TAS-303, a Novel Selective Norepinephrine Reuptake Inhibitor that Increases Urethral Pressure in Rats, Indicating Its Potential as a Therapeutic Agent for Stress Urinary Incontinence

Stress urinary incontinence (SUI) is characterized by involuntary leakage associated with exertion, effort, sneezing, coughing, or lifting. Duloxetine, a serotonin norepinephrine reuptake inhibitor, is approved for the treatment of patients with SUI in some European countries, but not in the United States. There is currently no globally approved pharmacological drug for the treatment of patients with SUI. Therefore, a new pharmacological treatment option is required. TAS-303 [4-piperidinyl 2,2-diphenyl-2-(propoxy-1,1,2,2,3,3,3-day7)acetate hydrochloride] is a novel small-molecule selective norepinephrine reuptake inhibitor that displays significant norepinephrine transporter (NET) inhibitory activity toward the serotonin or dopamine transporters. In this report, we describe the pharmacological properties of TAS-303 and its effects on urethral function, using preclinical in vitro and in vivo studies. Radioligand-binding studies showed that TAS-303 selectively and potently inhibited [3H]norepinephrine binding to the human NET. Oral administration of TAS-303 (3 mg/kg) significantly increased norepinephrine levels in the plasma, whereas it did not significantly affect epinephrine, dopamine, and serotonin levels. TAS-303 (0.3, 1, and 3 mg/kg) dose-dependently increased basal urethral pressure in normal rats and leak point pressure in vaginal distention rats, exhibiting a maximal effect comparable to duloxetine. In the forced swimming test, TAS-303 (100 mg/kg) showed no significant effects on immobility time in rats, raising the possibility that this agent would have minimal central nervous system side effects at an effective dose for urethral function. These results demonstrate that TAS-303 has therapeutic potential for the treatment of patients with SUI.


Introduction
Urinary incontinence (UI) is defined as an involuntary leakage of urine and may cause adverse effects on social interactions and psychologic health (Sarkar and Ritch, 2000). UI can be classified into the following three groups: urge UI, stress UI (SUI), and mixed UI. Urge UI is characterized by a sudden desire to void with involuntary voiding and leakage of significant amounts of urine, and is frequently caused by an overactive bladder (OAB). Several pharmacologic treatment options, such as muscarinic M 3 receptor antagonists and b 3 -adrenoreceptor agonists, are available for OAB. These are recognized to be effective in the improvement of OAB symptoms and to have a good safety profile (Maman et al., 2014). SUI is characterized by involuntary leakage on effort or exertion, or on sneezing or coughing (Abrams et al., 2002). SUI commonly occurs in adults, particularly woman. It has been reported that the prevalence of SUI is 21.5%-38.2% in woman older than 20 years and 52.5% for those older than 50 years (Hannestad et al., 2000;Minassian et al., 2008;Coyne et al., 2012). Previously, some SUI studies have attempted to assess the impact of incontinence on quality of life. In these studies, it was demonstrated that woman suffering from severe SUI hesitated to go outdoors (Grimby et al., 1993;Swithinbank and Abrams, 1999). Although SUI is not considered a life-threatening disease, its impact on patients' quality of life is often devastating and results in social isolation for many. The main cause of SUI is thought to be the damage of muscles and nerves near the urethral sphincter during pregnancy and delivery, leading to the impairment of urethral resistance. In this context, the basic concept of SUI treatment is to increase urethral resistance. Current treatment options for SUI are as follows: surgical therapy, physical therapy, and pharmacological therapy. Surgical therapy is usually conducted for patients with moderate to severe SUI. A review of the literature demonstrated that in women 65 years or younger who underwent mid-urethral sling procedures, SUI cure rates ranged from 73% to 95% (Gerten et al., 2008). However, surgical intervention is invasive and has been associated with vaginal erosion, infection, and de novo urge symptoms (Gerten et al., 2008). In physical therapy, https://doi.org/10. 1124/jpet.118.248039. s This article has supplemental material available at jpet.aspetjournals.org.
In pharmacological therapy, duloxetine, a serotonin (5hydroxytryptamine: 5-HT) and norepinephrine (NE) reuptake inhibitor (SNRI) has demonstrated clinical efficacy for patients with SUI (Norton et al., 2002;Dmochowski et al., 2003). Duloxetine is reported to act in both the central nervous system (CNS) and the peripheral nervous system (PNS) (Thor, 2003;Miyazato et al., 2008). In the CNS, it was shown that duloxetine enhanced noradrenergic and serotonergic excitatory control of external urethral sphincter (EUS) spinal motoneurons and evoked contractions of the EUS. In the PNS, duloxetine increased NE levels at sympathetic postganglionic nerve terminals, leading to the activation of peripheral adrenergic a 1 receptors in urethral smooth muscles. These actions are therefore considered to be the underlying mechanism responsible for its efficacy in women with SUI. However, duloxetine is labeled with a "black box warning" about the increased risk of suicide (Friedman and Leon, 2007). It has been reported that the serotonin system is associated with the onset of the above-mentioned side effect (Oelke et al., 2006;Mann, 2013). In other words, the inhibition of serotonin transporter (SERT) by duloxetine may be involved in the increased risk of suicide. Consequently, duloxetine is not approved for the treatment of SUI in the United States. Because there is no globally approved drug for the treatment of patients with SUI, a new pharmacological treatment option is required. For these reasons, a selective NE reuptake inhibitor (NRI) is a potential therapeutic drug for patients with SUI that lack the side effects associated with SERT inhibition. We created the novel selective NRI TAS-303 hydrochloride [4-piperidinyl 2,2-diphenyl-2-(propoxy-1,1,2,2,3,3,3-day 7 )acetate hydrochloride]. In this study, we report that TAS-303 selectively inhibited NE transporter (NET) over SERT or dopamine transporter (DAT) and also inhibited the uptake of [ 3 H]norepinephrine in vitro. We also demonstrate that TAS-303 exhibited a dose-dependent increase in basal urethral pressure in normal rats, and in leak point pressure (LPP) in vaginal distention (VD) rats in vivo.

Materials and Methods
Ethics Statement. All animal experiments were executed according to the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health and approved by the local Committee of Animal Use and Care of Taiho Pharmaceutical Co. Ltd. (Tsukuba, Japan). Female Sprague-Dawley rats at 10-13 weeks of age (Charles River Laboratories Japan, Inc., Kanagawa, Japan) were used for this study and housed in a controlled environment (24 6 1°C, 12-hour light/dark cycle) with free access to food and water.
Transporter Binding. Dilutions of each test compound were made for concentrations ranging from 3 to 3000 nM for TAS-303, and 0.3 to 300 nM for duloxetine. Each assay was performed according to the protocols of Eurofins Pharma Discovery Services Taiwan Ltd. (Taipei, Taiwan). Briefly, Madin-Darby canine kidney cells expressing human NET were used for binding assays in assay buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM leupeptin, 10 mM phenylmethane sulfonyl fluoride). A 40-mg aliquot of membranes was incubated with 0.2 nM [ 125 I]RTI-55 (NEX-272; PerkinElmer Inc., MA, USA) for 3 hours at 4°C. Nonspecific binding was estimated in the presence of the tricyclic antidepressant (TCA) desipramine at 10 mM. Membranes were filtered and washed three times, and the filters were then counted to determine the specific binding of [ 125 I]RTI-55. For binding to SERT, human embryonic kidney 293 cell membranes stably transfected with a plasmid encoding the human SERT were prepared in assay buffer using standard techniques. A 9-mg aliquot of membranes was incubated with 0.4 nM [ 3 H]paroxetine for 60 minutes at 25°C. Nonspecific binding was estimated in the presence of the TCA imipramine at 10 mM. Membranes were filtered and washed three times, and the filters were counted to determine [ 3 H]paroxetine-specific binding. DAT binding assays were conducted using Chinese hamster ovary-S cells expressing human DAT in assay buffer. A 40-mg aliquot of membranes was incubated with 0.15 nM [ 125 I]RTI-55 for 3 hours at 4°C. Nonspecific binding was estimated in the presence of the dopamine reuptake inhibitor nomifensine at 10 mM. Membranes were filtered and washed three times, and the filters were then counted to determine [ 125 I]RTI-55-specific binding. Competition studies were carried out to determine the potencies of unlabeled compounds to displace [ 125 I]RTI-55, [ 3 H]paroxetine, or [ 125 I]RTI-55 for NET, SERT, or DAT binding to membranes, respectively. All assays were performed in triplicate.
Monoamine Uptake. Each assay was performed according to the protocols of Eurofins Pharma Discovery Services Taiwan Ltd. Briefly, NE uptake was assessed in Madin-Darby Canine Kidney cells expressing human recombinant NET that were plated overnight. Test compounds (TAS-303, 3-3000 nM; duloxetine, 0.3-300 nM) were preincubated with cells (2 Â 10 5 cells/ml) in assay buffer (5 mM Tris-HCl, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl 2 , 1.2 mM MgSO 4 , 5 mM D-glucose, 1 mM ascorbic acid, pH 7.1) for 20 minutes at 25°C; 25 nM [ 3 H]norepinephrine was then added and incubated for an additional 15 minutes. After the assay buffer was removed, the cells were washed in assay buffer to remove free [ 3 H]norepinephrine. A lysate was obtained from the solubilized cells and counted to determine [ 3 H]norepinephrine uptake. Reduction of [ 3 H]norepinephrine uptake by $50%, relative to 10 mM desipramine, indicated significant inhibitory activity. Serotonin uptake was assessed in human embryonic kidney 293 cells expressing human recombinant SERT that were plated overnight. Test compounds were preincubated with cells (1 Â 10 5 cells/ml) in assay buffer for 20 minutes at 25°C; 65 nM [ 3 H]serotonin was then added and incubated for an additional 15 minutes. Intracellular [ 3 H]serotonin is trapped on the filters. The filters were then rinsed with assay buffer and counted to determine [ 3 H]serotonin uptake. Reduction of [ 3 H]serotonin uptake by $50%, relative to 1 mM fluoxetine, indicated significant inhibitory TAS-303, a Novel NRI, Increases Urethral Pressure in Rats activity. Dopamine uptake was assessed in Chinese hamster ovary-S/human DAT clone 28 cells expressing human recombinant DAT that were plated overnight. Test compounds were preincubated with cells (4 Â 10 4 cells/ml) in assay buffer for 20 minutes at 25°C; 50 nM [ 3 H]dopamine was then added and incubated for an additional 10 minutes. After the assay buffer was removed, the cells were washed in assay buffer to remove free [ 3 H]dopamine. A lysate was obtained from the solubilized cells and counted to determine [ 3 H]dopamine uptake. Reduction of [ 3 H]dopamine uptake by $50%, relative to 10 mM nomifensine, indicated significant inhibitory activity. If significant inhibition of uptake was observed, the possibility of compound-induced cytotoxicity was evaluated by applying the same concentrations of compounds described above to a separate group of untreated cells.
Measurement of Isolated Urethral Contractions. Female Sprague-Dawley rats 11-13 weeks of age were euthanized by decapitation, and the urethra was isolated. The proximal urethra was cut open and then cut in the traverse direction. Specimens 1.5 mm in width and 2 mm in length were prepared. The prepared specimens were transferred to a 37°C organ bath (10 ml) filled with Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl 2 , 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , 1.2 mM MgSO 4 , 10 mM glucose) saturated with 95% O 2 -5% CO 2 gas. One end of the specimen was connected to an FD pickup transducer (TB-612T; Nihon Kohden Co., Ltd., Tokyo, Japan) and the other end was fixed to the organ bath. Urethral tension was recorded and analyzed using a PowerLab 16/30 data acquisition system (ADInstruments Pty Ltd, Sydney, NSW, Australia) via an FD pickup transducer and strain pressure amplifier (AP-601G; Nihon Kohden Co., Ltd.). NE-induced urethral contractions were assessed in reference to the previously described method (Pérez-Martínez et al., 2011). A resting tension of 1.0 g was applied to the isolated urethras, and the specimens were equilibrated for approximately 40 minutes. Ultrapure water (100 ml) was added to the organ bath. Ten minutes later, 100 ml of a 100 mM NE solution was added to the vessel and NE-induced urethral contractions were measured. Urethral specimens were washed with Krebs-Henseleit solution, and recovery of tension to the original equilibration level was confirmed 40 minutes after the previous measurement of NE-induced contractions. Then, 100 ml of ultrapure water was added to the organ bath. The final concentrations of NE in the organ bath were 1 nM to 1 mM. This procedure was repeated until the urethral specimen stabilized, and the data obtained after stabilization were used as the control data. After measurement of the control data, the urethral specimen was washed with Krebs-Henseleit solution. Recovery of resting tension to the original equilibration level was confirmed 50 minutes after previous measurement of NE-induced contractions. A series of solutions (five concentrations) of each test substance was assessed from low concentration to high concentration using the same specimen. The NE (1 mM)-induced maximum contraction when examined with vehicle alone (ultrapure water) prior to the evaluation of test substances because control data were designated as 100% contraction, and the urethral contractions for each test solution were expressed as relative values (%). The EC 50 value for NE was obtained from a sigmoidal curve under variable maximum effect conditions.
Determination of TAS-303 Levels in Rat Plasma. A single dose of TAS-303 (1, 3, or 10 mg/kg) was administered by gavage. Blood samples were collected from the jugular vein into heparinized tubes at 0, 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. These samples were centrifuged at 4°C, and the supernatants were collected as plasma. Plasma concentrations of TAS-303 were determined using a high-performance liquid chromatographic method combined with tandem mass spectrometry.
Measurement of Monoamine Levels in Plasma. Female Sprague-Dawley rats (10 weeks of age) were divided into a control group (N 5 10) and a TAS-303 group (N 5 9). Rats were treated once daily with distilled water or TAS-303 (3 mg/kg) orally. Rats were anesthetized by intraperitoneal injection of 1.2 g/kg urethane 30 minutes after the final administration. Blood samples were collected from the inferior vena cava into tubes containing EDTA as the anticoagulant. A portion of the blood was kept at 270°C for serotonin analysis. The remaining blood was centrifuged at 4°C, and the supernatant was collected as plasma. The plasma samples were stored in a freezer at 270°C for the analysis of epinephrine, norepinephrine, and dopamine. Monoamine levels were measured by BML Inc. (Tokyo, Japan).
Measurement of Urethral Pressure. Female Sprague-Dawley rats at 11 weeks of age were anesthetized by intraperitoneal injection of 1.2 g/kg urethane. After confirming the loss of extremity reflex, an abdominal median incision was performed. Both ureters were ligated with a 4-0 silk ligature, and the ureter was cut at the ligated position near to the kidney. Then, the urethra was ligated tightly with a 4-0 silk ligature. The dome of the bladder was cut open and a catheter (PE-50; Nippon Becton, Dickinson and Company, Ltd., Tokyo, Japan) was placed within, and the opening of the bladder was then sutured with a purse string suture. A three-way stopcock was attached to the other end of the catheter, and the stream was divided into two paths. One was connected to a pressure transducer (DX-360; Nihon Kohden Co., Ltd.), and the other was connected to a syringe filed with physiologic saline and connected to a continuous infusion device (KDS200; KD Scientific Inc., Holliston, MA). An additional catheter was inserted into the urethra from the external urethral orifice. A three-way stopcock was attached to the outer end of this catheter, and the stream was divided into two paths. One was connected to a pressure transducer, and the other was connected to a syringe, as described above. After catheterization, physiologic saline was infused into the bladder at a rate of 10 ml/h via a microsyringe pump connected to the bladder on a heating board (PS-53; Sakura Finetek Japan Co., Tokyo, Japan). The infusion was stopped when rhythmic contractions of the bladder were confirmed. Physiologic saline was then infused into the urethra at a rate of 3 ml/h, and the inner pressure of the bladder and urethral pressure were measured with a pressure amplifier (AP-641G; Nihon Kohden Co., Ltd.) connected to a pressure transducer, and recorded using the PowerLab 16/30 data acquisition system. TAS-303 at 0.3, 1, or 3 mg/kg or vehicle (ultrapure water) was administered by gavage to rats 30 minutes prior to urethane anesthesia. For evaluation of duloxetine (1 mg/kg), rats were subjected to the operation described above and treated intravenously after equilibration of intravesical pressure and intraurethral pressure. Urethral pressure was analyzed by its waveform using Chart version 5.2.2 (ADInstruments Pty Ltd.). The primary evaluation point was set at the urethral baseline pressure, and the effects of duloxetine were assessed relative to the baseline values. After oral treatment, individual urethral baseline pressure was calculated as an average pressure during a period of 30 minutes from 1 hour after administration. For intravenous administration, individual urethral baseline pressure was calculated as an average pressure during the 30-minute period after administration.
Measurement of LPP. Female Sprague-Dawley rats 11 weeks of age were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital. After confirming the loss of body reflex, a balloon catheter (JU-LB1005; Terumo Corporation, Tokyo, Japan) was inserted into the vagina. A 4-ml aliquot of physiologic saline was injected into the balloon to expand the catheter. The rats were then placed on a heating board warmed to 37°C for 3 hours to maintain VD. Next, the distention was stopped, and the rats were returned to normal housing conditions with free access to food and water. LPP was assessed 4 days after VD model preparation. Rats were anesthetized by intraperitoneal injection of 1.0-1.2 g/kg urethane. Rats were placed in a prone position, and the spinal cord was completely severed between thoracic vertebrae 9 and 10. The rats were then inverted to a supine position, and an abdominal median incision was performed. A vinyl-tube (SV45; Natsume Seisakusho Co., Ltd., Tokyo, Japan) was then inserted and indwelled in the bladder from the dome of bladder. A three-way stopcock was attached to the other end of the catheter; one path was connected to a syringe filed with physiologic saline, and the other was connected to a pressure transducer. Next, the bladder was emptied and 200-350 ml of physiologic saline was injected into the 324 bladder. After the instillation of saline, a gentle slow manual pressure increase was applied to the abdomen of the rat by a trained investigator until urethral leakage occurred, stimulating a mild Credé's maneuver, as previously described (Woo et al., 2009). Intravesical pressure at the occurrence time of urine leakage was designated as LPP. Intravesical pressure was measured by a pressure amplifier connected to a pressure transducer, and recorded and analyzed using PowerLab 16/30 data acquisition system. TAS-303 at 0.3, 1, or 3 mg/kg or vehicle (ultrapure water) was orally administered to the rats 30 minutes prior to urethane anesthesia, and the animals were subjected to surgery for LPP measurement as described above. LPP was measured 1 hour after dosing. For intravenous administration, pre-LPP values were determined prior to the administration of vehicle (physiologic saline) or 1 mg/kg duloxetine, and LPP was measured again 5 minutes after intravenous administration. The measurement of LPP was conducted in a blinded manner.
Forced Swimming Test. The forced swimming test (FST) was conducted according to the previously described method (Porsolt et al., 1978). A cylindrical acrylic tank (20 cm in diameter and 50 cm in height) was filled with water (at room temperature) to a depth of 25 cm. For acclimatization to the test system, 11-week-old female Sprague-Dawley rats were subjected to a forced swimming trial for 15 minutes 1 day prior to the administration of test substances. The immobility time of rats in the water during the 15-minute acclimatization trial was measured. Any rat showing an immobility time of less than 180 seconds during the 15-minute trial, indicating a lack of apparent depressive condition, was excluded from the study. Based on the immobility time in the trial, rats were categorized as follows. "Swimming" was designated as "the state when rats were moving both fore-and rear-extremities," and "the time of not swimming" was designated as "immobility time." The day after the swimming acclimatization trial, vehicle, TAS-303 (10, 30, 100 mg/kg), or imipramine (60 mg/kg) was orally administered to rats. One hour after dosing, individual rats were placed in the tank and the FST was conducted for 5 minutes. Immobility time was measured as described above. Evaluation of the results was conducted in a blinded manner.
Statistical Analysis. Results are reported as the mean 6 S.E.M. Statistical analysis was carried out using a Student's t test or Williams' test. A P value ,0.05 was considered to show statistical significance. All data analyses were performed using SAS version 9.2 and EXSUS version 8.0.0 (CAC Exicare Corporation, Tokyo, Japan).

Results
Affinity to Monoamine Transporters and Inhibition of Monoamine Uptake by TAS-303. The inhibitory potential of TAS-303 and duloxetine on ligand binding to monoamine transporters was assessed using human NET-, human SERT-, and human DAT-expressing cells (Table 1). TAS-303 selectively inhibited binding of the NE transporter ligand to membranes from cells transfected with human NET relative to the SERTs and DATs. Duloxetine selectively inhibited binding of NE and SERTs relative to the DAT. In functional cell-based assays, the inhibitory effects of TAS-303 and duloxetine on the uptake of NE, serotonin, and dopamine were also evaluated ( Fig. 2

; Table 2). TAS-303 selectively inhibited the cellular uptake of [ 3 H]norepinephrine relative to [ 3 H]serotonin and [ 3 H]dopamine. Duloxetine selectively inhibited the uptake of [ 3 H]norepinephrine and [ 3 H]serotonin relative to [ 3 H]dopamine.
Effect of TAS-303 on Norepinephrine-Induced Contractions of Rat Isolated Urethra. The effect of TAS-303 on NE-induced contractions of isolated urethral strips was assessed to confirm the efficacy of TAS-303. The reproducibility of consecutive NE-induced contraction-response curves on the same tissue was evaluated in the presence of the vehicle for TAS-303 and duloxetine (ultrapure water). The EC 50 values for the concentration-response curves for vehicle are shown in Table 3. Five consecutive vehicle treatments produced no significant change in EC 50 values through six trials ( Fig. 3; Table 3). Duloxetine shifted the concentration-response curve to the left. Significant differences were observed at concentrations of 10 nM or greater (P , 0.05 for 10 nM, P , 0.01 for more than 30 nM). TAS-303 also shifted the concentration-response curve to the left. A significant difference was observed at a concentration of 100 nM or greater (P , 0.01).
Plasma Monoamine Concentration. To determine the functional effects of TAS-303, rats were administered TAS-303 and its ability to increase plasma monoamines was evaluated (Fig. 4). Plasma norepinephrine levels were significantly higher in the TAS-303 group than in the vehicle group. In contrast, there were no differences in plasma epinephrine, dopamine, or serotonin levels between the two groups.
Effect of TAS-303 on Urethral Pressure in Rats. A previous study showed that intravenous injection of duloxetine (1 mg/kg) significantly increased the basal urethral pressure in rats (Kamo and Hashimoto, 2007). To assess the pharmacological potential of TAS-303 for regulating urethral resistance in vivo, the basal urethral pressure of rats was measured after oral administration (Fig. 5). Intravenous administration of physiologic saline induced no apparent changes in urethral baseline pressure (data not shown). Intravenous administration of duloxetine (1 mg/kg) significantly increased urethral baseline pressure by 15% compared with the vehicle group (P , 0.05). Oral administration of TAS-303 (0.3, 1, and 3 mg/kg) resulted in a dose-dependent increase in urethral pressure. At greater than 1 mg/kg dose of TAS-303, urethral baseline pressure significantly increased compared with the vehicle group (P , 0.05), and increased by 38% at a dose of 3 mg/kg. Effect of TAS-303 on LPP in VD Model Rats. SUI has been reported to mainly occur as a result of pregnancy and delivery. The VD model was selected because it is commonly used to simulate vaginal delivery, and imitates the urethral damage occurring after delivery (Lin et al., 1998;Cannon et al., 2002). A previous report (Miyazato et al., 2009) showed that intravenous injection of 1 mg/kg duloxetine significantly increased sneezeinduced LPP in VD rats. To investigate the effect of TAS-303 on urethral function in an SUI model, LPP in the rat VD model was measured after oral administration (Fig. 6). LPP was defined as the intravesical pressure at the occurrence of incontinence induced by abdominal pressure loading, and the experiment was conducted in a blinded manner. The mean LPP of VD rats was 32.5 6 1.6 cmH 2 O and was significantly lower than that of shamoperated rats (50.6 6 2.6 cmH 2 O, P , 0.01), indicating the successful establishment of the VD model. Intravenous administration of duloxetine (1 mg/kg) significantly increased LPP by 20% compared with the vehicle group (P , 0.01). Oral administration of TAS-303 (0.3, 1, and 3 mg/kg) resulted in a dose-dependent increase in LPP. At a greater than 1 mg/kg dose of TAS-303, LPP was significantly increased compared with the vehicle group (P , 0.05), and increased by 26% at a dose of 3 mg/kg.
Effect of TAS-303 on Blood Pressure and Heart Rate in Dogs. It has been reported that antidepressants are associated with the potential cardiovascular risks (Mago et al., 2014). Thus, the effects of TAS-303 on cardiovascular parameters in dogs were assessed to clarify whether TAS-303 would have the   Fig. 1). The dog is selected because this is one of the most generally used animal models for assessing the effects of drugs on blood pressure and heart rate (HR). Time course of the effects of a single oral administration of TAS-303 (1, 3, or 10 mg/kg) on systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and HR was evaluated in conscious dogs. Predrug values for SBP, DBP, and MBP in the vehicle-treated group were 137 6 4, 90 6 8, and 106 6 6 mm Hg, respectively, these values being similar to those in each TAS-303-treated group. Predrug value for HR in the vehicle-treated group was 79 6 6 beats/min, which was similar to that in each TAS-303-treated group. No significant difference between the TAS-303 group (at each dose) and the vehicle group was detected in the evaluation of SBP, DBP, MBP, and HR. Effect of TAS-303 on Immobility Time of Rats in the FST. The FST is one of the most frequently used animal models for assessing antidepressant-like effects of centrally acting antidepressants, such as selective serotonin reuptake inhibitors, SNRIs, and TCAs. It is well established that treatment with such drugs reduces the duration of immobility in the FST (Rénéric and Lucki, 1998;Guan et al., 2014). To clarify whether TAS-303 affects the CNS, the immobility time of rats in the FST was assessed. The effects of treatment with TAS-303 and the TCA imipramine on immobility time in the FST are shown in Fig. 7. Imipramine was selected as a positive control, as the effect of this agent on immobility time in the FST is well described (Guan et al., 2014). The immobility time in the imipramine group was significantly lower than that in the vehicle group (P , 0.01). In contrast, no significant difference between the TAS-303 group (at each dose) and the vehicle group was detected.

Discussion
In this article, we report on the pharmacological properties of the novel NRI inhibitor TAS-303. The pharmacological profile of TAS-303 (potency, selectivity, and in vivo efficacy) and its pharmacokinetic profile were evaluated. To further support the rationale for norepinephrine reuptake inhibition, we also examined the effect of TAS-303 on plasma norepinephrine levels in rats. Moreover, we estimated the antidepressant-like effect of TAS-303 using the FST. These studies characterize TAS-303 as an orally available, potent, and selective NRI with a lesser risk of CNS adverse events.
We investigated the direct effect of TAS-303 on monoaminergic and cholinergic receptors. The percentage of the agonistic activity of TAS-303 (10 mM) on the adrenergic a 1A receptor, a 1B receptor, or 5-HT 2c receptor was 24%, 4%, or 0%, respectively. The ligand-binding inhibition of TAS-303 (10 mM) on the adrenergic a 1D receptor, nicotinic acetylcholine receptor, or 5-HT 1A receptor was 7%, 12%, 7%, respectively (data not shown). Therefore, TAS-303 is considered to have almost no direct effect on the receptors associated with urethral smooth muscle and EUS contraction. Among the three monoamine transporters (NET, SERT, and DAT), TAS-303 selectively inhibited NET [inhibition constant (K i ), 50.9 nM] and also inhibited the uptake of [ 3 H]norepinephrine (IC 50 , 38.9 nM). Furthermore, the concentration-response curve for NE-induced urethral contractions was shifted to the left after treatment with TAS-303 in the urethral muscle strip experiment. The effect of TAS-303 on plasma monoamine levels was evaluated, and the elevation of norepinephrine level, but not the other monoamines tested, in the plasma was observed after administration of 3 mg/kg TAS-303. In the rat efficacy study, treatment with 3 mg/kg TAS-303 produced an increase in urethral pressure, where the C max of TAS-303 was above the IC 50 of the in vitro uptake assay (38.9 nM). We have confirmed that TAS-303 is a substrate for P-glycoprotein, indicating that this agent would have a low ability to penetrate the blood-brain barrier (data not shown). Moreover, we have confirmed that total 14 C radioactivity in the plasma for orally administered 14 C-labeled TAS-303 (3 mg/kg) peaked at 1 hour after administration using quantitative whole-body autoradiography, and no 14 C radioactivity was observed in the cerebrum, cerebellum, or spinal cord (data not shown). Therefore, TAS-303 is not likely to enter the CNS. These findings suggest that the peripheral elevation of NE levels induced by TAS-303 administration underlies the increase in urethral pressure in rats.
Previous animal studies have suggested that NE and serotonin are involved in the neural control of lower urinary tract function (Gajewski et al., 1984;Downie and Bialik, 1988;Thor et al., 1990;Danuser and Thor, 1996;Espey et al., 1998;Conlon et al., 2009). Miyazato et al. (2008 reported that duloxetine acted in both the CNS and the PNS and stated that duloxetine seems to be superior to the NRI nisoxetine in enhancing active urethral closure, possibly because of the additional activation of the serotonergic system by duloxetine. In contrast to duloxetine, it is suggested that TAS-303 peripherally inhibited NET without affecting NET in the CNS. Concern remains about the efficacy of TAS-303 for patients with SUI, because this agent has inhibitory activity against peripheral NET alone. To estimate the efficacy of TAS-303, we compared its effect to that of duloxetine on urethral resistance in vivo. In this study, TAS-303 significantly increased urethral pressure by 38% in normal rats. In the same assessment, the clinically approved agent duloxetine produced a 15% increase in urethral pressure. Furthermore, TAS-303, a Novel NRI, Increases Urethral Pressure in Rats TAS-303 significantly increased LPP by 26% in VD rats. In the same assessment, intravenous administration of duloxetine produced a 20% increase of LPP in VD rats. Therefore, TAS-303 increased the urethral pressure of normal rats and increased the LPP of VD rats to levels comparable to those with duloxetine. These results suggest that the inhibition of peripheral NET may be sufficient to increase urethral resistance. This suggestion is supported by a previous report (Klarskov et al., 2009), in which the selective NRI esreboxetine exhibited clinical efficacy in a US study of patients with SUI. In addition, (Fujimori et al., 2015) evaluated the in vivo potency of the peripheral-selective NRI compound 12 on normal rat LPP compared with esreboxetine. Compound 12 produced an elevation in LPP, and the maximum response was comparable to that for esreboxetine. Considering the negative CNS-penetrating property of compound 12, the increasing effect on urethral pressure in rats was suggested to be mediated mainly by the inhibition of peripheral NET. In this study, we selected 1 mg/kg as the dose of duloxetine, because this dose is often used to produce maximal effects on urethral pressure (Kamo and Hashimoto, 2007;Miyazato et al., 2015). It has been reported that the C max of duloxetine after intravenous administration of radiolabeled duloxetine (5 mg/kg) to rats was 14 mM (Application Brochure; Eli Lilly Japan K.K., Kobe, Japan), and this concentration is sufficiently higher than the C max (116 nM) after oral administration of the clinical dose (40 mg) in humans (Interview Form for Cymbalta, revised in 2017, Eli Lilly Japan K.K.). Thus, the plasma level of duloxetine after intravenous administration of 1 mg/kg would reach a value equivalent to or greater than the clinical dose. These findings indicate that TAS-303 has therapeutic potential as an SUI treatment. However, a limitation of this study is the difference in the administration route between TAS-303 and duloxetine, which did not allow adequate assessment of the effect of both drugs in the in vivo study. Accordingly, the efficacy of TAS-303 in patients with SUI needs to be elucidated in clinical trials. NE is well known to bind to adrenergic receptors in the blood vessels and myocardium, which caused increase in blood pressure and heart rate. Thus, NRIs are considered to have potential cardiovascular risks. However, previous reports (Tanum 2000;Versiani et al., 2000) have shown that in patients with depression receiving a selective NRI, reboxetine, there was a tendency toward orthostatic changes in blood pressure, but this was not clinically significant. Moreover, a review of the literature about the cardiovascular safety profile

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of duloxetine has demonstrated that the use of duloxetine does not appear to be associated with significant cardiovascular risks in patients with depression or diabetic neuropathic pain (Wernicke et al., 2007). Therefore, it is considered that NET inhibition would have little risk of clinically significant cardiovascular side effects. In this report, a single dose of TAS-303 (10 mg/kg) to dogs resulted in no significant change in blood pressure and heart rate, where the C max (431 nM; data not shown) of TAS-303 was above the C max (49 nM) in the measurement of rat urethral pressure. These findings suggested that NRIs have the selectivity for urethral efficacy over cardiovascular events. There is a need for future studies to elucidate the selectivity. It remains to be confirmed in clinical trials of TAS-303 whether increases in urethral pressure can be achieved in the absence of cardiovascular events with NET inhibition.
Antidepressants are required to be labeled with a black box warning about the increased risk of suicide (Friedman and Leon, 2007). It has been reported that the serotonin system is associated with the onset of the side effect mentioned above (Oelke et al., 2006;Mann, 2013). Consequently, duloxetine is not approved for the treatment of SUI in the United States. To assess the risk of suicide associated with TAS-303, the FST was conducted in rats. It is well established that treatment with centrally acting antidepressants, including SNRIs and TCAs, reduce the immobility time in the FST (Guan et al.,Fig. 5. Effects of duloxetine and TAS-303 on urethral baseline pressure in rats. (A) Vehicle or duloxetine (1 mg/kg) was administered intravenously, and the urethral baseline pressure was measured for 30 minutes after administration. (B) Vehicle or TAS-303 (0.3, 1, or 3 mg/kg) was administered orally, and the urethral baseline pressure was measured for a period of 30 minutes beginning 1 hour after administration. Results are shown as the mean 6 S.E.M. of five rats in each group. *P , 0.05 vs. vehicle (physiologic saline) by a paired Student's t test. #P , 0.05 vs. vehicle by Williams' test. TAS-303, a Novel NRI, Increases Urethral Pressure in Rats 2014). As described previously, the TCA imipramine (60 mg/kg) reduced the immobility time of rats in the FST. A previous study (Rénéric and Lucki, 1998) showed that subcutaneous administration of duloxetine at greater than 10 mg/kg significantly decreased the immobility time of rats in the FST. These findings suggest that duloxetine exhibits an antidepressant-like effect in the FST. Clinically, duloxetine is often prescribed for both major depressive disorder and SUI at a dose of 40 mg/d. Therefore, it is assumed that the anti-SUI effect and antidepressant effect are produced in the same dose range. In contrast to imipramine, TAS-303 had no effect on the immobility time of rats in the FST, even at a high dose (100 mg/kg). These findings suggest that TAS-303 presents little risk of central adverse effects at an effective dose for urethral function.
TAS-303 demonstrated selective NET inhibitory activity and produced a significant increase in urethral resistance in rats, with a maximum effect comparable to that of the clinically effective drug duloxetine, suggesting that this agent has a therapeutic potential for patients with SUI. Moreover, TAS-303 showed no effect on the immobility time of rats in the FST test, indicating that this agent would have a lesser risk of central adverse effects. Recently, a double-blind, single-dose, placebo-controlled crossover study of TAS-303 at a dose of 18 mg was conducted in 16 patients with SUI (Yono et al., 2017). There were no reports of serious adverse events, significant changes in blood pressure, or abnormal urinalysis results, suggesting that this agent was well tolerated. At present, a phase II randomized clinical study of TAS-303 in female patients with SUI is being conducted (NCT02906683). Quantification of LPP in sham or VD rats. (E) Vehicle or duloxetine (1 mg/kg) was administered intravenously, and LPP was measured at 5 minutes after administration. (F) Vehicle or TAS-303 (0.3, 1, or 3 mg/kg) was administered orally, and LPP was measured at 1 hour after administration. Results are shown as the mean 6 S.E.M. of 10 rats in each group. **P , 0.01 vs. vehicle (physiologic saline) by a paired Student's t test. #P , 0.05 vs. vehicle by Williams' test.