Ketamir-2 is a new molecular entity and novel ketamine analog designed to have an improved oral bioavailability, and offer a superior safety profile compared to existing ketamine treatments. Unlike ketamine, which acts broadly as an N-Methyl-D-aspartic acid (NMDA) receptor antagonist, influencing multiple receptor sites including opioid and monoaminergic systems (Zorumski et al., 2016), Ketamir-2 selectively targets the phencyclidine (PCP) site of the NMDA receptor with lower affinity. This selective binding reduces the risk of adverse effects, such as sedation and dissociation, commonly seen with ketamine (Orhurhu et al., 2023), while aiming to maximize therapeutic outcomes for conditions like depression, anxiety, and neuropathic pain.

The binding of ketamine to the PCP site within the NMDA receptor is crucial as it allows for noncompetitive antagonism of the receptor, effectively blocking its activity (Sharp, 1996). This binding mechanism contributes to ketamine’s analgesic and antidepressant effects, as it disrupts normal glutamatergic neurotransmission (Mion and Villevieille, 2013). Additionally, the high trapping capability of ketamine at the PCP site enhances its potency and duration of action in modulating synaptic activity. In addition to the PCP site on the NMDA receptor, ketamine also binds to the sigma-1 receptor, which is implicated in various neuroprotective and antidepressant effects (Robson et al., 2012). Furthermore, ketamine has been shown to interact with opioid receptors, particularly the mu-opioid receptor, contributing to its analgesic properties (Sarton et al., 2001). These multiple binding sites highlight ketamine’s complex pharmacological profile and its potential therapeutic applications beyond anesthesia. Ketamine and its metabolites also interact with several neurotransmitter transporters, including the serotonin transporter (SERT) (Spies et al., 2017) and norepinephrine transporter (NET) (Liebe et al., 2018). Ketamine acts as an uptake inhibitor for SERT, with reported IC50 values indicating significant inhibition of serotonin uptake, which may contribute to its antidepressant effects (Spies et al., 2017). Additionally, it has been shown to inhibit dopamine and norepinephrine uptake, further influencing neurotransmitter dynamics in the central nervous system (Hess et al., 2021).

The connection to schizophrenia arises from ketamine’s ability to disrupt normal brain signaling, particularly in regions like the prefrontal cortex, nucleus accumbens, and hippocampus, which are also implicated in schizophrenia (Abdallah et al., 2018). Research shows that ketamine increases dopamine turnover in the nucleus accumbens, a brain area involved in regulating movement and reward. This dopamine surge aligns with the dopamine hypothesis of schizophrenia, which suggests that excessive dopamine activity contributes to psychotic symptoms (Hunt et al., 2005)​. Additionally, ketamine reduces the activity of inhibitory GABAergic interneurons, leading to a disinhibition of excitatory neurons. This imbalance creates a state of neural hyperactivity, potentially mirroring the sensory and perceptual distortions seen in schizophrenia. Studies also indicate that ketamine-induced hyperlocomotion can be blocked by dopamine receptor antagonists like haloperidol, reinforcing the link between this behavior and dopaminergic mechanisms relevant to schizophrenia (Irifune et al., 1991)​. However, the effect is not solely dopamine-driven—ketamine’s impact on glutamate and other systems, like endocannabinoids and serotonin, suggests a broader disruption of neural networks (Xu, et al., 2020)​. In this respect, it was interesting to assess the effect of Ketamir-2 in the same model.

Recently, the S-enantiomer of Ketamine, esketamine, has shown efficacy as an adjunct therapy in treatment resistance depression receiving approval for use in combination with an oral antidepressant (Daly et al., 2018). Rat models have been extensively used to assess the antidepressant and anxiolytic effects of ketamine. These models provide valuable insights into ketamine’s rapid-acting and long-lasting effects on mood and anxiety-related behaviors. Several studies have investigated the effects of ketamine using the Open Field Test (OFT) (Fitzgerald et al., 2021), Elevated Plus Maze (EPM), and Forced Swimming Test (FST) in rodent models. The elevated plus maze (EPM) is a widely used behavioral test to assess anxiety-like behavior in rodents, and it has been employed in several studies to investigate the effects of ketamine. Acute administration of ketamine was shown to increase time spent in open arms of the EPM in both saline- and morphine-conditioned mice, indicating an anxiolytic-like effect (McKendrick et al., 2020)​. Also, Ketamine induced antidepressant-like effects in the FST at 30 min and 24 h post-administration, reducing immobility time (Parise et al., 2013)​. We have employed these models to assess the potential antidepressant and anxiolytic effects of Ketamir-2.

Ketamir-2 is a five membered ring analogue of ketamine (Figure 1). In the in vivo studies we have used the hemipamoate salt of Ketamir-2, also known as 1-(2-chlorophenyl)-N-methyl-2-oxocyclopentan-1-aminium 4,4′- hemi-salt of methylenebis (3-hydroxy-2-naphthoate); Ketamir pamoate; Ketamir hemipamoate; M209. Its chemical formula is C₄₇H₄₄Cl₂N₂O₈ with a molecular weight of 835.77 g/mol. The chemical structure is provided in Figure 1. This drug is a racemic mixture, with 1:1 ratio between the R- and S- enantiomers.

Ketamine’s role as an antidepressant in human and in animal models has been extensively studied, offering insights into its rapid-acting effects and mechanisms, which differ from traditional antidepressants. In preclinical research, ketamine is typically administered to rodents (mice or rats) to evaluate its ability to reverse depression-like behaviors, often in models mimicking human depressive symptoms. Common animal models include the Forced Swim test (FST) and tail suspension test (TST). In the FST and TST, animals are placed in inescapable situations (e.g., a water tank or suspended by the tail), and immobility time is measured as an indicator of behavioral despair—analogous to hopelessness in depression. Ketamine, significantly reduces immobility within 30 min to a few hours, unlike traditional antidepressants like SSRIs (e.g., fluoxetine), which require weeks to show effects. The mechanism behind this rapid action involves ketamine’s antagonism of NMDA receptors, which triggers a cascade of neurobiological changes.

Unlike its hyperlocomotion effects at higher doses, which seem to be mediated through increased Dopamine release, lower antidepressant doses (e.g., 10 mg/kg) enhance synaptic plasticity. Ketamine’s metabolite, (2R, 6R)-hydroxynorketamine (HNK), also contributes. Unlike ketamine itself, HNK lacks NMDA antagonism but still exerts antidepressant effects, possibly via AMPA receptor activation, further stimulating synaptic plasticity. In mice, HNK reduces FST immobility at doses as low as 5–10 mg/kg, suggesting it may underlie some of ketamine’s benefits without its dissociative side effects. These findings align with human studies, where ketamine (e.g., 0.5 mg/kg IV) relieves depressive symptoms within hours, paving the way for derivatives like esketamine, now FDA-approved.

While Ketamir-2 is likely binding at PCP-site of NMDA, like ketamine, it markedly differs from Ketamine in its lower affinity to this site, but also in its marked selectivity towards numerous other receptors sites and transporters that Ketamine also targets. Also, the main metabolite of Ketamir-2, unlike the metabolite of ketamine, retains low PCP binding affinity and lacks interaction with the NMDA site.

In other related studies (manuscript in preparation), Ketamir-2 given orally at similar dose ranges as in the current study, was shown to be effective in reducing pain in several models, including spinal nerve ligation, paclitaxel or diabetes induced neuropathic models (data not shown). Ketamine is widely studied for its analgesic properties, particularly in acute pain, chronic pain, and neuropathic pain. Its mechanisms involve NMDA receptor blockade, modulation of glutamatergic signaling, and interactions with other systems (e.g., opioid, monoaminergic) (Niesters et al., 2014; Hirota and Lambert, 2011).

The similarity of anti-depressant and pain reducing activities between Ketamir-2 and Ketamine, albeit the observed low affinity of Ketamir-2 and its metabolite nor-Ketamir on the NMDA PCP-site, and its lack of activity at other sites, compared with Ketamine, suggest that the PCP-site has the most important role for these specific activities.

We have a series of informational ADME assessments typical for a novel therapeutic classified as a small molecule. In model Caco-2 permeation system, it was shown that Ketamir-2 is permeable at a dose of 10 µM (A-B and B-A) and is not highly bound to plasma proteins in mouse, rat, dog, cynomolgus monkey, or human plasma (data not shown). Substrate interaction and metabolism studies indicate that Ketamir-2 is not a substrate of P-glycoprotein (data not shown). In addition, the pharmacokinetics of Ketamir-2 have been characterized in a series of in vivo PK/TK studies conducted in Sprague Dawley rats and Beagle dogs. General PK data indicate that Ketamir-2 does cross the blood brain barrier with longer exposure times and greater levels of nor-Ketamir present in the brain. Moreover, the PK profile of Ketamir-2 following oral administration is characterized by rapid absorption, a short half-life and high clearance (data not shown). Brain levels of the drug are expected to affect both its pharmacological activity and its selectivity towards other potential side-effects. Studies conducted in rats at high doses have recently confirmed the lack of such side effects, further confirming its good selectivity.

For Ketamine, there are evidence that ketamine is a P-gp substrate, with implications for its absorption, distribution, and efficacy in pain management (e.g., neuropathic pain) and other therapeutic contexts. The findings suggest that P-gp-mediated efflux may contribute to ketamine’s low oral bioavailability and variable central effects, which are critical for its analgesic applications (Tomkins et al., 2018). In this respect, the absence of P-gp interaction and the better oral bioavailability of Ketamir-2 (and its metabolite), compare with Ketamine, may explain that even at much lower affinity towards the PCP-site, and better selectivity, some of the pharmacological activities, such as antidepressant and pain-reducing actions can be maintained.

To further confirm the potential consequences of the lower affinity and selectivity of Ketamir-2 in vivo, we have evaluated the effects of Ketamir-2 administration on locomotor activity in male C57BL6/J mice. Measurements of spontaneous locomotor activity by actimetry are widely used for in vivo safety evaluation of new drugs on the central nervous system (CNS) in rodents.

In this study, vehicle-treated animals displayed normal time-dependent activity during the 1-h recording period. Animals treated with Ketamir-2 20 mg/kg ip and 50 mg/kg po showed a similar pattern of locomotor activity compared to the vehicle group. On the other hand, Ketamir-2 50 mg/kg ip decreased locomotor activity compared to vehicle-treated animals. Ketamine 20 mg/kg ip increased locomotor activity. The differences between the sedative effect observed upon intraperitoneal activity, compared with the lack of effect upon oral administration, may be explained by the relatively low bioavailability of ketamir-2 under these experimental conditions.

The mechanisms underlying ketamine-induced hyperlocomotion involve disruptions in glutamatergic, dopaminergic, and other neurotransmitter systems, which parallel neurochemical imbalances implicated in schizophrenia (Irifune et al., 1991)​. As studies indicate that ketamine-induced hyperlocomotion can be blocked by dopamine receptor antagonists like haloperidol, it is suggested that this behavior is mainly dopamine-linked. However, the effect is not solely dopamine-driven—ketamine’s impact on glutamate and other systems, like endocannabinoids and serotonin, suggests a broader disruption of neural networks (Xu, et al., 2020)​. The absence of effect of Ketamir-2 in this model, reflects its lack of interaction at adrenergic reuptake sites and/or dopaminergic neurons reinforcing the selectivity of Ketamir-2 for the NMDA receptor.

The Open Field Test (OFT), Elevated Plus Maze (EPM), and Forced Swimming Test (FST) are commonly employed behavioral assays used to evaluate the efficacy of anxiety and depression medications. In the Open Field Test (OFT), mice treated with antidepressants typically exhibit greater locomotor activity, moving more extensively and exploring a larger area of the open field compared to control mice. These treated mice are also more inclined to spend time in the central region of the open field, which is usually avoided by anxious animals. Such behavior indicates a decrease in anxiety and an increase in exploratory tendencies. According to the results presented in Figure 5, mice treated with all tested concentrations of Ketamir-2 moved larger distance than vehicle treated mice. Mice treated with 100 and 200 mg/kg Ketamir-2 also were significantly faster, with higher velocity compared to control. Mice treated with Ketamir-2 also showed a trend of more time spent in the center, however it was not statistically significant.

In the Elevated Plus Maze (EPM), mice treated with antidepressants typically demonstrate increased exploratory behavior and reduced anxiety. These mice are more likely to enter and spend more time in the open arms of the maze, areas that anxious animals usually avoid. This increased presence in the open arms suggests an anxiolytic effect and enhanced willingness to explore novel environments. According to the results presented in Figure 6, mice treated with 100 or 300 mg/kg Ketamir-2 moved larger distance than vehicle treated mice. Mice treated with 300 mg/kg Ketamir-2 also significantly were faster, with higher velocity, and spent more time in the center of the maze, compared to control. However, there was no significant difference in time spent in the open arms only (without the center zone) of Ketamir-2 treated mice compared with control. Ketamine treated mice did spend significantly more time in the open arms, in the center is included.

In the Forced Swimming Test (FST), mice treated with antidepressants are generally more mobile. Antidepressant-treated mice show reduced immobility time compared to control mice. Instead of remaining immobile, they exhibit increased active behaviors such as swimming and climbing. This increased activity is interpreted as a sign of decreased behavioral despair and an antidepressant-like effect. According to the results presented in Figure 7, mice treated with Ketamir-2 show reduction in traveled distance and lower velocity (300 mg/kg Ketamir concentration shows similar trend to the rest of the concentrations, however it is not statistically significant). When mobility state was analyzed, 300 mg/kg of Ketamir-2 is the only Ketamir-2 treatment that reduced hyper-mobility, and increased mobility significantly. No treatment significantly induced immobility compared to vehicle treated, however-100 and 200 mg/kg Ketamir-2 and Ketamine showed a trend of reduced immobility.

Ketamine was used as a positive control in this experiment however, in many parameters it did not differ from vehicle treatment, or its response was opposite to that of Ketamir-2. The lasting antidepressant effects of ketamine may vary depending on the treatment regimen, concentration, and the time studied after administration. Therefore, these parameters should be carefully examined in future studies.