Efficacy of SAL0114, an Oral NMDA Receptor Antagonist with Multimodal Activity, in Major Depressive Disorder

Efficacy of SAL0114, an Oral NMDA Receptor Antagonist with Multimodal Activity, in Major Depressive Disorder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Efficacy of SAL0114, an Oral NMDA Receptor Antagonist with Multimodal Activity, in Major Depressive Disorder Xuefeng Hu, Wei Xing, Ruhuan Wang, Xiaoqing Li, Jiahuan Li, Jie Yan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4119597/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The first FDA-approved rapid-acting antidepressant was esketamine, but its use was limited because of the risk of addiction, and although the subsequent combination of dextromethorphan (DM) + bupropion (BUP) has alleviated some of the problems, there is still a clinical need for safer and more effective compounds. In this paper, we use a strategy of deuterated substitution of DM to improve stability and reduce metabolites to improve safety and efficacy. We analyzed the effects of deuterium substitution on the in vitro activities of DM and deDX (deDM metabolite) by radioligand competition binding assay, evaluated the antidepressant and synergistic effects of deDM and BUP by a mouse behavioral despair model, and further assessed the synergistic mechanism of deDM and BUP by a reserpine-induced hypothermia rats model and an ammonia-induced cough mice model, which showed that deuterium substitution does not change the DM and deDX (deDM metabolite) in vitro activity, but can improve the in vivo effectiveness of DM, suggesting that deDM has the potential to be more stable in vivo with fewer metabolites, i.e., fewer side effects, and therefore, deDM and BUP is a safer and more potent combination for depressed patients than the combination of DM and BUP. Biological sciences/Drug discovery/Pharmacology/Pharmacodynamics Health sciences/Neurology/Neurological disorders Major depressive disorder NMDAR receptors Sigma-1 receptor Dextromethorphan Bupropion Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Major depressive disorder (MDD) is a severe mental illness that affects millions of people globally 1 . Currently, there are five main classes of antidepressants, including aminoketones, triazolopyridines, monoamine oxidase inhibitors, tricyclic antidepressants, and selective serotonin reuptake inhibitors. However, a common problem with these drugs is that they are slow to start working, often take weeks to take effect, and more than 30% of patients do not improve after treatment, putting them at risk of self-harm 2 . Fortunately, the 2019 FDA-approved esketamine addresses the slow onset of depression by providing rapid relief of depression within 24 hours 3 . However, esketamine is highly addictive and currently limited in use in healthcare settings, which makes long-term medication management difficult for patients; as a result, there is an urgent clinical need for safer and faster-acting antidepressants. Dextromethorphan (DM) is an over-the-counter antitussive drug with a similar mechanism of action to that of esketamine, both of which block NMDA receptors 4,5 . Moreover, dextromethorphan has been used as an antitussive agent for more than 40 years, so it has better safety potential than esketamine. However, there are a number of issues that must be addressed before administering dextromethorphan to depressed patients. Dextromethorphan has a strong hepatic first-pass effect, which decreases its bioavailability and ultimately leads to diminished antidepressant effects 6,7 . There are two ways to overcome this. First, a CYP2D6 inhibitor was added to increase the stability of dextromethorphan. The U.S. Food and Drug Administration (FDA) has approved combination medications such as AVP-923 (DM+quinidine) and AXS-05 (DM+bupropion) based on similar tactics 8,9 . Second, deuterium substitution was applied to dextromethorphan. Deuterium is a naturally occurring isotope of hydrogen, and by deuterium substitution at the appropriate metabolic site, the half-life of DM can be prolonged. By adopting these two strategies, we altered and combined various compounds, ultimately selecting the deDM+BUP combination. This paper emphasizes in vitro and in vivo pharmacological experiments. These studies assessed the impact of deuterium substitution on the in vitro activity and in vivo stability of DM, the antidepressant effects and mechanisms of deDM and BUP, and the synergistic effect of combining deDM and BUP on antidepressant effects. These pharmacological studies pave the way for clinical trials of the combination of deDM and BUP. The findings outlined in this paper also provide valuable insights into the synergistic effects, safety, and potential mechanisms of action of this combination compound, which ultimately benefits patients with depression. Methods Animals Male Sprague–Dawley rats weighing 200–230 g and male C57BL/6J mice weighing 19–22 g were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang, China), and male ICR mice weighing 18–22 g were obtained from Shanghai Lingchang Biology Science and Technology Co., Ltd. (Shanghai, China). All rats and mice were housed 4-5 per cage under an 8:00 am/8:00 pm light/dark schedule at controlled temperature and humidity with free access to food and water. All procedures were carried out in accordance with guidelines approved by the Animal Ethics Committee of Shenzhen Salubris Pharmaceuticals Co., Ltd. (Shenzhen, China) or Wuxi Apptec Co., Ltd. Reagents and drugs Deuterated dextromethorphan (deDM), deuterated dextrorphan (deDX), bupropion (BUP), dextromethorphan (DM) and dextrorphan (DX) were obtained from Shenzhen salubris Pharmaceuticals Co., Ltd. (Shenzhen, China). Reserpine and imipramine were obtained from Sigma‒Aldrich (Shanghai, China); ammonium hydroxide was purchased from Ailan (Shanghai) Chemical Technology Co., Ltd. (Shanghai, China); and 3 H-MK801, 3 H-hydroxytryptamine creatine sulfate, 3 H-norepinephrine hydrochloride, 3 H-imipramine, nisoxetine 3H-hydrochloride, serotonin transporter membrane and norepinephrine transporter were purchased from PerkinElmer, Inc. (Massachusetts, USA). ); NMDA membranes were obtained from Pharmron (Beijing, China); and memantine hydrochloride and paroxetine were purchased from Adooq Bioscience (California, USA). Radioligand competition binding NMDAR binding Membrane preparation was performed as previously described (Awtry and Werling, 2003) 10 . Briefly, the brains of male Sprague‒Dawley rats were dissected for tissue collection. The tissue was then homogenized in ice-cold extraction solution, centrifuged at 40,000 × g for 10 min, the supernatant was discarded, the precipitate was resuspended in extraction solution, and the above steps were repeated 2 times. The precipitate was resuspended in resuspension solution and centrifuged at 40,000×g for 10 min. The supernatant was discarded, and the above steps were repeated 2 times. Finally, the precipitate was resuspended in a 10-fold volume of resuspension solution and stored in portions at -80 °C. The protein concentration was determined with a Pierce TM BCA protein assay kit (Thermo Fisher Scientific, Massachusetts, USA). The competitive binding of the compound and 3 H-MK801 on the NMDA receptor was assessed using the filtration binding method, which involves washing the radioligands that are not bound to the NMDA receptor and examining the signal values of the radioligands bound to the NMDA receptor with a Microbeta instrument (Perkin Elmer, Massachusetts, USA). Serotonin transporter (SERT) binding The experiments were carried out in the Pharmaron laboratory. The competitive binding of the samples and 3 H-hydroxytryptamine creatine sulfate on SERT was assessed using a radioligand binding method. The ability of the compound to bind competitively to SERT (Ki) was determined by detecting the signal value of the radioligand bound to the transporter via a Microbeta instrument (Perkin Elmer, Massachusetts, USA). Norepinephrine transporter (NET) binding The experiments were carried out in the Pharmaron laboratory. The competitive binding of the compound and 3 H-Nisoxetine on NETs (PerkinElmer, Massachusetts, USA) was assessed using a radioligand binding method. The ability of the compound to bind competitively to the NET (Ki) was determined by detecting the signal value of the radioligand bound to the transporter via a Microbeta instrument (Perkin Elmer, Massachusetts, USA). Sigma 1 receptor binding The experiments were carried out in the Eurofins Discovery laboratory. The competitive binding of the samples and 3 H-pentazocine to the Sigma-1 receptor of human-derived Jurkat cells was assessed using a radioligand binding method. The ability of the compound to bind competitively to the Sigma-1 receptor (Ki) was determined by detecting the signal value of the radioligand bound to the receptor. Nicotinic acetylcholine receptor (nAchR) α3β4 binding The experiments were carried out in the Eurofins Discovery laboratory. The competitive binding of the samples and 125 I-epibatidine to the nAchR α3β4 of human recombinant CHO-K1 cells was assessed using a radioligand binding method. The ability of the compound to bind competitively to the nAchR α3β4 (Ki) was determined by detecting the signal value of the radioligand bound to the receptor. Forced swim test The mouse forced swim test was built in a way similar to what was published previously by Nguyen et al. 4 with some minor modifications. Mice were placed in individual cylinders of water (18 cm deep) for a total of 6 min for the forced swim test. The first two minutes were for acclimatization and were not scored. The remaining 4 minutes were used to measure immobility time with ANY-Maze Version 4.63 video tracking software (Stoelting Co., Illinois, USA). The only movement needed to keep the animal's head above the water's surface was considered immobility. The ANY-Maze software settings were as follows: accustomization period = 120 s, test duration = 240 s, minimum immobility time = 2 s, and immobility sensitivity =75%. Tail suspension test The TST was adapted from the behavioral despair test described by Nguyen et al. 4 , with some modifications. Each mouse was hung up by its tail and attached to a metal rod using adhesive tape (2 cm from the tip of the tail). The distance between the animals and any object was kept at least 15 cm. The ANY-maze version 4.63 video tracking program was used to record individual mice for a total of 6 minutes. The first two minutes were for acclimatization and were not scored. The remaining 4 minutes were used to measure immobility time with ANY-Maze Version 4.63 video tracking software (Stoelting Co., Wood Dale, IL). When the mice hung passively and motionlessly, they were considered immobile. Reserpine-induced hypothermia in rats The reserpine-induced hypothermia test was performed according to the method described by Rojas-Corrales et al. 11,12 , with some modifications. In brief, eighteen hours after reserpine treatment (4 mg/kg, ip), rectal temperature was measured in degrees centigrade with a rectal probe connected to a thermometer (Omron Healthcare, Dalian, China), and only rats showing severe hypothermia (< 34.5 °C) were selected for treatment. Reserpinized rats were randomly assigned to experimental groups, and rectal temperature was recorded 0.5 h after drug treatment. Ammonia-induced cough mouse model Antitussive effects were investigated by using a classical mouse cough model induced by ammonia liquor with minor modifications 13,14 . To measure the latency period and cough frequency, the mice were exposed to a 500 mL glass jar containing 0.2 mL of 13% ammonium hydroxide solution. Mice that had a cough frequency of more than three times in one minute and a latent latency of less than one minute were selected as test subjects. After recovering for 24 hours, the qualified mice were randomly assigned to groups and given one oral dose of the test medications. Each mouse was placed in a 500 mL glass jar filled with 0.2 mL 13% ammonia liquor for 1 minute after the medication had been administered for 0.5 hours. The mice were then removed from the chamber, and a trained observer recorded the frequency of coughing that occurred within 3 minutes. The following equation expresses the percentage of suppression of coughing time: Inhibition = [(To-T)/To x 100%], where To = Control Group Cough Time and Tt = Treatment Group Cough Time. Statistical analysis Differences between experimental groups were evaluated for statistical significance using one-way ANOVA. A P value less than 0.05 was considered to indicate statistical significance. The data are presented as the means ± standard errors of the means (SEMs). IC 50 values were calculated using GraphPad Prism, version 5 (Graph Pad Software Inc., San Diego, CA, USA). Ethics approval This work was approved by the Animal Ethics Committee of Shenzhen Salubris Pharmaceuticals Co., Ltd. (Shenzhen, China) or Wuxi Apptec Co., Ltd. (Shanghai, China) Feasibility and rationality We confirm that all procedures were carried out in accordance with relevant guidelines and regulations. Animal experiments in our study were conducted according to ARRIVE 2.0 guidelines. Results Radioligand competition binding We evaluated the competitive binding capabilities of four compounds to four CNS target sites chosen for their association with drug-induced depression. The drugs tested were DM, DX (a metabolite of DM), deDM, and deDX (a metabolite of deDM). Tables 1-5 present the comparative binding profiles. Table 1-5 shows that the affinity of DM for SERT (Ki = 40 nM ± 7 nM) was greater than that for NMDA receptors (Ki = 1.6 μM), Sigma-1 receptors (Ki = 1.7 μM), nAch α3β4 receptors (Ki = 14.7 μM), and NETs (Ki = 20.5 μM). deDM exhibited similar affinities to these targets as DM. DX, a metabolite of DM, displayed a 17-fold lower affinity for SERT (Ki = 144.2 nM) than DM. However, the affinity of DX for NMDA receptors (Ki = 0.22 μM) and NETs (Ki = 5.7 μM) was greater than that of DM. The affinity profile of DX was similar to that of deDX across the five targets. Behavioral despair model As illustrated in Figure 1, in the mouse FST model, deDM tended to reduce immobility time in the FST compared to that in the vehicle control group, although the difference was not statistically significant. On the other hand, bupropion significantly decreased immobility time in the FST. Notably, the combination of deDM and bupropion further reduced the immobility time in the FST in a dose-dependent manner, with a significant difference observed between the BUP and deDM+BUP (18+50 mg/kg) dose groups, indicating a synergistic effect of deDM and BUP in reducing the immobility time in the FST. As depicted in Figure 2, similar to the results of the FST experiment, no significant difference in immobility time was observed between the vehicle control group and the deDM group in the TST experiment. However, bupropion significantly decreased the immobility time of the mice, and the combination of bupropion and deDM further reduced the immobility time of the mice in a dose-dependent manner. Rat Reserpine-Induced Hypothermia Model As depicted in Figure 3, the body temperature of the rats in the model group was significantly lower than that of the rats in the normal control group. Notably, deDM dose-dependently increased the body temperature of model animals, with deDM (10 mg/kg) achieving statistical significance. Furthermore, the combination of deDM and bupropion further increased the body temperature of the rats in a dose-dependent manner, with the body temperature of the rats in the deDM and BUP (10+40 mg/kg) groups significantly different from that of the rats in the bupropion group. At the same dose, the combination of deDM and BUP (10+40 mg/kg) had a more potent effect on warming in rats than did the combination of DM and BUP (10+40 mg/kg). Ammonia-Induced Mouse Cough Model As shown in Figure 4, both BUP (40 mg/kg) and deDM (5 mg/kg and 10 mg/kg) demonstrated certain antitussive effects, although the difference was not statistically significant. However, the combination of bupropion and deDM effectively inhibited ammonia-induced cough in mice in a dose-dependent manner, with the combination of deDM and BUP (10+40 mg/kg) showing a greater inhibitory effect on cough in mice than the combination of DM and BUP (10+40 mg/kg) at the same dose. Discussion In this paper, we first investigated the effect of deuterium substitution on the activity of DM and DX via in vitro assays to determine whether deuterium substitution affects their antidepressant effects in vivo by altering their activity. Previous studies 15 have reported that DM can bind to multiple targets, including NMDARs, sigma-1 receptors, nACh receptors, SERT, and NETs. We investigated the impact of deuteration on DM activity at these targets and found that deuteration did not affect DM activity. Similarly, we examined the activity of the DM metabolite DX and the deuterated metabolite of deDM, deDX, and found that deuteration did not affect the activity of DX at these targets. However, deuteration enhances the stability of the in vivo metabolism of the compounds, further increasing the safety and efficacy of the compounds, as has been demonstrated in several studies 16–18 . In addition, in our pharmacological studies using a reserpine-induced rat hypothermia model and an ammonia-induced mouse cough model, we found that at equivalent doses, deDM+BUP had better pharmacological effects than DM+BUP, further demonstrating this point. We employed the FST and TST to evaluate the antidepressant effects of deDM, BUP, and their combination. The FST and TST are classic models for screening antidepressant drugs, allowing for rapid assessment of antidepressant activity, and are widely used in drug screening and development 19,20 . Our results indicated that within a dose range of 10-18 mg/kg, deDM tended to reduce immobility time in mice during the FST and TST, but the differences were not significant. BUP (50 mg/kg) significantly reduced immobility time in both tests. When BUP was combined with deDM, it further reduced immobility time in both the FST and TST, suggesting a synergistic antidepressant effect. There are two reasons for this synergistic effect. First, BUP can increase the stability of deDM. DeDM is mainly metabolized in vivo by CYP2D6, and BUP is a CYP2D6 inhibitor 21 that can inhibit the metabolism of deDM or DM, thus increasing deDM exposure in vivo . Second, there is also the possibility of mechanistic synergism between deDM and BUP. BUP is an aminoketone compound whose mechanism mainly involves the inhibition of presynaptic dopamine (DA) and norepinephrine (NE) reuptake, with the blockade of DA reuptake being more potent 22 , which enhances dopaminergic transmission and exerts antidepressant effects. In contrast, deDM, similar to DM, can exert antidepressant effects by antagonizing NMDA receptors, agonist sigma-1 receptors, and both SERTs and NETs. In addition, we further investigated the mechanism of the antidepressant effects of deDM+BUP using a reserpine-induced hypothermia model in rats and an ammonia-induced cough model in mice. Patients with depression often experience dysregulation of body temperature control. The reserpine-induced hypothermia rat model can simulate this symptom, aiding researchers in observing whether antidepressant drugs can correct the temperature decrease caused by reserpine. This can indirectly reflect the drug's effect on improving certain depressive symptoms. Compared to traditional tests such as the FST and TST, which assess depression based on behavioral responses under inescapable stress conditions typically related to behavioral despair, the reserpine-induced hypothermia model offers a physiological perspective for evaluating antidepressant efficacy involving changes in the endocrine system and temperature regulation. In the reserpine-induced rat hypothermia model, reserpine is a vesicular uptake inhibitor that leads to depression symptoms by inhibiting the reabsorption of monoamine neurotransmitters such as NA, DA, and 5-HT into vesicles, preventing them from being degraded by monoamine oxidase 23 . BUP exerts its antidepressant effect by inhibiting dopamine and norepinephrine transporters, suppressing their reuptake and increasing neurotransmitter concentrations in the synaptic cleft. Similarly, DM and deDM can act as antidepressants by inhibiting 5-HT and norepinephrine transporters. The results showed that both BUP and deDM significantly increased the body temperature of the rats, i.e., exerting an antidepressant effect. Moreover, compared to BUP and deDM alone, the combination further increased rat body temperature, indicating a synergistic antidepressant effect. Through the previous target of action analysis of deDM and BUP, the targets of action of BUP and deDM are complementary; in other words, the effects on elevated levels of neurotransmitters are complementary, which contributes to their synergistic effect. In addition, at the same dose, the body temperature of the rats in the deDM+BUP group was greater than that in the DM+BUP group, suggesting that deDM+BUP has a more stable material basis, i.e., a greater degree of deDM exposure. Furthermore, we utilized a mouse model of cough induced by ammonia. Recent research indicates that sigma-1 receptors regulate the cough reflex by influencing the release of neurotransmitters and neural excitability. Consequently, this model enables the evaluation of agonistic activity targeting sigma-1 receptors. In the ammonia-induced mouse cough model, the results of the study showed that BUP and deDM had a certain antitussive trend, but there was no significant difference from the model control group. Although deDM shares the same mechanism as DM, it does not exhibit an antitussive effect, possibly due to lower exposure levels in mice. When combined with BUP, the exposure level of deDM increased, and the results showed that deDM+BUP significantly inhibited mouse coughing, indicating synergistic enhancement in the model. Furthermore, at the same dosage, the suppression rate of coughing in mice was greater for deDM+BUP than for DM+BUP, suggesting that compared with DM, deDM has higher exposure levels. In addition to its involvement in the cough process, sigma-1R is also involved in the pathogenesis of depression. Early investigations 5 have shown that sigma 1 receptor agonists modulate neurotransmitter networks, signaling pathways, and brain area activity related to the physiology of depression, and sigma 1 receptor knockout animals display depression-like characteristics. Compared with typically prescribed antidepressant medications, Sigma 1 receptor agonists may promote a quicker onset of antidepressant efficacy. This is supported by the fact that sigma 1 receptor agonists, such as SA 4503, improved serotonergic neuronal activity in the dorsal spinal nucleus after just two days of treatment, whereas conventional antidepressants typically require at least two weeks of treatment. In addition, Sigma-1 receptors further regulate the synaptic transmission of NMDA receptors 24 . Together, these data suggest that sigma-1 receptors can work independently and/or in conjunction with other pathways (e.g., monoaminergic systems) to produce more rapid antidepressant effects and that DM, by exploiting these mechanisms, may produce faster effects than conventional antidepressants in depressed patients. DM is rapidly metabolized to DX in the body, which is associated with adverse psychiatric side effects and addiction potential. Because DX has a greater binding rate to NMDARs, these adverse effects are more directly associated with DX. Further stabilization of DM through deuteration may lead to a reduction in metabolites and hence a decrease in negative effects. Currently, deDM+BUP is in phase 1 clinical trials after submitting its IND file in China. In these settings, this medicine has demonstrated good safety and tolerability. We believe it might provide patients with depression with a safer and more efficient approach to treatment. Conclusion Our in vitro and in vivo data collectively demonstrated that deuterium substitution does not change the activity of DM on the target but enhances the metabolic stability of DM. DeDM and BUP can synergistically enhance the antidepressant effect through the complementarity of the target of action and the enhancement of in vivo exposure of deDM via the inhibition of CYP2D6 by BUP and, at the same time, can also reduce the risk of deDX-induced clinical side effects due to the reduction of the metabolites of deDM. Compared with DM+BUP, deDM+BUP is therefore expected to become a safer and more effective antidepressant drug combination in the future. Declarations Data availability The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request. Acknowledges The authors are very grateful to all the partcipants. Author contributions Ying Xiao and Xuefeng Hu conceived the study. Xuefeng Hu, Wei Xing, Ruhuan Wang, Xiaoqing Li and Jiahuan Li performed the experiments. Ying Xiao, Xuefeng Hu and Wei XingYing analyzed the data. Xuefeng Hu wrote the manuscript. Ying Xiao revised the manuscript. Funding This research was funded by a grant from the "Research on Key Technologies for Clinically Science Major New Drugs" with grant number XMHT20220104041. Competing interests The authors declare no competing interests. References Li, Z., Ruan, M., Chen, J. & Fang, Y. Major Depressive Disorder: Advances in Neuroscience Research and Translational Applications. Neurosci Bull 37 , 863–880 (2021). Preskorn, S. H. Recent pharmacologic advances in antidepressant therapy for the elderly. Am J Med 94 , 2S-12S (1993). FDA Approves New Nasal Spray Medication for Treatment-Resistant Depression; Available Only at a Certified Doctor’s Office or Clinic . Nguyen, L., Scandinaro, A. L. & Matsumoto, R. R. Deuterated (d6)-dextromethorphan elicits antidepressant-like effects in mice. Pharmacology Biochemistry and Behavior 161 , 30–37 (2017). Nguyen, L., Robson, M. J., Healy, J. R., Scandinaro, A. L. & Matsumoto, R. R. Involvement of Sigma-1 Receptors in the Antidepressant-like Effects of Dextromethorphan. PLoS ONE 9 , e89985 (2014). Pope, L. E. et al. Pharmacokinetics of dextromethorphan after single or multiple dosing in combination with quinidine in extensive and poor metabolizers. J Clin Pharmacol 44 , 1132–1142 (2004). Taylor, C. P., Traynelis, S. F., Siffert, J., Pope, L. E. & Matsumoto, R. R. Pharmacology of dextromethorphan: Relevance to dextromethorphan/quinidine (Nuedexta®) clinical use. Pharmacology & Therapeutics 164 , 170–182 (2016). Vecera, C. M., C Courtes, A., Jones, G., Soares, J. C. & Machado-Vieira, R. Pharmacotherapies Targeting GABA-Glutamate Neurotransmission for Treatment-Resistant Depression. Pharmaceuticals (Basel) 16 , 1572 (2023). Olney, N. & Rosen, H. AVP-923, a combination of dextromethorphan hydrobromide and quinidine sulfate for the treatment of pseudobulbar affect and neuropathic pain. IDrugs 13 , 254–265 (2010). Werling, L. L., Keller, A., Frank, J. G. & Nuwayhid, S. J. A comparison of the binding profiles of dextromethorphan, memantine, fluoxetine and amitriptyline: Treatment of involuntary emotional expression disorder. Experimental Neurology 207 , 248–257 (2007). Rojas-Corrales, M. O., Berrocoso, E., Gibert-Rahola, J. & Micó, J. A. Antidepressant-like effect of tramadol and its enantiomers in reserpinized mice: comparative study with desipramine, fluvoxamine, venlafaxine and opiates. J Psychopharmacol 18 , 404–411 (2004). Dhamija, I., Parle, M. & Kumar, S. Antidepressant and anxiolytic effects of Garcinia indica fruit rind via monoaminergic pathway. 3 Biotech 7 , 131 (2017). Jiang, K. et al. Antitussive, expectorant and anti-inflammatory activities of different extracts from Exocarpium Citri grandis. J Ethnopharmacol 156 , 97–101 (2014). Adejayan, A. A., Ozolua, R. I., Uwaya, D. O., Eze, G. I. & Ezike, A. C. Evaluation of the anti-asthmatic and antitussive potentials of methanol leaf extract of Napoleona vogelii in rodents. Biomedicine & Pharmacotherapy 109 , 120–126 (2019). Stahl, S. M. Dextromethorphan/Bupropion: A Novel Oral NMDA (N-methyl-d-aspartate) Receptor Antagonist with Multimodal Activity. CNS Spectr. 24 , 461–466 (2019). Di Martino, R. M. C., Maxwell, B. D. & Pirali, T. Deuterium in drug discovery: progress, opportunities and challenges. Nat Rev Drug Discov 22 , 562–584 (2023). Belete, T. M. Recent Updates on the Development of Deuterium-Containing Drugs for the Treatment of Cancer. DDDT Volume 16 , 3465–3472 (2022). Cargnin, S., Serafini, M. & Pirali, T. A primer of deuterium in drug design. Future Medicinal Chemistry 11 , 2039–2042 (2019). Yankelevitch-Yahav, R., Franko, M., Huly, A. & Doron, R. The forced swim test as a model of depressive-like behavior. J Vis Exp 52587 (2015) doi:10.3791/52587. O’Leary, O. F. & Cryan, J. F. The Tail-Suspension Test: A Model for Characterizing Antidepressant Activity in Mice. in Mood and Anxiety Related Phenotypes in Mice (ed. Gould, T. D.) vol. 42 119–137 (Humana Press, Totowa, NJ, 2009). Kotlyar, M. et al. Inhibition of CYP2D6 Activity by Bupropion. Journal of Clinical Psychopharmacology 25 , 226–229 (2005). Bupropion . Strawbridge, R., Javed, R. R., Cave, J., Jauhar, S. & Young, A. H. The effects of reserpine on depression: A systematic review. J Psychopharmacol 37 , 248–260 (2023). Martina, M., Turcotte, M.-E. B., Halman, S. & Bergeron, R. The sigma-1 receptor modulates NMDA receptor synaptic transmission and plasticity via SK channels in rat hippocampus. J Physiol 578 , 143–157 (2007). Tables Table 1 DM, deDM, DX, and deDX binding effects on NMDA receptors Compound Ki (nM) DM 1590.3±374.6 deDM 1897.0±281.3 DX 220.4±31.2 deDX 422.5±46.4 Results expressed as mean ± SEM, n=3. Table 2 Binding effects of DM, deDM, DX, and deDX on the Sigma-1 receptor Compoud Ki (μM) DM 1.7 deDM 1.7 DX 2.9 deDX 2.9 Results are expressed as mean. Table 3 Binding effects of DM, deDM, DX, and deDX on α3β4 nicotinic acetylcholine receptors Compound Ki (μM) DM 14.7 deDM 15.8 DX 10.0 deDX 15.0 Results expressed as mean. Table 4 Binding effects of DM, deDM, DX, and deDX on serotonin transporter (SERT) Compound Ki(nM) DM 8.4 deDM 8.3 DX 144.2 deDX 120.8 Results expressed as mean. Table 5 Binding effects of DM, deDM, DX, and deDX on norepinephrine transporters (NETs). Compound Ki(μM) DM 20.5 deDM 17.1 DX 5.7 deDX 7.9 Results expressed as mean. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4119597","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":289849562,"identity":"c74dbefe-1b43-45a5-9945-d9d610f5e9a6","order_by":0,"name":"Xuefeng Hu","email":"","orcid":"","institution":"Shenzhen Salubris Pharmaceuticals Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Xuefeng","middleName":"","lastName":"Hu","suffix":""},{"id":289849563,"identity":"07b91c1a-7836-45f2-89cf-8281ddc0f8ba","order_by":1,"name":"Wei Xing","email":"","orcid":"","institution":"Shenzhen Salubris Pharmaceuticals Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Xing","suffix":""},{"id":289849564,"identity":"97904b1e-d756-48cd-b4e5-d8fa21319286","order_by":2,"name":"Ruhuan Wang","email":"","orcid":"","institution":"Shenzhen Salubris Pharmaceuticals Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Ruhuan","middleName":"","lastName":"Wang","suffix":""},{"id":289849565,"identity":"883f9480-5502-42ea-b650-08fd4b37650e","order_by":3,"name":"Xiaoqing Li","email":"","orcid":"","institution":"Shenzhen Salubris Pharmaceuticals Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Xiaoqing","middleName":"","lastName":"Li","suffix":""},{"id":289849566,"identity":"57ace7ed-db55-4eff-94f1-6ecabd908153","order_by":4,"name":"Jiahuan Li","email":"","orcid":"","institution":"Shenzhen Salubris Pharmaceuticals Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Jiahuan","middleName":"","lastName":"Li","suffix":""},{"id":289849567,"identity":"248b92f4-7e98-4217-8755-1dab2ffa67d9","order_by":5,"name":"Jie Yan","email":"","orcid":"","institution":"Shenzhen Salubris Pharmaceuticals Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Yan","suffix":""},{"id":289849568,"identity":"38db1528-9f91-4eb6-8e63-14c56cdf5cdb","order_by":6,"name":"Junjun Wu","email":"","orcid":"","institution":"Shenzhen Salubris Pharmaceuticals Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Junjun","middleName":"","lastName":"Wu","suffix":""},{"id":289849569,"identity":"e0ef1bd4-a2fb-4150-abb2-0e543b2c4719","order_by":7,"name":"Ying Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYDACZhBhwMDAz8DAeICxgYGHeC2SDQwMRGqBAYMDEC1EqDzO/PAxT8Edu83nDz84zLvjnox8A/PDRzfwaJFsZjM25jF4lrztRprBYd4zxTwGB4AiOXi08DMzmEnzGBxONrvBANTSlsBjwMDDJo1PCxsz+zewFuP+4x/AWuQbCGjhZ+YB22JnwJADsYXhAAEtks08xYZzDA4nSNzIKTg49wzQYYcJ+MXg/PGND978OWzP3w9kvN2RYC/f3vzwMT4tIMAEjL7EBjiXmYByEGD8wcBgT4S6UTAKRsEoGKkAAGNnSSQ0UPvnAAAAAElFTkSuQmCC","orcid":"","institution":"Shenzhen Salubris Pharmaceuticals Co., Ltd","correspondingAuthor":true,"prefix":"","firstName":"Ying","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2024-03-18 03:18:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4119597/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4119597/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54594873,"identity":"dd43c700-3ded-4213-9120-ab3d8abe6f46","added_by":"auto","created_at":"2024-04-12 18:30:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":149769,"visible":true,"origin":"","legend":"\u003cp\u003eAntidepressant-like effects of compounds in the forced swim test in mice. The data shown are expressed as the mean ± S.E.M. *P\u0026lt;0.05, ***P\u0026lt;0.001, compared with the saline-treated group; ##P\u0026lt;0.01, compared with the BUP-treated group; one-way ANOVA followed by post hoc Dunnett’s test.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4119597/v1/e3aa3f6c14e9c16fb32a82a6.png"},{"id":54595103,"identity":"7244a7ae-9511-45d1-acb3-82320c609080","added_by":"auto","created_at":"2024-04-12 18:38:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":277869,"visible":true,"origin":"","legend":"\u003cp\u003eAntidepressant-like effects of compounds in the tail suspension test in mice. The data shown are expressed as the mean ± S.E.M. ***P\u0026lt;0.001, compared with the saline-treated group; one-way ANOVA followed by post hoc Dunnett’s test.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4119597/v1/9a3f86e067963cb2f69a7a76.png"},{"id":54594875,"identity":"93c5f8dc-51f1-4ceb-89b0-f1cce0d09372","added_by":"auto","created_at":"2024-04-12 18:30:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":162394,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of the compounds on reversing hypothermia in a rat reserpine-induced hypothermia model. The data shown are expressed as the mean ± S.E.M. *P\u0026lt;0.05, **P\u0026lt;0.01, compared with the saline-treated group; ##P\u0026lt;0.05, ##P\u0026lt;0.01, compared with the BUP-treated group; one-way ANOVA followed by post hoc Dunnett’s tests.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4119597/v1/15842f7fa999ced4ef2a9356.png"},{"id":54594876,"identity":"997400fe-c196-460d-ba0d-a12e6bcce08f","added_by":"auto","created_at":"2024-04-12 18:30:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38760,"visible":true,"origin":"","legend":"\u003cp\u003eAntitussive effects of compounds in an ammonia-induced cough mouse model. The data shown are expressed as the mean ± S.E.M. *P\u0026lt;0.05, **P\u0026lt;0.01, compared with the saline-treated group; one-way ANOVA followed by post hoc Dunnett’s test.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4119597/v1/cd10d623412c665aff5ec72b.png"},{"id":57667271,"identity":"88c7862b-3089-4049-821d-54b8934ec0bc","added_by":"auto","created_at":"2024-06-04 05:29:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2152964,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4119597/v1/4e50da4d-fc56-4ad6-bbdc-ad3e3f105fc4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy of SAL0114, an Oral NMDA Receptor Antagonist with Multimodal Activity, in Major Depressive Disorder","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMajor depressive disorder (MDD) is a severe mental illness that affects millions of people globally\u003csup\u003e1\u003c/sup\u003e. Currently, there are five main classes of antidepressants, including aminoketones, triazolopyridines, monoamine oxidase inhibitors, tricyclic antidepressants, and selective serotonin reuptake inhibitors. However, a common problem with these drugs is that they are slow to start working, often take weeks to take effect, and more than 30% of patients do not improve after treatment, putting them at risk of self-harm\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFortunately, the 2019 FDA-approved esketamine addresses the slow onset of depression by providing rapid relief of depression within 24 hours \u003csup\u003e3\u003c/sup\u003e. However, esketamine is highly addictive and currently limited in use in healthcare settings, which makes long-term medication management difficult for patients; as a result, there is an urgent clinical need for safer and faster-acting antidepressants.\u003c/p\u003e\n\u003cp\u003eDextromethorphan (DM) is an over-the-counter antitussive drug with a similar mechanism of action to that of esketamine, both of which block NMDA receptors \u003csup\u003e4,5\u003c/sup\u003e. Moreover, dextromethorphan has been used as an antitussive agent for more than 40 years, so it has better safety potential than esketamine.\u003c/p\u003e\n\u003cp\u003eHowever, there are a number of issues that must be addressed before administering dextromethorphan to depressed patients. Dextromethorphan has a strong hepatic first-pass effect, which decreases its bioavailability and ultimately leads to diminished antidepressant effects \u003csup\u003e6,7\u003c/sup\u003e. There are two ways to overcome this. First, a CYP2D6 inhibitor was added to increase the stability of dextromethorphan. The U.S. Food and Drug Administration (FDA) has approved combination medications such as AVP-923 (DM+quinidine) and AXS-05 (DM+bupropion) based on similar tactics\u003csup\u003e8,9\u003c/sup\u003e. Second, deuterium substitution was applied to dextromethorphan. Deuterium is a naturally occurring isotope of hydrogen, and by deuterium substitution at the appropriate metabolic site, the half-life of DM can be prolonged.\u003c/p\u003e\n\u003cp\u003eBy adopting these two strategies, we altered and combined various compounds, ultimately selecting the deDM+BUP combination. This paper emphasizes \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e pharmacological experiments. These studies assessed the impact of deuterium substitution on the\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e activity and \u003cem\u003ein vivo\u003c/em\u003e stability of DM, the antidepressant effects and mechanisms of deDM and BUP, and the synergistic effect of combining deDM and BUP on antidepressant effects. These pharmacological studies pave the way for clinical trials of the combination of deDM and BUP. The findings outlined in this paper also provide valuable insights into the synergistic effects, safety, and potential mechanisms of action of this combination compound, which ultimately benefits patients with depression.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale Sprague\u0026ndash;Dawley rats weighing 200\u0026ndash;230 g and male C57BL/6J mice weighing 19\u0026ndash;22 g were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang, China), and male ICR mice weighing 18\u0026ndash;22 g were obtained from Shanghai Lingchang Biology Science and Technology Co., Ltd. (Shanghai, China). All rats and mice were housed 4-5 per cage under an 8:00 am/8:00 pm light/dark schedule at controlled temperature and humidity with free access to food and water. All procedures were carried out in accordance with guidelines approved by the Animal Ethics Committee of Shenzhen Salubris Pharmaceuticals Co., Ltd. (Shenzhen, China) or Wuxi Apptec Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagents and drugs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeuterated dextromethorphan (deDM), deuterated dextrorphan (deDX), bupropion (BUP), dextromethorphan (DM) and dextrorphan (DX) were obtained from Shenzhen salubris Pharmaceuticals Co., Ltd. (Shenzhen, China). Reserpine and imipramine were obtained from Sigma‒Aldrich (Shanghai, China); ammonium hydroxide was purchased from Ailan (Shanghai) Chemical Technology Co., Ltd. (Shanghai, China); and \u003csup\u003e3\u003c/sup\u003eH-MK801, \u003csup\u003e3\u003c/sup\u003eH-hydroxytryptamine creatine sulfate, \u003csup\u003e3\u003c/sup\u003eH-norepinephrine hydrochloride, \u003csup\u003e3\u003c/sup\u003eH-imipramine, nisoxetine 3H-hydrochloride, serotonin transporter membrane and norepinephrine transporter were purchased from PerkinElmer, Inc. (Massachusetts, USA). ); NMDA membranes were obtained from Pharmron (Beijing, China); and memantine hydrochloride and paroxetine were purchased from Adooq Bioscience (California, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRadioligand competition binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMDAR binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMembrane preparation was performed as previously described (Awtry and Werling, 2003)\u003csup\u003e10\u003c/sup\u003e. Briefly, the brains of male Sprague‒Dawley rats were dissected for tissue collection. The tissue was then homogenized in ice-cold extraction solution, centrifuged at 40,000 \u0026times; g for 10 min, the supernatant was discarded, the precipitate was resuspended in extraction solution, and the above steps were repeated 2 times. The precipitate was resuspended in resuspension solution and centrifuged at 40,000\u0026times;g for 10 min. The supernatant was discarded, and the above steps were repeated 2 times. Finally, the precipitate was resuspended in a 10-fold volume of resuspension solution and stored in portions at -80 \u0026deg;C. The protein concentration was determined with a Pierce\u003csup\u003eTM\u003c/sup\u003e BCA protein assay kit (Thermo Fisher Scientific, Massachusetts, USA). The competitive binding of the compound and \u003csup\u003e3\u003c/sup\u003eH-MK801 on the NMDA receptor was assessed using the filtration binding method, which involves washing the radioligands that are not bound to the NMDA receptor and examining the signal values of the radioligands bound to the NMDA receptor with a Microbeta instrument (Perkin Elmer, Massachusetts, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerotonin transporter (SERT) binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were carried out in the Pharmaron laboratory. The competitive binding of the samples and \u003csup\u003e3\u003c/sup\u003eH-hydroxytryptamine creatine sulfate on SERT was assessed using a radioligand binding method. The ability of the compound to bind competitively to SERT (Ki) was determined by detecting the signal value of the radioligand bound to the transporter via a Microbeta instrument (Perkin Elmer, Massachusetts, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNorepinephrine transporter (NET) binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were carried out in the Pharmaron laboratory. The competitive binding of the compound and \u003csup\u003e3\u003c/sup\u003eH-Nisoxetine on NETs (PerkinElmer, Massachusetts, USA) was assessed using a radioligand binding method. The ability of the compound to bind competitively to the NET (Ki) was determined by detecting the signal value of the radioligand bound to the transporter via a Microbeta instrument (Perkin Elmer, Massachusetts, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSigma 1 receptor binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were carried out in the Eurofins Discovery laboratory. The competitive binding of the samples and \u003csup\u003e3\u003c/sup\u003eH-pentazocine to the Sigma-1 receptor of human-derived Jurkat cells was assessed using a radioligand binding method. The ability of the compound to bind competitively to the Sigma-1 receptor (Ki) was determined by detecting the signal value of the radioligand bound to the receptor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNicotinic acetylcholine receptor (nAchR) \u0026alpha;3\u0026beta;4 binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were carried out in the Eurofins Discovery laboratory. The competitive binding of the samples and \u003csup\u003e125\u003c/sup\u003eI-epibatidine to the nAchR \u0026alpha;3\u0026beta;4 of human recombinant CHO-K1 cells was assessed using a radioligand binding method. The ability of the compound to bind competitively to the nAchR \u0026alpha;3\u0026beta;4 (Ki) was determined by detecting the signal value of the radioligand bound to the receptor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eForced swim test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mouse forced swim test was built in a way similar to what was published previously by Nguyen et al.\u003csup\u003e4\u003c/sup\u003e with some minor modifications. Mice were placed in individual cylinders of water (18 cm deep) for a total of 6 min for the forced swim test. The first two minutes were for acclimatization and were not scored. The remaining 4 minutes were used to measure immobility time with ANY-Maze Version 4.63 video tracking software (Stoelting Co., Illinois, USA). The only movement needed to keep the animal\u0026apos;s head above the water\u0026apos;s surface was considered immobility. The ANY-Maze software settings were as follows: accustomization period = 120 s, test duration = 240 s, minimum immobility time = 2 s, and immobility sensitivity =75%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTail suspension test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TST was adapted from the behavioral despair test described by Nguyen et al. \u003csup\u003e4\u003c/sup\u003e, with some modifications. Each mouse was hung up by its tail and attached to a metal rod using adhesive tape (2 cm from the tip of the tail). The distance between the animals and any object was kept at least 15 cm. The ANY-maze version 4.63 video tracking program was used to record individual mice for a total of 6 minutes. The first two minutes were for acclimatization and were not scored. The remaining 4 minutes were used to measure immobility time with ANY-Maze Version 4.63 video tracking software (Stoelting Co., Wood Dale, IL). When the mice hung passively and motionlessly, they were considered immobile.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReserpine-induced hypothermia in rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reserpine-induced hypothermia test was performed according to the method described by Rojas-Corrales et al.\u003csup\u003e11,12\u003c/sup\u003e, with some modifications. In brief, eighteen hours after reserpine treatment (4 mg/kg, ip), rectal temperature was measured in degrees centigrade with a rectal probe connected to a thermometer (Omron Healthcare, Dalian, China), and only rats showing severe hypothermia (\u0026lt; 34.5 \u0026deg;C) were selected for treatment. Reserpinized rats were randomly assigned to experimental groups, and rectal temperature was recorded 0.5 h after drug treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmmonia-induced cough\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntitussive effects were investigated by using a classical mouse cough model induced by ammonia liquor with minor modifications\u003csup\u003e13,14\u003c/sup\u003e. To measure the latency period and cough frequency, the mice were exposed to a 500 mL glass jar containing 0.2 mL of 13% ammonium hydroxide solution. Mice that had a cough frequency of more than three times in one minute and a latent latency of less than one minute were selected as test subjects. After recovering for 24 hours, the qualified mice were randomly assigned to groups and given one oral dose of the test medications. Each mouse was placed in a 500 mL glass jar filled with 0.2 mL 13% ammonia liquor for 1 minute after the medication had been administered for 0.5 hours. The mice were then removed from the chamber, and a trained observer recorded the frequency of coughing that occurred within 3 minutes.\u003c/p\u003e\n\u003cp\u003eThe following equation expresses the percentage of suppression of coughing time: Inhibition = [(To-T)/To x 100%], where To = Control Group Cough Time and Tt = Treatment Group Cough Time.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferences between experimental groups were evaluated for statistical significance using one-way ANOVA. A P value less than 0.05 was considered to indicate statistical significance. The data are presented as the means \u0026plusmn; standard errors of the means (SEMs). IC\u003csub\u003e50\u003c/sub\u003e values were calculated using GraphPad Prism, version 5 (Graph Pad Software Inc., San Diego, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was approved by the Animal Ethics Committee of Shenzhen Salubris Pharmaceuticals Co., Ltd. (Shenzhen, China) or Wuxi Apptec Co., Ltd. (Shanghai, China)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFeasibility and rationality\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confirm that all procedures were carried out in accordance with relevant guidelines and regulations. Animal experiments in our study were conducted according to ARRIVE 2.0 guidelines.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eRadioligand competition binding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe evaluated the competitive binding capabilities of four compounds to four CNS target sites chosen for their association with drug-induced depression. The drugs tested were DM, DX (a metabolite of DM), deDM, and deDX (a metabolite of deDM). Tables 1-5 present the comparative binding profiles.\u003c/p\u003e\n\u003cp\u003eTable 1-5 shows that the affinity of DM for SERT (Ki = 40 nM \u0026plusmn; 7 nM) was greater than that for NMDA receptors (Ki = 1.6 \u0026mu;M), Sigma-1 receptors (Ki = 1.7 \u0026mu;M), nAch \u0026alpha;3\u0026beta;4 receptors (Ki = 14.7 \u0026mu;M), and NETs (Ki = 20.5 \u0026mu;M). deDM exhibited similar affinities to these targets as DM. DX, a metabolite of DM, displayed a 17-fold lower affinity for SERT (Ki = 144.2 nM) than DM. However, the affinity of DX for NMDA receptors (Ki = 0.22 \u0026mu;M) and NETs (Ki = 5.7 \u0026mu;M) was greater than that of DM. The affinity profile of DX was similar to that of deDX across the five targets.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBehavioral despair model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 1, in the mouse FST model, deDM tended to reduce immobility time in the FST compared to that in the vehicle control group, although the difference was not statistically significant. On the other hand, bupropion significantly decreased immobility time in the FST. Notably, the combination of deDM and bupropion further reduced the immobility time in the FST in a dose-dependent manner, with a significant difference observed between the BUP and deDM+BUP (18+50 mg/kg) dose groups, indicating a synergistic effect of deDM and BUP in reducing the immobility time in the FST.\u003c/p\u003e\n\u003cp\u003eAs depicted in Figure 2, similar to the results of the FST experiment, no significant difference in immobility time was observed between the vehicle control group and the deDM group in the TST experiment. However, bupropion significantly decreased the immobility time of the mice, and the combination of bupropion and deDM further reduced the immobility time of the mice in a dose-dependent manner.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRat Reserpine-Induced Hypothermia Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs depicted in Figure 3, the body temperature of the rats in the model group was significantly lower than that of the rats in the normal control group. Notably, deDM dose-dependently increased the body temperature of model animals, with deDM (10 mg/kg) achieving statistical significance. Furthermore, the combination of deDM and bupropion further increased the body temperature of the rats in a dose-dependent manner, with the body temperature of the rats in the deDM and BUP (10+40 mg/kg) groups significantly different from that of the rats in the bupropion group. At the same dose, the combination of deDM and BUP (10+40 mg/kg) had a more potent effect on warming in rats than did the combination of DM and BUP (10+40 mg/kg).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmmonia-Induced Mouse Cough Model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 4, both BUP (40 mg/kg) and deDM (5 mg/kg and 10 mg/kg) demonstrated certain antitussive effects, although the difference was not statistically significant. However, the combination of bupropion and deDM effectively inhibited ammonia-induced cough in mice in a dose-dependent manner, with the combination of deDM and BUP (10+40 mg/kg) showing a greater inhibitory effect on cough in mice than the combination of DM and BUP (10+40 mg/kg) at the same dose.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this paper, we first investigated the effect of deuterium substitution on the activity of DM and DX via \u003cem\u003ein vitro\u003c/em\u003e assays to determine whether deuterium substitution affects their antidepressant effects \u003cem\u003ein vivo\u003c/em\u003e by altering their activity.\u003c/p\u003e\n\u003cp\u003ePrevious studies\u003csup\u003e15\u003c/sup\u003e have reported that DM can bind to multiple targets, including NMDARs, sigma-1 receptors, nACh receptors, SERT, and NETs. We investigated the impact of deuteration on DM activity at these targets and found that deuteration did not affect DM activity. Similarly, we examined the activity of the DM metabolite DX and the deuterated metabolite of deDM, deDX, and found that deuteration did not affect the activity of DX at these targets. However, deuteration enhances the stability of the \u003cem\u003ein vivo\u003c/em\u003e metabolism of the compounds, further increasing the safety and efficacy of the compounds, as has been demonstrated in several studies\u003csup\u003e16\u0026ndash;18\u003c/sup\u003e. In addition, in our pharmacological studies using a reserpine-induced rat hypothermia model and an ammonia-induced mouse cough model, we found that at equivalent doses, deDM+BUP had better pharmacological effects than DM+BUP, further demonstrating this point.\u003c/p\u003e\n\u003cp\u003eWe employed the FST and TST to evaluate the antidepressant effects of deDM, BUP, and their combination. The FST and TST are classic models for screening antidepressant drugs, allowing for rapid assessment of antidepressant activity, and are widely used in drug screening and development\u003csup\u003e19,20\u003c/sup\u003e. Our results indicated that within a dose range of 10-18 mg/kg, deDM tended to reduce immobility time in mice during the FST and TST, but the differences were not significant. BUP (50 mg/kg) significantly reduced immobility time in both tests. When BUP was combined with deDM, it further reduced immobility time in both the FST and TST, suggesting a synergistic antidepressant effect.\u003c/p\u003e\n\u003cp\u003eThere are two reasons for this synergistic effect. First, BUP can increase the stability of deDM. DeDM is mainly metabolized \u003cem\u003ein vivo\u003c/em\u003e by CYP2D6, and BUP is a CYP2D6 inhibitor\u003csup\u003e21\u003c/sup\u003e that can inhibit the metabolism of deDM or DM, thus increasing deDM exposure \u003cem\u003ein vivo\u003c/em\u003e. Second, there is also the possibility of mechanistic synergism between deDM and BUP. BUP is an aminoketone compound whose mechanism mainly involves the inhibition of presynaptic dopamine (DA) and norepinephrine (NE) reuptake, with the blockade of DA reuptake being more potent\u003csup\u003e22\u003c/sup\u003e, which enhances dopaminergic transmission and exerts antidepressant effects. In contrast, deDM, similar to DM, can exert antidepressant effects by antagonizing NMDA receptors, agonist sigma-1 receptors, and both SERTs and NETs.\u003c/p\u003e\n\u003cp\u003eIn addition, we further investigated the mechanism of the antidepressant effects of deDM+BUP using a reserpine-induced hypothermia model in rats and an ammonia-induced cough model in mice.\u003c/p\u003e\n\u003cp\u003ePatients with depression often experience dysregulation of body temperature control. The reserpine-induced hypothermia rat model can simulate this symptom, aiding researchers in observing whether antidepressant drugs can correct the temperature decrease caused by reserpine. This can indirectly reflect the drug\u0026apos;s effect on improving certain depressive symptoms. Compared to traditional tests such as the FST and TST, which assess depression based on behavioral responses under inescapable stress conditions typically related to behavioral despair, the reserpine-induced hypothermia model offers a physiological perspective for evaluating antidepressant efficacy involving changes in the endocrine system and temperature regulation.\u003c/p\u003e\n\u003cp\u003eIn the reserpine-induced rat hypothermia model, reserpine is a vesicular uptake inhibitor that leads to depression symptoms by inhibiting the reabsorption of monoamine neurotransmitters such as NA, DA, and 5-HT into vesicles, preventing them from being degraded by monoamine oxidase\u003csup\u003e23\u003c/sup\u003e. BUP exerts its antidepressant effect by inhibiting dopamine and norepinephrine transporters, suppressing their reuptake and increasing neurotransmitter concentrations in the synaptic cleft. Similarly, DM and deDM can act as antidepressants by inhibiting 5-HT and norepinephrine transporters. The results showed that both BUP and deDM significantly increased the body temperature of the rats, i.e., exerting an antidepressant effect. Moreover, compared to BUP and deDM alone, the combination further increased rat body temperature, indicating a synergistic antidepressant effect. Through the previous target of action analysis of deDM and BUP, the targets of action of BUP and deDM are complementary; in other words, the effects on elevated levels of neurotransmitters are complementary, which contributes to their synergistic effect. In addition, at the same dose, the body temperature of the rats in the deDM+BUP group was greater than that in the DM+BUP group, suggesting that deDM+BUP has a more stable material basis, i.e., a greater degree of deDM exposure.\u003c/p\u003e\n\u003cp\u003eFurthermore, we utilized a mouse model of cough induced by ammonia. Recent research indicates that sigma-1 receptors regulate the cough reflex by influencing the release of neurotransmitters and neural excitability. Consequently, this model enables the evaluation of agonistic activity targeting sigma-1 receptors. In the ammonia-induced mouse cough model, the results of the study showed that BUP and deDM had a certain antitussive trend, but there was no significant difference from the model control group. Although deDM shares the same mechanism as DM, it does not exhibit an antitussive effect, possibly due to lower exposure levels in mice. When combined with BUP, the exposure level of deDM increased, and the results showed that deDM+BUP significantly inhibited mouse coughing, indicating synergistic enhancement in the model. Furthermore, at the same dosage, the suppression rate of coughing in mice was greater for deDM+BUP than for DM+BUP, suggesting that compared with DM, deDM has higher exposure levels.\u003c/p\u003e\n\u003cp\u003eIn addition to its involvement in the cough process, sigma-1R is also involved in the pathogenesis of depression. Early investigations\u003csup\u003e5\u003c/sup\u003e have shown that sigma 1 receptor agonists modulate neurotransmitter networks, signaling pathways, and brain area activity related to the physiology of depression, and sigma 1 receptor knockout animals display depression-like characteristics. Compared with typically prescribed antidepressant medications, Sigma 1 receptor agonists may promote a quicker onset of antidepressant efficacy. This is supported by the fact that sigma 1 receptor agonists, such as SA 4503, improved serotonergic neuronal activity in the dorsal spinal nucleus after just two days of treatment, whereas conventional antidepressants typically require at least two weeks of treatment. In addition, Sigma-1 receptors further regulate the synaptic transmission of NMDA receptors\u003csup\u003e24\u003c/sup\u003e. Together, these data suggest that sigma-1 receptors can work independently and/or in conjunction with other pathways (e.g., monoaminergic systems) to produce more rapid antidepressant effects and that DM, by exploiting these mechanisms, may produce faster effects than conventional antidepressants in depressed patients.\u003c/p\u003e\n\u003cp\u003eDM is rapidly metabolized to DX in the body, which is associated with adverse psychiatric side effects and addiction potential. Because DX has a greater binding rate to NMDARs, these adverse effects are more directly associated with DX. Further stabilization of DM through deuteration may lead to a reduction in metabolites and hence a decrease in negative effects.\u003c/p\u003e\n\u003cp\u003eCurrently, deDM+BUP is in phase 1 clinical trials after submitting its IND file in China. In these settings, this medicine has demonstrated good safety and tolerability. We believe it might provide patients with depression with a safer and more efficient approach to treatment.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e data collectively demonstrated that deuterium substitution does not change the activity of DM on the target but enhances the metabolic stability of DM. DeDM and BUP can synergistically enhance the antidepressant effect through the complementarity of the target of action and the enhancement of \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eexposure of deDM via the inhibition of CYP2D6 by BUP and, at the same time, can also reduce the risk of deDX-induced clinical side effects due to the reduction of the metabolites of deDM. Compared with DM+BUP, deDM+BUP is therefore expected to become a safer and more effective antidepressant drug combination in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledges\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are very grateful to all the partcipants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYing Xiao and Xuefeng Hu conceived the study. Xuefeng Hu, Wei Xing, Ruhuan Wang, Xiaoqing Li and Jiahuan Li performed the experiments. Ying Xiao, Xuefeng Hu and Wei XingYing analyzed the data. Xuefeng Hu wrote the manuscript. Ying Xiao revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by a grant from the \"Research on Key Technologies for Clinically Science Major New Drugs\" with grant number XMHT20220104041.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi, Z., Ruan, M., Chen, J. \u0026amp; Fang, Y. Major Depressive Disorder: Advances in Neuroscience Research and Translational Applications. \u003cem\u003eNeurosci Bull\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 863\u0026ndash;880 (2021).\u003c/li\u003e\n\u003cli\u003ePreskorn, S. H. Recent pharmacologic advances in antidepressant therapy for the elderly. \u003cem\u003eAm J Med\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 2S-12S (1993).\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eFDA Approves New Nasal Spray Medication for Treatment-Resistant Depression; Available Only at a Certified Doctor\u0026rsquo;s Office or Clinic\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eNguyen, L., Scandinaro, A. L. \u0026amp; Matsumoto, R. R. Deuterated (d6)-dextromethorphan elicits antidepressant-like effects in mice. \u003cem\u003ePharmacology Biochemistry and Behavior\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e, 30\u0026ndash;37 (2017).\u003c/li\u003e\n\u003cli\u003eNguyen, L., Robson, M. J., Healy, J. R., Scandinaro, A. L. \u0026amp; Matsumoto, R. R. Involvement of Sigma-1 Receptors in the Antidepressant-like Effects of Dextromethorphan. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e89985 (2014).\u003c/li\u003e\n\u003cli\u003ePope, L. E. \u003cem\u003eet al.\u003c/em\u003e Pharmacokinetics of dextromethorphan after single or multiple dosing in combination with quinidine in extensive and poor metabolizers. \u003cem\u003eJ Clin Pharmacol\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 1132\u0026ndash;1142 (2004).\u003c/li\u003e\n\u003cli\u003eTaylor, C. P., Traynelis, S. F., Siffert, J., Pope, L. E. \u0026amp; Matsumoto, R. R. Pharmacology of dextromethorphan: Relevance to dextromethorphan/quinidine (Nuedexta\u0026reg;) clinical use. \u003cem\u003ePharmacology \u0026amp; Therapeutics\u003c/em\u003e \u003cstrong\u003e164\u003c/strong\u003e, 170\u0026ndash;182 (2016).\u003c/li\u003e\n\u003cli\u003eVecera, C. M., C Courtes, A., Jones, G., Soares, J. C. \u0026amp; Machado-Vieira, R. Pharmacotherapies Targeting GABA-Glutamate Neurotransmission for Treatment-Resistant Depression. \u003cem\u003ePharmaceuticals (Basel)\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1572 (2023).\u003c/li\u003e\n\u003cli\u003eOlney, N. \u0026amp; Rosen, H. AVP-923, a combination of dextromethorphan hydrobromide and quinidine sulfate for the treatment of pseudobulbar affect and neuropathic pain. \u003cem\u003eIDrugs\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 254\u0026ndash;265 (2010).\u003c/li\u003e\n\u003cli\u003eWerling, L. L., Keller, A., Frank, J. G. \u0026amp; Nuwayhid, S. J. A comparison of the binding profiles of dextromethorphan, memantine, fluoxetine and amitriptyline: Treatment of involuntary emotional expression disorder. \u003cem\u003eExperimental Neurology\u003c/em\u003e \u003cstrong\u003e207\u003c/strong\u003e, 248\u0026ndash;257 (2007).\u003c/li\u003e\n\u003cli\u003eRojas-Corrales, M. O., Berrocoso, E., Gibert-Rahola, J. \u0026amp; Mic\u0026oacute;, J. A. Antidepressant-like effect of tramadol and its enantiomers in reserpinized mice: comparative study with desipramine, fluvoxamine, venlafaxine and opiates. \u003cem\u003eJ Psychopharmacol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 404\u0026ndash;411 (2004).\u003c/li\u003e\n\u003cli\u003eDhamija, I., Parle, M. \u0026amp; Kumar, S. Antidepressant and anxiolytic effects of Garcinia indica fruit rind via monoaminergic pathway. \u003cem\u003e3 Biotech\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 131 (2017).\u003c/li\u003e\n\u003cli\u003eJiang, K. \u003cem\u003eet al.\u003c/em\u003e Antitussive, expectorant and anti-inflammatory activities of different extracts from Exocarpium Citri grandis. \u003cem\u003eJ Ethnopharmacol\u003c/em\u003e \u003cstrong\u003e156\u003c/strong\u003e, 97\u0026ndash;101 (2014).\u003c/li\u003e\n\u003cli\u003eAdejayan, A. A., Ozolua, R. I., Uwaya, D. O., Eze, G. I. \u0026amp; Ezike, A. C. Evaluation of the anti-asthmatic and antitussive potentials of methanol leaf extract of Napoleona vogelii in rodents. \u003cem\u003eBiomedicine \u0026amp; Pharmacotherapy\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e, 120\u0026ndash;126 (2019).\u003c/li\u003e\n\u003cli\u003eStahl, S. M. Dextromethorphan/Bupropion: A Novel Oral NMDA (N-methyl-d-aspartate) Receptor Antagonist with Multimodal Activity. \u003cem\u003eCNS Spectr.\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 461\u0026ndash;466 (2019).\u003c/li\u003e\n\u003cli\u003eDi Martino, R. M. C., Maxwell, B. D. \u0026amp; Pirali, T. Deuterium in drug discovery: progress, opportunities and challenges. \u003cem\u003eNat Rev Drug Discov\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 562\u0026ndash;584 (2023).\u003c/li\u003e\n\u003cli\u003eBelete, T. M. Recent Updates on the Development of Deuterium-Containing Drugs for the Treatment of Cancer. \u003cem\u003eDDDT\u003c/em\u003e \u003cstrong\u003eVolume 16\u003c/strong\u003e, 3465\u0026ndash;3472 (2022).\u003c/li\u003e\n\u003cli\u003eCargnin, S., Serafini, M. \u0026amp; Pirali, T. A primer of deuterium in drug design. \u003cem\u003eFuture Medicinal Chemistry\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 2039\u0026ndash;2042 (2019).\u003c/li\u003e\n\u003cli\u003eYankelevitch-Yahav, R., Franko, M., Huly, A. \u0026amp; Doron, R. The forced swim test as a model of depressive-like behavior. \u003cem\u003eJ Vis Exp\u003c/em\u003e 52587 (2015) doi:10.3791/52587.\u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Leary, O. F. \u0026amp; Cryan, J. F. The Tail-Suspension Test: A Model for Characterizing Antidepressant Activity in Mice. in \u003cem\u003eMood and Anxiety Related Phenotypes in Mice\u003c/em\u003e (ed. Gould, T. D.) vol. 42 119\u0026ndash;137 (Humana Press, Totowa, NJ, 2009).\u003c/li\u003e\n\u003cli\u003eKotlyar, M. \u003cem\u003eet al.\u003c/em\u003e Inhibition of CYP2D6 Activity by Bupropion. \u003cem\u003eJournal of Clinical Psychopharmacology\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 226\u0026ndash;229 (2005).\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eBupropion\u003c/em\u003e.\u003c/li\u003e\n\u003cli\u003eStrawbridge, R., Javed, R. R., Cave, J., Jauhar, S. \u0026amp; Young, A. H. The effects of reserpine on depression: A systematic review. \u003cem\u003eJ Psychopharmacol\u003c/em\u003e \u003cstrong\u003e37\u003c/strong\u003e, 248\u0026ndash;260 (2023).\u003c/li\u003e\n\u003cli\u003eMartina, M., Turcotte, M.-E. B., Halman, S. \u0026amp; Bergeron, R. The sigma-1 receptor modulates NMDA receptor synaptic transmission and plasticity via SK channels in rat hippocampus. \u003cem\u003eJ Physiol\u003c/em\u003e \u003cstrong\u003e578\u003c/strong\u003e, 143\u0026ndash;157 (2007).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e DM, deDM, DX, and deDX binding effects on NMDA receptors\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e\u003cstrong\u003eKi (nM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e1590.3\u0026plusmn;374.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e1897.0\u0026plusmn;281.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e220.4\u0026plusmn;31.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e422.5\u0026plusmn;46.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults expressed as mean \u0026plusmn; SEM, n=3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Binding effects of DM, deDM, DX, and deDX on the Sigma-1 receptor\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompoud\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e\u003cstrong\u003eKi (\u0026mu;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults are expressed as mean.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eBinding effects of DM, deDM, DX, and deDX on \u0026alpha;3\u0026beta;4 nicotinic acetylcholine receptors\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e\u003cstrong\u003eKi (\u0026mu;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e14.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e15.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults expressed as mean.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u0026nbsp;\u003c/strong\u003eBinding effects of DM, deDM, DX, and deDX on serotonin transporter (SERT)\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e\u003cstrong\u003eKi(nM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e8.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e144.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e120.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults expressed as mean.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u0026nbsp;\u003c/strong\u003eBinding effects of DM, deDM, DX, and deDX on norepinephrine transporters (NETs).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\"\u003e\n \u003cp\u003e\u003cstrong\u003eKi(\u0026mu;M)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e20.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e17.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003eDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"42.42424242424242%\"\u003e\n \u003cp\u003edeDX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"57.57575757575758%\" valign=\"top\"\u003e\n \u003cp\u003e7.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eResults expressed as mean.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Major depressive disorder, NMDAR receptors, Sigma-1 receptor, Dextromethorphan, Bupropion","lastPublishedDoi":"10.21203/rs.3.rs-4119597/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4119597/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The first FDA-approved rapid-acting antidepressant was esketamine, but its use was limited because of the risk of addiction, and although the subsequent combination of dextromethorphan (DM) + bupropion (BUP) has alleviated some of the problems, there is still a clinical need for safer and more effective compounds. In this paper, we use a strategy of deuterated substitution of DM to improve stability and reduce metabolites to improve safety and efficacy. We analyzed the effects of deuterium substitution on the in vitro activities of DM and deDX (deDM metabolite) by radioligand competition binding assay, evaluated the antidepressant and synergistic effects of deDM and BUP by a mouse behavioral despair model, and further assessed the synergistic mechanism of deDM and BUP by a reserpine-induced hypothermia rats model and an ammonia-induced cough mice model, which showed that deuterium substitution does not change the DM and deDX (deDM metabolite) in vitro activity, but can improve the in vivo effectiveness of DM, suggesting that deDM has the potential to be more stable in vivo with fewer metabolites, i.e., fewer side effects, and therefore, deDM and BUP is a safer and more potent combination for depressed patients than the combination of DM and BUP.","manuscriptTitle":"Efficacy of SAL0114, an Oral NMDA Receptor Antagonist with Multimodal Activity, in Major Depressive Disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 18:30:24","doi":"10.21203/rs.3.rs-4119597/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"52c3d1e3-43c4-4857-93a6-f37ae23ab2bc","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30525445,"name":"Biological sciences/Drug discovery/Pharmacology/Pharmacodynamics"},{"id":30525446,"name":"Health sciences/Neurology/Neurological disorders"}],"tags":[],"updatedAt":"2024-06-04T05:21:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-12 18:30:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4119597","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4119597","identity":"rs-4119597","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}