketamine (20 mg/kg) elicited antidepressant effects in the same model. In the present study, the pharmacokinetic data demonstrated that the plasma, brain, and CSF concentrations of (R)-norketamine and (2 R,6 R)-HNK following the administration of each compound (10 mg/kg) were higher than those generated after the administration of (R)-ketamine (10 mg/kg). Therefore, a sufficient exposure to (R)-norketamine and (2 R,6 R)-HNK was achieved at the dose used. On the basis of the pharmacokinetic/pharmacodynamic relationship, both (R)-norketamine and (2 R,6 R)-HNK might not contribute to the antidepressant actions of (R)-ketamine in the LPS-induced inflammation model, at least at 3 h after drug administration. Of note, Suzuki et al. [20] recently claimed that (2 R,6 R)-HNK may mediate the sustained antidepressant effects of (R,S)-ketamine via NMDA receptor inhibition. However, the present pharmacokinetic data indicated that the plasma concentrations of (2 R,6 R)-HNK declined at a rate similar to those of (R)-ketamine following (R)-ketamine administration. Therefore, from a pharmacokinetic point of view, it is unlikely that (2 R,6 R)-HNK mediates the sustained antidepressant effects of (R)-ketamine.

Next, we investigated the involvement of (2 R,6 R)-HNK in the actions of (R)-ketamine by inhibiting the metabolism of (R)-ketamine to (2 R,6 R)-HNK. In humans, (R,S)-ketamine has been proposed to be metabolized to (2 S,6 S;2 R,6 R)-HNK by N-demethylation with multiple CYP isoforms including CYP2B6, followed by hydroxylation with CYP2A6, CYP3A5, and CYP2B6 [12]. On the basis of this information, we successfully validated this condition, demonstrating that a combination of 1-ABT, a multiple CYP inhibitor [21,22,23], and ticlopidine, a CYP2B6 inhibitor [24, 25], prevented the generation of (2 R,6 R)-HNK from (R)-ketamine. Indeed, plasma levels of (2 R,6 R)-HNK after the administration of (R)-ketamine were not detected (in the LPS-treated mice) or were barely detected (in naive mice) by pretreatment with the CYP inhibitors. In contrast, in the presence of the CYP inhibitors, the plasma levels of (R)-ketamine after the administration of ineffective doses of (R)-ketamine (3 mg/kg for the LPS-treated mice and 10 mg/kg for the naive mice) were increased to approximately the same levels obtained after the administration of effective doses of (R)-ketamine (10 mg/kg for the LPS-treated mice and 30 mg/kg for the naive mice). Therefore, if (R)-ketamine exerts the antidepressant effects, at the ineffective doses, in the presence of the CYP inhibitors, it indicates that (R)-ketamine itself, and not (2 R,6 R)-HNK, is responsible for its antidepressant effects. In the present study, we obtained the results that in the presence of the CYP inhibitors, (R)-ketamine, at an ineffective dose (3 mg/kg), exerted the antidepressant effects in the FST of LPS-treated mice at 3 h after the administration. Likewise, (R)-ketamine, at an ineffective dose (10 mg/kg), exerted the antidepressant effects in the TST of naive mice at 24 h after the administration in the presence of the CYP inhibitors. Therefore, the antidepressant effects of (R)-ketamine paralleled the plasma levels of (R)-ketamine, but not (2 R,6 R)-HNK. These results provide direct evidence that the generation of (2 R,6 R)-HNK is not essential for (R)-ketamine’s acute and sustained antidepressant effects, at least in two different mouse models.

Although (R)-norketamine may not be involved in the antidepressant actions of (R)-ketamine, based on the results of the pharmacokinetic/pharmacodynamic relationship, we did not provide direct evidence for the role of (R)-norketamine in the antidepressant actions of (R)-ketamine in the present study. Therefore, the involvement of (R)-norketamine in the antidepressant effects of (R)-ketamine needs to be further investigated under conditions in which the generation of (R)-norketamine is prevented.

The present results contradict previously reported results [7]. Zanos et al. [7] claimed that the formation of (2 S,6 S;2 R,6 R)-HNK is essential and sufficient to exert the antidepressant effects of (R,S)-ketamine and that (2 R,6 R)-HNK, in particular, has a critical role. In their study, they demonstrated that deuterated (R,S)-ketamine, which reduced the formation of (2 S,6 S;2 R,6 R)-HNK, prevented the sustained (24 h) antidepressant activity of (R,S)-ketamine. The discrepancy between these previously reported results and the present results requires some discussion. In their study, although the exposure levels of (2 S,6 S;2 R,6 R)-HNK in the brain were reduced when the deuterated (R,S)-ketamine was administered, the levels of (R,S)-ketamine and (R,S)-norketamine were not changed. In addition, metabolism to (2 S,6 S;2 R,6 R)-HNK was only partially, and not completely, prevented. In contrast, the presently reported experimental conditions almost completely prevented the formation of (2 R,6 R)-HNK, resulting in reasonable increases in the plasma levels of both (R)-ketamine and (R)-norketamine. Thus, the present condition can investigate the roles of (2 R,6 R)-HNK in the antidepressant effects of (R)-ketamine more clearly and adequately. Although they demonstrated that (2 R,6 R)-HNK induced the sustained antidepressant effects in several animal models, these results do not necessarily mean that the formation of (2 R,6 R)-HNK is essential for the antidepressant effects of (R)-ketamine. The antidepressant effects of (2 R,6 R)-HNK may occur at exposure levels higher than those obtained after the effective doses of (R)-ketamine administration. Indeed, we observed in the present study that (2 R,6 R)-HNK tended to reduce the increased immobility time in LPS-treated mice. (2 R,6 R)-HNK and (R,S)-ketamine, at the same dose, have recently been reported to show similar effects on serotonin release in the medial prefrontal cortex [26] as well as on AMPA receptor functions in the nucleus accumbens and dopamine neurons in the ventral tegmental area [27]. These findings are underpinned by the recent report that both (R,S)-ketamine and (2 R,6 R)-HNK inhibit NMDA receptor at rest and eukaryotic elongation factor 2 phosphorylation, while higher concentration is required for (2 R,6 R)-HNK than (R,S)-ketamine [20]. However, Pham et al. [26] also reported that local injection of (R,S)-ketamine (2 nmol) and (2 R,6 R)-HNK (2 nmol) into the medial prefrontal cortex exerted the antidepressant effects at 24 h after injection in the FST, indicating that both (R,S)-ketamine and (2 R,6 R)-HNK show the sustained antidepressant effects on their own, presumably through different mechanisms. Moreover, this result also indicates that biotransformation to (2 R,6 R)-HNK is not necessary for (R,S)-ketamine’s antidepressant effect.

It should be noted that there are some limitations in the present study. To fully elucidate the roles of (2 R,6 R)-HNK in the antidepressant effects of (R)-ketamine and to clarify discrepancy between results of other laboratories, studies in other behavioral models including chronic stress models are warranted. Moreover, although we investigated the antidepressant effects of (R)-ketamine at 24 h after the administration, the roles of metabolites of (R)-ketamine may differ at discrete time points. For example, the role of (2 R,6 R)-HNK in the long-lasting antidepressant effects of (R)-ketamine (e.g., at 7 days) needs to be clarified.

In conclusion, we demonstrated that (2 R,6 R)-HNK, and possibly (R)-norketamine, are not essential for the antidepressant actions of (R)-ketamine. Because (2 R,6 R)-HNK is formed from only (R)-ketamine, the present results also suggest that metabolism to (2 R,6 R)-HNK is not involved in the antidepressant effects of (R,S)-ketamine. The present study should provide an important insight into the active substance responsible for the antidepressant effects of (R)-ketamine, thereby facilitating further studies on this important issue in ketamine research.