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Under healthy condition, more than one urethra-closing reflex, including both bladder afferent-independent and -dependent actions function during momentary elevation of intravesical (bladder) pressure to prevent urinary incontinence. In the current study, the effects of a novel selective 5-hydroxytryptamine type 2C (5-HT2C) receptor agonist, TAK-233, on evoked momentary urethra-closing functions were investigated in female rats and humans to elucidate 5-HT2C receptor functions. In anesthetized female rats, TAK-233 dose-dependently and significantly increased the urethral resistance during sneezing in rats with distended vaginas and bilaterally transected pelvic nerves. The drug also dose-dependently and significantly increased urethral resistance during momentary intravesical pressure elevation by electrical stimulation of abdominal muscles in rats with a transected spinal cord at the T8-9 level and intact pelvic nerves. The increased effects observed during electrical stimulation were abolished by either an intravenously administered selective 5-HT2C receptor antagonist, SB 242084, or bilateral transection of the pelvic nerves or somatic nerves innervating the external urethral sphincter and pelvic floor muscles. In the spinal cord-transected and pelvic nerve-intact rats, TAK-233 enlarged the urethra-closing responses induced by both passive and abrupt intravesical pressure elevation measured by a micro-tip transducer located in the middle urethra. Additionally, the effects of TAK-233 on the stimulus threshold of urethral contractile responses induced by transcranial magnetic stimulation were investigated in healthy female volunteers. The drug dose-dependently and significantly lowered this stimulus threshold, indicating an increased sensitivity of the response. These results demonstrate that 5-HT2C receptor stimulation enhances the evoked momentary urethra-closing functions in both female rats and humans. Significance Statement 5-hydroxytryptamine (serotonin) type 2C (5-HT2C) receptor stimulation by TAK-233 enhanced urethral resistance in rats during an evoked momentary event in which the bladder afferent-independent or -dependent reflex functions via striated muscle-mediated mechanisms. The increases in sensitivity of transcranial magnetic stimulation-evoked urethral contractile responses in healthy female subjects indicates that this mechanism also functions in humans. The evoked momentary conditions activating these reflexes provide a suitable model to demonstrate the exerting effects of 5-HT2C receptor stimulation.

The voltage-gated sodium channel, Nav1.1, is predominantly expressed in parvalbumin-positive fast spiking interneurons and has been genetically linked to Dravet syndrome. Starting from a high throughput screening hit isoxazole derivative 5, modifications of 5 via combinations of IonWorks and Q-patch assays successfully identified the nicotinamide derivative 4. Its increasing decay time constant (tau) of Nav1.1 currents at 0.03 μM along with significant selectivity against Nav1.2, Nav1.5, and Nav1.6 and acceptable brain exposure in mice was observed. Compound 4 is a promising Nav1.1 activator that can be used to analyze pathophysiological functions of the Nav1.1 channel towards treating various central nervous system diseases.

Drug induced kidney injury (DIKI) is a common reason for compound attrition in drug development pipelines with proximal tubule epithelial cells (PTECs) most commonly associated with DIKI. Here, we investigated freshly isolated human (hPTECs) as an in vitro model for assessing renal tubular toxicity. The freshly isolated hPTECs were first characterized to confirm gene expression of important renal transporters involved in drug handling which was further corroborated by confirming the functional activity of organic cation transporter 2 and organic anion transporter 1 by using transporter specific inhibitors. Additionally, functionality of megalin/cubilin endocytic receptors was also confirmed. A training set of 36 compounds was used to test the ability of the model to classify them using six different endpoints which included three biomarkers (Kidney Injury Molecule-1, Neutrophil gelatinase-associated lipocalin, and Clusterin) and three non-specific injury endpoints (ATP depletion, LDH leakage, and barrier permeability via transepithelial electrical resistance) in a dose-dependent manner across two independent kidney donors. In general, biomarkers showed higher predictivity than non-specific endpoints, with Clusterin showing the highest predictivity (Sensitivity/Specificity - 65.0/93.8 %). By using the thresholds generated from the training set, nine candidate internal Takeda compounds were screened where PTEC toxicity was identified as one of the findings in preclinical animal studies. The model correctly classified four of six true positives and two of three true negatives, showing validation of the in vitro model for detection of tubular toxicants. This work thus shows the potential application of freshly isolated primary hPTECs using translational biomarkers in assessment of tubular toxicity within the drug discovery pipeline.

With the aim to discover a gastric antisecretory agent more potent than the existing proton pump inhibitors, novel 3,4-dihydro-1H-spiro(naphthalene-2,2'-piperidin)-1-one derivatives, which could occupy two important lipophilic pockets (described as LP-1 and LP-2) of H+,K+-ATPase and can strongly bind to the K+-binding site, were designed based on a docking model. Among the compounds synthesized, compound 4d showed a strong H+,K+-ATPase-inhibitory activity and a high stomach concentration in rats, resulting in potent inhibitory action on histamine-stimulated gastric acid secretion in rats. Furthermore, 4d exerted significant inhibitory action on histamine-stimulated gastric-acid secretion in rats with a rapid onset and moderate duration of action after the administration. These findings may lead to a new insight into the drug design of potassium-competitive acid blockers.

The voltage-gated sodium channel, Na

The main purpose of the systemic energy metabolism is to provide a source of energy, mainly glucose, for the brain; therefore, blood glucose levels would be expected to correlate with exercise performance. The individual training status may also affect the blood glucose levels. The aim of the present study was to assess the association between blood glucose levels and running velocity during prolonged running in athletes with different training statuses. Two female college athletes, a triathlete and a tennis player, ran a course that was 247.4 m in circumference for 5 h while wearing a continuous glucose monitoring system. Blood was obtained at time-points of -1, 1, 3 and 5 h. The athletes had free access to food and fluids throughout the run. The athletes ran at almost the same pace without a sudden decrease in pace. The blood glucose levels increased and remained high in the triathlete, whereas the tennis player remained hypoglycemic throughout the run. Carbohydrate ingestion did not affect the blood glucose levels. The magnitude of hormonal changes, e.g. insulin, adrenaline and cortisol, was greater in the tennis player. The blood glucose concentration did not correlate with the running velocity or the carbohydrate ingestion; however, a discrepancy in blood glucose transition was observed between the triathlete and the tennis player, indicating a possible association between the adaptation to endurance exercise and the blood glucose kinetics during prolonged running.

With the aim to discover a novel excellent potassium-competitive acid blocker (P-CAB) that could perfectly overcome the limitations of proton pump inhibitors (PPIs), we tested various approaches based on pyrrole derivative 1 as a lead compound. As part of a comprehensive approach to identify a new effective drug, we tried to optimize the duration of action of the pyrrole derivative. Among the compounds synthesized, fluoropyrrole derivative 20j , which has a 2-F-3-Py group at position 5, fluorine atom at position 4, and a 4-Me-2-Py sulfonyl group at the first position of the pyrrole ring, showed potent gastric acid-suppressive action and moderate duration of action in animal models. On the basis of structural properties including a slightly larger C log P value (1.95), larger log D value (0.48) at pH 7.4, and fairly similar pKa value (8.73) compared to those of the previously optimized compound 2a , compound 20j was assumed to undergo rapid transfer to the stomach and have a moderate retention time there after single administration. Therefore, compound 20j was selected as a new promising P-CAB with moderately long duration of action.

With the aim to find a novel long-lasting potassium-competitive acid blocker (P-CAB) that would perfectly overcome the limitations of proton pump inhibitors (PPIs), we tried various approaches based on pyrrole derivative 1b as a lead compound. As part of a comprehensive approach to identification of a new drug, we explored excellent compounds that have low lipophilicity by introducing a polar hetero-aromatic group at position 5 of the pyrrole ring. Among the compounds synthesized, fluoropyrrole derivative 37c, which has a 2-F-3-Py group at the fifth position, lower pKa, and much lower ClogP and logD values than 1b dose, showed potent gastric-acid suppressive action resulting from gastric H+,K+-ATPase inhibition in animal models. Its maximum intragastric pH elevation effect was strong in rats, and its duration of action was much longer than that of either lansoprazole or lead compound 1b in dogs. Therefore, compound 37c can be considered a promising new P-CAB with long duration of action.

To discover a gastric antisecretory agent more potent than existing proton pump inhibitors, novel pyrrole derivatives were synthesized, and their H(+),K(+)-ATPase inhibitory activities and inhibitory action on histamine-stimulated gastric acid secretion in rats were evaluated. Among the compounds synthesized, compound 17a exhibited selective and potent H(+),K(+)-ATPase inhibitory activity through reversible and K(+)-competitive ionic binding; furthermore, compound 17c exhibited potent inhibitory action on histamine-stimulated gastric acid secretion in rats and Heidenhain pouch dogs.

In our pursuit of developing a novel and potent potassium-competitive acid blocker (P-CAB), we synthesized pyrrole derivatives focusing on compounds with low log D and high ligand-lipophilicity efficiency (LLE) values. Among the compounds synthesized, the compound 13e exhibited potent H(+),K(+)-ATPase inhibitory activity and potent gastric acid secretion inhibitory action in vivo. Its maximum efficacy was more potent and its duration of action was much longer than those of proton pump inhibitors (PPIs). Therefore, compound 13e (1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine fumarate, TAK-438) was selected as a drug candidate for the treatment of gastroesophageal reflux disease (GERD), peptic ulcer, and other acid-related diseases.

The synthesis and biological evaluation of a series of novel 3-phenylpiperidine-4-carboxamide derivatives are described. These compounds are generated by hybridization of the substructures from two types of tachykinin NK 1 receptor antagonists. Compound 42 showed high metabolic stability and excellent efficacy in the guinea-pig GR-73637-induced locomotive activity assay at 1 and 24 h after oral administration. It also exhibited good pharmacokinetic profiles in four animal species, and a low potential in a pregnane X receptor induction assay.

We attempted to establish and validate an in vivo pharmacodynamic (PD) rabbit model to screen tachykinin NK2 receptor (NK2-R) antagonists using pharmacological and pharmacokinetic (PK)/PD analyses. Under urethane anesthesia, changes in intracolonic pressure associated with intravenous (i.v.) administration of a selective NK2-R agonist, βAla8-neurokinin A(4-10) (βA-NKA), was monitored as a PD marker. The analgesic effects of NK2-R antagonists were evaluated by monitoring visceromotor response (VMR) to colorectal distension in a rabbit model of visceral hypersensitivity induced by intracolonic treatment of acetic acid. Intravenous administration of βA-NKA induced transient colonic contractions dose-dependently, which were inhibited by the selective NK2-R antagonists in dose- and/or plasma concentration–dependent manners. The correlation between PD inhibition and plasma concentration normalized with the corresponding in vitro binding affinity was relatively high (r2 = 0.61). Furthermore, the minimum effective doses on the VMR and ID50 values calculated in the PD model were highly correlated (r2 = 0.74). In conclusion, we newly established and validated a rabbit model of agonist-induced colonic contractions as a screening tool for NK2-R antagonists. In a drug discovery process, this PD model could enhance the therapeutic candidate selection for irritable bowel syndrome, pharmacologically connecting in vitro affinity for NK2-R with in vivo therapeutic efficacy. Keywords:: NK2-receptor antagonist, colonic contraction, pharmacodynamic (PD) marker, pharmacokinetic (PK) / pharmacodynamic (PD), colorectal distension

On the basis of a series of novel and potent potassium-competitive acid blockers represented by 1-sulfonylpyrrole derivative 7 , we prepared several five-membered heterocyclic analogues ( 8 ) and evaluated their H + ,K + -ATPase activities in vitro. We also assessed the role of the methylaminomethyl side chain by comparison with methylamino and ethylamino derivatives. We observed that the five-membered core ring and its orientation affect inhibitory activity and that the methylaminomethyl moiety is the best side chain. On the basis of potency and ligand-lipophilicity efficiency, compound 7 remains the most drug-like of the compounds studied to date. This study revealed the factors necessary for potent H + ,K + -ATPase inhibition, such as differences in electron density, the properties of the lone pair at each apical position of the heteroaromatic ring, and the geometry of the substituents.

TAK-480, 4-(difluoromethoxy)-N-((1R,2S)-2-(((3aR,4R,9bR)-4-(methoxymethyl)-2, 3,3a,4,5,9b-hexahydro-1H-pyrrolo[3,2-c]quinolin-1-yl)carbonyl)cyclohexyl)benzamide, is a novel tachykinin NK2–receptor antagonist. In this study, we investigated its antagonistic activity and efficacy in animal models of visceral hypersensitivity and stimulated bowel function which have been implicated to underlie the symptoms in irritable bowel syndrome (IBS). TAK-480 showed potent binding affinity for human NK2 receptors with a marked species difference and a 10,000-fold selectivity versus NK1 and NK3 receptors. TAK-480 dose-dependently antagonized colonic contractions induced by administration of the NK2 receptor–selective agonist beta-Ala8-NKA(4-10) (βA-NKA) in anesthetized rabbits. In a rabbit model of intracolonic zymosan-induced visceral hypersensitivity, TAK-480 markedly inhibited the visceromotor response to colorectal distension, in contrast to the moderate inhibition by the serotonin 5-HT3–receptor antagonist alosetron. In addition, TAK-480 suppressed ricinoleic acid–induced defecation without affecting spontaneous defecation in guinea pigs, whereas alosetron suppressed both. Furthermore, TAK-480 inhibited smooth muscle contractions produced by natural tachykinins (substance P, neurokinin A, and neurokinin B) as well as βA-NKA in an isolated human colon. In conclusion, the novel NK2-receptor antagonist TAK-480 improved visceral hypersensitivity and accelerated defecation without causing constipation in experimental animals. Furthermore, the potent functional blockade of NK2 receptors in human colon might suggest the potential effectiveness of TAK-480 in IBS patients. Keywords:: NK2-receptor antagonist, TAK-480, irritable bowel syndrome, visceral hypersensitivity, stimulated defecation

We examined the involvement of endogenous calcium-regulating hormones in the anabolic action of PTH, especially in relation to vitamin D. When normal rats (4 weeks old) were given a subcutaneous injection of hPTH(1-34) everyday or one, two or three times a week for 4 weeks adjusting the total weekly dose to 350µg/kg, femoral dry weight increased in an injection frequency-dependent manner. Daily injection of hPTH(1-34) into normal rats for 12 days caused a dose-dependent increase in serum concentration of 1,25(OH)2D but not calcitonin, associated with a dose-dependent increase in femoral dry weight. Therefore, the effect of hPTH(1-34) was examined under the condition of vitamin D (VD)-deficiency. In VD-deficient rats (13 weeks old), the change in serum immunoreactive PTH after the injection of hPTH(1-34) was the same as that observed in normal rats, although the basal level of serum PTH was significantly higher. Furthermore, VD-deficient rats treated with hPTH(1-34) for 4 weeks showed a dose-dependent increase in femoral dry weight without affecting circulating 1,25(OH)2D. These results suggest the involvement of systemic or local factor(s) independent of the action of VD in the anabolic action of daily PTH treatment.

Running for an extended period of time can cause severe stress on the body, subsequently damaging skeletal muscle and resulting in changes in blood components. However, few reports have examined vital responses during and after running. This study analyzed inflammatory responses during and after running and changes in stress responses as determined by serial changes in blood components. Venous blood was obtained before starting, 6 h after starting, 12 h after starting, and immediately after finishing 24 h of continuous running. Samples were analyzed for high-sensitivity C-reactive protein (hsCRP), pentraxin 3 (ptx3), white blood cells (WBC), myoglobin, creatine kinase (CK), and hormones. Diet and physical activity were standardized 24 h before and after running. Subjects comprised 16 men who agreed to participate in experimental running on November 8 and 9, 2008, at Tokyo Gakugei University. Mean running distance was 151.32 +/- 32.1 km (range, 83.6-210.0 km) in 24 h. A significant increase in hsCRP was seen from 12 h after starting to completion. Compared to hsCRP, ptx3 gradually increased from before starting to after completion, showing a significant difference between pre and post-run ptx3 levels. WBC count increased significantly until 6 h after starting. Neutrophils in leukocytosis increased significantly during the first 6 h. Eosinophils decreased significantly over the course of the 24 h. Cortisol increased, and testosterone decreased significantly from 6 h after starting. Dehydroepiandrosterone sulfate (DHEA-S), myoglobin, and CK increased over the course of the 24 h. Reactive oxygen metabolites (d-ROMs) changed within the normal range though there was a significant decrease, and biological anti-oxidant potential (BAP) stabilized. Active natural killer cells decreased significantly after 24 h running. Biopyrrin (BPn) increased significantly. Changes in stress oxide were small both during and after running, and adaptation for antioxidation was good. DHEAS, a biomarker of aging, was found to increase over the course of the 24 h, suggesting that controlling decreases in DHEA-S may be possible using exercise, particularly in males. The key finding was that DHEA S levels tended to increase with continuous aerobic exercise.

In this study, we attempted to clarify the mechanism of tachykinin-induced motor response in isolated smooth muscle preparations of the human colon. Fresh specimens of normal colon were obtained from patients suffering from colonic cancer. Using mucosa-free smooth muscle strips, smooth muscle tension with circular direction was monitored isometrically. Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) produced marked contraction. All of these contractions were inhibited by saredutant, a selective NK2-R antagonist, but not by CP122721, a selective NK1-R antagonist or talnetant, a selective NK3-R antagonist. βAla8-NKA(4-10) induced concentration-dependent contraction similar to NKA, but Sar9-Met11-SP and Met-Phe7-NKB did not cause marked contraction. Colonic contraction induced by βAla8-NKA(4-10) was completely blocked by saredutant, but not by atropine. Tetrodotoxin or NG-nitro-l-arginine methyl ester pretreatment significantly enhanced βAla8-NKA(4-10)–induced contraction. Immunohistochemical analysis showed that the NK2-R was expressed on the smooth muscle layers and myenteric plexus where it was also co-expressed with neuronal nitric oxide synthase in the myenteric plexus. These results suggest that the NK2-R is a major contributor to tachykinin-induced smooth muscle contraction in human colon and that the NK2-R–mediated response consists of an excitatory component via direct action on the smooth muscle and an inhibitory component possibly via nitric oxide neurons. Keywords:: NK1 receptor, NK2 receptor, NK3 receptor, human colon, smooth muscle

Proton pump inhibitors (PPIs) are widely used in the treatment of acid-related diseases. However, several unmet medical needs, such as suppression of night-time acid secretion and rapid symptom relief, remain. In this study, we investigated the pharmacological effects of 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1 H -pyrrol-3-yl]- N -methylmethanamine monofumarate (TAK-438), a novel potassium-competitive acid blocker (P-CAB), on gastric acid secretion in comparison with lansoprazole, a typical PPI, and SCH28080 [3-(cyanomethyl)-2-methyl,8-(phenylmethoxy)imidazo(1,2- a )pyridine], a prototype of P-CAB. TAK-438, SCH28080, and lansoprazole inhibited H + ,K + -ATPase activity in porcine gastric microsomes with IC 50 values of 0.019, 0.14, and 7.6 μM, respectively, at pH 6.5. The inhibitory activity of TAK-438 was unaffected by ambient pH, whereas the inhibitory activities of SCH28080 and lansoprazole were weaker at pH 7.5. The inhibition by TAK-438 and SCH28080 was reversible and achieved in a K + -competitive manner, quite different from that by lansoprazole. TAK-438, at a dose of 4 mg/kg (as the free base) orally, completely inhibited basal and 2-deoxy-d-glucose-stimulated gastric acid secretion in rats, and its effect on both was stronger than that of lansoprazole. TAK-438 increased the pH of gastric perfusate to a higher value than did lansoprazole or SCH28080, and the effect of TAK-438 was sustained longer than that of lansoprazole or SCH28080. These results indicate that TAK-438 exerts a more potent and longer-lasting inhibitory action on gastric acid secretion than either lansoprazole or SCH28080. TAK-438 is a novel antisecretory drug that may provide a new option for the patients with acid-related disease that is refractory to, or inadequately controlled by, treatment with PPIs.

To examine risk factors for coronary artery sclerosis in patients with diabetes mellitus.Patients with diabetes were divided into two groups based on whether their electrocardiogram (ECG) showed ischemic changes. In addition to traditional risk factors, other parameters (ie, serum levels of serotonin, homocysteine, thrombomodulin, plasminogen activator inhibitor-1, tissue plasminogen activator, vascular endothelial growth factor, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and endothelial-leukocyte adhesion molecule-1) were measured in both groups.In the group with ischemic ECG changes (n=13), systolic blood pressure was significantly higher than in the group with no changes (n=18). There were no significant differences in the serum levels of other factors; however, body mass index, hemoglobin A1c, total cholesterol, low density lipoprotein cholesterol and triglyceride levels tended to be higher in the group with ischemic ECG changes.Although the sample of patients was limited, these results suggest that strict control of traditional risk factors, especially high blood pressure, is important for preventing coronary artery sclerosis in patients with diabetes mellitus.

The present study was conducted to clarify the effects of ultra-marathon (ultra long-term aerobic exercise in which people run long distances) on the brain; examine the issue of central fatigue; verify the serotonin hypothesis of exercise-induced brain fatigue, and ascertain relationships between central fatigue and oxidative stress.Subjects consisted of 15 individuals (12 men, 3 women) who ran continuously for 24 h. Mean age was 44 +/- 9 years (range, 31 approximately 64 years). Blood tests were conducted: (1) before starting to run (around 09:00); (2) 16h after starting (02:00 the next day); and (3) just after the finish (around 10:00 the next day) to measure the serum levels of serotonin, melatonin, free tryptophan (f-Tp) and free fatty acid. At the same time, urine samples were collected to measure levels of urinary biopyrrins (BPn). Subjective symptoms were investigated using the Japanese version of the Profile of Mood States (POMS) instrument.(1) Participants ran a mean (+/- SD) distance of 162.6 +/- 18.3 km. (2) There were not marked changes in serum serotonin levels. Serum melatonin levels at 3 time points were 3.4 +/- 0.6 pg/ml, 57.2 +/- 15.2pg/ml and 7.8 +/- 8.9pg/ml, respectively(p0.01 before start vs. 16h after start). Serum f-Trp levels at the 3 time points were 5.4 +/- 0.9 nmol/ml, 9.7 +/- 2.1 nmol/ml and 11.5 +/- 4.9 nmol/ml, respectively (p0.05 before start vs. just before finish). Free fatty acid levels were 0.42 +/- 0.10 nmol/ml, 1.26 +/- 0.11 nmol/ml and 1.39 +/- 0.23 nmol/ml, respectively (p0.01 before start vs. 16 hours after start) (p0.05 before start vs. just after finish). (3) Urinary BPn levels increased with time, from 1.2 +/- 0.7 nmol/ml to 2.6 +/- 1.0 nmol/ml to 4.0 +/- 1.5 nmol/ml, respectively (p0.01 before the start vs. 16 hours after the start). (4) In terms of POMS scores, fatigue score (Factor F) increased, but vitality score (Factor V) was high at all time points and did not demonstrate any marked changes. Scores for anger and hostility were low (Iceberg profile-type: convex type). Urinary BPn levels were correlated significantly with both serum f-Trp level and Factor F:(y = 8.41x + 2.5, r = 0.708, n = 42) and (y = 2.82x + 5.9, r = 0.568, n = 42), respectively. Urinary BPn thus reflected the degree of subjective fatigue with a high level of sensitivity.The present results suggest that running continuously for 24h induces brain fatigue and that oxidative stress may be involved.