Main Research Findings

1) Usmarapride was found to have disease modifying potential and may act as a promising therapeutic intervention for alleviating cognitive dysfunctional related to Alzheimer’s disease.

2) Activation of 5-HT4 receptors such as usmarapride was shown to facilitate neurogenesis from transplanted neural stem cells.

 

 

Selected Data
1) The study performed by the research team of Nirogi et al evaluated the behavioral, neurochemical, and pharmacological profile of usmarapride (SUVN-D4010), a selective 5-HT4 receptor partial agonist, to determine its therapeutic potential for the treatment of Alzheimer’s disease. A series of complementary in vivo behavioral experiments, neurochemical analyses, pharmacological validation studies, and biochemical assays were performed to assess the compound’s effects on multiple domains of cognition, cholinergic neurotransmission, and amyloid precursor protein processing. Adult Wistar rats, Swiss mice, and C57BL/6J mice were housed under controlled environmental conditions with unrestricted access to food and water. Usmarapride was synthesized in-house, while comparator compounds including scopolamine, MK-801, donepezil, and the selective 5-HT4 receptor antagonist GR-125487 were obtained from commercial or institutional sources [1].
To evaluate episodic memory, the investigators employed the object recognition task (ORT) under several experimental conditions. Animals underwent habituation followed by familiarization with two identical objects before being challenged with either a delayed retention interval or pharmacologically induced cognitive impairment using scopolamine or MK-801. During the choice trial, one familiar object was replaced with a novel object, and the amount of time spent exploring each object was recorded. A discrimination index was calculated to quantify recognition memory performance. Additional experiments examined whether pretreatment with the selective 5-HT4 receptor antagonist GR-125487 blocked the cognitive effects of usmarapride, thereby confirming receptor specificity. Separate ORT studies also evaluated whether co-administration of low-dose usmarapride with donepezil enhanced cognitive performance beyond either treatment alone [1].
Working memory was assessed using the radial arm maze (RAM). Rats were habituated to an eight-arm maze over several days before undergoing testing under scopolamine-induced cognitive impairment. Food pellets were placed at the ends of each arm, and animals were allowed to retrieve rewards while investigators recorded repeated arm entries and omissions. Percentage choice accuracy was calculated from the number of correct arm selections relative to total entries, providing an objective measure of short-term spatial working memory. Usmarapride was administered orally before scopolamine treatment to determine its ability to reverse cholinergic-dependent working memory deficits.
The investigators next examined social recognition memory using the social recognition task (SRT). Adult male rats were exposed to juvenile conspecifics during an initial learning session followed by a second trial after either a 24-hour delay or a brief interval combined with scopolamine administration. During the testing session, animals encountered both the previously familiar juvenile and a novel juvenile, and investigators measured the duration of social investigation directed toward each animal. A discrimination index quantified preference for the novel juvenile. Additional experiments evaluated combination therapy with donepezil and determined whether usmarapride enhanced existing cholinergic treatment. Emotional and associative memory were assessed using the fear conditioning response (FCR) model. Rats received paired tone-foot shock conditioning followed by scopolamine administration, and freezing behavior during later testing without shock served as an indicator of learned associative memory [1].
To investigate the neurochemical mechanisms underlying the behavioral effects, the researchers measured extracellular acetylcholine concentrations within the frontal cortex using in vivo microdialysis. Guide cannulas were stereotaxically implanted into the frontal cortex of anesthetized rats, after which microdialysis probes continuously perfused artificial cerebrospinal fluid. Baseline dialysate samples were collected before administration of vehicle or usmarapride, followed by serial sample collection for up to four hours after treatment. Acetylcholine concentrations were quantified using tandem mass spectrometry, and area-under-the-curve analyses were performed to assess overall neurotransmitter release. Additional experiments incorporated pretreatment with GR-125487 to determine whether changes in acetylcholine release were mediated specifically through 5-HT4 receptor activation.
Finally, the investigators evaluated the potential disease-modifying effects of usmarapride by measuring soluble amyloid precursor protein-alpha (sAPP-α), a product of non-amyloidogenic APP processing associated with neuroprotection. C57BL/6J mice received acute administration of vehicle or usmarapride before sacrifice at predetermined time points. Frontal cortex tissue was isolated, homogenized, and processed for enzyme-linked immunosorbent assay (ELISA) to quantify sAPP-α concentrations. Collectively, these integrated behavioral paradigms, pharmacological validation studies, neurochemical measurements, and biochemical assays provided a comprehensive assessment of the cognitive-enhancing and potential disease-modifying properties of usmarapride in preclinical models of Alzheimer’s disease [2].
2) The study by the research team of Goto et al investigated whether activation of serotonin 5-HT4 receptors enhances neurogenesis from transplanted neural stem cells (NSCs) following intestinal surgery and whether combining pharmacological stimulation with stem cell transplantation could improve regeneration of the enteric nervous system. Specifically, the researchers examined the effects of the selective 5-HT4 receptor agonist mosapride citrate (MOS) on the survival, migration, and neuronal differentiation of transplanted embryonic NSCs in a mouse model of ileal transection and anastomosis. The study also evaluated whether these effects were mediated specifically through 5-HT4 receptor activation by administering the selective antagonist SB-207266. To visualize newly generated neurons within the thick granulation tissue that develops after intestinal surgery, the investigators employed in vivo two-photon excitation fluorescence microscopy (2PM), which provides substantially greater tissue penetration than conventional fluorescence microscopy [2].
As an initial quality assessment, commercially obtained neural stem cells isolated from the hippocampus and subventricular zone of embryonic day-12.5 C57BL/6 mouse embryos were cultured in neural stem cell growth medium for four days to confirm their viability and differentiation potential before transplantation. The cultured NSCs formed neurospheres under standard conditions. Additional culture experiments examined the effects of brain-derived neurotrophic factor (BDNF), mosapride, and the selective 5-HT4 receptor antagonist GR113808 on neurite outgrowth. Microscopic evaluation compared untreated control cultures with cultures exposed to BDNF alone, mosapride alone, or mosapride combined with GR113808, allowing investigators to determine whether activation of 5-HT4 receptors directly influenced neuronal differentiation before initiating the animal experiments [2].
Prior to transplantation, the NSCs were labeled with the long-lasting fluorescent membrane dye PKH26, enabling the transplanted cells and their differentiated progeny to be identified within recipient tissues for several weeks after transplantation. The labeling protocol involved incubating the cells with PKH26 dye, terminating the reaction with bovine serum albumin, and performing repeated washing and centrifugation steps to remove excess dye while preserving cell viability. After labeling, 2 × 105 NSCs suspended in phosphate-buffered saline were injected into the tail vein of each recipient mouse immediately following intestinal surgery.
Adult Thy1 promoter yellow fluorescent protein (YFP) transgenic mice served as recipients because endogenous enteric neurons express cytoplasmic YFP, allowing investigators to distinguish neurons generated from endogenous host stem cells (YFP-positive) from those derived from transplanted PKH26-labeled NSCs (PKH26-positive). Under anesthesia, the terminal ileum was surgically transected approximately 5–6 cm proximal to the ileocecal junction, followed by end-to-end anastomosis. Immediately after surgery, PKH26-labeled NSCs were transplanted intravenously. Animals were then assigned to one of three treatment groups receiving daily oral administration for two weeks of vehicle (0.1% DMSO), 100 μM mosapride, or mosapride plus the selective 5-HT4 receptor antagonist SB-207266. Mice were fasted for the first two postoperative days before returning to unrestricted feeding for the remainder of the treatment period.
Fourteen days after transplantation, the anastomotic region was examined using in vivo two-photon excitation microscopy. Following laparotomy under anesthesia, the ileum was stabilized in an imaging chamber while spontaneous intestinal contractions were suppressed using papaverine. A Ti-sapphire laser tuned to 950 nm simultaneously excited YFP and PKH26 fluorescence, enabling visualization of neurons originating from both endogenous and transplanted stem cells. Image stacks consisting of approximately 200 optical sections acquired at 1-μm intervals were collected to depths approaching 400 μm beneath the serosal surface. Three-dimensional reconstructions were generated using specialized imaging software, and investigators quantified the number and spatial distribution of PKH26-positive and YFP-positive neurons throughout multiple predefined fields surrounding the anastomosis [2].
Following live imaging, intestinal tissues were harvested for confocal microscopy and immunohistochemical analysis. Frozen longitudinal sections containing the anastomotic region were prepared to examine localization of transplanted cells within the granulation tissue. Immunostaining with the neuronal marker protein gene product 9.5 (PGP9.5) confirmed whether PKH26-labeled cells had differentiated into mature enteric neurons and formed ganglion-like structures. Quantitative analyses evaluated neuron numbers within individual imaging fields, different tissue depths, and the entire anastomotic region. This integrated experimental design allowed the investigators to determine whether pharmacological activation of 5-HT4 receptors enhances enteric neurogenesis from both transplanted and endogenous neural stem cells during intestinal repair [2].

 

Discussion
1) The study conducted by Köhler-Forsberg et al demonstrated that usmarapride (SUVN-D4010) produced robust procognitive effects across multiple behavioral models that assessed episodic, working, social, and emotional memory while simultaneously enhancing cholinergic neurotransmission and promoting non-amyloidogenic processing of amyloid precursor protein. Across nearly all behavioral paradigms, usmarapride consistently improved cognitive performance in both naturally induced memory deficits and pharmacologically impaired models, supporting its potential as both a symptomatic cognitive enhancer and a disease-modifying therapeutic candidate for Alzheimer’s disease. Importantly, many of these effects occurred over a broad dose range and were shown to depend specifically on activation of 5-HT4 receptors through antagonist validation experiments [1].
In the ORT, usmarapride significantly improved episodic memory under multiple experimental conditions. In animals challenged with a 24-hour retention interval, vehicle-treated rats failed to distinguish between familiar and novel objects, demonstrating normal forgetting. In contrast, rats treated with usmarapride at doses ranging from 0.3 to 10 mg/kg spent significantly more time exploring the novel object, indicating successful retention of object memory. Correspondingly, discriminative index values increased significantly, particularly at doses of 1 and 10 mg/kg. Similar improvements were observed in pharmacologically impaired animals. Both scopolamine- and MK-801-treated rats exhibited marked deficits in object recognition, but usmarapride dose-dependently restored recognition memory by increasing exploration of the novel object and improving discrimination indices. The receptor specificity of these effects was confirmed by pretreatment with the selective 5-HT4 receptor antagonist GR-125487, which abolished the memory-enhancing actions of usmarapride. These findings demonstrated that the compound’s procognitive effects were mediated directly through activation of 5-HT4 receptors rather than nonspecific behavioral stimulation [1].

 

Figure 1: Changes in A) exploration times and B) discrimination index as a measure of time-induced episodic memory deficits in response to treatment with experimental compounds
Combination studies further demonstrated that usmarapride potentiated the cognitive effects of donepezil, the current standard treatment for Alzheimer’s disease. When administered individually at low doses, neither donepezil nor usmarapride significantly improved episodic memory. However, combined treatment produced significantly greater discrimination between familiar and novel objects and yielded higher discrimination index scores than either drug alone. Similar synergistic effects were observed in the SRT, where combined treatment significantly enhanced recognition of novel juvenile animals compared with either monotherapy. These findings suggest that usmarapride may augment existing cholinergic therapies and potentially permit lower therapeutic doses of acetylcholinesterase inhibitors while maintaining cognitive efficacy [1].
Usmarapride also produced significant improvements in additional domains of cognition. In the radial arm maze, scopolamine markedly impaired working memory by increasing repeated arm entries and reducing percentage choice accuracy. Treatment with usmarapride significantly reversed these deficits at all tested doses, with the greatest improvements observed at 1 and 3 mg/kg. In the social recognition task, both naturally induced time-delay deficits and scopolamine-induced impairments were significantly attenuated by usmarapride, as treated animals consistently spent more time investigating unfamiliar juvenile rats than familiar ones. Discrimination indices increased significantly across multiple doses, demonstrating preservation of social recognition memory. Likewise, in the fear conditioning response model, scopolamine substantially reduced freezing behavior, indicating impaired associative and emotional memory. Usmarapride dose-dependently restored freezing responses toward normal levels, demonstrating improved retention of conditioned fear memory and suggesting efficacy across multiple cognitive domains that are commonly disrupted during Alzheimer’s disease progression.
Neurochemical analyses provided mechanistic support for the observed behavioral improvements. In vivo microdialysis demonstrated that usmarapride significantly increased extracellular acetylcholine concentrations within the frontal cortex at doses of 3 and 10 mg/kg. Area-under-the-curve analyses confirmed sustained elevations in acetylcholine release throughout the sampling period. Pretreatment with GR-125487 completely prevented these increases, demonstrating that enhancement of cholinergic neurotransmission resulted specifically from 5-HT4 receptor activation. Because cortical acetylcholine plays a critical role in attention, learning, and memory, these findings provide a neurochemical explanation for the widespread cognitive improvements observed throughout the behavioral studies [1].
Finally, usmarapride demonstrated evidence of potential disease-modifying activity by significantly increasing sAPP-α concentrations in the frontal cortex. Acute administration of usmarapride produced significant elevations in sAPP-α at both 3 and 10 mg/kg, although the timing of peak responses differed between doses. Since sAPP-α is generated through the non-amyloidogenic processing pathway of amyloid precursor protein and possesses well-established neuroprotective, neurotrophic, and synaptic plasticity-promoting properties, its elevation suggests that usmarapride may shift APP metabolism away from the production of neurotoxic amyloid-β peptides. Collectively, the behavioral, neurochemical, and biochemical findings demonstrate that usmarapride consistently enhances multiple forms of learning and memory through selective 5-HT4 receptor activation, increases cortical acetylcholine release, potentiates the therapeutic effects of donepezil, and promotes neuroprotective APP processing. These combined actions support the compound’s potential to provide both symptomatic cognitive improvement and disease-modifying benefits in Alzheimer’s disease and justify continued clinical development [1].
2) The study conducted by the research team of Goto et al demonstrated that activation of 5-HT4 receptors with MOS markedly enhanced enteric neurogenesis following intestinal surgery by promoting the differentiation of neurons from both transplanted NSCs and endogenous host stem cells. Initial in vitro experiments established that the transplanted NSCs remained viable and responsive to pharmacological stimulation before transplantation. After four days in culture, untreated NSCs formed characteristic neurospheres with minimal neurite extension. Treatment with BDNF produced only modest outgrowth of neuronal projections, whereas exposure to 1 μM mosapride resulted in substantially greater neurite extension from the neurospheres. When the selective 5-HT4 receptor antagonist GR113808 was added together with mosapride, this enhanced neurite outgrowth was abolished, indicating that the trophic effects of mosapride on embryonic neural stem cells were mediated specifically through activation of 5-HT4 receptors. These preliminary findings confirmed both the functional competence of the cultured NSCs and the ability of 5-HT4 receptor stimulation to directly promote neuronal differentiation before transplantation [2].
Two weeks after intestinal transection, anastomosis, and intravenous transplantation of PKH26-labeled NSCs, in vivo two-photon excitation microscopy revealed extensive neurogenesis within the granulation tissue surrounding the surgical anastomosis. In mice treated with mosapride, investigators observed abundant YFP-positive neurons, representing neurons derived from endogenous host neural stem cells, together with numerous PKH26-positive neurons, which originated from the transplanted embryonic NSCs. Both neuronal populations were detected throughout the thick granulation tissue at depths ranging from approximately 1 to over 200 μm beneath the serosal surface. Three-dimensional reconstructions demonstrated that many PKH26-positive neurons formed interconnected clusters resembling newly developing enteric ganglia, with visible neuronal processes extending between neighboring cells. The similar spatial distribution of transplanted and host-derived neurons suggested that both cell populations migrated into the injured tissue and participated simultaneously in reconstruction of the enteric nervous system following surgery [2].

 

Figure 2: Changes in the average number of A) PKH26-positive and B) YEP-positive neurons in the anastomotic region following treatment with the experimental compounds.
Confocal microscopy and immunohistochemical analyses confirmed that the transplanted cells differentiated into mature enteric neurons. Examination of longitudinal tissue sections showed that PKH26-positive cells accumulated specifically within the granulation tissue surrounding the anastomosis and were largely absent from adjacent normal intestinal tissue. Immunostaining for the neuronal marker PGP9.5 demonstrated that many PKH26-labeled cells were also PGP9.5-positive, confirming successful neuronal differentiation of the transplanted stem cells. Furthermore, clusters of PKH26-positive neurons were incorporated into newly formed ganglion-like structures, providing anatomical evidence that transplanted embryonic neural stem cells not only survived after systemic administration but also integrated into the regenerating enteric neural network. These histological findings closely paralleled the observations obtained with live two-photon imaging.
Quantitative analysis demonstrated that mosapride significantly increased neurogenesis throughout the anastomotic region. In representative animals treated with mosapride, investigators counted 59 PKH26-positive neurons and 693 YFP-positive neurons within the analyzed imaging volume surrounding the surgical knot, whereas animals receiving both mosapride and the selective antagonist SB-207266 exhibited only 26 PKH26-positive neurons and 136 YFP-positive neurons in comparable tissue volumes. Statistical analyses performed across all experimental animals showed that mosapride significantly increased neuron numbers within multiple predefined imaging fields distributed around the anastomosis [2].
The stimulatory effects were observed for both transplanted and host-derived neurons, although endogenous YFP-positive neurons consistently outnumbered transplanted PKH26-positive neurons by approximately tenfold. Analysis according to tissue depth further demonstrated that mosapride significantly increased transplanted neuron density primarily between 60 and 80 μm beneath the serosal surface, while endogenous neurogenesis was enhanced predominantly between 40 and 80 μm, indicating that the greatest regenerative activity occurred within the middle regions of the granulation tissue rather than at superficial or deep locations.

 

Figure 2: Changes in the average number of A) PKH26-positive and B) YFP-positive neurons in the anastomotic region following treatment with the experimental compounds.
The most compelling evidence for the regenerative effects of mosapride came from comparisons of total neuron numbers across treatment groups. Relative to vehicle-treated controls, mosapride increased the total number of PKH26-positive neurons approximately 2.5-fold, from 23 ± 4 to 48 ± 9 cells per 0.8649 mm². A nearly identical enhancement was observed for endogenous neurogenesis, with YFP-positive neurons increasing approximately 2.5-fold, from 206 ± 24 to 522 ± 163 cells per 0.8649 mm². Importantly, co-administration of the selective 5-HT4 receptor antagonist SB-207266 almost completely abolished these regenerative effects, reducing transplanted neuron numbers to 17 ± 8 cells and endogenous neuron numbers to 206 ± 126 cells per 0.8649 mm², values comparable to vehicle-treated controls. Collectively, these findings demonstrate that activation of 5-HT4 receptors significantly enhances enteric neurogenesis from both transplanted embryonic neural stem cells and endogenous host stem cells after intestinal injury. The results further indicate that combining 5-HT4 receptor agonist therapy with stem cell transplantation may represent a promising regenerative strategy for disorders involving enteric nervous system degeneration, including Hirschsprung’s disease and related gastrointestinal neuropathies [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Nirogi R, Grandhi VR, Medapati R, et al. Usmarapride (SUVN-D4010), a 5-HT4 receptor partial agonist for the potential treatment of Alzheimer’s disease: Behavioural, neurochemical and pharmacological profiling. Eur J Pharmacol. 2023;947:175625. doi:10.1016/j.ejphar.2023.175625

[2] Goto K, Kawahara I, Inada H, et al. Activation of 5-HT4 receptors facilitates neurogenesis from transplanted neural stem cells in the anastomotic ileum. J Physiol Sci. 2016;66(1):67-76. doi:10.1007/s12576-015-0396-1

 

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