More than 350 million people across the globe suffer from major depressive disorder (MDD), which is a debilitating and refractory disease that is ranked second globally in terms of disease burden (Smith, 2014). Depression has become a serious global public health challenge due to its characteristics of high morbidity incidence, high suicidal rate, high recurrence rate and high disability rate (Otte et al., 2016). Several different classes of antidepressants have been used to treat depression such as tricyclic antidepressants (TCAs), selective serotonin (5-HT) reuptake inhibitors (SSRIs), and serotonin-norepinephrine (NE) reuptake inhibitors (SNRIs). In general, currently available antidepressants are effective in the treatment of depression but most have various shortcomings such as delayed onset of action, limited therapeutic efficacy with modest response rate (50–70%), lack of cognitive improvement, causing sexual dysfunction and suicidal tendencies (Dupuy et al., 2011). For example, venlafaxine was the first marketed SNRI antidepressant and its main active metabolite, desvenlafaxine (O-desmethylvenlafaxine, ODV; its succinate monohydrate salt [DVS] is marketed as Pristiq®) which is also a SNRI, was later marketed as an improved antidepressant than its parent compound, but both still have significant unwanted effects such as gastrointestinal discomfort and sexual dysfunction (Shelton, 2019). Therefore, safer and effective antidepressants are in high demand to meet the clinical needs.

In recent years, the triple monoamine reuptake inhibition hypothesis has been increasingly recognized as a potentially beneficial strategy to treat depression (Subbaiah, 2018). Dopamine (DA) plays an essential role in the etiology of depression (Dunlop BW and Nemeroff, 2007; Belujon and Grace, 2017). Increasing the synaptic dopamine level in the mesolimbic cortex can improve the late onset effect of antidepressants. Elevation of the dopamine level in the hypothalamus enhances the dopaminergic neural tone in the nucleus accumbens, which can improve anhedonia, cognitive function, rewarding motivation and goal-directed behaviors, and reduce sexual dysfunction related to the use of SSRIs and SNRIs (Belujon and Grace, 2017). For example, bupropion is a weak inhibitor of DA and NE transporters and, when combined with SSRIs or SNRIs, it can shorten the onset of antidepressant action and reduce the occurrence of sexual dysfunction (Trivedi et al., 2006; Zisook et al., 2006). In addition, the SSRI citalopram has been found to significantly improve the onset of action when combined with the dopaminergic agent methylphenidate (Lavretsky et al., 2006). Combined, these results suggest that triple reuptake inhibitors which can enhance the functionality of all three monoamines may have improved therapeutic profile to treat depression such as faster onset, improved cognitive function, less anhedonia and sexual dysfunction (Prins et al., 2011).

Toludesvenlafaxine hydrochloride ((±)-4-(2-(dimethylamino)-1-(1-hydroxycyclohexyl) ethyl) phenyl 4-methylbenzoate hydrochloride) (also known as LPM570065 or ansofaxine) is a new chemical entity and a potential triple reuptake inhibitor (Figure 1). It is a prodrug that can quickly converts to the SNRI desvenlafaxine under the hydrolysis of ubiquitous esterase in vivo (Zhang et al., 2014). Microdialysis study shows that toludesvenlafaxine can increase the serotonin (5-HT), norepinephrine (NE) and dopamine (DA) levels in the striatum after oral drug administration while, as expected, desvenlafaxine does not increase the striatal DA level (Zhang et al., 2014). These results suggest that toludesvenlafaxine may have unique pharmacological effects beyond its conversion to desvenlafaxine. The goals of the current study were three-fold: 1) despite the neurochemical evidence that toludesvenlafaxine was able to increase the central 5-HT, NE and DA levels (Zhang et al., 2014), conclusive in vitro binding and functional data supporting toludesvenlafaxine as a triple reuptake inhibitor have not been reported, which will be examined in this study; 2) although toludesvenlafaxine has been shown to reduce the immobility time in the forced swim test (Zhang et al., 2014), forced swim test as a rodent model of depression has been controversial and it is more appropriate to be used as a quick screening assay for potential antidepressant-like activities (Nestler and Hyman, 2010); as such, this study further evaluated the antidepressant-like activity of toludesvenlafaxine using two rodent models of depression with higher validity: chronic unpredictable mild stress model and olfactory bulbectomized model; 3) although toludesvenlafaxine has been shown to be able to enter the brain after oral and intravenous administration (Zhang et al., 2014), detailed pharmacokinetic profile remains unknown, which will be examined in this study.

The primary findings of the current study were that toludesvenlafaxine demonstrated characteristic tri-reuptake inhibitor profile in vitro assays, which showed highest inhibition activity at SERT. In preclinical rat models of depression, toludesvenlafaxine demonstrated dose-dependent and significant antidepressant-like activity. In preclinical pharmacokinetic study, toludesvenlafaxine was well-absorbed into the brain with highest drug concentration detected at hypothalamus. Overall, these results reveal toludesvenlafaxine as a tri-uptake inhibitor and has the potential to act as a safe antidepressant agent.

One major issue with currently wide prescribed antidepressants including SSRIs and SNRIs is the slow onset of action and limited therapeutic efficacy. Many patients with MDD only partially respond to and some have no clinically meaningful response to these antidepressants. In addition, potent 5-HT reuptake inhibition often leads to hypodopaminergic adverse effects such as decreased sexual function, weight gain and emotion blunt (Nutt, 2008). Mesolimbic dopaminergic system is crucially involved in cognition, motivation and reward-related behaviors (Nestler and Carlezon, 2006). Therefore, the inclusion of DA reuptake inhibition may elevate the mesolimbic dopaminergic activity and ameliorate some 5-HT reuptake inhibition-related adverse effects, which is a primary rationale for the proposed tri-reuptake inhibitors as potential treatments of MDD. Clinical evidence that the combination therapy with the DA transporter blocker bupropion and SSRIs is effective in SSRI-resistant MDD patients (Zisook et al., 2006) seems in support of this notion. Over the years, several TRIs with varying potencies in inhibiting monoamine transporters have been developed and tested in preclinical and clinical studies with some success and interest remains strong in developing TRIs for the treatment of MDD (Sharma et al., 2015).

In this study, toludesvenlafaxine demonstrated high affinities at SERT, NET and DAT and differential inhibition potencies at the three transporters in cell-based in vitro assays, with the binding affinities ratio of 2.7: 1: 10.4, respectively. The reuptake inhibition ratio of toludesvenlafaxine at 5-HT, NE and DA was 23.3: 1.2: 1, showing clear preference for inhibiting 5-HT reuptake. As a comparison, DVS only shows reuptake inhibition for 5-HT and NE (IC₅₀ values were 53.5 ± 7.6 nM and 568.7 ± 29.9 nM, respectively), with an inhibition ratio of 10.6:1, similar to that of toludesvenlafaxine (Table.1). However, 3.9-fold higher affinity of toludesvenlafaxine to DAT compared to SERT, whereas 23.3-fold higher reuptake inhibition of toludesvenlafaxine to 5-HT compared to DAT. This discrepancy may be due to different transporter expression levels, different methodology or in different laboratories (Ilic et al., 2020). These results are consistent with a previous study showing that toludesvenlafaxine increases the striatal 5-HT, DA and NE levels while DVS only increases the striatal 5-HT and NE levels (Zhang et al., 2014). Together, these findings strongly suggest that toludesvenlafaxine is a triple reuptake inhibitor at 5-HT, NE and DA.

CUMS model is a well-validated animal model that mirrors many aspects of human depression symptomology such as anhedonia, decreased motivation and locomotion, and elevated stress hormone level (Willner, 2016). Likewise, olfactory bulbectomized model is well-validated and has decades of research literature to support its utility as a valuable animal model of depression (Harkin et al., 2003; Morales-Medina et al., 2017). In this study, it was found that although both DVS and toludesvenlafaxine significantly prevented hypoactivity and anhedonia in the CUMS rats, toludesvenlafaxine seems to be more effective than DVS. In contrast, DVS was largely ineffective in the rat olfactory bulbectomy model, but chronic treatment with toludesvenlafaxine was able to significantly prevent the hyperactivity, anhedonia and elevated serum corticosterone level (Tables 3 and 4). Together, these results suggest that toludesvenlafaxine shows significant antidepressant-like activity and may be more effective than DVS at similar doses.

Conventional antidepressants such as SSRIs or SNRIs usually need several weeks to exert antidepressant effect, and maximal effect may occurred in 6–8 weeks of treatment (F Artigas et al., 2018). The activation of mesolimbic dopaminergic neurons plays central role in the control of motivation and reward related behaviors that are frequently blunted in depression (Naranjo et al., 2001). Thus, immediate increases in synaptic dopamine levels (via inhibition of dopamine reuptake) may result in a more rapid relief of symptoms associated with anhedonia than produced by SSRIs or SNRIs (Skolnick et al., 2006). Toludesvenlafaxine treatment at 4–16 mg/kg significantly and dose-dependently reduced the hyperactivity on the 7th day of dosing. These results suggest that toludesvenlafaxine may have fast-acting antidepressant-like activity, and are consistent with the previous findings of tri-reuptake inhibitor (Prins et al., 2011).

One common side effect with the use of antidepressants in general, and SSRIs in particular, is sexual dysfunction. Although the exact mechanisms that cause sexual dysfunction remain elusive, it is suggested that the inhibitory effects of serotonergic antidepressants lead to blunted dopamine release in hypothalamic and mesolimbic areas and neuroendocrine imbalance such as elevated prolactin secretion and reduced testosterone level (Lyons et al., 2016; Bijlsma et al., 2014). In this study, while the rats that received olfactory bulbectomy surgery showed marked reduction of serum testosterone level, the difference did not reach statistical significance primarily due to the large variability in the sham-operated rats. Nonetheless, chronic toludesvenlafaxine treatment dose-dependently and significantly prevented the testosterone reduction while DVS showed no significant effect (Table 4). This result is consistent with and may be explained by the high concentration of both toludesvenlafaxine and its primary metabolite ODV in hypothalamus after oral dosing. In our previous studies, repeated treatment with toludesvenlafaxine (30–300 mg/kg) for 63 days dose-dependently increased sperm density and count in male rats for up to 24.1% (Guo et al., 2018). In addition, prolonged treatment with toludesvenlafaxine (30–300 mg/kg) for 91 days dose-dependently reduced the serum prolactin level by as much as 78% and increased the serum testosterone level by up to 37% (Li et al., 2018). Given the strong correlation between low testosterone level and depression symptomology and the effectiveness of testosterone supplement therapy in depressed men (Walther et al., 2019), these results strongly suggest that toludesvenlafaxine may have low risk of leading to sexual dysfunction but instead its effect on androgen level may benefit its antidepressant effect and improve the sexual function in patients with depression, which ultimately will improve the quality of life of these patients.

In an effort to understand the bioavailability and pharmacokinetic profile of oral toludesvenlafaxine administration, rats were used and toludesvenlafaxine was found to be quickly converted into ODV after drug absorption. Only trace amount of toludesvenlafaxine was detected in the rat plasma after 8 mg/kg toludesvenlafaxine administration. The analysis of tissue distribution found that after intragastric toludesvenlafaxine administration, the drug quickly entered the central nervous system and highest concentrations of both toludesvenlafaxine and ODV were found in the hypothalamus at much higher level than in the plasma. This finding suggests that, for reasons currently unclear, portion of toludesvenlafaxine was not converted to ODV after absorption but quickly redistributed to certain tissues including hypothalamus. Although it is currently unclear the implications of this distribution profile in the therapeutic effects of toludesvenlafaxine, the drug and its metabolite ODV were eliminated quite slowly in the hypothalamus, detectable even 12 h after drug dosing. In a separate study using Beagle dogs as experimental subjects, it was found that after oral administration of 80 mg toludesvenlafaxine extended release tablets the drug was quickly converted into ODV and the plasma toludesvenlafaxine level was below detection limit. However, toludesvenlafaxine was detectable in the dog hypothalamus and the whole brain tissue (unpublished data). Together, these results suggest that toludesvenlafaxine can easily pass the blood brain barrier and enter the brain, with highest drug concentrated in the hypothalamus tissue.

In summary, this study characterized the in vitro binding affinity and reuptake inhibiting activities of toludesvenlafaxine at DAT, NET and SERT, which confirms its triple reuptake inhibitor profile. In two widely-used rat depression models, toludesvenlafaxine demonstrated dose-dependent and significant antidepressant-like activity with notable effect on preventing testosterone decrease. Toludesvenlafaxine readily enters the brain and, along with its major metabolite ODV, concentrates at specific brain regions such as the hypothalamus. The toxicity studies confirmed toludesvenlafaxine had good safety and tolerability properties in acute, subchronic oral toxicity (13-weeks), and genotoxicity evaluations (Li et al., 2018). fertility and early embryonic development (Guo et al., 2018). No CNS-stimulating (e.g., hyperactivity, irritation or hyperreactivity) or CNS-depressing (e.g., sedation, hypolocomotion, muscle relaxation) effects were observed after acute treatment or repeated dosing treatment with toludesvenlafaxine (Li et al., 2018). Preclinical abuse potential evaluations have been systematically studied which suggest that toludesvenlafaxine has no abuse potential (unpublished results).

Combined, this study for the first time systematically characterized a novel tri-reuptake inhibitor, toludesvenlafaxine, and lays the foundation for future studies to translate preclinical results to therapeutic efficacy in patients with major depressive disorder. Toludesvenlafaxine has finished all preclinical studies and is currently under phase III clinical trial for the treatment of major depressive disorder (clinical trial registration identifier: NCT04853407).