Martyn D. Wood

SB 242084, a selective and brain penetrant 5-HT2C receptor antagonist.

SB 242084 has a high affinity (pKi 9.0) for the cloned human 5-HT2C receptor and 100- and 158-fold selectivity over the closely related cloned human 5-HT2B and 5-HT2A subtypes respectively. SB 242084 had over 100-fold selectivity over a range of other 5-HT, dopamine and adrenergic receptors. In studies of 5-HT-stimulated phosphatidylinositol hydrolysis using SH-SY5Y cells stably expressing the cloned human 5-HT2C receptor, SB 242084 acted as an antagonist with a pKb of 9.3, which closely resembled its corresponding receptor binding affinity. SB 242084 potently inhibited m-chlorophenylpiperazine (mCPP, 7 mgkg i.p. 20 min pre-test)-induced hypolocomotion in rats, a model of in vivo central 5-HT2C receptor function, with an ID50 of 0.11 mg/kg i.p., and 2.0 mg/kg p.o. SB 242084 (0.1-1 mg/kg i.p.) exhibited an anxiolytic-like profile in the rat social interaction test, increasing time spent in social interaction, but having no effect on locomotion. SB 242084 (0.1-1 mg/kg i.p.) also markedly increased punished responding in a rat Geller-Seifter conflict test of anxiety, but had no consistent effect on unpunished responding. A large acute dose of SB 242084 (30 mg/kg p.o.) had no effect on seizure susceptibility in the rat maximal electroshock seizure threshold test. Also, while SB 242084 (2 and 6 mg/kg p.o. 1 hr pre-test) antagonized the hypophagic response to mCPP, neither acute nor subchronic administration of the drug, for 5 days at 2 or 6 mg/kg p.o. twice daily, affected food intake or weight gain. The results suggest that SB 242084 is the first reported selective potent and brain penetrant 5-HT2C receptor antagonist and has anxiolytic-like activity, but does not possess either proconvulsant or hyperphagic properties which are characteristic of mutant mice lacking the 5-HT2C receptor.

In vitro and in vivo profile of SB 206553, a potent 5‐HT2C/5‐HT2B receptor antagonist with anxiolytic‐like properties

1 SB 206553 (5‐methyl‐1‐(3‐pyridylcarbamoyl)‐1,2,3,5‐tetrahydropyrrolo[2,3‐f]indole) displays a high affinity (pK1 7.9) for the cloned human 5‐HT2C receptor expressed in HEK 293 cells and the 5‐HT2B receptor (pA2 8.9) as measured in the rat stomach fundus preparation. SB 206553 has low affinity for cloned human 5‐HT2A receptors expressed in HEK 293 cells (pK1 5.8) and (pK1 <6) for a wide variety of other neurotransmitter receptors. 2 SB 206553 appears to be a surmountable antagonist of 5‐HT‐stimulated phosphoinositide hydrolysis in HEK 293 cells expressing the human 5‐HT2C receptor (pKB 9.0). 3 The compound potently (ID50 5.5 mg kg−1, p.o., 0.27 mg kg−1, i.v.) inhibited the hypolocomotor response to m‐chlorophenylpiperazine (mCPP), a putative model of 5‐HT2C/5‐HT2B receptor function in vivo. 4 At similar doses (2–20 mg kg−1, p.o.) SB 206553 increased total interaction scores in a rat social interaction test and increased punished responding in a rat Geller‐Seifter conflict test. These effects are consistent with the possession of anxiolytic properties. 5 SB 206553 also increased suppressed responding in a marmoset conflict model of anxiety at somewhat higher doses (15 and 20 mg kg−1, p.o.) but also reduced unsuppressed responding. 6 These results suggest that SB 206553 is a potent mixed 5‐HT2C/5‐HT2B receptor antagonist with selectivity over the 5‐HT2A and all other sites studied and possesses anxiolytic‐like properties.

In vivo properties of SB 200646A, a 5-HT2C/2B receptor antagonist.

1 SB 200646A, N‐(1‐methyl‐5‐indolyl)‐N′‐(3‐pyridyl) urea hydrochloride, the first reported selective 5‐HT2C/2B over 5‐HT2A receptor antagonist, (pK1 rat 5‐HT2C receptor 6.9, pA2 rat 5‐HT2B receptor 7.5, pKi rat 5‐HT2A receptor 5.2) dose‐dependently blocked a putative rat model of 5‐HT2C receptor activation; 1‐(3‐chlorophenyl)piperazine (mCPP, 5 mg kg−1, i.p. 20 min pretest)‐induced hypolocomotion (estimated ID50 19.2 mg kg−1, p.o.). 2 SB 200646A also blocked another putative in vivo model of 5‐HT2C receptor function; mCPP (5 mg kg−1, i.p. 20 min pretest)‐induced hypophagia in 23 h food‐deprived rats (estimated ID50 18.3 mg kg−1, p.o.). 3 SB 200646A did not antagonize 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane (DOI)‐induced head shakes in rats at doses up to 200 mg kg−1, p.o., an effect thought to be mediated by 5‐HT2A receptors for which SB 200646A has its next highest affinity (50 fold less) after the 5‐HT2C and 5‐HT2B sites. 4 SB 200646A (20, 40 mg kg−1, p.o., 1 h pretest) also reversed mCPP (0.5 mg kg−1, i.p., 30 min pretest)‐induced anxiety in the social interaction test, under low light familiar conditions. 5 When given alone, under high light unfamiliar conditions, SB 200646A (2–40 mg kg−1, p.o.) increased active social interaction without affecting locomotor activity in the rat social interaction test. This is consistent with an anxiolytic action of SB 200646A. 6 These results indicate that SB 200646A has in vivo efficacy and that 5‐HT2C or 5‐HT2B receptors are indeed likely to mediate mCPP‐induced hypolocomotion, hypophagia and anxiogenesis. They also suggest that 5‐HT2C/2B receptor blockade induces anxiolysis.

Gabapentin is not a GABAB receptor agonist.

Recent experiments have demonstrated that formation of functional type B gamma-aminobutyric acid (GABA(B)) receptors requires co-expression of two receptor subunits, GABA(B1) and GABA(B2). Despite the identification of these subunits and a number of associated splice variants, there has been little convincing evidence of pharmacological diversity between GABA(B) receptors comprising different subunit combinations. However, Ng et al. [Mol. Pharmacol., 59 (2000) 144] have recently suggested a novel and important pharmacological difference between GABA(B) receptor heterodimers expressing the GABA(B1a) and GABA(B1b) receptor subunits. This study suggested that the antiepileptic GABA analogue gabapentin (Neurontin) is an agonist at GABA(B) receptors expressing the GABA(B1a) but not the GABA(B1b) receptor subunit. The importance of this finding with respect to identifying novel GABA(B) receptor subunit specific agonists prompted us to repeat these experiments in our own [35S]-GTPgammaS binding and second messenger assay systems. Here we report that gabapentin was completely inactive at recombinant GABA(B) heterodimers expressing either GABA(B1a) or GABA(B1b) receptor subunits in combination with GABA(B2) receptor subunits. In addition, in both CA1 and CA3 pyramidal neurones from rodent hippocampal slices we were unable to demonstrate any agonist-like effects of gabapentin at either pre- or post-synaptic GABA(B) receptors. In contrast, gabapentin activated a GABA(A) receptor mediated chloride conductance. Our data suggest that gabapentin is not a GABA(B)-receptor agonist let alone a GABA(B) receptor subunit selective agonist.

Structure-function studies of allosteric agonism at M2 muscarinic acetylcholine receptors

The M2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues 172EDGE175, Tyr177, and Thr423. However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M2 mAChRs in which the charge of the 172EDGE175 sequence had been neutralized and each Tyr177 and Thr423 was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide (C7/3-phth) but minimal effects on the orthosteric antagonist [3H]N-methyl scopolamine. In contrast, the allosteric agonists 4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride (McN-A-343), 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), and the novel AC-42 derivative 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) demonstrated an increased affinity or proportion of high-affinity sites at the combined EDGE-YT mutation, indicating a different mode of binding to the prototypical modulators. Subsequent functional assays of extracellular signal-regulated kinase (ERK)1/2 phosphorylation and guanosine 5′-(γ-[35S]thio)triphosphate ([35S]GTPγS) binding revealed minimal effects of the mutations on the orthosteric agonists acetylcholine (ACh) and pilocarpine but a significant increase in the efficacy of McN-A-343 and potency of 77-LH-28-1. Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr177 and Thr423, rather than the 172EDGE175 sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M2 mAChRs.

Characterization of a CNS penetrant, selective M1 muscarinic receptor agonist, 77-LH-28-1

M1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimers disease and schizophrenia. However, the discovery of subtype‐selective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh‐binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC‐42 (4‐n‐butyl‐1‐[4‐(2‐methylphenyl)‐4‐oxo‐1‐butyl]‐piperidine), which bind to an allosteric site and selectively activate the M1 mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs.

SB-243213; a selective 5-HT2C receptor inverse agonist with improved anxiolytic profile: lack of tolerance and withdrawal anxiety

SB-243213 (5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-trifluoromethylindoline hydrochloride) is a new, selective 5-hydroxytryptamine (5-HT)2C receptor inverse agonist. SB-243213 has high affinity for the human 5-HT2C receptor (pK(i) 9.37) and greater than a 100-fold selectivity over a wide range of neurotransmitter receptors, enzymes and ion channels. In in vitro functional studies, SB-243213 acted as an inverse agonist at the human 5-HT2C receptor with a pK(b) of 9.8. In in vivo studies, SB-243213 was a potent inhibitor of central 5-HT2C receptor-mediated function in rats, blocking meta-chlorophenylpiperazine-induced hypolocomotion with an ID50 of 1.1 mg/kg p.o. and a long duration of action (>8 h). In rats, SB-243213 exhibited anxiolytic-like activity in both the social interaction and Geller-Seifter conflict tests. Importantly, unlike diazepam, chronic administration of SB-243213 did not result in the development of either tolerance to the anxiolytic-like effects or withdrawal anxiogenesis. Furthermore, in rodents, SB-243213 did not affect seizure threshold, did not increase body weight or induce catalepsy, but attenuated the haloperidol-induced catalepsy. SB-243213 did not affect amphetamine-, MK-801- or phencyclidine-induced hyperactivity. In conclusion, SB-243213 may possess an improved anxiolytic profile compared to benzodiazepines. SB-243213 also modulates dopaminergic transmission, lacks pro-psychotic properties and may have utility in the treatment of schizophrenia and motor disorders.

Pharmacological characterisation of human 5-HT2 receptor subtypes.

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

Probing the Molecular Mechanism of Interaction between 4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42) and the Muscarinic M1 Receptor: Direct Pharmacological Evidence That AC-42 Is an Allosteric Agonist

4-n-Butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride (AC-42) is a selective agonist of the muscarinic M1 receptor previously suggested to interact with an “ectopic” site on this receptor. However, the pharmacological properties of this site (i.e., whether it overlaps to any extent with the classic orthosteric site or represents a novel allosteric site) remain undetermined. In the present study, atropine or pirenzepine significantly inhibited the ability of either carbachol or AC-42 to stimulate inositol phosphate accumulation or intracellular calcium mobilization in Chinese hamster ovary (CHO) cells stably expressing the human M1 receptor. However, the interaction between either of these antagonists and AC-42 was characterized by Schild slopes significantly less than unity. Increasing the concentrations of atropine revealed that the Schild regression was curvilinear, consistent with a negative allosteric interaction. More direct evidence for an allosteric mode of action of AC-42 was obtained in [3H]N-methylscopolamine ([3H]NMS) binding studies, in that both AC-42 and the prototypical modulator gallamine failed to fully inhibit specific [3H]NMS binding in a manner that was quantitatively described by an allosteric model applied to both modulator data sets. Furthermore, AC-42 and gallamine significantly retarded the rate of [3H]NMS dissociation from CHO-hM1 cell membranes, conclusively demonstrating their ability to bind to a topographically distinct site to change M1 receptor conformation. These data provide the first direct evidence that AC-42 is an allosteric agonist that activates M1 receptors in the absence of the orthosteric agonist.

Identification of Multiple Binding Sites for [3H]5-Hydroxytryptamine in the Rat CNS

Abstract: Recent studies indicate that there may be multiple subtypes of [3H]5‐hydroxytryptamine ([3H]5‐HT) binding sites. Mianserin and spiperone inhibited the specific binding of [3H]5‐HT (2–3 nM) to rat brain cortical membranes with shallow displacement curves. The displacement data for spiperone were best described by the presence of three independent binding sites, for which spiperone had high, medium, and low affinities. The displacement data for mianserin were best fitted by two independent, high‐ and low‐affinity sites. The inclusion of mianserin (250 nM) to inhibit [3H]5‐HT binding to the mianserin‐sensitive site selectively blocked one of the sites discriminated by spiperone. These results suggest the presence of three binding sites for [3H]]5‐HT. one blocked by low concentrations of spiperone (5‐HT1A), one blocked by low concentrations of mianserin (5‐HT1C), and one blocked only by high concentrations of both mianserin and spiperone (5‐HT1B). Regional differences in the relative densities of the three sites were observed. The hippocampus was rich in 5‐HT1A sites, whereas the striatum contained mainly 5‐HT1B and 5‐HT1C sites. Selective degeneration of 5‐HT‐containing nerve terminals induced by the neurotoxin 5,7‐dihydroxytryptamine increased binding to all three sites in the cerebral cortex. Binding of [3H]5‐HT to the three sites was differentially modulated by CaCl2 and guanylimidodiphosphate. The present data suggest the presence of three independent 5‐HT1 binding sites having different affinities for mianserin and spiperone and having different regional distributions.