Arketamine, a new rapid-acting antidepressant: A historical review and future directions

The N-methyl-d-aspartate receptor (NMDAR) antagonist (R,S)-ketamine causes rapid onset and sustained antidepressant actions in treatment-resistant patients with major depressive disorder (MDD) and other psychiatric disorders, such as bipolar disorder and post-traumatic stress disorder. (R,S)-ketamine is a racemic mixture consisting of (R)-ketamine (or arketamine) and (S)-ketamine (or esketamine), with (S)-enantiomer having greater affinity for the NMDAR. In 2019, an esketamine nasal spray by Johnson & Johnson was approved in the USA and Europe for treatment-resistant depression. In contrast, an increasing number of preclinical studies show that arketamine has greater potency and longer-lasting antidepressant-like effects than esketamine in rodents, despite the lower binding affinity of arketamine for the NMDAR. Importantly, the side effects, i.e., psychotomimetic and dissociative effects and abuse liability, of arketamine are less than those of (R,S)-ketamine and esketamine in animals and humans. An open-label study demonstrated the rapid and sustained antidepressant effects of arketamine in treatment-resistant patients with MDD. A phase 2 clinical trial of arketamine in treatment-resistant patients with MDD is underway. This study was designed to review the brief history of the novel antidepressant arketamine, the molecular mechanisms underlying its antidepressant actions, and future directions.

(R,S)-ketamine (Ki = 0.53 μM for NMDAR) is a racemic mixture consisting of (R)-ketamine (or arketamine: Ki = 1.4 μM) and (S)-ketamine (or esketamine: Ki = 0.30 μM) ( Fig. 1) (Ebert et al., 1997). We compared the antidepressant-like effects of the two enantiomers in several animal models of depression. We found that arketamine has more potent and longer-lasting antidepressant-like effects than esketamine in rodent models of depression, despite its lower affinity for the NMDAR (Fig. 2) (Yang et al., 2015;Zhang et al., 2014). Furthermore, note that the side effects of arketamine are less than those of (R,S)-ketamine and esketamine (Chang et al., 2019;Hashimoto et al., 2017;Yang et al., 2015). Taken together, arketamine would be a novel rapid-acting antidepressant without the side effects of (R,S)-ketamine and esketamine. This study aimed to review the brief history of arketamine, the molecular mechanisms underlying its antidepressant actions, and future directions.
In 2000, Dr. Berman et al. (2000) first conducted a double-blind, placebo-controlled study of (R,S)-ketamine in medication-free patients with MDD (Fig. 2). A single intravenous infusion of (R,S)-ketamine (0.5 mg/kg) produced rapid and sustained antidepressant effects in patients with MDD. In 2006, Dr. Zarate et al. (2006 reported that a single infusion of (R,S)-ketamine (0.5 mg/kg) produced rapid and sustained antidepressant effects in treatment-resistant patients with MDD (Fig. 2). As mentioned earlier, the robust antidepressant effects of (R,S)-ketamine in treatment-resistant patients with MDD or BD have been replicated by many research groups worldwide. At present, (R,S)-ketamine has been widely used as an off-label treatment for psychiatric disorders, such as MDD, BD, and PTSD (Mathai et al., 2022;O'Brien et al., 2022).

Comparison of two enantiomers of (R,S)-ketamine in humans
In 1985, White et al. (1985) compared the clinical and electroencephalographic effects of two enantiomers of (R,S)-ketamine on healthy control subjects. They found that esketamine has more potent anesthetic  effects than arketamine, suggesting a role of NMDAR in anesthetic action. Therefore, esketamine has been used as an anesthetic agent in several countries, such as the European Union and China. Mathisen et al. (1995) reported a higher incidence of psychotomimetic side effects in patients with orofacial pain treated with esketamine than in those treated with arketamine. Subsequently, Vollenweider et al. (1997) reported that esketamine caused psychotic reactions, i.e., depersonalization and hallucinations, in healthy control subjects; however, arketamine did not produce any psychotic symptoms in the same subjects at the same dose, and most of them experienced a state of relaxation. These findings suggest that esketamine contributes to the acute side effects of (R,S)-ketamine, whereas arketamine may not be associated with these side effects (Hashimoto, 2019(Hashimoto, , 2020aZanos et al., 2018). It was recognized that NMDAR inhibition could play a key role in the antidepressant actions of (R,S)-ketamine for depressive symptoms in patients with MDD or BD (Krystal et al., 2019). Therefore, Johnson & Johnson developed an esketamine nasal spray for treatment-resistant depression because esketamine has a higher affinity for the NMDAR than arketamine. In 2019, the esketamine nasal spray by Johnson & Johnson was approved for treatment-resistant depression in the USA and Europe ( Fig. 2) (Kim et al., 2019;Mahase, 2019). However, there are several concerns about its efficacy and safety (Turner, 2019).
In 2020, Leal et al. (2021) conducted the first open-label study of arketamine in treatment-resistant patients with MDD (Fig. 2). A single intravenous infusion of arketamine (0.5 mg/kg) produced rapid-acting and sustained antidepressant effects in treatment-resistant patients with MDD, and the treated patients showed very low psychotomimetic and dissociative symptoms (Leal et al., 2021). The data from this pilot study support the preclinical and previous clinical findings. Nonetheless, further randomized controlled trial is needed to compare the antidepressant and side effects of arketamine with those of esketamine in patients with MDD.

Roles of the NMDAR and α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor (AMPAR)
Although the precise mechanisms underlying the antidepressant effects of (R,S)-ketamine remain unclear, its rapid antidepressant effects are considered to occur via the blockade of NMDARs located in inhibitory interneurons, leading to the disinhibition of pyramidal cells and the burst of glutamatergic transmission (Krystal et al., 2019). Cumulative preclinical studies using the two aforementioned enantiomers showed that arketamine has stronger and longer-lasting antidepressant-like effects than esketamine in several models of depression (Yang et al., 2015(Yang et al., , 2018bYao et al., 2022;Zhang et al., 2014Zhang et al., , 2020, although arketamine was less potent for the NMDAR than esketamine (Fig. 1).
It has also been reported that AMPAR antagonists block the antidepressant-like effects of (R,S)-ketamine and its enantiomers in rodents (Autry et al., 2011;Fukumoto et al., 2017;Li et al., 2010;Yang et al., 2015;Zanos et al., 2016), suggesting that AMPAR activation contributes to the antidepressant-like effects of ketamine and its enantiomers. In contrast, it is unlikely that AMPAR activation may play a role in the antidepressant-like actions of (S)-norketamine, a major metabolite of esketamine, in rodent models of depression Yang et al., 2018a).
Using functional magnetic resonance imaging (fMRI), Masaki et al. (2019) reported that the selective and potent NMDAR antagonist (+)-MK-801 (Ki = 0.0019 μM for the NMDAR) ( Fig. 1) (Ebert et al., 1997), (R,S)-ketamine, and esketamine produced a significant positive response in the cortex, nucleus accumbens, and striatum of conscious rats. In contrast, arketamine produced a negative response in these brain regions. These data suggest that the inhibition of the NMDAR does not play a role in brain activation after a single injection of arketamine (Masaki et al., 2019). Notably, (+)-MK-801 did not have antidepressant effects in patients with depression (unpublished data by Merck; Hashimoto, 2020a), although it showed rapid-acting antidepressant-like effects in a model of chronic social defeat stress (CSDS) (Yang et al., 2016a). Furthermore, the pharmacokinetic profiles of the two enantiomers are similar (Fukumoto et al., 2017). Taken all together, it is unlikely that NMDAR inhibition plays a major role in the antidepressant-like actions of arketamine (Hashimoto, 2019(Hashimoto, , 2020a(Hashimoto, , 2020bYang et al., 2019;Wei et al., 2022a).

Extracellular signal-regulated kinase (ERK) and ERK-NRBP1-CREB-BDNF signaling
Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family, has been deemed as a biochemical signal integrator and coincidence detector for coordinating the secondary pathways that respond to extracellular signals (Sweatt, 2001). The phosphorylation of ERK activates cAMP-response element binding (CREB), an intranuclear regulator regulating transcription through autophosphorylation, and plays a central role in regulating and maintaining mood and memory (Bilecki et al., 2004;Walters et al., 2005). CREB is widely distributed in the hippocampus and cerebral cortex (Mantamadiotis et al., 2002). When CREB is phosphorylated by signal stimulation, CREB can recognize the corresponding site of CREB and activate the transcription of CREB downstream target brain-derived neurotrophic factor (BDNF) (Bambah-Mukku et al., 2014;Esvald et al., 2022;Martinowich et al., 2003;Wang et al., 2017). Both ERK and CREB-BDNF pathways are well known to play a major role in depression (Hashimoto et al., 2004;Hashimoto, 2010;Nestler et al., 2002;Wang and Mao, 2019;Zhang et al., 2016).
The mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase that forms the catalytic subunits of two distinct protein complexes, known as mTOR complex 1 and 2 (Saxton and Sabatini, 2017). In 2010, Li et al. (2010) demonstrated the role of mammalian target of rapamycin (mTOR) in the antidepressant-like actions of (R, S)-ketamine in rodents because the mTOR inhibitor rapamycin inhibits the antidepressant-like effects of (R,S)-ketamine. Two mTOR inhibitors, namely, rapamycin and AZD8055, blocked the antidepressant-like effects of esketamine (not arketamine) in CSDS-susceptible mice (Yang et al., 2018b), whereas the ERK inhibitor SL327 blocked the antidepressant-like effects of arketamine (not esketamine) in CSDS-susceptible mice (Yang et al., 2018b). These findings suggest that ERK signaling activation contributes to the antidepressant-like effects of arketamine (Hashimoto, 2019;Scotton et al., 2022;Yang et al., 2018b).
Since ERK contributes to the long-lasting antidepressant effects of arketamine (Yang et al., 2018b), we hypothesize that the mechanism of the long-lasting antidepressant effects of arketamine may be achieved by activating ERK, which induces CREB phosphorylation and subsequent BDNF transcription in the microglia (Fig. 3). Using the BV2 cell line and primary microglia, we found that both arketamine and esketamine could activate Bdnf exon IV promoter via CREB phosphorylation. Importantly, arketamine was more potent than esketamine. Using isobaric tags for relative and absolute quantification, we identified nuclear receptor-binding protein 1 (NRBP1) as a differentially expressed protein for the two enantiomers in the medial prefrontal cortex (mPFC) of CSDS-susceptible mice (Yao et al., 2022). NRBP1 was expressed in the neurons (for Camk2α + and GABA + ) and microglia (for CD11b + ), but not in the astrocytes (for glial fibrillary acidic protein) of the mPFC of mice (Yao et al., 2022). Since the phosphorylation of ERK could activate the transcription factor CREB, resulting in the regulation of BDNF transcription (Bambah-Mukku et al., 2014;Esvald et al., 2022;Martinowich et al., 2003;Wang et al., 2017), we examined the relationship between NRBP1 and ERK-CREB signaling. Immunoprecipitation assay showed that NRBP1 and CREB bind to each other under physiological function and after arketamine (or esketamine) treatment (Yao et al., 2022). Western blotting showed that siRNA-NRBP1 caused the downregulation of NRBP1 and decreased the ratio of phospho-CREB/CREB in BV2 cells in a concentration-dependent manner. Arketamine significantly increased the expression of NRBP1 and the phospho-CREB/CREB ratio in the primary microglia in a concentration-dependent manner. Fig. 3. Proposed signaling pathways underlying the antidepressant-like actions of arketamine Arketamine binds to an unknown protein in the microglia, resulting in ERK activation. ERK activation results in CREB activation, promoting CREB binding with BDNF exon IV promoter through interaction with NRBP1, resulting in BDNF transcription. Subsequently, released BDNF binds to its receptor TrkB on the neuron, resulting in ameliorating the decreased dendritic spine density. The proposed hypothesis is from the article (Yao et al., 2022).
Interestingly, the ERK inhibitor SL327 significantly attenuated the increased expression of NRBP1 and BDNF and the increased ratio of phospho-ERK/ERK and phospho-CREB/CREB in arketamine-treated primary microglia (Yao et al., 2022). Collectively, arketamine could activate the phosphorylation of CREB by activating NRBP1 and ERK, resulting in BDNF upregulation in microglia (Fig. 3).
DNA/RNA heteroduplex oligonucleotide (HDO) is a novel technology for gene silencing (Asada et al., 2021;Cao et al., 2022aCao et al., , 2022bNishina et al., 2015;Yoshioka et al., 2019). Using CREB-HDO and BDNF exon IV-HDO that specifically targets CREB and CREB-binding sequences of BDNF exon IV, we found that CREB-HDO and BDNF exon IV-HDO significantly attenuated the antidepressant-like effects of arketamine in CSDS-susceptible mice. Moreover, CREB-HDO significantly attenuated the increased ratio of phospho-CREB/CREB and BDNF protein, the decreased CD11b immunoreactivity, and the increased phospho-CREB immunoreactivity in the mPFC of arketamine-treated CSDS-susceptible mice. BDNF exon IV-HDO significantly attenuated the increased BDNF protein and the decreased CD11b immunoreactivity in the mPFC of arketamine-treated CSDS-susceptible mice (Yao et al., 2022). Therefore, the antidepressant-like effects of arketamine in rodents might be produced by activating CREB, resulting in BDNF upregulation in the microglia (Fig. 3).
Mannosylated clodronate liposomes (MCLs) are anti-inflammatory phenotypes of microglial inhibitor (Miron et al., 2013). Interestingly, MCLs significantly attenuated the antidepressant-like effects of arketamine and attenuated the increased BDNF protein, arginase 1 (a marker of the anti-inflammatory phenotype of microglia), and phospho-CREB immunoreactivity in the mPFC of arketamine-treated CSDS-susceptible mice (Yao et al., 2022). Therefore, these results suggest that arketamine has antidepressant-like effects by upregulating BDNF expression in anti-inflammatory microglial phenotypes.

Transforming growth factor β1 (TGF-β1) signaling
Transforming growth factor β1 (TGF-β1) is a member of the TGF-β superfamily of cytokines which control proliferation, differentiation, and other functions. Furthermore, TGF-β1 plays a role in the immune system which is involved in depression (Li et al., 2006;Sanjabi et al., 2017). Using RNA sequencing in the PFC of a CSDS model, we identified TGF-β1 as a differentially expressed gene for two enantiomers. We found that TGF-β1 contributes to the antidepressant-like effects of arketamine in CSDS-susceptible mice (Zhang et al., 2020). TGF-β1 and its receptors are mainly expressed in microglia. The partial depletion of microglia in the mPFC by PLX3397 (an inhibitor of colony-stimulating factor 1 receptor), blocked the antidepressant-like effects of arketamine in a CSDS model, suggesting that microglial TGF-β1 contributes to the antidepressant-like effects of arketamine (Zhang et al., 2020). Moreover, intranasal administration of TGF-β1 produced rapid and sustained antidepressant effects in the CSDS model, and the TrkB inhibitor ANA-12 significantly blocked the antidepressant-like effects of TGF-β1 (Wei et al., 2021;Zhang et al., 2020). Collectively, it is likely that microglial TGF-β1 could contribute to the antidepressant-like effects of arketamine and that TGF-β1 could be a novel antidepressant agent (Wei et al., 2022a). A recent study using blood samples discovered microRNA-144-3p and several genes such as Tgfb1, Bdnf, and Creb1 as upstream regulators of the predicted target genes following (R,S)-ketamine treatment to CSDS-susceptible mice (van der Zee et al., 2022), suggesting microRNA-144-3p as potential biomarker for depression. These genes such as Tgfb1, Bdnf, and Creb1 are shown to associated with antidepressant actions of (R,S)-ketamine and arketamine (Yao et al., 2022;Zhang et al., 2020). Therefore, it is also of interest to investigate whether TGF-β1 has antidepressant effects in patients with MDD.

Opioid receptors
Opioid receptors distributed throughout the brain play an important role in modulation of mood and reward (Hess et al., 2022;. In 2018, Williams et al. (2018) reported that the opioid receptor antagonist naloxone blocked the antidepressant actions of (R,S)-ketamine (0.5 mg/kg) in treatment-resistant patients with MDD. Subsequently, the same group reported that naloxone attenuated the anti-suicidal effects of (R,S)-ketamine (0.5 mg/kg) in treatment-resistant patients with MDD (Williams et al., 2019). These data suggest that the antidepressant and anti-suicidal effects of (R, S)-ketamine require opioid receptor activation; however, the sample size of this study was small.
In contrast, pretreatment with naloxone did not block the antidepressant-like effects of (R,S)-ketamine in CSDS or LPS-treated depression models (Zhang and Hashimoto, 2019b). Bonaventura et al. (2021) also examined the effects of the two enantiomers on a selected panel of 98 receptors and enzymes. At 10 μM, the PCP-binding site of NMDAR was identified as a hit for the two enantiomers. Moreover, at 10 μM, mu-opioid receptor was identified as a hit for esketamine, but not for arketamine (Bonaventura et al., 2021), consistent with the previous report (Hirota et al., 1999). No other hits were identified at 10 μM. If opioid receptors play a key role in the antidepressant effects of (R, S)-ketamine, esketamine must be more potent than arketamine (Zhang and Hashimoto, 2019b). Collectively, it is unlikely that the opioid receptor system plays a major role in the antidepressant actions of (R, S)-ketamine and its enantiomers (Hashimoto, 2020b).

KCNQ2 channel
KCNQ2 (voltage-gated potassium channel subfamily Q member 2), a voltage-gated potassium channel subunit, has been associated with neurodevelopmental disorders such as benign familial neonatal seizures and developmental and epileptic encephalopathy (Kuersten et al., 2020;Springer et al., 2021). Using single-cell RNA-sequencing data, Lopez et al. (2022) recently reported the role of the cell type-specific regulation of KCNQ2 (voltage-gated potassium channel subfamily Q member 2) in the sustained antidepressant-like effects of (R,S)-ketamine. It is, therefore, of interest to investigate the effects of the two enantiomers of ketamine and selective NMDAR antagonist (+)-MK-801 to confirm the role of KCNQ2 and NMDAR in antidepressant-like effects of (R,S)-ketamine and its enantiomers .
Furthermore, the KCNQ activator retigabine, also known as ezogabine, might augment the antidepressant-like effects of (R,S)-ketamine in mice (Lopez et al., 2022). Although the findings reported in this study are new, this study used control-naïve mice without depression-like behaviors. The use of animals without a depression-like phenotype may result in the misinterpretation of the antidepressant-like effects of new candidates, such as (R,S)-ketamine and its metabolites (Hashimoto and Shirayama, 2018). Further studies using rodents with depression-like behaviors are needed to confirm the role of KCNQ2 in the antidepressant-like effects of (R,S)-ketamine . A randomized controlled trial showed that ezogabine had antidepressant effects in patients with MDD , although ezogabine did not show (R,S)-ketamine-like robust antidepressant effects in patients with depression. Therefore, it is of interest to investigate whether a combination of retigabine with a low dose of (R, S)-ketamine, i.e., 0.25 mg/kg, could produce antidepressant effects and reduce (R,S)-ketamine-induced side effects in patients with depression .

Conclusion and future directions
As discussed above, arketamine would be a new antidepressant agent without the side effects of (R,S)-ketamine and esketamine. Clinical trials of arketamine are underway by Perception Neuroscience (USA), Otsuka Pharmaceutical Co., Ltd. (Japan), Jiangsu HengRui Medicine Co., Ltd. (China), and Jiangsu Enhua Pharmaceutical Co., Ltd. (China). We are looking forward to seeing the effects of arketamine in patients with MDD in the near future.
In addition to its antidepressant-like effects, arketamine has beneficial effects on various animal models of neurological disorders, such as Parkinson's disease and multiple sclerosis Wang et al., 2021Wang et al., , 2022aWang et al., , 2022b. Since depression is a common psychiatric symptom in patients with neurological disorders, arketamine could improve depressive symptoms in patients with neurological disorders Wei et al., 2022a). Moreover, (R,S)-ketamine and arketamine have anti-shock, anti-inflammatory, bronchodilating, and neuroprotective effects (Hirota and Lambert, 2022;Wan et al., 2022;Zhang et al., 2021aZhang et al., , 2021b. Therefore, it is possible that arketamine could be a new prophylactic or therapeutic drug for patients with neurological disorders or inflammatory diseases Wei et al., 2022a).
Finally, the precise molecular and cellular mechanisms involved in the antidepressant effects of (R,S)-ketamine and its enantiomers remain unclear (Hashimoto, 2020a(Hashimoto, , 2022Jelen et al., 2021;Scotton et al., 2022;Wei et al., 2022a). Brain-body communication such as gut-microbiota-brain axis and brain-spleen axis may play a role in the beneficial effects of arketamine Qu et al., 2017;Wan et al., 2022;Wang et al., 2022a;Wei et al., 2022b;Yang et al., 2017;Zhang et al., 2021c) although further study is needed. Notably, the classical psychedelics, such as psilocybin and N, N-dimethyltryptamine, are reported to produce rapid-acting and sustained antidepressant actions in patients with MDD (Bender and Hellerstein, 2022;Carhart-Harris et al., 2016;James et al., 2022;McClure and Roth, 2022). However, it remains unclear whether the molecular mechanisms underlying the antidepressant effects of psychedelics are similar to the antidepressant effects of (R, S)-ketamine. Further detailed study is needed to fully understand the molecular and cellular mechanisms underlying the rapid-acting antidepressant actions of arketamine and psychedelics.

Declaration of competing interest
Dr. Hashimoto is the inventor of filed patent applications on "The use of R-ketamine in the treatment of psychiatric diseases", "(S)-norketamine and salt thereof as pharmaceutical", "R-ketamine and derivative thereof as prophylactic or therapeutic agent for neurodegeneration disease or recognition function disorder", "Preventive or therapeutic agent and pharmaceutical composition for inflammatory diseases or bone diseases", "R-ketamine and its derivatives as a preventive or therapeutic agent for a neurodevelopmental disorder", and "TGF-β1 in the treatment of depression" by the Chiba University. Dr. Hashimoto has also received speakers' honoraria, consultant fee, or research support from Abbott, Boehringer Ingelheim, Daiichi-Sankyo, Meiji Seika Pharma, Seikagaku Corporation, Sumitomo-Pharma, Taisho, Otsuka, Murakami Farm and Perception Neuroscience. The other authors have no conflict of interest.

Data availability
No data was used for the research described in the article.