David G. Trist

Pharmacological characterization of thromboxane and prostanoid receptors in human isolated urinary bladder

Cumulative concentration‐response curves (CRC) to prostaglandin E1 (PGE1), PGE2, PGD2 and PGF2α (0.01–30 μM) and to the thromboxane A2 (TXA2) receptor agonist U‐46619 (0.01–30 μM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation. All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF2α>U‐46619>PGE2 whereas weak contractile responses were obtained with PGD2 and PGE1. Any of the agonists tested was able to induce a clear plateau of response even at 30 μM. The selective TXA2 antagonist, GR 32191B (vapiprost), antagonized U‐46619‐induced contractions with an apparent pKB value of 8.27±0.12 (n=4 for each antagonist concentration). GR 32191B (0.3 μM) did not antagonize the contractile responses to PGF2α and it was a non‐surmountable antagonist of PGE2 (apparent pKB of 7.09±0.04; n=5). The EP receptor antagonist AH 6809 at 10 μM shifted to the right the CRC to U‐46619 (apparent pKB value of 5.88±0.04; n=4). Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 μM) and atropine (1 μM). U‐46619 (0.01–3 μM) potentiated the twitch contraction in a dose‐dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pKB of 8.54±0.14 (n=4 for each antagonist concentration). PGF2α in the range 0.01–10 μM (n=7), but not PGE2 and PGE1 (n=3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5±0.3% of KCl 100 mM‐induced contraction) but this potentiation was unaffected by 0.3 μM GR 32191B (n=5). Cumulative additions of U‐46619 (0.01–30 μM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 μM; n=3). Moreover, pretreatment of the tissue with 0.3 μM U‐46619 did not potentiate the smooth muscle response to 7 μM bethanecol (n=2). We concluded that TXA2 can induce direct contraction of human isolated urinary bladder through the classical TXA2 receptor. Prostanoid receptors, fully activated by PGE2 and PGF2α are also present. All these receptors are probably located post‐junctionally. The rank order of agonist potency and the fact that GR32191B, but not AH6809, antagonized responses to PGE2 seem to indicate the presence of a new EP receptor subtype. Moreover, we suggest the presence of prejuctional TXA2 and FP receptors, potentiating acetylcholine release from cholinergic nerve terminals.

Pharmacological analysis of 5-hydroxytryptamine effects on electrically stimulated human isolated urinary bladder

1 5‐Hydroxytryptamine (5‐HT) is able to potentiate the contractions induced by electrical field stimulation of pieces of human isolated urinary bladder. On the basis of available selective 5‐HT agonists and antagonists, we have further investigated the receptors involved and their site of action. 2 5‐HT produced a concentration‐dependent increase of the contractile response to electrical field stimulation from 0.1 nm to μm. At higher concentrations (up to 100 μm) the effect decreased. These activities were mimicked by a variety of 5‐HT agonists, for which the following rank order of potency was found: 5‐HT > α‐methyl 5‐HT > 5‐methoxytryptamine > 5‐carboxamidotryptamine > 2‐methyl 5‐HT ≫ GR 43175. In addition the gastro‐prokinetics agents metoclopramide, cisapride and the 5‐HT3 antagonist ICS 205–930 behaved as 5‐HT agonists, their EC50 values being 2.3, 0.3, and 0.5 (μm) respectively. 3 The 5‐HT potentiating effect was resistant to antagonism by ondansetron (1 μm) and cyanopindolol (1 μm), selective 5‐HT3 and 5‐HT1A/1B antagonists respectively. The 5‐HT2 antagonists ketanserin (1 μm), spiperone (1 μm) and methysergide (1 μm) also showed a weak inhibitory activity. Methiothepin (0.1–1 μm) antagonized only the inhibitory effect of 5‐HT. Metoclopramide (0.1–1 μm), cisapride (0.01–0.1 μm) and ICS 205–930 (0.3–3 μm) all produced a rightward displacement of the 5‐HT response curve with concomitant reduction of the maximum response. The pA2 values calculated were 7.4, 8.5 and 7.0 respectively. The antagonism of metoclopramide was receptor specific and was not apparently related to interactions with dopaminergic activity since domperidone showed no antagonism of 5‐HT, and metoclopramide, itself, did not antagonize the potentiating effect of prostaglandin F2α. 4 The receptor involved in the potentiating effect of 5‐HT may be located prejunctionally because 5‐HT did not potentiate responses to acetylcholine (ACh) or electrical field stimulation with the parameters of direct muscle excitation. Also, since the 5‐HT potentiating effect was blocked by atropine, it may be attributed to a release of ACh. 5 This study suggests that in the human urinary bladder 5‐HT causes two opposite effects on the contractile response to electrical field stimulation. A potentiating effect at low concentrations due to an interaction with an atypical receptor, different from the classical 5‐HT1, 5‐HT2 or 5‐HT3 subtypes and an inhibitory effect at greater concentrations probably due to an interaction with 5‐HT1‐like receptors. The possibility that this atypical receptor possesses some characteristics of those found in other isolated preparations like guinea‐pig ileum, rat oesophagus and mouse embryo colliculi neurones is discussed.

Differential Effects of the CRF-R1 Antagonist GSK876008 on Fear-Potentiated, Light- and CRF-Enhanced Startle Suggest Preferential Involvement in Sustained versus Phasic Threat Responses

The amplitude of the acoustic startle response is increased when elicited in the presence of brief cues that predict shock (fear-potentiated startle) and also when elicited during sustained exposure to bright light (light-enhanced startle). Although both effects are thought to reflect fear or anxiety, their neuroanatomical substrates differ. Although fear-potentiated startle is disrupted by reversible inactivation of the central nucleus of the amygdala (CeA) but not the closely related bed nucleus of the stria terminalis (BNST), light-enhanced startle is disrupted by BNST inactivation but not by CeA inactivation. Intraventricular infusions of corticotropin-releasing factor (CRF) also increase startle (CRF-enhanced startle) and this effect is mediated by CRF receptors within the BNST, with no involvement of the CeA. Together, these observations suggest that CeA- and BNST-dependent fear and anxiety may be differentially sensitive to CRF receptor blockade. We tested this by orally administering the novel, potent, and selective CRF-R1 antagonist GSK876008 to rats before CRF-enhanced, light-enhanced, or fear-potentiated startle testing. GSK876008 disrupted CRF-enhanced startle with a linear dose–response curve, and light-enhanced startle with a U-shaped dose–response curve, but did not disrupt fear-potentiated startle to a visual stimulus at any dose tested, and even augmented the response in some animals. GSK876008 also disrupted shock-related ‘baseline’ startle increases, which may have reflected context conditioning (shown elsewhere to also be BNST-dependent). Overall, these results suggest that short-duration CeA-dependent threat responses can be pharmacologically dissociated from longer duration BNST-dependent responses in terms of their sensitivity to CRF1 receptor antagonists.

Results from 2 randomized, double-blind, placebo-controlled studies of the novel NK1 receptor antagonist casopitant in patients with major depressive disorder.

Clinical study results for neurokinin (NK) receptor antagonists in the treatment of depression have been mixed, with Phase III studies failing to fulfill the early promise demonstrated in Phase II studies. Casopitant, a selective NK1 antagonist that achieves nearly complete receptor occupancy was studied in 2 randomized, placebo-controlled, double-blind, Phase II trials in depressed outpatients to test the hypothesis that nearly complete NK1 receptor occupancy is required to achieve antidepressant efficacy. Study 092 used an interactive voice response system to recruit depressed patients with baseline Hamilton Depression (17-item, HAMD17) total scores higher than 24 who were randomized to fixed-dose casopitant 30 mg/d, 80 mg/d, or placebo for 8 weeks (n = 356). Study 096 required Carroll Depression Scale-Revised self-assessment scores of higher than 24 for randomization to casopitant 120 mg/d, paroxetine 30 mg/d (both reached via forced titration), or placebo for 8 weeks (n = 362). In study 092, casopitant 80 mg but not 30 mg achieved statistically significant improvement versus placebo on the primary outcome measure, week 8 last observation carried forward change from baseline HAMD17 (difference = −2.7; 95% confidence interval, −5.1 to −0.4, P = 0.023). In study 096, neither casopitant nor paroxetine achieved statistical separation from placebo at end point on HAMD17 (casopitant difference = −1.7; 95% CI, −3.8 to 0.4, P = 0.282). Casopitant and paroxetine were generally well tolerated in most patients. These studies suggest that NK1 antagonists that have nearly complete receptor occupancy may be effective in the treatment of depression.

Discovery process and pharmacological characterization of 2-(S)-(4-fluoro-2-methylphenyl)piperazine-1-carboxylic acid [1-(R)-(3,5-bis-trifluoromethylphenyl)ethyl]methylamide (vestipitant) as a potent, selective, and orally active NK1 receptor antagonist.

In an effort to discover novel druglike NK(1) receptor antagonists a new series of suitably substituted C-phenylpiperazine derivatives was identified by an appropriate chemical exploration of related N-phenylpiperazine analogues, with the specific aim to maximize their in vitro affinity and optimize in parallel their pharmacokinetic profile. Among the compounds synthesized, 2-(S)-(4-fluoro-2-methylphenyl)piperazine-1-carboxylic acid [1-(R)-(3,5-bis-trifluoromethylphenyl)ethyl]methylamide (vestipitant) was identified as one of the most in vitro potent and selective NK(1) receptor antagonists ever discovered, showing appropriate pharmacokinetic properties and in vivo activity. On the basis of its preclinical profile, this compound was selected as a drug candidate.

Pharmacological analysis of carboxyphenylglycines at metabotropic glutamate receptors

Three carboxyphenylglycine derivatives were examined for their activity on glutamate metabotropic receptors negatively linked to adenylate cyclase. Chinese hamster ovary cells stably expressing mGlu2 and mGlu4 were utilised for this study. A receptor binding analysis was also performed for the main classes of glutamate ionotropic receptors and for the glycine binding site on the NMDA-receptor complex. In mGlu2 expressing cells (S)4-carboxy-3-hydroxyphenylglycine and (S)4-carboxy-phenylglycine antagonized forskolin-stimulated cAMP levels, with EC50 of 21 and 970 microM, respectively, acting as agonists at this receptor subtype, whereas (RS) alpha-methyl-4-carboxyphenylglycine antagonized glutamate response in these cells. None of these compounds showed any agonistic or antagonistic activity on mGlu4 expressing cells. No affinity for the ionotropic receptors (NMDA, AMPA and kainate) and for the glycine site of the NMDA-receptor complex was found using the receptor binding approach, except for (RS)4-carboxy-3-hydroxyphenylglycine which showed a pKi of 5.68 in ((+/-)2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding for NMDA receptor, although this can be ascribed to the (R) form of the racemic mixture.

Synthesis and pharmacological characterization of novel druglike corticotropin-releasing factor 1 antagonists.

To identify new CRF(1) receptor antagonists, an attempt to modify the bis-heterocycle moiety present in the top region of the dihydropyrrole[2,3]pyridine template was made following new pharmacophoric hypothesis on the CRF(1) receptor antagonists binding pocket. In particular, the 2-thiazole ring, present in the previous series of compounds, was replaced by more hydrophilic non aromatic heterocycles able to make appropriate H-bond interactions with amino acid residues Thr192 and Tyr195. This exploration, followed by an accurate analysis of the substitution of the pendant aryl ring, enabled to identify in vitro potent compounds showing excellent pharmacokinetics and outstanding in vivo activity in animal models of anxiety, both in rodents and primates.

Synthesis and pharmacological characterisation of 2,4-Dicarboxy-pyrroles as selective non-Competitive mGluR1 antagonists

Metabotropic glutamate receptors (mGluRs) are an unusual family of G-protein coupled receptor (GPCR), and are characterised by a large extracellular N-terminal domain that contains the glutamate binding site. We have identified a new class of non-competitive metabotropic glutamate receptor 1 (mGluR1) antagonists, 2,4-dicarboxy-pyrroles which are endowed with nanomolar potency. They interact within the 7 transmembrane (7TM) domain of the receptor and show antinociceptive properties when tested in a number of different animal models.

A selective neurokinin-1 receptor antagonist in chronic PTSD: A randomized, double-blind, placebo-controlled, proof-of-concept trial

The substance P-neurokinin-1 receptor (SP-NK(1)R) system has been extensively studied in experimental models of stress, fear, and reward. Elevated cerebrospinal fluid (CSF) SP levels were reported previously in combat-related PTSD. No medication specifically targeting this system has been tested in PTSD. This proof-of-concept randomized, double-blind, placebo-controlled trial evaluated the selective NK(1)R antagonist GR205171 in predominately civilian PTSD. Following a 2-week placebo lead-in, 39 outpatients with chronic PTSD and a Clinician-Administered PTSD Scale (CAPS) score ≥50 were randomized to a fixed dose of GR205171 (N=20) or placebo (N=19) for 8weeks. The primary endpoint was mean change from baseline to endpoint in the total CAPS score. Response rate (≥50% reduction in baseline CAPS) and safety/tolerability were secondary endpoints. CSF SP concentrations were measured in a subgroup of patients prior to randomization. There was significant improvement in the mean CAPS total score across all patients over time, but no significant difference was found between GR205171 and placebo. Likewise, there was no significant effect of drug on the proportion of responders [40% GR205171 versus 21% placebo (p=0.30)]. An exploratory analysis showed that GR205171 treatment was associated with significant improvement compared to placebo on the CAPS hyperarousal symptom cluster. GR205171 was well-tolerated, with no discontinuations due to adverse events. CSF SP concentrations were positively correlated with baseline CAPS severity. The selective NK(1)R antagonist GR205171 had fewer adverse effects but was not significantly superior to placebo in the short-term treatment of chronic PTSD. (ClinicalTrials.gov Identifier: NCT 00211861, NCT 00383786).

Carbon fibre micro-electrodes for concomitant in vivo electrophysiological and voltammetric measurements: no reciprocal influences

Differential pulse voltammetry and more recently cyclic voltammetry have been successfully used to monitor basal levels of endogenous chemicals by means of treated carbon fibre microbiosensors inserted in specific brain regions. In this study, feasibility of concomitant in vivo recordings of stable electrophysiological signals and basal ascorbate, catecholaminergic and indolaminergic voltammetric peaks at the same cerebral site by means of a single electrically treated carbon fibre micro electrode (microbiosensor) is presented. The results indicate that these two independent techniques can be combined in vivo at a single electrode, and that voltammetric measurements of unstimulated levels of extracellular compounds do not alter concomitant basal cell firing for a period long enough (more than 6 h) to allow pharmacological manipulations.