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Sorafenib is an oral multikinase inhibitor approved for the treatment of differentiated thyroid carcinoma (DTC), renal cell carcinoma, and hepatocellular carcinoma. In the phase III DECISION trial in patients with DTC, sorafenib exposure and the incidence of some adverse events (AEs) were higher than in previous trials; therefore, we analyzed exposure-response relationships, including progression-free survival (PFS) and selected AEs in patients with DTC. A novel, stratified prediction-corrected visual predictive check (pc-VPC) was developed to show robustness of the exposure-response relationships. Time-to-event simulations confirmed the benefit of the recommended dosing schedule of 800 mg/day: initial doses of 800 mg/day were associated with the highest PFS, whereas lower doses (600 or 400 mg/day) were associated with improved tolerability but reduced PFS. A simulated dose-reduction strategy of 800 mg/day for an initial two cycles followed by dose reductions seemed likely to maintain efficacy while possibly mitigating selected AEs (e.g., diarrhea and hand-foot skin reactions)., (© 2019 Bayer AG. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Quantification of different levels of 5-hydroxytryptamine4 (5-HT4) receptor agonism expression across animal species as well as across organs within the same animal species offers substantial potential for the separation of desired gastrointestinal versus undesired cardiac pharmacological activity of compounds in development. Since a detailed investigation of such properties is lacking to date, we set out to quantify gastric and cardiac effects of 5-HT4 receptor ligands in the pig, a model considered to be representative for the human situation. An in vitro test was developed to study the potentiating effect of 5-HT, prucalopride, tegaserod, R149402 (4-amino-5-chloro-2,2-dimethyl-2,3-dihydro-benzofuran-7-carboxylic acid [3-hydroxy-1-(3-methoxy-propyl)-piperidin-4ylmethyl]-amide), and R199715 (4-amino-5-chloro-2,3-dihydro-benzofuran-7-carboxylic acid [3-hydroxy-1-(3-methoxy-propyl)-piperidin-4ylmethyl]-amide) on electrically induced cholinergic contractions in longitudinal muscle strips of the proximal stomach. The results were compared with inotropic and chronotropic effects of these compounds in the electrically paced left atrium and spontaneously beating right atrium, respectively. To quantify the observed tissue-dependent responses, a nonlinear mixed-effects model based on the operational model of agonism was developed and successfully fitted to the data. The model quantified the tissue-dependent partial agonism of the selective 5-HT4 receptor agonists prucalopride, R149402, and R199715, whereas tegaserod and 5-HT were equiefficacious. The model was further extended to incorporate the responses to prucalopride in the presence of the 5-HT4 receptor antagonist GR113808 ([1-[2-[(methylsulphonyl)amino]ethyl]-4-piperidinyl-]methyl 1-methyl-1H-indole-3-carboxylate). The results indicate that these interactions do not follow a simple competitive pattern and that they differ between stomach and left atrium.

Pharmacokinetic (PK) studies of oxaliplatin, using a dose regimen of 85 mg/m, are lacking. A PK model may be used in future studies to investigate the relationship between pharmacokinetics and dose limiting toxicity. The purpose of this study was to construct a population PK model to describe platinum (Pt) concentrations in plasma in 33 patients with colorectal cancer. The secondary objective was to determine the relationship between the amount of Pt in 24-hour urine and the amount of Pt in fractionated urine collection periods. Plasma and urine samples were collected from patients during their first oxaliplatin treatment course. Population PK analysis was performed with WinNonMix. The model that best described the Pt concentrations in plasma was a two-compartment PK model. The elimination clearance (CL) and the elimination clearance of the peripheral compartment (CL2) (median +/- SE) were 25.2 +/- 6.3 L/hr and 68 +/- 24.8 L/hr, respectively. The median volume of distribution (V1) was determined to be 41.6 +/- 9.4 L and the median volume of distribution of the peripheral compartment (V2) was 452.5 +/- 96.4 L. The relationship between the cumulative amount of Pt in urine in the first 12 hours compared with the amount of Pt in 24 hours urine was reflected by a correlation coefficient (r2) of 0.95. The cumulative Pt concentration in urine in the first 10 hours and the first 8 hours compared with 24 hours was reflected by correlation coefficients r2 = 0.93 and r2 = 0.897, respectively. This PK model could be useful in identifying predictors for PK and pharmacodynamic variability to individualize dosing. The results of this study suggest that fractionated urine samples can replace 24-hour urine collection.

We aimed to evaluate the gastric relaxant capacity of the 5-HT(1/7)-receptor agonist 5-carboxamidotryptamine (5-CT) in conscious dogs and to clarify the mechanism of action by use of selective antagonists, vagotomy, and in vitro experiments. A barostat enabled us to monitor the intragastric volume in response to different treatments (intravenously administered) before and after supradiaphragmatic vagotomy [results presented as the maximum volume change after treatment (mean; n = 5-11)]. In vitro experiments were performed with isolated muscle strips cut from four different stomach regions of the vagotomized dogs [results were fitted to the operational model of agonism to determine the efficacy parameter tau (n = 5)]. 5-CT (0.5-10 microg/kg) caused a dose-dependent gastric relaxation (29-267 ml) that was completely blocked by the selective 5-HT(7)-receptor antagonist SB-269970 (50 microg/kg). After vagotomy, the relaxation to 10 microg/kg 5-CT was significantly less pronounced (73 vs. 267 ml; P < 0.05) but still blocked by SB-269970, whereas the response to the nitric oxide donor nitroprusside was similar to that before vagotomy (178 vs. 218 ml). In vitro, 5-CT concentration dependently inhibited the PGF(2alpha)-contracted muscle strips before and after vagotomy. Although before and after vagotomy the response in every region was mediated by 5-HT(7) receptors (apparent affinity dissociation constant: SB-269970, 8.2-8.6 vs. 8.3-8.6, respectively), the response after vagotomy was less efficacious (log tau: 1.9 to 0.5 vs. 1.4 to -0.1). The results indicate that the 5-CT-induced proximal stomach relaxation in conscious dogs before and after vagotomy is mediated via 5-HT(7) receptors. The decreased efficacy of 5-CT in vitro after vagotomy is probably related to vagotomy-induced changes in receptor density or coupling efficiency and provides a possible explanation for the decreased in vivo response to 5-CT after vagotomy.

5-Hydroxytryptamine 4 (5-HT4) receptor agonists promote colonic propulsion. The alteration of circular muscle (CM) motility underlying this involves inhibition of contractility via smooth muscle 5-HT4 receptors and proximal colonic motility stimulation, the mechanism of the latter not having been characterized. Our aim was to identify and characterize a 5-HT4 receptor-mediated stimulation of human colon CM contractile activity. 5-HT4 receptor ligands were tested on electrical field stimulation (EFS)-induced contractions of human colonic muscle strips cut in the circular direction (called 'whole tissue' strips). Additionally, after incubation of tissues with [3H]-choline these compounds were tested on EFS-induced release of tritium in whole tissue strips and in 'isolated' CM strips, obtained by superficial cutting in the CM layer. Tetrodotoxin and atropine blocked EFS-induced contractions of whole tissue CM strips. Prucalopride (0.3 micromol L-1) evoked a heterogenous response on EFS-induced contraction, ranging from inhibition (most frequently observed) to enhancement. In the release experiments, EFS-induced tritium efflux was blocked by tetrodotoxin. Prucalopride increased EFS-induced tritium and [3H]-acetylcholine efflux in whole tissue and in isolated CM strips. All effects of prucalopride were antagonized by the selective 5-HT4 receptor antagonist GR113808. The results obtained indicate the presence of excitatory 5-HT4 receptors on cholinergic nerves within the CM of human colon.

This study aimed to determine, quantify and explain regional differences in the relaxant response to the selective 5-HT(1) and 5-HT(7) receptor agonist 5-carboxamidotryptamine (5-CT) throughout the canine stomach. Longitudinal muscle strips from eight gastric corpus regions and six antrum regions were mounted for isotonic measurement. The 5-CT-induced relaxation was examined on a prostaglandin F(2alpha)-induced submaximal response, expressed as percentage of this response and fitted to the operational model of agonism (OMOA). 5-HT(7) receptor messenger RNA (mRNA) expression was compared by means of quantitative PCR. 5-CT inhibited PGF(2alpha)-induced tonic contraction (corpus) and increase of phasic contraction amplitude (antrum). The consistent antagonism produced by the selective 5-HT(7) receptor antagonist SB-269970 (10 nm, pA(2) estimates 8.2-8.9) confirmed that in every region, the inhibition by 5-CT was 5-HT(7) receptor mediated. However, variation in the maximum effect (61-108%) and pEC(50) (6.4-8.6) was observed throughout the different regions. The OMOA explained these differences as differences in the efficacy parameter tau (ratio of receptor density and coupling efficiency; log tau estimates ranging from 0.1 to 2.1). The log tau gradient decreases going from the lesser to the greater curvature. A proportional difference (68%) in the relative expression of 5-HT(7) receptor mRNA between the lesser and the greater curvature indicates that differences in receptor density contribute to the observed functional differences. This study illustrates that 5-HT(7) receptors are present throughout the ventral wall of the canine stomach, but the efficacy (expressed as log tau) is clearly greater close to the lesser curvature. Differences in 5-HT(7) receptor expression at least partially explain the functional differences.

Accumulating data have been published emphasizing the important role of 5-hydroxytryptamine (5-HT) receptors in proximal stomach relaxation. However, a proper in vivo characterization of 5-HT receptors mediating gastric relaxation is still missing. In the current study, we focus on the in vivo characterization of 5-HT1A receptors mediating relaxation of the proximal stomach in conscious dogs. Beagle dogs were equipped with a gastric fistula. In the conscious state, volume changes within an intragastric bag were measured at constant pressure by means of a barostat. Results are presented as the maximum volume increase after treatment (mean+/-s.e.m.). All drugs were injected intravenously. The 5-HT1A receptor agonist flesinoxan (10, 50, 100 and 150 microg kg-1) induced a dose-dependent relaxation of the canine proximal stomach (50+/-10, 230+/-51, 290+/-38 and 275+/-33 ml, respectively; n=9-11). The selective 5-HT1A receptor antagonist WAY-100635 dose-dependently inhibited the flesinoxan-induced relaxation. NG-nitro-l-arginine methyl ester did not affect this relaxation, suggesting that nitrergic nerves are not involved. After supradiaphragmatic vagotomy, the baseline of the intragastric volume was larger compared to that before vagotomy (317+/-50 vs 142+/-28 ml, respectively; n=5). Compensation for this by either reduction of the intraballoon pressure or infusion of a contractile dose of bethanechol did not establish a condition in which flesinoxan was able to relax the stomach. In contrast, nitroprusside induced a significant gastric relaxation when tone was increased by bethanechol. It is concluded that flesinoxan induces proximal gastric relaxation in conscious dogs via 5-HT1A receptors. The response is mediated through a vagal pathway without involvement of nitrergic nerves.

1. The aim of this study was to characterize the 5-HT receptors involved in the 5-HT-induced contraction of longitudinal muscle (LM) strips of porcine proximal stomach. This was done in a classical organ bath set-up for isotonic measurement. 2. The concentration-contraction curve to 5-HT was not modified by 5-HT(3) and 5-HT(4) receptor antagonism. Methysergide, ketanserin and mesulergine antagonized the curve to 5-HT. Concomitantly, increasing concentrations of ketanserin and mesulergine progressively revealed a biphasic nature of the 5-HT curve. Ketanserin antagonized the low-affinity receptor while it did not modify the high-affinity receptor. 3. Tetrodotoxin did not influence the concentration-contraction curve to 5-HT neither in the absence nor presence of ketanserin, indicating that nerves are not involved. 4. Ketanserin competitively antagonized the monophasic concentration-response curve to alpha-Methyl-5-HT, yielding a Schild slope that was not significantly different from unity. After constraining the Schild slope to unity, a pK(B) estimate of 8.23+/-0.90 was obtained. This affinity estimate of ketanserin closely approximates previously reported affinities at 5-HT(2A) receptors. 5. In the presence of ketanserin (0.1 microM; exposing the high-affinity receptor), a wide range of 5-HT receptor antagonists covering all 5-HT receptors known, was tested. Only methysergide and ritanserin inhibited the response to 5-HT, thus expressing affinity for the high-affinity receptor. This did not reveal the identity of the receptor involved. 6 It can be concluded that 5-HT induces pig proximal stomach (LM) contraction via 5-HT(2A) receptors located on smooth muscle. A ketanserin-insensitive phase of contractions could not be characterized between the actually known classes of 5-HT receptors with the pharmacological tools that were used.

1. We aimed to characterize 5-HT receptors mediating contraction and relaxation to 5-HT in dog proximal stomach longitudinal muscle (LM) strips. 2. Of the tryptamine analogues tested, 5-HT was the most potent contractile agent at basal length, while 5-CT was the most potent relaxant of PGF(2alpha)-induced contraction. Neither the contractions to 5-HT, nor the relaxations to 5-CT were influenced by tetrodotoxin, illustrating that action potential propagation is not involved. 3. The 5-HT-induced contraction was antagonized by mesulergine (0.03 to 0.3 microM) and ketanserin (2 - 20 nM), but the antagonism was not of a simple competitive nature, indicating multiple receptor involvement. Ketanserin (3 to 30 nM) and mesulergine (30 nM) competitively antagonized the alpha-Me-5-HT-induced contraction (pK(B): 8.83+/-0.09 and pA(2): 8.25+/-0.06 respectively). These affinity values are in line with literature affinities of ketanserin and mesulergine at 5-HT(2A) receptors in various bioassays. 4. The 5-CT-induced inhibition of PGF(2alpha)-induced contraction was competitively antagonized by mesulergine (pK(B) estimate: 8.52+/-0.12) and by the selective 5-HT(7) receptor antagonist SB-269970 (pK(B) estimate: 9.36+/-0.14). Both pK(B) estimates are in line with literature affinities of these compounds for 5-HT(7) receptors. Mesulergine (30 nM) and SB-269970 (10 nM) shifted the relaxant curve to 5-HT parallel to the right in the presence of ketanserin (0.3 microM) (pA(2) estimates of 8.08+/-0.10 and 8.75+/-0.14 respectively), indicative of 5-HT(7) receptor involvement. 5. It is concluded that 5-HT induces dog proximal stomach (LM) contraction via smooth muscle 5-HT(2A) receptors and relaxation via smooth muscle 5-HT(7) receptors.

1. We aimed to characterize the 5-HT receptors involved in the 5-HT-induced effect on electrically induced contractions of dog antrum longitudinal muscle in vitro. 2. In the presence of L-NOARG (0.1 mM), electrical field stimulation (EFS) induced atropine- and tetrodotoxin-sensitive contractions. Tetrodotoxin or atropine left any agonist tested ineffective. These EFS-induced contractions were on average enhanced by 5-HT (0.3 microM), however, pronounced variation in the response to 5-HT was observed. There were non-significant trends of the selective 5-HT3 receptor antagonist granisetron (1 microM), and methysergide (1 microM; preventing interactions of 5-HT with 5-HT1, 5-HT2, 5-ht5, 5-HT6 and 5-HT7 receptors) to increase the response to 5-HT. The selective 5-HT4 receptor antagonist GR 113808 (0.1 microM) displayed a non-significant trend to inhibit the 5-HT-induced increase. 3. Combination experiments with methysergide (1 microM), granisetron (1 microM) and GR 113808 (0.1 microM) revealed that the 5-HT (0.3 microM)-induced response consisted of (1) an excitatory component blocked by GR 113808, (2) excitatory and inhibitory components both blocked by methysergide. 4. The selective 5-HT4 receptor agonist prucalopride (0.3 microM) increased EFS-induced contractions, an effect prevented by GR 113808 (0.1 microM). 5. The increase of EFS-induced contractions by the preferential 5-HT2 receptor agonist alpha-Me-5-HT (0.3 microM) was antagonized by 5-HT2B receptor antagonists. 6. The 5-HT1/5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT; 0.3 microM) inhibited EFS-induced contractions. This was prevented by methysergide (1 microM), the 5-HT7 receptor antagonist mesulergine (0.3 microM) and the selective 5-HT7 receptor antagonist SB-269970 (0.3 microM). 7. In the presence of GR 113808 (0.1 microM), alpha-Me-5-HT (1 microM) increased EFS-induced contractions. The 5-HT (0.3 microM)-induced inhibition of the stimulation by alpha-Me-5-HT was prevented by SB-269970 (0.3 microM). 8. In conclusion, dog antral longitudinal muscle is endowed with (1) excitatory neuronal 5-HT4 receptors and 5-HT2B receptors and (2) inhibitory smooth muscle 5-HT7 receptors.

The novel enterokinetic drug prucalopride was tested at various intravenous and oral doses in fasted dogs to assess: (i) the effects on colonic contractile motility patterns; and (ii) the mediation of these effects by 5-hydroxytryptamine (5-HT4) receptors. Colonic motility patterns were assessed in conscious dogs with four chronically implanted strain-gauge force transducers that were sutured on the serosal side of the colon. Prucalopride altered colonic contractile motility patterns in a dose-dependent fashion by stimulating high-amplitude clustered contractions in the proximal colon and by inhibiting contractile activity in the distal colon. Prucalopride was equipotent after oral and intravenous administration, as reflected by the values for the effective dose that induced 50% of maximum effect (95% confidence limits): 0.04 mg kg(-1) p.o. (0.01-0.1 mg kg(-1)) and 0.01 mg kg(-1) i.v. (0.006-0.04 mg kg(-1)). Prucalopride also caused a dose-dependent decrease in the time to the first giant migrating contraction (GMC); at higher doses of prucalopride, the first GMC generally occurred within the first half-hour after treatment. Subcutaneous pretreatment with the 5-HT4 receptor antagonist GR125487 (40 microg kg(-1) bodyweight) completely prevented the effects of orally administered prucalopride (0.31 mg kg(-1) bodyweight). Prucalopride, given orally or intravenously, alters colonic motility in the fasted conscious dog in a dose-dependent fashion. It induces GMCs and causes proximal colon stimulation and distal colon inhibition of contractile motility patterns by stimulating 5-HT4 receptors.

Prucalopride is a novel enterokinetic compound and is the first representative of the benzofuran class. We set out to establish its pharmacological profile in various receptor binding and organ bath experiments. Receptor binding data have demonstrated prucalopride's high affinity to both investigated 5-HT(4) receptor isoforms, with mean pK(i) estimates of 8.60 and 8.10 for the human 5-HT(4a) and 5-HT(4b) receptor, respectively. From the 50 other binding assays investigated in this study only the human D(4) receptor (pK(i) 5.63), the mouse 5-HT(3) receptor (pK(i) 5.41) and the human sigma(1) (pK(i) 5.43) have shown measurable affinity, resulting in at least 290-fold selectivity for the 5-HT(4) receptor. Classical organ bath experiments were done using isolated tissues from the rat, guinea-pig and dog gastrointestinal tract, using various protocols. Prucalopride was a 5-HT(4) receptor agonist in the guinea-pig colon, as it induced contractions (pEC(50)=7.48+/-0.06; insensitive to a 5-HT(2A) or 5-HT(3) receptor antagonist, but inhibited by a 5-HT(4) receptor antagonist) as well as the facilitation of electrical stimulation-induced noncholinergic contractions (blocked by a 5-HT(4) receptor antagonist). Furthermore, it caused relaxation of a rat oesophagus preparation (pEC(50)=7.81+/-0.17), in a 5-HT(4) receptor antagonist sensitive manner. Prucalopride did not cause relevant inhibition of 5-HT(2A), 5-HT(2B), or 5-HT(3), motilin or cholecystokinin (CCK(1)) receptor-mediated contractions, nor nicotinic or muscarinic acetylcholine receptor-mediated contractions, up to 10 microM. It is concluded that prucalopride is a potent, selective and specific 5-HT(4) receptor agonist. As it is intended for treatment of intestinal motility disorders, it is important to note that prucalopride is devoid of anti-cholinergic, anticholinesterase or nonspecific inhibitory activity and does not antagonise 5-HT(2A), 5-HT(2B) and 5-HT(3) receptors or motilin or CCK(1) receptors.

We aimed to study 5-HT(4) receptors in canine stomach contractility both in vivo and in vitro. In anaesthetized Beagle dogs, the selective 5-HT(4) receptor agonist prucalopride (i.v.) induced dose-dependent tonic stomach contractions under isobaric conditions, an effect that was antagonized by the selective 5-HT(4) receptor antagonist GR 125487 (10 microg kg(-1), i.v.). Electrical field stimulation (EFS) of corpus longitudinal muscle strips resulted in atropine- and tetrodotoxin-sensitive contractions (L-NOARG (0.1 mM) present in all organ bath solutions). Prucalopride increased these contractions (maximal response after single-dose addition (0.3 microM): 165% of initial value, or after cumulative addition: 188%). In the presence of methysergide (3 microM), 5-HT also increased EFS-contractions (after single-dose addition (0.3 microM): increase to 192%, after cumulative addition: 148%). The selective 5-HT(4) receptor antagonists GR 113808 (0.1 microM) or GR 125487 (10 nM) antagonized the prucalopride (0.3 microM)-induced contraction increments. When EFS-induced contractions were blocked by atropine or tetrodotoxin, prucalopride was ineffective. In the presence of methysergide (3 microM), the contraction increases to 5-HT (0.3 microM) were prevented by GR 113808 (0.1 microM). The prucalopride curve (pEC(50) 7.9) was shifted in parallel to the right by GR 113808 3 nM (pA(2) 9.4). In the presence of methysergide (3 microM), the curve to 5-HT (pEC(50) 8.1) was competitively antagonized by GR 113808, yielding a Schild slope of 0.8+/-0.2 (pK(B) of 9.1 with unit Schild slope). In corpus circular muscle strips, the prucalopride (0.3 microM)-induced augmentation of EFS-contractions (258%) was also prevented by GR 113808 (0.1 microM) (124%). In conclusion, the effects of 5-HT(4) receptor agonists on proximal stomach motor activity in vivo can be explained by an effect on 5-HT(4) receptors on cholinergic nerves within the gastric muscle wall.

5-HT(4) receptors mediate circular muscle relaxation in both human and canine large intestine, but this phenomenon alone can not explain the improvement in colonic motility induced by selective 5-HT(4) receptor agonists in vivo. We set out to characterize 5-HT(4) receptor-mediated effects in longitudinal muscle strips of canine and human large intestine. Electrical field stimulation (EFS) was applied providing submaximal isotonic contractions. L-NOARG (0.1 mM) was continuously present in the organ bath to preclude nitric oxide-induced relaxation to EFS. The selective 5-HT(4) receptor agonist prucalopride (0.3 microM) enhanced EFS-evoked contractions, that were antagonized in both preparations by the selective 5-HT(4) receptor antagonist GR 113808 (0.1 microM). The prucalopride-induced increase was present in canine ascending and descending colon, but absent in rectum. Regional differences in response to prucalopride were not observed in human ascending and sigmoid colon and rectum. Incubation with atropine (1 microM) or tetrodotoxin (0.3 microM) inhibited EFS-induced contractions, which were then unaffected by prucalopride (0.3 microM) in both tissues. In the presence of methysergide (3 microM; both tissues) and granisetron (0.3 microM; only human tissues), 5-HT (0.3 microM) enhanced EFS-induced contractions, an effect that was antagonized by GR 113808 (0.1 microM). In the presence of atropine or tetrodotoxin, EFS-induced contractions were inhibited, leaving 5-HT (0.3 microM) ineffective in both preparations. This study demonstrates for the first time that in human and canine large intestine, 5-HT(4) receptors are located on cholinergic neurones, presumably mediating facilitating release of acetylcholine, resulting in enhanced longitudinal muscle contractility. This study and previous circular muscle strip studies suggest that 5-HT(4) receptor agonism facilitates colonic propulsion via a coordinated combination of inhibition of circumferential resistance and enhancement of longitudinal muscle contractility.

Recently, it was demonstrated that 5-HT induces relaxation of human colon circular muscle through activation of 5-HT(4) receptors and 5-HT(7) receptors. The aim of the current study was to develop a new in vitro bioassay of human colon that would facilitate the pharmacological analysis of 5-HT responses mediated solely by 5-HT(4) receptors. Contracting circular muscle strips with KCl (80 mM) yielded a stable contractile tension and, in contrast to muscarinic cholinoceptor agonists and histamine, a profound reduction of spontaneous contractility. This allowed the establishment of reproducible, fully-defined, agonist concentration-response curves by cumulative dosing. Under these conditions, 5-HT induced a concentration-dependent relaxation (pEC(50) 7.31, Hill slope 0.91). Neither methysergide (10 microM) nor granisetron (1 microM) affected the 5-HT-induced relaxation, suggesting that 5-HT(1), 5-HT(2), 5-HT(3), 5-ht(5), 5-HT(6) or 5-HT(7) receptors are not involved. The lack of effect of tetrodotoxin (0.3 microM) indicated a direct effect of 5-HT on the smooth muscle. The selective 5-HT(4) receptor antagonists GR 113808, GR 125487 and RS 39604 competitively antagonized the 5-HT-induced relaxation (pK(B) 9.43, 10.12 and 8.53, respectively). SB 204070 (1 nM) produced a rightward shift (pA(2) 10.34) and depression of the 5-HT curve. These affinity estimates are similar to those previously reported for 5-HT(4) receptors. The selective 5-HT(4) receptor agonists, prucalopride and R076186, induced relaxations (pEC(50) 7.50 and 7.57, respectively), that were blocked by GR 113808 (3 nM), yielding pA(2) estimates of 9.31 and 9.21, respectively. To summarise, in KCl (80 mM)-contracted muscle strips, 5-HT induces relaxation through activation of a homogeneous smooth muscle 5-HT(4) receptor population. This new bioassay allows the focused, pharmacological characterization of human colonic 5-HT(4) receptors in vitro.

5-HT4 receptors mediate relaxation of human colon circular muscle. However, after 5-HT4 receptor blockade (SB 204070 10 nM), 5-HT still induced a relaxation (pEC50 6.3). 5-HT4 receptors were sufficiently blocked, as the curves to 5-HT obtained in the presence of 10 and 100 nM SB 204070 were indistinguishable. This 5-HT-induced relaxation was tetrodotoxin-insensitive, indicative of a smooth muscle relaxant 5-HT receptor. This, and the rank order of potency (5-CT=5-MeOT=5-HT) suggested involvement of 5-HT1 or 5-HT7 receptors. Mesulergine, a 5-HT7 receptor antagonist at nanomolar concentrations, and a 5-HT1 receptor antagonist at micromolar concentrations, competitively antagonized the 5-HT-induced relaxation (pKB 8.3) and antagonized the relaxation to 5-CT. Methysergide antagonized the 5-HT-induced relaxation (pA2 7.6). It is concluded that the profile of the smooth muscle inhibitory 5-HT receptor resembles that of the 5-HT7 receptor. These data provide the first evidence for functional human 5-HT7 receptors.

This study aimed to characterize for the first time in vitro 5-HT4 receptors in the canine gastrointestinal tract. For this purpose, we used circular muscle strips of the canine isolated rectum. In the presence of methysergide (60 microM), 5-HT induced relaxation of methacholine (1 microM)-precontracted muscle strips, yielding a monophasic sigmoidal concentration-relaxation curve (pEC50 7.2+/-0.07). Tetrodotoxin (0.3 microM) did not affect the curve to 5-HT, suggesting the inhibitory 5-HT receptor is located on the smooth muscle. Granisetron (0.3 microM) did also not affect the curve to 5-HT, which excludes the 5-HT3 receptor mediating the relaxation to 5-HT. The presence of methysergide rules out the involvement of 5-HT1, 5-HT2 or 5-HT7 receptors. 5-HT, the selective 5-HT4 receptor agonists R076186, prucalopride (R093877) and SDZ HTF-919 and the 5-HT4 receptor agonists cisapride and 5-MeOT relaxed the muscle strips with a rank order of potency R076186 = 5-HT > cisapride > prucalopride > or = SDZ HTF-919 > 5-MeOT. The selective 5-HT4 receptor antagonists GR 125487, RS 39604 and GR 113808 competitively antagonized the relaxations to 5-HT, yielding pK(B) estimates of 9.7, 7.9 and 9.1, respectively. The selective 5-HT4 receptor antagonist SB 204070 shifted the curve to 5-HT rightward and depressed the maximal response (apparent pA2 10.6). GR 113808 (10 nM) produced a parallel rightward shift of the curve to the selective 5-HT4 receptor agonists R076186 (pA2 8.8). It is concluded that 5-HT induces relaxation of the canine rectum circular muscle through stimulation of a single population of smooth muscle 5-HT4 receptors. For the first time, a nonhuman species was shown to exhibit relaxant 5-HT4 receptors in the large intestine.

1. Although conscious dogs have often been used for colonic motility studies with 5-hydroxytryptamine (5-HT), the effects of 5-HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5-HT of the canine isolated colon longitudinal muscle. 2. Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5-HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5-HT re-uptake, monoamine oxidase or blockade of alpha-adrenoceptors. Tetrodotoxin (0.3 microM) did not affect the responses to 5-HT, suggesting that smooth muscle 5-HT receptors are involved. The selective 5-HT4 receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5-HT and therefore it was included in the organ bath solution in all further experiments. The 5-HT1 and 5-HT2 receptor antagonist methysergide (0.1 microM) depressed the curve to 5-HT, but the selective 5-HT3 receptor antagonist granisetron (0.3 microM) had no effect. 3. Besides 5-HT, alpha-methyl-5-HT (alpha-Me-5-HT), 5-methoxytryptamine (5-MeOT), 2-methyl-5-HT (2-Me-5-HT) and 5-carboxamidotryptamine (5-CT) also induced contractions, with the following rank order of potency (pEC50 values in parentheses): 5-HT (6.9) = alpha-methyl-5-HT (6.9) > 2-Me-5-HT (5.8) = 5-MeOT (5.7) = 5-CT (5.6), indicative of 5-HT2 receptor involvement, alpha-Me-5-HT produced a bell-shaped curve, which was not affected by alpha-adrenoceptor blockade. 5-HT, 5-MeOT, 2-Me-5-HT and 5-CT produced a monophasic concentration-response curve, consistent with an interaction with a single receptor site. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and tryptamine only induced contractions at a concentration exceeding 1 microM. 4. The selective 5-HT2B receptor antagonist SB 204741 (0.3 microM) did not affect the curve to 5-HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with pKb values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 microM) shifted the curve to 5-MeOT rightward yielding an apparent pA2 of 7.1. Other antagonists at 5-HT2A receptors also surmountably inhibited the contractions to 5-HT (apparent pA2 value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of "pseudo-irreversible' antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5-HT2A receptors. 5. It was concluded that 5-HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5-HT2A receptors. The presence of inhibitory 5-HT4 receptors cannot be ruled out.