B. R. Tuladhar

Evidence for a 5‐HT3 receptor involvement in the facilitation of peristalsis on mucosal application of 5‐HT in the guinea pig isolated ileum

1 The 5‐HT receptor involved in the effect of mucosal application of 5‐HT to facilitate peristalsis was investigated in the isolated guinea pig ileum. 2 An application of 5‐HT (3–100 μM) to the mucosal surface (by inclusion of 5‐HT in the Krebs‐Henseleit solution passing through the lumen of the ileum) caused a concentration related facilitation of peristalsis characterized by a reduction in the peristaltic threshold. 3 Peristalsis was not modified by methiothepine (0.1 μM), ritanserin (0.1 μM), ondansetron (5 μM), granisetron (1 μM) or SB 204070 (0.1 μM) administered alone to the mucosal surface. 4 The concentration–response curve to mucosally applied 5‐HT was not altered by the mucosally applied 5‐HT1/2 receptor antagonist methiothepine (0.1 μM), the 5‐HT2 receptor antagonist ritanserin (0.1 μM) or the 5‐HT4 receptor antagonist SB 204070 (0.1 μM). However, the mucosally applied 5‐HT3 receptor antagonists ondansetron (5 μM) and granisetron (1 μM) shifted the response curves to mucosally applied 5‐HT to the right in a parallel and surmountable manner. The pD2 values in the absence and presence of ondansetron were 5.42±0.07 and 4.12±0.10, respectively, (n=6) and that of granisetron were 5.45±0.12 and 4.50±0.10 respectively, (n=5). 5 Serosally applied ondansetron (5 μM) or granisetron (1 μM) had no effect on the concentration–response curve to mucosally applied 5‐HT. However, the serosally applied ondansetron and granisetron antagonised the facilitatory effect of serosally applied 5‐HT (10 μM) when administered in the presence of serosally applied SB 204070 (0.1 μM). 6 It is concluded that the facilitatory effect of mucosally applied 5‐HT to reduce the peristaltic threshold in the guinea pig ileum is mediated via a 5‐HT3 receptor located on the mucosal and not the serosal side of the ileum.

5-HT4 receptor mediated facilitation of the emptying phase of the peristaltic reflex in the guinea-pig isolated ileum

1 The influence of 5‐hydroxytryptamine (5‐HT) receptor agonists and antagonists on the emptying phase (circular muscle contraction) of the peristaltic reflex was investigated in the guinea‐pig isolated ileum. 2 The effect of drug application to the serosal surface was measured as the changes in threshold pressure required to trigger the peristaltic reflex and the interval between the peristaltic strokes. A facilitation or inhibition of peristalsis was defined as a reduction or increase in threshold pressure respectively. 3 Peristalsis was not modified by the inclusion of methysergide (1 μm) and/or ondansetron (2 μm) in the bathing medium. 5‐HT (0.1–1.0 μm) caused a facilitation of the perstaltic reflex; the response curve to 5‐HT was not altered by the presence of methysergide (1 μm) and ondansetron (2 μm). 4 In the presence of methysergide (1 μm) plus ondansetron (2 μm), 5‐HT (7.36 ± 0.06), 5‐methoxytryptamine (7.01 ± 0.17), 5‐carboxamidotryptamine (5.43 ± 0.06), renzapride (6.09 ± 0.17), (S)‐zacopride (5.99 ± 0.11), (R)‐zacopride (5.61 ± 0.13) and metoclopramide (4.8 ± 0.65) caused a concentration‐related facilitation of the peristaltic reflex, the pEC50 values (mean ± s.e.mean) being shown in parentheses. 2‐Methyl‐5‐HT was ineffective up to 10 μm. 5 The administration of SDZ 205–557 (1 μm) alone failed to modify the peristaltic reflex, but caused a parallel dextral shift in the concentration‐effect curve to 5‐HT (apparent pKB 7.38 ± 0.30). It failed to modify the effect of acetylcholine to enhance the peristaltic reflex. 6 It is concluded that the rank order of potency of the 5‐HT agonists from the indole and substituted benzamide series to facilitate the emptying phase of the peristaltic reflex in the guinea‐pig ileum closely correlates with their published actions as 5‐HT4 agonists in other systems. An agonist action on the 5‐HT4 receptor is also supported by the potency of the 5‐HT3/5‐HT4 antagonist SDZ 205–557 (but not the 5‐HT3 antagonist ondansetron) to inhibit the effects of 5‐HT.

The tachykinin NK1 receptor antagonist PD 154075 blocks cisplatin-induced delayed emesis in the ferret

The activity of a selective tachykinin NK1 receptor antagonist, PD 154075 ([(2-benzofuran)-CH2OCO]-(R)-alpha-MeTrp-(S)-NHCH(CH3) Ph), was examined in radioligand binding studies, in a [Sar9,Met(O2)11]substance P-induced foot-tapping model in the gerbil, and in cisplatin-induced acute and delayed emesis in the ferret. In radioligand binding studies, PD 154075 showed nanomolar affinity for the human, guinea-pig, gerbil, dog and ferret NK1 receptors with an approximate 300 times lower affinity for the rodent NK1 receptor. Using NK2,NK3 receptors and a range of other receptor ligands, PD 154075 was shown to exhibit a high degree of selectivity and specificity for the human type NK1 receptor. Following subcutaneous administration PD 154075 dose dependently (1-100 mg/kg) antagonised the centrally mediated [Sar9,Met(O2)11] substance P-induced foot tapping in the gerbil with a minimum effective dose (MED) of 10 mg/kg. The ability of PD 154075 to readily penetrate into the brain following oral administration was confirmed by its extraction and high performance liquid chromatography assay from the rat brain. PD 154075 was shown to achieve a relatively fast and sustained brain concentration (brain/plasma ratios ranged from 0.27 to 0.41 during the time period of 0.25-12 h). Further pharmacokinetic studies revealed that the absolute oral bioavailability of PD 154075 in the rat was (mean +/- S.D.) 49 +/- 15%. PD 154075 (1-30 mg/kg, i.p.) dose dependently antagonised the acute vomiting and retching in the ferret measured for 4 h following administration of cisplatin (10 mg/kg, i.p.) with a MED of 3 mg/kg. The administration of a lower dose of cisplatin (5 mg/kg, i.p.) in the ferret induces both an acute (day 1) and delayed (days 2 and 3) phase of emesis. The i.p. administration of PD 154075, 10 mg/kg three times a day for 3 days, almost completely blocked both the acute and delayed emetic responses. In the same study, the 5-HT3 receptor antagonist ondansetron (1 mg/kg, i.p., t.i.d.) was also very effective against the acute emetic response observed during the first 4 h following cisplatin, but it was only weakly active against the delayed response. In conclusion, PD 154075 is a selective and specific high affinity NK1 receptor antagonist with good oral bioavailability which is effective against both acute and delayed emesis induced by cisplatin in the ferret.

5‐HT7 receptors mediate the inhibitory effect of 5‐HT on peristalsis in the isolated guinea‐pig ileum

The study was undertaken to investigate the 5‐HT receptor mediating the inhibitory effect of 5‐HT on peristalsis in the guinea‐pig isolated ileum. The facilitatory and inhibitory effects were measured as the decrease and increase, respectively, in the intraluminal pressure required to trigger peristalsis. In the presence of 5‐HT2/3&4 receptor antagonists ketanserin (0.1 μM), granisetron (1 μM) and SB‐204070 (1 μM), a cumulative addition (0.1–100 μM) of 5‐HT or 5‐carboxamidotryptamine, but not 2‐methyl‐5‐HT produced a concentration‐dependent increase in the threshold required to trigger peristalsis. The 5‐HT7 receptor selective antagonist SB‐269970‐A (0.01–1 μM) or methiothepin (0.01–0.1 μM) concentration‐dependently antagonised this response to 5‐HT. SB‐269970‐A (1 μM) and methiothepin (1 μM) were also able to restore peristalsis in tissues in which peristalsis was inhibited by a prior addition of 30 μM of 5‐HT. The results indicate an involvement of 5‐HT7 receptors in the inhibitory effect of 5‐HT on peristalsis in the guinea‐pig ileum.

The role of endogenous opioids in the control of gastrointestinal motility: predictions from in vitro modelling

Gastrointestinal motility can be assessed in vitro by investigating the effects of drugs or gene knockouts on intestinal propulsion, and on neurone‐mediated responses evoked by electrical field stimulation (EFS). The latter predominantly measure enteric motor activity and can detect prokinetic activity of exogenous agents. Some evidence suggests that naloxone has prokinetic activity when evaluated for an ability to modulate responses to EFS, but the effects are inconsistent across different species or intestinal regions. Models of intestinal peristalsis measure an integrated sensory‐motor nerve function and possess more intact neuro‐neuronal connections. In such preparations, the effects of naloxone also suggest a prokinetic property but again, this is inconsistent. By contrast, consistent prokinetic activity of naloxone is apparent in models where peristalsis is compromised by drug‐induced suppression of motor nerve activity or by modulation of endogenous processes using receptor antagonists or inappropriate intraluminal distension. These data suggest that endogenous opioids play little or no role in normal intestinal physiology, but suppress intestinal motility when motor function is compromised. Consequently, drugs that antagonize opioid receptors may exert prokinetic activity in conditions where intestinal motility is reduced, such as constipation. Further work is required to elucidate the opiate receptor(s) involved.

Pharmacological characterization of the 5-HT receptor-mediated contraction in the mouse isolated ileum

The pharmacological characterization of a 5‐HT receptor‐mediated contractile response in the mouse isolated ileum is described. In the presence of methysergide (1 μM), 5‐hydroxytryptamine (5‐HT, 0.3–100 μM) produced phasic concentration‐dependent contractions of segments of the mouse isolated ileum with a pEC50 value of 5.47±0.09. The 5‐HT3 receptor selective agonists m‐chlorophenylbiguanide (0.3–100 μM, pEC50 5.81±0.04), 1‐phenylbiguanide (3–100 μM, pEC50 5.05±0.06) and 2‐methyl‐5‐HT (3–100 μM, pEC50 5.00±0.07) acted as full agonists to induce contractile responses. 5‐methoxytryptamine (0.1–100 μM), RS 67506 (0.1–100 μM) and α‐methyl‐5‐HT (0.1–100 μM) failed to mimic the 5‐HT responses. The contractile response to 5‐HT was not antagonized by either 5‐HT2 receptor antagonists ritanserin (0.1 μM) or ketanserin (1 μM) nor the 5‐HT4 receptor antagonist SB 204070 (0.1 μM). The 5‐HT3 receptor selective antagonists granisetron (0.3–1 nM), tropisetron (1–10 nM), ondansetron (10 nM–1 μM) and MDL 72222 (10 nM–1 μM) caused rightward displacement of the concentration‐response curves to 5‐HT. The lower concentrations of the antagonists caused approximate parallel rightward shifts of the concentration‐response curves to 5‐HT with apparent pKB values for granisetron (9.70±0.39), tropisetron (9.18±0.20), ondansetron (8.84±0.24) and MDL 72222 (8.65±0.35). But higher concentrations of antagonists resulted in a progressive reduction in the maximum responses. The contractile response to 5‐HT was abolished by tetrodotoxin (0.3 μM); atropine (0.1 and 1 μM) decreased the maximum response of the 5‐HT concentration‐response curve by approximately 65%. It is concluded that a neuronally located 5‐HT3 receptor mediates a contractile response to 5‐HT in the mouse ileum. The 5‐HT3 receptor in the mouse ileum has a different pharmacological profile to that reported for the guinea‐pig ileum.

Pharmacological characterization of the 5-hydroxytryptamine receptor mediating relaxation in the rat isolated ileum.

1 The aim of the present study was to investigate a 5‐HT4 receptor involvement in the mediation of a 5‐HT‐induced relaxation response in the rat isolated ileum in vitro. 2 Ileal segments were taken at regular intervals from the ileo‐caecal junction to duodenum. 5‐HT (1 μm) induced a relaxation or contraction response in segments taken from the terminal ileum: the relaxation decreased and finally disappeared as contractions dominated in the proximal tissues. The 5‐HT‐induced relaxations were enhanced in the terminal segments and the contractions attenuated in both terminal and proximal segments, in the presence of methysergide (1 μm) and atropine (0.1 μm). 3 In the presence of methysergide (1 μm) and atropine (0.1 μm), a cumulative addition of 5‐HT (0.01‐1 μm) induced a concentration‐dependent relaxation in the terminal (1–20 cm from the ileo‐ceacal junction) ileal segments which at higher concentrations of 5‐HT (3–30 μm) reverted to contraction. 4 The rank order of potency of indole agonists in inducing a concentration‐related relaxation response in tissues of the terminal ileum (pretreated with pargyline (100 μm) and in the presence of methysergide (1 or 100 μm) and atropine (0.1 μm)) was 5‐hydroxytryptamine (6.97 ± 0.06), 5‐methoxytryptamine (6.50 ± 0.07), α‐methyl‐5‐hydroxytryptamine (5.53 ± 0.17), 5‐carboxamidotryptamine (5.51 ± 0.12) and 2‐methyl‐5‐hydroxytryptamine (< 5), the pEC50 values (mean ± s.e.mean) being shown in parentheses. 5 Pretreatment of tissues with pargyline (100 μm) selectively enhanced the potency of 5‐methoxytryptamine by a factor of 19 but failed to modify the potency of the other indole agonists. 6 The 5‐HT4 receptor antagonists, tropisetron, SDZ 205–557 and GR 113808 antagonized the relaxation response to 5‐HT (in the presence of methysergide (1 or 10 μm) and atropine (0.1 μm)) with pKB values (95% CL) of 6.09 (5.94‐6.24), 7.0 (6.9‐7.09) and 8.95 (8.81‐9.1) respectively. Apparent pKB values estimations for tropisetron (1 μm) and GR 113808 (10 nM) using the agonists 5‐methoxytryptamine and 5‐carboxamidotryptamine were 6.37 ± 0.31, 5.91 ± 0.38 and 8.83 ± 0.11, 8.82 ± 0.22 respectively. 7 Tropisetron (10 μm), SDZ 205–557 (3 μm) and GR 113808 (10–100 nM) caused an increase in basal tone of the rat terminal ileum when administered in the presence of methysergide and atropine. 8 The relaxation response to 5‐HT in the rat terminal ileum was not antagonized by ritanserin (1 μm), ondansetron (1 μm) or Nω‐nitro‐l‐arginine methyl ester (100 μm) and with only a twofold dextral shift of the concentration‐response curve by tetrodotoxin (1 μm). 9 It is concluded that the relaxant response to 5‐HT in the terminal region of the ileum is mediated directly at the smooth muscle; a ranked indole agonist potency and selective antagonism by 5‐HT4 receptor antagonists tropisetron, SDZ 205–557 and GR 113808 indicate a 5‐HT4 receptor involvement in the relaxation response.

5‐HT3 and 5‐HT4 receptor‐mediated facilitation of the emptying phase of the peristaltic reflex in the marmoset isolated ileum

1 The patterns of intestinal motility and the effect of an increase in intraluminal pressure were studied in vitro on segments obtained from the marmoset small intestine. 2 Segments obtained from the distal half of the marmoset small intestine exhibited segmentation, consisting of narrow annular contractions (lasting for 2–3 s) interposed between the relaxed segments of varying length. The subsequent contractions occurred slightly distal to the previous contraction, with ring‐like contractions appearing to move in the aboral direction. Such segmentation was infrequent or absent in the segments obtained from the proximal half of the small intestine. An increase in intraluminal pressure inhibited segmentation and finally produced peristalsis in most of the tissues. 3 The influence of 5‐hydroxytryptamine (5‐HT) receptor agonists and antagonists on the threshold of the peristaltic reflex was investigated in the segments obtained from the distal half of the intestine after segmentation had subsided. The effect of drug application to the serosal surface was measured as a change in threshold pressure required to trigger the peristaltic reflex. A facilitation was defined in two ways (a) as a reduction in the threshold pressure required to trigger the reflex and (b) in those tissues that failed to respond with peristalsis on raising intraluminal pressure to the maximum attainable (1 kPa), as a reduction in threshold pressure compared to this value. 4 5‐HT (7.85±0.19), 5‐methoxytryptamine (7.79±0.24), 5‐carboxamidotryptamine (6.66±0.13) and 2‐methyl‐5‐HT (6.24±0.16) caused a concentration related facilitation of the peristaltic reflex, the pD2 values (mean±s.e.mean) being shown in parentheses. 5 The concentration‐response curves to both 5‐HT and 5‐methoxytryptamine were dextrally shifted in a surmountable manner in the presence of GR 113808 (30 nM). pD2 values for 5‐HT and 5‐methoxytryptamine were significantly decreased to 6.98±0.24 and 6.83±0.36 respectively in the presence of GR 113808 (30 nM). 6 In the presence of a high concentration of (10 μm) 5‐methoxytryptamine the subsequent addition of 2‐methyl‐5‐HT (3–10 μm) but not 5‐methoxytryptamine (10 μm) facilitated peristalsis; the effect of 3 μm 2‐methyl‐5‐HT was significantly decreased by 2 μm ondansetron. 7 It is concluded that the facilitation of the peristaltic reflex in the marmoset intestine induced by 5‐HT at submicromolar concentrations involves a 5‐HT4 receptor stimulation with an additional 5‐HT3 receptor activation at higher concentrations.

Aryl sulfonamides as selective PDE4 inhibitors

A series of novel selective phosphodiesterase 4 (PDE4) inhibitors has been developed which displays activity both in vitro and in vivo. These compounds possess good selectivity for the catalytic site of PDE4 over the high affinity Rolipram binding site. In vivo studies demonstrate a reduced propensity to display the emetic side effects which are commonly observed with PDE4 inhibitors.

PDE4 inhibitors: New xanthine analogues

Novel xanthine analogues are described which are selective PDE4 inhibitors with improved therapeutic potential over theophylline.