Jacqueline A.M. Smith

The role of IP prostanoid receptors in inflammatory pain

Prostanoid receptor-mediated sensitization of sensory nerve fibres is a key contributor to the generation of hyperalgesia. It is generally thought that prostaglandin (PG) E2 is the principal pro-inflammatory prostanoid. Consequently, prostanoid EP receptors on sensory neurones have been identified as potential therapeutic targets. However, IP prostanoid receptors are also present on sensory neurones, and recent data from transgenic mice lacking the IP receptor demonstrate its importance in the induction of oedema and pain behaviour. PGI2, the primary endogenous agonist for the IP receptor, is rapidly produced following tissue injury or inflammation; thus, it may be of equal, or greater, importance than PGE2 during episodes of inflammatory pain. In this review, Keith Bley, John Hunter, Richard Eglen and Jacqueline Smith compare the roles of EP and IP receptors in nociception and suggest that the IP receptor constitutes a novel target for anti-nociceptive agents.

The 5-HT4 receptor agonist, tegaserod, is a potent 5-HT2B receptor antagonist in vitro and in vivo

Tegaserod (Zelnorm®) is a potent 5‐hydroxytryptamine4 (5‐HT4) receptor agonist with clinical efficacy in disorders associated with reduced gastrointestinal motility and transit. The present study investigated the interaction of tegaserod with 5‐HT2 receptors, and compared its potency in this respect to its 5‐HT4 receptor agonist activity. Tegaserod had significant binding affinity for human recombinant 5‐HT2A, 5‐HT2B and 5‐HT2C receptors (pKi=7.5, 8.4 and 7.0, respectively). The 5‐HT2B receptor‐binding affinity of tegaserod was identical to that at human recombinant 5‐HT4(c) receptors (mean pKi=8.4) in human embryonic kidney‐293 (HEK‐293) cells stably transfected with the human 5‐HT4(c) receptor. Tegaserod (0.1–3 μM) inhibited 5‐HT‐mediated contraction of the rat isolated stomach fundus potently (pA2=8.3), consistent with 5‐HT2B receptor antagonist activity. Tegaserod produced, with similar potency, an elevation of adenosine 3′,5′ cyclic monophosphate in HEK‐293 cells stably transfected with the human 5‐HT4(c) receptor (mean pEC50=8.6), as well as 5‐HT4 receptor‐mediated relaxation of the rat isolated oesophagus (mean pEC50=8.2) and contraction of the guinea‐pig isolated colon (mean pEC50=8.3). Following subcutaneous administration, tegaserod (0.3 or 1 mg kg−1) inhibited contractions of the stomach fundus in anaesthetized rats in response to intravenous dosing of α‐methyl 5‐HT (0.03 mg kg−1) and BW 723C86 (0.3 mg kg−1), selective 5‐HT2B receptor agonists. At similar doses, tegaserod (1 and 3 mg kg−1 subcutaneously) evoked a 5‐HT4 receptor‐mediated increase in colonic transit in conscious guinea‐pigs. The data from this study indicate that tegaserod antagonizes 5‐HT2B receptors at concentrations similar to those that activate 5‐HT4 receptors. It remains to be determined whether this 5‐HT2B receptor antagonist activity of tegaserod contributes to its clinical profile.

Increased acid-sensing ion channel ASIC-3 in inflamed human intestine.

Objectives Acid-sensing ion channels (ASICs) are expressed by rat sensory neurons and may mediate pain associated with tissue acidosis after inflammation or injury. Our aim was to examine the molecular forms and localization of ASICs in human intestine and dorsal root ganglia using immunochemical techniques, and to measure the effects of inflammation and injury. Design and methods Inflamed Crohns disease intestine and injured human dorsal root ganglia, with appropriate controls, were studied by Western blotting and immunohistochemistry, using specific affinity-purified ASIC antibodies. Results In the Western blot, there was a significant three-fold increase in the mean relative optical density of the ASIC-3 55-kDa band (but not ASIC-1 or ASIC-2) in full-thickness inflamed intestine, as well as in separated muscle and mucosal layers. There was a corresponding trend for an increased immunoreactive density and increased number of ASIC-3-positive neurons in the myenteric and sub-mucous plexus of inflamed intestine. In dorsal root ganglia, immunoreactivity for all ASICs was restricted to a sub-population (about 50%) of small-diameter (nociceptor) sensory neurons, and was generally less intense after injury. Conclusions Increased ASIC-3 in inflamed intestine suggests a role in pain or dysmotility, for which ASICs represent new therapeutic targets.

Characterization of prostanoid receptor-evoked responses in rat sensory neurones.

Prostanoid receptor‐mediated sensitization, or excitation, of sensory nerve fibres contributes to the generation of hyperalgesia. To characterize the prostanoid receptors present on sensory neurones, biochemical assays were performed on primary cultures of adult rat dorsal root ganglia (DRG) and the F‐11 (embryonic rat DRG×neuroblastoma hybrid) cell line. In DRG cultures, the IP receptor agonists, cicaprost and carbaprostacyclin (cPGI2) stimulated cyclic AMP accumulation. Prostaglandin E2 (PGE2) also increased cyclic AMP levels, but to a lesser extent, while carbocyclic thromboxane A2 (cTxA2), PGD2 and PGF2α had negligible effects. The rank order of agonist potency was cicaprost >PGE2=BMY45778=cPGI2=PGI2. In the F‐11 cells, the rank order of agonist potency for the stimulation of cyclic AMP accumulation was: cicaprost>iloprost=cPGI2=PGI2=BMY45778>PGE2=cTXA2. In DRG cultures, cicaprost induced significantly more accumulation of inositol phosphates than PGE2. To examine the effects of prostanoids on C‐fibre activity, extracellular recordings of d.c. potentials from the rat isolated vagus nerve were made with the ‘grease‐gap’ technique. PGI2 (0.1 nM–10 μM) produced the largest depolarizations of the nerve. The rank order of agonist potency was: PGI2=cPGI2=PGE1>cTXA2>PGE2=PGD2=TXB2>PGF2α. Prior depolarization of nerves with either forskolin (10 μM) or phorbol dibutyrate (1 μM) alone significantly reduced the response to PGI2 (10 μM), while simultaneous application of both forskolin and phorbol dibutyrate attenuated PGI2 responses almost completely. Putative EP1 and/or TP receptor‐selective antagonists had no effect on the responses to PGI2, cPGI2 or PGE2 in the three preparations studied. Collectively, these data are consistent with a positive coupling of IP receptors to both adenylyl cyclase and phospholipase C in sensory neurones. These findings suggest that IP receptors play a major role in the sensitization of rat sensory neurones.

A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M2 Muscarinic Receptor

THRX-160209 is a potent antagonist at the M2 muscarinic acetylcholine (ACh) receptor subtype that was designed using a multivalent strategy, simultaneously targeting the orthosteric site and a nearby site known to bind allosteric ligands. In this report, we describe three characteristics of THRX-160209 binding that are consistent with a multivalent interaction: 1) an apparent affinity of the multivalent ligand for the M2 receptor subtype (apparent pKI = 9.51 ± 0.22) that was several orders of magnitude greater than its two monovalent components (apparent pKI values < 6.0), 2) specificity of THRX-160209 for the M2 receptor subtype compared with the closely related M4 (apparent pKI = 8.78 ± 0.24) and M1,M3, and M5 receptors (apparent pKI values ≤ 8.0), and 3) acceleration (>10-fold) of the dissociation rate of tritium-labeled THRX-160209 from M2 receptors by competing monovalent ligands that are known to interact with either the orthosteric site (e.g., atropine) or a well characterized allosteric site (e.g., obidoxime) on the receptor. In complementary kinetic studies assessing allosteric modulation of the receptor, unlabeled THRX-160209 retarded dissociation of [3H]N-methyl scopolamine (NMS). The effects of THRX-160209 on retardation of [3H]NMS dissociation were competitively inhibited by obidoxime, suggesting that obidoxime and THRX-160209 bind to an overlapping region coincident with other typical muscarinic allosteric agents, such as 3-methyl-5-[7-[4-[(4S)-4-methyl-1,3-oxazolidin-2-yl]phenoxy]heptyl]-1,2-oxazole (W84) and gallamine. Taken together, these data are consistent with the hypothesis that THRX-160209 binds in a multivalent manner to the M2 receptor, simultaneously occupying the orthosteric site and a spatially distinct allosteric site.

THRX-198321 is a bifunctional muscarinic receptor antagonist and β2-adrenoceptor agonist (MABA) that binds in a bimodal and multivalent manner

Biphenyl-2-yl-carbamic acid 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)-ethylamino]-nonyl}-piperidin-4-yl ester (THRX-198321) is a single molecule composed of a muscarinic acetylcholine receptor (mAChR) antagonist moiety, represented by the fragment MA, linked by a C9 polymethylene chain to a β2-adrenoceptor (β2AR) agonist moiety, represented by the fragment 8-hydroxy-5-((R)-1-hydroxy-2-methylamino-ethyl)-1H-quinolin-2-one (BA). THRX-198321 exhibited high affinity for mAChR (M2 pKI,App = 10.57 ± 0.09; M3 pKI,App = 10.07 ± 0.11) and β2AR (pKI,App = 9.54 ± 0.15), with potent mAChR antagonist (M2 pKI,Fn = 9.69 ± 0.23; M3 pKI,Fn = 10.05 ± 0.17) and β2AR agonist (pEC50 = 9.25 ± 0.02) activities. Consistent with multivalent interactions, THRX-198321 binding affinity was >300-fold higher at mAChR and 29-fold higher at β2AR relative to its monovalent fragments biphenyl carbamic acid piperidinyl ester (MA) and BA, respectively. THRX-198321 was a competitive antagonist at mAChR (M2 pKB = 9.98 ± 0.13; M3 pKB = 10.31 ± 0.89), whereas THRX-198321 agonist activity at β2AR was competitively inhibited by propranolol. Interactions of THRX-198321 with an allosteric site on mAChR and a novel extracellular allosteric site on β2AR, respectively, were detected by measuring THRX-198321-evoked changes in the dissociation rates for the orthosteric radioligands, [N-methyl-3H]scopolamine methyl chloride (M2 pEC50,diss = 6.73 ± 0.10; M3 pEC50,diss = 5.02 ± 0.14) and [4,6-propyl-3H]dihydroalprenolol (β2AR pEC50,diss = 3.82 ± 0.25). The carbostyril-linker fragment (BA-L) binds to the allosteric site of mAChR (M2 pEC50,diss = 5.06 ± 0.03; M3 pEC50,diss = 4.15 ± 0.25), whereas the MA fragment binds to the allosteric site of β2AR (pEC50,diss = 3.60 ± 0.18). Collectively, these observations suggest that THRX-198321 exhibits a multivalent bimodal orientation in the orthosteric and allosteric binding pockets of mAChR and β2AR, a phenomenon that may be unique to this class of molecule.

P2Y receptor regulation of cultured rat cerebral cortical cells: calcium responses and mRNA expression in neurons and glia.

We have investigated increases in cytosolic Ca2+ in response to nucleotides in mixed rat cerebrocortical cultures (neurons and glia in similar numbers) and in essentially neuron‐free glial cultures. In both cultures, the agonist‐response profile was 2‐methylthioADP(2MeSADP)>2‐methylthioATP(2MeSATP)>ADP>ATP>adenosine 5′‐O‐(3‐thiotriphosphate), consistent with a P2Y1 receptor. The maximal responses to 2MeSADP, 2MeSATP and ADP were identical, but that to ATP was higher. Suramin, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, reactive blue 2 (RB2), and adenosine biphosphate (A3P5P) were antagonists with apparent pA2 values of 5.5 for suramin, 6.4 for RB2, and 4.7 for A3P5P. Single cell imaging divided the cells from the mixed neuronal–glial cultures into two populations: responsive (neurons) and unresponsive (glial cells) to high [K+]. The response of cells to nucleotides was almost exclusively limited to those not responsive to high K+. In the presence of extracellular Mn2+, the response of the mixed cultures to 30 mM K+ and 20 μM Bay K 8644 was attenuated. However, when 2MeSADP was added there was no reduction in response in cultures previously loaded with Mn2+. This further indicated that the 2MeSADP response was not in the neurons. Reverse transcriptase–polymerase chain reaction studies detected transcripts for P2Y1, P2Y4 and P2Y6 in RNA preparations from embryonic rat cortex, and from both mixed and glial cultures. P2Y2 transcripts were not detected in the embryonic cortex. Based on this and previous work, it is proposed that the principal P2Y influences in the brain are on cytosolic Ca2+ in glial cells and presynaptic sites on neurons.

Influence of ligand binding kinetics on functional inhibition of human recombinant serotonin and norepinephrine transporters.

INTRODUCTION Monoamine reuptake inhibitors treat a wide range of CNS disorders, including depression, obesity, and pain. The in vitro pharmacological properties of these inhibitors are determined routinely using radioligand binding and/or neurotransmitter uptake assays. Measurements from such studies can be influenced by assay design and ligand-specific characteristics, both of which may contribute to discrepancies in literature reports. METHODS We modified traditional methodologies to identify and account for factors that can confound in vitro potency determinations. Apparent equilibrium binding affinities (pK(i) values) were determined in either HEK293 cells stably-transfected with human recombinant serotonin (SERT) or norepinephrine (NET) transporters, or membranes prepared from these cell lines. Care was taken to ensure that apparent affinities were measured under conditions that minimized ligand depletion and established equilibrium for both the radioligand and the compound of interest. An unlabelled ligand kinetic method was used to approximate inhibitor binding kinetic constants and corresponding dissociation half lives. To measure inhibitory effects on substrate uptake, both radiolabeled neurotransmitter ([(3)H]-5-HT or [(3)H]-NE) and fluorescence-based assays were used. The time-dependent nature of functional inhibition was examined using a fluorescent substrate uptake assay which provided real-time measurements of NET and SERT function. RESULTS SERT and NET inhibitors displayed a range of affinities, potencies, and inhibition modes by binding and functional uptake assays. Binding kinetic profiles for this panel of inhibitors were diverse, and affected in vitro measures using the former techniques. DISCUSSION In the present study we describe key features of in vitro assay methodology that can influence the apparent pharmacological profiles of standard SERT and/or NET inhibitors. Such information can serve as a foundation for understanding the in vitro profiles of monoamine reuptake inhibitors in the context of their clinical efficacy and tolerability.

Prediction of Human Serotonin and Norepinephrine Transporter Occupancy of Duloxetine by Pharmacokinetic/Pharmacodynamic Modeling in the Rat

Translation of central nervous system occupancy and clinical effect from animal models to humans has remained elusive for many pharmacological targets. The current studies evaluate the ability of a rodent pharmacokinetic/pharmacodynamic (PK/PD) modeling approach to translate ex vivo occupancy determined in rats to that observed after positron emission tomography (PET) imaging in humans for the dual serotonin transporter (SERT) and norepinephrine transporter (NET) inhibitor duloxetine. Ex vivo transporter occupancy in rat spinal cord was evaluated after single oral doses of 0.3 to 60 mg/kg. A novel methodology, based on the initial rates of association of transporter selective radioligands to tissue homogenates, was developed and validated for the assessment of ex vivo transporter occupancy. Duloxetine exhibited selectivity for occupancy of SERT over NET in rat spinal cord with ED50 values of 1 and 9 mg/kg, respectively. Corresponding EC50 values for the inhibition of SERT and NET based on unbound duloxetine spinal cord concentrations were 0.5 and 8 nM. An effect compartment PK/PD modeling approach was used to analyze the relationship between the time course of duloxetine plasma concentration and SERT and NET occupancy. Duloxetine inhibited SERT and NET in rat spinal cord with a plasma EC50 of 2.95 and 59.0 ng/ml, respectively. Similar plasma EC50 values for the inhibition of SERT (2.29–3.7 ng/ml) have been reported from human PET studies. This study illustrates the value of translational PK/PD modeling approaches and suggests that the preclinical modeling approach used in the current study is capable of predicting plasma concentrations associated with 50% occupancy of SERT in the human central nervous system.

A comparison of the pharmacological properties of guinea-pig and human recombinant 5-HT4 receptors

5‐HT4 receptor agonists are used therapeutically to treat disorders of reduced gastrointestinal motility. Since such compounds are evaluated in guinea‐pigs, we cloned, expressed and pharmacologically characterized the guinea‐pig 5‐HT4 and human 5‐HT4(b) splice variant, which share 95% homology. The functional properties of guinea‐pig 5‐HT4(b) receptors were compared with native receptors in guinea‐pig colon.