The Novel TRPV1 Agonist ST‐6631 Improves Swallowing Function in Rat Models of Stroke‐Related Dysphagia and Demonstrates Pharmaceutical Advantage Over Capsaicin, by Exhibiting Minimal Pungent and Irritant Properties

ABSTRACT

Dysphagia, often associated with neurological disorders such as stroke, not only damages patients' quality of life, like the joy of engaging with meals, but also adversely affects mortality among patients by increasing risks of getting aspiration pneumonia. There are currently no approved drug‐based treatments, leaving dysphagic symptom management as unmet clinical needs. Nevertheless, transient receptor potential vanilloid 1 (TRPV1) channel has gained clinical interest to enhance the sensitivity of sensory nerves that facilitate swallowing reflexes in the pharynx. Here, a novel TRPV1 agonist and antedrug candidate ST‐6631 has been introduced, with its therapeutic efficacy comparable to the natural ligand capsaicin, in rats operated with permanent bilateral carotid artery occlusion (BCAO), the animal model of stroke‐related dysphagia. The ST‐6631 antedrug properties have shown rapid metabolism by the hepatic S9 enzymatic fractions, and also shown extremely limited systemic bioavailability in blood circulation after the oral administration in vivo to rats. Furthermore, ST‐6631 has been described to possess minimal pungency and irritancy in behavioral sensory assessments in awake rats. ST‐6631 has been revealed to provide only a modest form of depolarization in rat sensory dorsal root ganglion (DRG) neurons. In addition, ST‐6631 moderately activates the TRPV1 current, characterized by slower but long‐lasting waveforms, in the channel‐expressing Chinese hamster ovary (CHO) cells. Hence, the present study collectively highlights the pharmaceutically desired product profile of ST‐6631, achieving TRPV1 agonism with minimal adverse effects, in clear contrast to the noxious reference ligand, capsaicin.

1. Introduction

Dysphagia, the difficulty in swallowing, is a debilitating medical condition that affects millions of individuals worldwide [1, 2, 3]. Dysphagia often results from various neurological and other disorders, including stroke, Parkinson's disease, Alzheimer's disease, cancer, and obstructive motility dysfunctions [4, 5, 6, 7]. Dysphagia is classified into three types, based on an affected phase of swallowing process: oral, pharyngeal, and esophageal [8]. Oral dysphagia refers to a difficulty in propelling boluses from the mouth to the pharynx, often due to impaired tongue controls. Esophageal dysphagia refers to an obstructive motility dysfunction that affects the esophagus. Pharyngeal dysphagia, a primary focus of the present study, refers to an impairment in the initiative phase of swallowing reflexes. This pharyngeal phase is extremely critical for propelling food and liquid boluses through the pharynx, while preventing an accidental aspiration into the airway. Pharyngeal dysphagia contributes to subsequent comorbidities such as aspiration pneumonia, malnutrition, and dehydration in patients [9, 10].

Current clinical practices in the management of dysphagia predominantly focus on symptomatic mitigations through compensatory eating exercises [11, 12, 13], although these practices seem strategically different from addressing the pathophysiology of dysphagia. Yet, dietary modifications, to cook texture‐thickened foods, are common, reducing risks of accidental aspiration [14]. Rehabilitation exercises employ some effortful swallowing [15], known as the Mendelsohn maneuver [16], and the Shaker exercise [17]. More advanced rehabilitation strategies, for example, electrical neuromuscular stimulation of the esophagus, have been exploited lately [18]. Nevertheless, the effectiveness of those rehabilitation exercises varies considerably among patients, depending on the etiology and severity of dysphagia. Furthermore, it is understood that these rehabilitation approaches often face resource limitations, as they require specialized medical support and long‐term commitment from both patients and healthcare professionals.

Despite substantial burdens that dysphagia imposes on modern healthcare environments, a pharmaceutical intervention on this disorder remains remarkably underdeveloped. Currently, there are no approved drug‐based treatments that address the underlying neurophysiology of dysphagia [19, 20]. Yet, some therapeutic approaches have been considered recently. One is to modulate the sensitivity of sensory and motor nerves that are involved in the reflex function of swallowing, which is found to improve the pharyngeal and esophageal motility [21, 22]. Here, transient receptor potential vanilloid 1 (TRPV1) channel gains an attention of therapeutic interests [23]. TRPV1 channel is a calcium‐permeable, mixed cation channel that is primarily expressed on sensory neurons [24]. TRPV1 channel functions as a molecular integrator of various noxious stimuli, including heat, acidity, and natural ligands such as capsaicin [25]. In addition, TRPV1 channel plays a modulatory role in the sensory and motor nerve pathways that are responsible for swallowing reflexes, where TRPV1 activation enhances nervous signaling, relayed from the pharyngeal motility sensor to the swallowing center of brainstem [22, 26]. The increased nerve sensitivity facilitates the initiation of pharyngeal swallowing, leading to the smoothened coordination of bolus propulsion and prevention of accidental aspiration [27]. Indeed, capsaicin, the most extensively studied TRPV1 agonist, has been shown to improve the swallowing reflex not only in animal models [28, 29, 30] but also, importantly, in human patients suffering from dysphagia [31, 32, 33, 34, 35].

However, despite its promising pharmacotherapeutic evaluation, clinical uses of capsaicin have been extremely limited, mostly due to the compound's perceived pungent and irritant sensory properties. The topical application of capsaicin to the skin and the mucosa induces strong burning and intolerable sensations. Furthermore, the systemic exposure of capsaicin and related TRPV1 agonists has been pointedly warned of some known adverse effects, including hypothermia, cardiovascular, and gastrointestinal concerns [36, 37, 38], which further restrict the tolerability and safety acceptance, not suitable for administration to human patients.

To overcome the adverse properties of capsaicin and related agonists, a novel TRPV1 channel activator with improved tolerability and safety characteristics is highly anticipated, as part of further drug developments and therapeutic interventions to manage dysphagia. Here, a novel synthetic compound ST‐6631 has been introduced as a prospective antedrug candidate that demonstrates efficacy comparable to capsaicin in rat models of stroke‐related dysphagia. The advantages and uniqueness of ST‐6631 over capsaicin are particularly evident in its minimal pungency and irritancy, served not only in awake, behaving animals but also in sensory neurons in vitro. The balanced ST‐6631 properties to pursue TRPV1 agonistic efficacy but minimal adverseness are likely to fulfill pharmaceutically desirable product profiles, in context of clinical interventions to restore the function of swallowing. Hence, the present study provides a new insight into potential drug‐based strategic approaches for future therapeutic management of dysphagia and related motility dysfunctions in human patients.

2. Materials and Methods

2.1. Synthesis of ST‐6631 and Its Hydrolyzed Metabolite ST‐6631‐M1

ST‐6631 (3‐(4‐(2‐(4‐hydroxy‐3‐methoxyphenyl)acetamido)pyridin‐2‐yl)phenyl 2‐methylpropanoate hydrochloride), a novel TRPV1 agonist derived from capsaicin, was synthesized exclusively by Sumitomo Pharma Co. Ltd. [39, 40] The synthesis began with an amide coupling between 4‐benzyloxy‐3‐methoxyphenylacetic acid and 4‐amino‐2‐bromopyridine using T3P as the coupling reagent, affording 2‐(4‐(benzyloxy)‐3‐methoxyphenyl)‐N‐(2‐bromopyridin‐4‐yl)acetamide in quantitative yield. The corresponding hydrochloride salt of 2‐(4‐(benzyloxy)‐3‐methoxyphenyl)‐N‐(2‐(3‐hydroxyphenyl)pyridin‐4‐yl)acetamide was obtained via Suzuki–Miyaura coupling with (3‐hydroxyphenyl)boronic acid in 72% yield. Final acylation with isobutyryl chloride, followed by catalytic hydrogenation and hydrochloride salt formation, afforded ST‐6631 as a white solid in 90% yield. The product was purified by recrystallization from ethanol. ST‐6631‐M1, identified as a hydrolyzed metabolite of ST‐6631, was chemically synthesized through hydrolysis of ST‐6631. This synthetic approach enabled definitive structural characterization of the metabolite.

2.2. Animal Usage

All ethical standards were followed to conduct the present study. The use of animals and subsequent experimental procedures were reviewed and approved by the Institutional Animal Use and Care Committee, Sumitomo Pharma Co. Ltd.

2.3. Dysphagia Model Rats With Permanent Bilateral Common Carotid Artery Occlusion (BCAO) and the Evaluation of Swallowing Reflexes

(BCAO surgery) Male Crl:CD (SD) rats (9 week old, The Jackson Laboratory Japan Inc.) were anesthetized by the subcutaneous injection of buprenorphine hydrochloride at a dose of 0.01 mg/kg (twice administration). Rats were then fixed in a supine position under the continuous inhalation of 2% isoflurane anesthesia. The common carotid arteries were surgically exposed and ligated bilaterally with sutures. Sham operation was to expose the bilateral common carotid arteries without ligation. After the surgery, antibiotics (oxytetracycline) and antiseptics (povidone‐iodine) were applied to the neck of animals.

(Swallowing evaluation) On post‐operative day 29, these rats were assessed with their swallowing reflexes. First, the rats were anesthetized with urethane (1–1.3 g/kg, i.p.) in a supine position, while their body temperature was kept using a heating pad. A ventral midline incision was made on the animal's neck, and a tracheal cannula was intubated to maintain breathing. A pair of bipolar enamel‐coated stainless‐steel wire electrodes (interelectrode distance approximately 5 mm) was placed on the left mylohyoid muscle to record electromyographic activities. A small infusion tube for the test substance application was placed in the pharynx with its proximal end opening at the roof of the soft palate. To induce the swallowing reflex, an aqueous solution of vehicle or the test substance was applied into the pharynx space for 9 s at a flow rate of 10 μL/s using an infusion pump. The number of swallowing reflexes was counted for 1 min from the beginning of substance infusion.

2.4. Metabolic Stability of ST‐6631, Incubated With the Human Liver S9 Enzymatic Fractions

ST‐6631 (30 nM) was incubated at 37°C in 200 μL reaction mixture that contained 50 mM phosphate buffer (pH 7.4) and the human liver S9 fractions (2.0 μg/mL, XenoTech, Kansas, USA). After the 37°C incubation for 5 or 60 min, the metabolic reaction was stopped by adding 800 μL acetonitrile. The reaction mixture was centrifuged to remove protein precipitations, and then the supernatant was immediately filtered. The filtrate was analyzed using high‐performance liquid chromatography, coupled with tandem mass spectrometry (HPLC/MS/MS).

2.5. Plasma Concentration of ST‐6631, to Follow the Oral Administration to Rats

To evaluate the pharmacokinetics of ST‐6631, female Crl:CD (SD) rats (6 week old, n = 3) were administered a single oral dose of 20 mg/kg of the compound, being suspended in 0.5 w/v % methyl cellulose solution. The test was performed as part of early‐stage toxicological evaluation, so that the use of female rats reduced the required total amount of the test compound, ST‐6631. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, and 24 h post‐dose on the day. Plasma concentrations of ST‐6631 and its hydrolyzed metabolite ST‐6631‐M1 were measured using a method of LC/MS/MS.

2.6. Eye‐Wiping Behavioral Test

To evaluate and compare the substance pungency of capsaicin and ST‐6631 in awake, behaving animals, male Crl:CD (SD) rats (9 week old) were exposed to 10 μL of the test substance solution, instilled into the animal's right eye using a micropipette. The animals were immediately returned to an observational cage, and their nocifensive eye‐wiping behavior was recorded for 1 min. A camera was set up on top of the cage, and its video footage was utilized where necessary to cross‐check the experimental quality and to minimize observer biases.

2.7. Oral Mucosal Irritation Test

The test was conducted to evaluate the severity of oral mucosal irritation, caused by ST‐6631 that was administered through a clinically intended intraoral route. The test was performed in accordance with Safety Testing Guidelines of the Cosmetic, Toiletry, and Fragrance Association of the United States (CTFA Safety Testing Guidelines, 1981). Male Crl:CD (SD) rats (13 week old, n = 3/group) were exposed to ST‐6631, at a concentration of 0 (control, 30% ethanol) or 30 μM, applied to the mandibular oral vestibule, with a cotton‐soaked volume of 0.1 mL test solution per animal. Each applied dose lasted approximately 30 s/time, 4 times/day for 4 consecutive days. An aqueous solution of 4 w/v % sodium dodecyl sulfate (SDS) was referred to as a positive irritant control. During this dosing period, the oral mucosa including labial junction was macroscopically observed once daily. Irritancy scores were noted, based on the CTFA criteria for oral mucosal irritation. In addition, the histopathological evaluation of ST‐6631 applied sites was performed, 3 days after the end of dosing period, by taking hematoxylin‐ and eosin‐stained examination specimens.

2.8. Manual Patch‐Clamp Recording on the Primary Cultured Rat Sensory DRG Neurons

(Animals and DRGs) Neonatal SD rats (3 day old) were purchased from Japan SLC (Shizuoka, Japan). Animals were decapitated and tissues containing dorsal root ganglions (DRGs) were isolated under the ice‐cold Hanks' Balanced Salt Solution (HBSS, Thermo Fisher Scientific, Japan). Connective tissues such as dura and pia matter were carefully trimmed. Once DRGs were collected, they were warm‐incubated in the HBSS with 0.05% trypsin–EDTA (Thermo Fisher Scientific) at 37°C for 15 min. DRGs were rinsed but again warm‐incubated in the HBSS with 0.6 mg/mL collagenase (Sigma‐Aldrich, Japan) at 37°C for 30 min.

(Primary culture) Sensory DRG neurons were suspended in the warm Neurobasal medium (Thermo Fisher Scientific) supplemented with 1:1 penicillin–streptomycin‐glutamine (PSG, Thermo Fisher Scientific), 1:1 N‐2 supplement (Thermo Fisher Scientific), and 1 μg/mL NGF 2.5S (Thermo Fisher Scientific). DRG cells were counted to make up 1 × 10^5 cells/ml, then cultured on laminin‐coated cover glasses at 37°C until the use (DIV 1–3) for patch‐clamp experiments.

(Manual patch‐clamp) The extracellular solution contained (in mM): 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 5 Glucose, and 10 HEPES. The internal solution contained (in mM): 123 K‐gluconate, 20 KCl, 1 MgCl₂, 1 EGTA, 10 HEPES, 4 Glucose, 2 ATP‐Na, and 0.5 GTP‐Na₂. Whole‐cell patch‐clamp recordings were performed with the Axopatch 200B amplifier and pClamp 9 software (Molecular Devices, California, USA). Once a stable recording of membrane potentials was established, the test substance (capsaicin or ST‐6631) was topically applied through a fine glass capillary tube positioned near the recorded cell. Changes in the resting membrane potential, whether depolarizing responses were accompanied with the evocation of action potentials, were assessed to infer the excitable and perhaps pungent properties of test substances.

2.9. Automated Patch‐Clamp Recording on TRPV1‐Expressing CHO Cells

(TRPV1‐expressing cells) Chinese Hamster Ovary (CHO) cells that stably express human TRPV1 (hTRPV1) channels under the control of tetracycline induction were established at Sumitomo Pharma Co. Ltd. hTRPV1 cells were stored in the −80°C liquid nitrogen deep‐freezer, then were cultivated where appropriate. The culture medium was Ham's F‐12 nutrient mixture (Sigma‐Aldrich) supplemented with 1:10 fetal bovine serum (Thermo Fisher Scientific), 2.5 μg/mL Blasticidin S (InvivoGen, California, USA) and 0.2 mg/mL Geneticin (Thermo Fisher Scientific). One day before the patch‐clamp experiments, cells were handled in the culture medium added with 2 μg/mL tetracycline hydrochloride (Sigma‐Aldrich) to induce the stable expression of hTRPV1 channels.

(Automated patch‐clamp system) Automated whole‐cell patch‐clamp recordings were performed on the syncropatch 768PE system (Nanion Technologies, Munich, Germany). hTRPV1 cells were first suspended in the 2 mM Ca buffer solution containing (in mM): 140 NaCl, 5 glucose, 10 HEPES, 4 KCl, 1 MgCl₂, and 2 CaCl₂. Once cells were captured on recording pores on the bottom glass plate, the buffer solution was replaced with the 5 mM Ca extracellular solution containing (in mM): 135 NaCl, 5 glucose, 10 HEPES, 4 KCl, 1 MgCl₂, and 5 CaCl₂, to enhance the tight sealing of cell membranes onto the recording pores. The intracellular solution was cesium fluoride based, to contain (in mM): 110 CsF, 10 CsCl, 20 EGTA, and 10 HEPES, which also acted to ensure the further enhancement of tight sealing for a better quality of patch‐clamp recordings. When the whole‐cell configuration was achieved, cells were voltage‐clamped at −80 mV throughout the recordings.

(Ligand application) The TRPV1 ligand (capsaicin or ST‐6631) was applied in a manner of brief puff (0.5 s) to reach the final concentration of 1 μM in the extracellular solution, made up with 0.1% DMSO. Upon the ligand binding, a clear TRPV1‐activated inward current was elicited on the cell, recorded for 20 s.

2.10. Data Presentation and Statistical Analysis

Numerically quantitative and graphical data were presented in a format of mean +/−standard error of the mean (s.e.m.) where applicable. The normality of collected data sets was assessed by the Shapiro–Wilk test, and then appropriate statistical tests were applied, using the GraphPad Prism 9 software (Massachusetts, USA).

3. Results

3.1. Chemical Structure of ST‐6631

ST‐6631 was synthesized exclusively by Sumitomo Pharma Co. Ltd., and its chemical structure is disclosed for the first time here (Figure 1). This compound is currently developed as a novel drug candidate that agonistically activates TRPV1 channel. Its pharmacological efficacy and safety profiles are described hereafter. In addition, the hydrolyzed metabolite ST‐6631‐M1, which loses the target potency considerably, is shown for clarification in chemistry.

FIGURE 1.

Chemical structure of ST‐6631. ST‐6631 (3‐(4‐(2‐(4‐hydroxy‐3‐methoxyphenyl)acetamido)pyridin‐2‐yl)phenyl 2‐methylpropanoate hydrochloride) has been synthesized and developed as a novel TRPV1 agonist, exclusively by Sumitomo Pharma Co. Ltd. Its hydrolyzed metabolite, ST‐6631‐M1, is also shown for the clarification in chemistry.

3.2. ST‐6631 Improved the Swallowing Reflex in Stroke‐Related Dysphagia Models of BCAO Rats

The rats with permanent bilateral common carotid artery occlusion (BCAO) were first developed as animal models of stroke. However, the BCAO model rats also exhibited impaired counts of the swallowing reflex, compared to the sham‐operated controls (n = 10 animals each, two‐sample independent t‐test, ***p < 0.001, Figure 2). Since the human patients who experienced stroke would often suffer from similar dysphagic problems, the BCAO surgery rats here were also regarded as animal models of stroke‐related dysphagia in the present study.

FIGURE 2.

ST‐6631 improved the counts of swallowing reflex in animal models of stroke‐related dysphasia, BCAO rats. Rats operated with permanent bilateral common carotid artery occlusion (BCAO) were used to assess the function of swallowing reflexes. The BCAO rats displayed impaired counts of the swallowing reflex, compared to sham‐operated rats (n = 10 animals each, two‐sample independent t‐test, ***p < 0.001), which was to validate the BCAO surgery rats as animal models of stroke‐related dysphasia. The pharyngeal perfusion of capsaicin or ST‐6631 (1, 10, and 100 μM) resulted to the improved counts of swallowing reflex, compared to vehicle treatment, in the BCAO model rats (n = 10 animas/each treatment, one‐way ANOVA F (6.63) = 13.96, p < 0.001, followed by post hoc Dunnett's test, ††p = 0.003, †††p < 0.001, and ‡‡‡p < 0.001).

To validate and evaluate the efficacy of TRPV1 agonism to intervene dysphagic symptom mitigations, either capsaicin or ST‐6631 was perfused to the pharynx of these BCAO model rats. Capsaicin and ST‐6631 both increased the counts of swallowing reflexes at concentrations perfused (1, 10 and 100 μM), compared to the vehicle treatment on BCAO model rats (n = 10 animals/each treatment, one‐way ANOVA F (6.63) = 13.96, p < 0.001, followed by post hoc Dunnett's test, Figure 2). The both compounds successfully enhanced the swallowing function in rat models of stroke‐related dysphagia, and ST‐6631 appeared to cause swallowing improvement in a dose‐dependent manner. Hence, it was strongly suggested that ST‐6631 would possess novel therapeutic potentials to improve dysphagic comorbidities, in human patients who experienced and suffered from stroke.

3.3. ST‐6631 Was Rapidly Metabolized by the Human Hepatic S9 Enzymatic Fractions

The metabolic stability test of ST‐6631 (30 nM), incubated with the enzymatic mixture of human liver S9 fractions (2.0 μg/mL), resulted in that approximately 85% of ST‐6631 was metabolized rapidly within 5 min (Figure 3). The efficient metabolic breakdown of ST‐6631 by the human hepatic S9 enzymatic fractions suggested a faster clearance of the compound from systemic circulation in the case of in vivo administration.

FIGURE 3.

Rapid metabolic breakdown of ST‐6631 by the human liver S9 enzymatic fractions. ST‐6631 (30 nM), incubated with the human liver S9 enzymatic fractions (2.0 μg/mL) at 37°C, was rapidly metabolized, approximately 85% broken down as fast as 5 min from the beginning of enzymatic reaction.

3.4. ST‐6631 Exhibited Very Low Systemic Availability, Following the Oral Administration to Rats, to Support Its Applicational Usage as an Antedrug

Given the rapid breakdown of ST‐6631 by the hepatic S9 metabolic enzymatic mixtures, the systemic bioavailability of ST‐6631 was considered next. Naive rats were orally administered the compound at a single dose of 20 mg/kg, and blood samples were collected at designated post‐dose time points. In analysis, ST‐6631 and its hydrolyzed metabolite ST‐6631‐M1 were compared in their plasma concentrations. The peak ST‐6631 concentration was sampled at 15 min post‐dose, however only reaching a level of 0.09 ng/mL. On the other hand, the plasma concentration of hydrolyzed metabolite ST‐6631‐M1 was substantially higher at the same 15 min post‐dose, reaching a level of 40.1 ng/mL (445‐fold, Figure 4). Since the test was performed as a part of early‐stage toxicological evaluation, a relatively high dose of ST‐6631 was administered. It was expected that ST‐6631 was hydrolyzed immediately after the oral administration, for example, by carboxylesterases present in saliva and mucus of the gastrointestinal tract, which gave rise to an immediate but variable accumulation of the inactive metabolite ST‐6631‐M1 as early as 15 min post‐dose. This pharmacokinetic profile of ST‐6631 indicated the compound, being rapidly hydrolyzed, showed extremely low availability in the systemic blood circulation. Importantly, this low systemic bioavailability of ST‐6631 would lead to support a plausible advantage that the compound would be developed as a promising antedrug candidate. Since ST‐6631 has been designed as a novel TRPV1 agonist, a potential antedrug capability will favor minimizing risks of systemic compound exposure and subsequent adverse concerns associated with TRPV1 agonism.

FIGURE 4.

Plasma concentrations of ST‐6631 and its hydrolyzed metabolite ST‐6631‐M1, following the oral administration in vivo to rats. To assess the systemic bioavailability of ST‐6631 administered orally (20 mg/kg) to rats, plasma concentrations of ST‐6631 and its hydrolyzed metabolite ST‐6631‐M1 were analyzed at designated post‐dose time points. ST‐6631 reached its peak concentration (0.09 ng/mL) at a post‐dose point of 15 min; meanwhile, ST‐6631‐M1 also reached its highest concentration (40.1 ng/mL) at the same time point. Such rapid metabolism and very little systemic bioavailability of ST‐6631 supported strongly an argument that the compound would pharmaceutically be advantageous and developed as an antedrug candidate.

3.5. ST‐6631 Did Not Elicit Pungency, Following the Eye Exposure in Behaving Rats

Since ST‐6631 has been developed as a novel TRPV1 agonist, potential safety and tolerable properties, such as compound pungency, must be evaluated and addressed, in particular contrast to the reference compound capsaicin. Rats were presented with the instillation of test compound solution, applied to the cornea of the right eye. Strong nocifensive, eye‐wiping behavior was counted, and the counts appeared to increase in response to the eye instillation of capsaicin, up to a dose of 100 μM, compared to the vehicle instillation (n = 5 animals/treatment, Kruskal–Wallis test p < 0.001, followed by post hoc Dun's test, p = 0.052, Figure 5). In contrast, the nocifensive eye‐wiping behavior was hardly observed after ST‐6631 instillation, even at concentrations elevated up to 300 μM. This finding would lead to the safety property of ST‐6631, to indicate the compound does not elicit painful, burning, or pungent sensations, unlike other reference agonists known to cause adverse behavioral responses of nociception, triggered upon TRPV1 activation in awake animals.

FIGURE 5.

ST‐6631 was sensory perceived as minimally pungent, in contrast to the noxious reference of capsaicin, demonstrated with the nocifensive eye‐wiping behavior from observed rats. Immediately after the instillation of 10 μL test compound solution, into the right eye of awake, behaving rats, the nocifensive eye‐wiping behavior was observed for 1 min. Capsaicin instillation (100 μM) appeared to increase the counts of eye‐wiping responses, compared to vehicle treatment (n = 5/each treatment, Kruskal–Wallis test p < 0.001, followed by post hoc Dun's test, p = 0.052). On the contrary, ST‐6631 instillation did not cause the nocifensive eye‐wiping responses, even with its concentrations elevated up to 300 μM.

3.6. ST‐6631 Was Not an Irritant Chemical at a Clinically Intended Application Route on the Oral Mucosa

ST‐6631 was topically applied at a concentration of 0 (control) or 30 μM on the mucosa of the rat oral cavity. The irritancy score assessed under the CTFA guidelines and macroscopic tissue observation was noted as ‘0’. Hence, ST‐6631 was classified to fall into the lowest chemical category of ‘very mild’. In addition, no histopathological alterations such as necrotic damages were observed at all at mucosal parts of the application site (Figure 6a,b). On the other hand, a positive irritant control of SDS application resulted in the irritancy score of ‘1.5’, being classified into the ‘moderate irritant’ chemical category (Figure 6c).

FIGURE 6.

ST‐6631 showed no oral mucosal irritancy. Representative gross images (top) and hematoxylin–eosin (H&E)–stained sections (bottom) of rat oral mucosa after topical application. (a) Vehicle control: And (b) ST‐6631 (30 μM): No gross abnormalities and no histopathological changes at the application site. (c) Positive irritant control (sodium dodecyl sulfate, SDS): Gross alterations with corresponding histopathological changes to show epithelial necrosis (arrows). Scale bar (H&E), 100 μm.

3.7. ST‐6631 Induced the Slow and Mild Depolarization, Whereas Capsaicin Induced the Rapid Depolarization With Robust Action Potentials Evoked on Rat Sensory Neurons of the Primary DRG Culture

To investigate possible physiological mechanisms that ST‐6631 appeared less pungent and irritable, in contrast to the noxious reference of capsaicin, whole‐cell patch‐clamp recordings were performed on rat sensory neurons of the primary DRG culture. Brief bath application of capsaicin (50 μM, n = 14 cells) induced a strong depolarizing response of the membrane voltage, which even led to the evocation of robust action potentials (Figure 7a). On the other hand, the application of ST‐6631 (50–100 μM, n = 9 cells) induced a depolarizing membrane voltage response; however, with somewhat slower and milder characteristics. The ST‐6631 induced slow depolarizing response was minimally associated with the following evocation of action potentials (Figure 7b). The finding highlighted a clear contrast between capsaicin and ST‐6631 in the membrane voltage responses of rat sensory DRG neurons. The collectively milder potency of ST‐6631 on rat sensory DRG neurons might be suggested as one of the characteristic properties that relate to the compound's less pungency and irritancy, which was substantially different from the noxious counterpart capsaicin.

FIGURE 7.

Capsaicin caused a rapid depolarizing membrane response, accompanied with the evocation of robust action potentials, whereas ST‐6631 caused a modest form of depolarizing response, in sensory neurons of the rat DRG primary culture. The rat DRG primary cultured sensory neurons were exposed to either capsaicin (50 μM, n = 14 cells) or ST‐6631 (50–100 μM, n = 9 cells). Changes in the membrane voltage, whether there were apparent depolarizing responses, accompanied with or without the evocation of action potential firing, were assessed to categorize types of neuronal responses from the recorded cells a and b. A representative trace (left) and the types of neuronal responses (right) due to the capsaicin and ST‐6631 exposure, respectively.

3.8. ST‐6631 Elicited the Slower but Long‐Lasting TRPV1 Activation Current, Characteristically Differentiated From the Channel Activation Current Elicited by Capsaicin

To wrap up the physiological investigation that potentially explained the less pungent and irritable compound property of ST‐6631, ionic current waveforms depicted through the activation of the TRPV1 channel were analyzed. In the system of syncropatch automated voltage‐clamp, capsaicin or ST‐6631 (1 μM) was bath applied to the TRPV1‐expressing CHO cells (n = 53 or 46 cells, respectively), and the resultant channel activation waveforms were compared and contrasted through a number of measured parameters. The capsaicin exposure elicited the TRPV1 activation current, described with a fast and strong peak amplitude, followed by a rapid decay characteristic, indicative of the common channel desensitization (Figure 8a). On the contrary, the ST‐6631 exposure elicited the channel activation current, described with somewhat slower, mild but long‐lasting characteristics (Figure 8a). The measured parameters supported the clear comparison of TRPV1 activation current waveforms induced by capsaicin and ST‐6631 (Figure 8b, two‐sample independent t‐test). The peak current amplitude was reached faster (***p < 0.001) and stronger (*p = 0.014) with capsaicin. However, the time to take 50% decay current amplitude was substantially slower with ST‐6631 (***p < 0.001), resulting in the enhancement of measured current AUC (*p = 0.040). Therefore, these differential characteristics of TRPV1 activation waveforms, depicted by capsaicin or ST‐6631, altogether suggested a novel machinery of TRPV1 channel agonism. ST‐6631 would serve yet to understand channel activation kinetics or machinery, where somewhat delayed opening of the TRPV1 channel subsequently stabilized conformational dynamics in favor of slowing down the channel desensitization. Such slower but long‐lasting TRPV1 agonism might underlie in part the balanced pharmacology of ST‐6631, for example, to play modulatory excitation over sensory DRG neurons. ST‐6631 would modestly facilitate sensory nerve sensitivity while preventing robust nerve firings that the noxious counterpart capsaicin would otherwise evoke too fast. These proposed contrasts gained from electrophysiology might open an interesting discussion on how the pungency and irritancy of these two compounds had been perceived so differently in behaving animals.

FIGURE 8.

ST‐6631 elicited an active current on TRPV1‐expressing CHO cells, which was characterized with slower, milder but long‐lasting waveforms, differentiated from activation waveforms depicted with capsaicin. (a) Representative traces of the TRPV1 activated current, in response to the exposure of capsaicin or ST‐6631 (1 μM) on the channel‐expressing CHO cells. (b) A number of measured parameters to differentiate the TRPV1 activation current waveforms, depicted either by capsaicin (n = 53 cells) or ST‐6631 (n = 49 cells), were shown: Time to Peak (sec), Peak Amplitude (pA), Time to take 50% Decay Amplitude (sec), and area under the curve (AUC, pA sec). These measured parameters all pointed towards the novel mechanistic TRPV1 agonistic machinery, served by ST‐6631 (two‐sample independent t‐test, *p < 0.05 and ***p < 0.001).

4. Discussion

The present study comprehensively evaluated the pharmaceutical potential and advantages of ST‐6631, a novel TRPV1 agonist and antedrug candidate with minimal pungency and irritability, demonstrated in both in vivo and in vitro findings. The key findings were as follows: (1) ST‐6631 improved the counts of swallowing reflexes in the stroke‐related dysphagia model of BCAO rats. (2) ST‐6631 was rapidly metabolized by the human hepatic S9 enzymatic fractions. Consequently, the compound exhibited minimal systemic bioavailability, supporting its development as an antedrug candidate. (3) ST‐6631 was found to have substantially reduced pungent and irritable sensory properties, distinctively compared to the natural TRPV1 agonist capsaicin, in administered, awake, and behaving animals. (4) The electrophysiological properties of ST‐6631 demonstrated that ST‐6631 did facilitate the excitation of sensory DRG neurons but in a much milder fashion, protecting those sensory neurons from the rapid evocation of robust firing responses. The milder sensory neuronal excitation observed was likely attributable to a unique mechanism of TRPV1 channel activation. The slower and long‐lasting TRPV1 activation current, characterized with ST‐6631, was a striking differentiation from the faster but rapidly decaying TRPV1 activation current characterized with the noxious reference counterpart of capsaicin.

It was so critical that ST‐6631 mitigated the stroke‐related dysphagic symptom, the impaired counts of swallowing reflexes, in the rat models of BCAO. Although the BCAO model rats had been reviewed as the animal models of stroke and subsequent neuropathological disorders such as cognitive dysfunctions [41, 42], the BCAO model rats did perform the impaired pharyngeal swallowing reflex in the present study. Since a pharmacological intervention to restore the function of swallowing reflexes in the BCAO model rats had been successfully reported in previous literature also [43], it became our main proposal that ST‐6631 would be anticipated enough as a prospective drug candidate, with its potency applied to the therapeutic management of stroke‐related dysphagia. Such therapeutic prospection of ST‐6631, assessed and developed from the preclinical examinations here, would also be in line with previous studies elsewhere that the activation of TRPV1 channel facilitated swallowing reflexes in other rodent models of dysphagia [28, 29, 30]. Furthermore, it must be mentioned with emphasis that the activation of TRPV1 channel in humans, including the topical usage of capsaicin, has been thoroughly considered as clinical application strategies in patients affected by dysphagia [31, 32, 33, 34, 35].

Further, to add to the efficacy profile of ST‐6631 obtained from the rat BCAO models of dysphagia, the compound has shown its advantageous antedrug profile over other TRPV1 channel agonists and related activators. ST‐6631 was metabolized so rapidly by the human hepatic S9 enzymatic fractions. Subsequently, to align the finding, ST‐6631 showed substantially low systemic blood availability as early as 15 min after the oral administration in rats. It needs to be pointed out that the pharmacokinetic measurement was so far performed on female rats, as a part of early‐stage toxicological evaluation of ST‐6631. To establish the overall toxicological profiles of ST‐6631, the authors acknowledge the necessity of further evaluation, which would include to find a dose of NOAEL (no observed adverse effect level). Nevertheless, the antedrug characteristics of ST‐6631 would supportively encourage better safety dose controls in case of the clinical administration for human patients in future trials. ST‐6631 would be considered unlikely to cause potential risks of systemic exposure and its adverse consequences, for instance, hypothermia, cardiovascular, and gastrointestinal concerns, all reported in association with TRPV1 channel activation [36, 37, 38].

Moreover, not only the antedrug profile of ST‐6631 but also its markedly reduced pungency and irritancy, as observed in behaving animals, would lead to ensuring the safer tolerability and acceptance in consideration of future clinical practices for human patients affected by dysphagia and related motility disorders. Indeed, the topical application of ST‐6631 to the eye and the oral mucosa in awake animals did not raise potential concerns about the safety or tolerability associated with TRPV1 agonisms. ST‐6631 was judged undoubtedly to fall into the lowest category of mild chemicals according to the authorized CTFA guidelines. These findings together emphasize the distinctive contrast and striking differentiation from the typical adverseness carried by capsaicin, which has been known to evoke strong nocifensive guarding behaviors, for example the eye‐wiping behavior in rodents [44, 45].

To wrap up the compound properties about least pungency and irritancy found with ST‐6631, the electrophysiological evaluation and comparison to the noxious counterpart capsaicin has been thoroughly considered. ST‐6631 was uniquely characterized, eliciting slow depolarizing membrane responses on sensory neurons of the rat primary cultured DRGs. However, such slow depolarization was hardly accompanied by the evocation of action potential firings. To define a clear electrophysiological contrast, capsaicin was characterized to elicit rapid depolarizing membrane responses, accompanied by the evocation of robust action potential firings. These strikingly contrasting phenomena about the depolarizing membrane response with or without fast evocation of action potentials might be developed into an open discussion, whether the typical noxiousness of capsaicin was conveyed through sensory nerves, in a fashion of generating repetitive firings, whereas ST‐6631 only modestly sensitized sensory nerves.

The modest depolarizing nature of ST‐6631 was further investigated by evaluating waveforms of the TRPV1 channel activation current recorded from the target expressing CHO cells. The critical essence was to reveal the ST‐6631 elicited TRPV1 agonistic current that was slow but long‐lasting, compared to the counter of capsaicin elicited TRPV1 current shown on the CHO cells. The ST‐6631 elicited TRPV1 current, characterized by considerably milder waveforms than the current elicited by capsaicin, has been perhaps proposed as one of the bottomline characteristics of ST‐6631 pharmacology described thus far. In other words, the slow but sustained agonistic TRPV1 channel activation by ST‐6631 would ensure the moderate enhancement of sensory nervous cells in function, whereas still the modest fashion of sensory nerve enhancement would prevent conversion and transmission of otherwise vigorous TRPV1 activation firings that may be related to the perceived pungency and irritancy in animals.

In fact, there have been studies to discuss a plausible relationship between TRPV1 agonism and noxiousness, with the channel activation kinetics and lipophilicity of ligand substances [46, 47]. The more lipophilic the ligand substances, the faster the kinetics of TRPV1 channel activation, which leads to substantial influx of calcium and other mixed cationic ions. These biological consequences would result in the rapid and robust excitation of TRPV1‐bearing sensory neurons, understood as reliable nerve signal conveyers. In the case of capsaicin, the vigorous fashion of neuronal excitement, such as rapid evocation of repetitive action potential firings, traveling through the sensory nervous system, may be collectively and informationally transformed in a possible relationship with the perceived pungency and irritancy, typically unavoidable of TRPV1 agonism in behaving animals. In contrast, the much milder activation of TRPV1 current by ST‐6631 would be considered in line with the modest form of neuronal depolarization, not to trigger repetitive action potentials. The moderate sensitization of sensory nerves would then facilitate functional enhancement of swallowing reflexes, yet there would not be a parallel observation to concern the perceived noxiousness in behaving animals.

Finally, it is worthwhile to address some pathophysiological aspects and associated pharmacological interventions, in the context of better clinical management of dysphagia. The key concept of importance has become the sensitization, enhancement and coordination of sensory and motor nerves to facilitate the function of swallowing reflexes in the pharynx through the esophagus [21, 22]. There have been attempted cases so far in clinical studies to utilize capsaicin and similar TRPV1 channel activators [23, 27, 31, 32, 33, 34] as well as the electrical stimulation of neuromuscular junctions of the esophagus [18]. These attempts therefore have created a promising caveat to develop pharmaceutical interventions. Indeed, the excitatory modulation of sensory and motor nerves that are responsible for swallowing function would provide a viable and practical opportunity to enhance the swallowing reflex during a course of dysphagia management in human patients. Here, ST‐6631, the novel TRPV1 agonist and antedrug candidate, has been thoroughly described and evaluated. It is still an early‐stage development; however, ST‐6631 has shown a desirable pharmacological potency of enhancing sensory nerve excitability, which is considered an underlying principle of facilitating swallowing function in animal models of dysphagia and perhaps in human patients too. At the same time, ST‐6631 has shown fundamentally advantageous product profiles over its noxious counterpart capsaicin, in that ST‐6631 is a novel synthetic compound, carrying the minimum pungency and irritancy and yet being rapidly metabolized to an inactive hydrolyzed form in animals. The authors have begun to consult key opinion leaders in academia and clinical research across the field of dysphagia treatment, asking them whether the compound would attract therapeutic interests and how good the compound would be elaborated as a pharmaceutical product. In fact, it has been often discussed that ST‐6631 would be manufactured in a format of orally dissolving film, to take a safety handle of topical application that also maximizes this ST‐6631 product achievement gained from the nature of least noxiousness and antedrugness. To summarize, ST‐6631 has represented a pharmaceutically desirable product profile by pursuing its TRPV1 agonistic efficacy while simultaneously minimizing its adverseness that the natural ligand capsaicin would otherwise evoke in animals and humans. This novel synthetic product of ST‐6631 must be welcomed to advance drug‐based therapeutic interventions and clinical care quality controls towards the management of dysphagia and related motility dysfunctions in future.

5. Conclusion

Here, the novel TRPV1 agonist ST‐6631 has been evaluated in aspects of both in vivo and in vitro pharmacoprofiles, to address its potential advantages over capsaicin and related compounds, discussed in a context of drug development and future healthcare management of dysphagia. ST‐6631 is a prospective antedrug candidate, with its therapeutic potency expected to enhance the function of swallowing reflexes in patients affected by stroke‐related dysphagia. The ST‐6631 antedrug potentials, characterized by its rapid metabolic breakdown and fast clearance from systemic blood circulation, ensure the safety of administration by reducing adverse considerations associated with TRPV1 agonism. In addition, the extremely minimized ST‐6631 perceived sensory pungency and irritancy further safeguard the tolerability and acceptance in patients, demonstrating the pharmaceutically desirable product profile, granted also with TRPV1 agonism. The present study represents the novelty of ST‐6631, to contribute to the advancement of future drug‐based therapeutic interventions and the clinical symptomatic management over dysphagia and other motility dysfunctions.

Author Contributions

Masanori Miyauchi: conceptualization, data curation, formal analysis, investigation, project administration, writing‐original draft. Hiroyuki Kitano: conceptualization, data curation, investigation, writing‐original and revised manuscript. Mami Kochi: conceptualization, data curation, formal analysis, investigation, writing‐original and revised manuscript. Itaru Natsutani: conceptualization, data curation, formal analysis, investigation, writing‐original and revised manuscript. Yuki Mizukami: data curation, investigation. Yasuyo Maezawa: data curation, formal analysis, investigation. Kozo Yoshida: conceptualization, investigation. Noboru Iwagaki: conceptualization, data curation, formal analysis, investigation, writing‐original and revised manuscript.

Conflicts of Interest

All authors of the present study are employees of Sumitomo Pharma Co. Ltd., Osaka, Japan.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.