Sathapana Kongsamut

Iloperidone binding to human and rat dopamine and 5-HT receptors.

Iloperidone (HP 873; 1-[4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidinyl]propoxy] -3- methoxyphenyl]ethanone) is a compound currently in clinical trials for the treatment of schizophrenia. Iloperidone displays affinity for dopamine D2 receptors and for 5-HT2A receptors and has a variety of in vivo activities suggestive of an atypical antipsychotic. Here we present an examination of the affinity of iloperidone to a variety of human and rat homologs of dopamine and 5-HT receptor subtypes. We employed receptor binding assays using membranes from cells stably expressing human dopamine D1, D2S, D2L, D3, D4 and D5 and 5-HT2A and 5-HT2C receptors and rat 5-HT6 and 5-HT7 receptors. Iloperidone displayed higher affinity for the dopamine D3 receptor (Ki = 7.1 nM) than for the dopamine D4 receptor (Ki = 25 nM). Iloperidone displayed high affinity for the 5-HT6 and 5-HT7 receptors (Ki = 42.7 and 21.6 nM, respectively), and was found to have higher affinity for the 5-HT2A (Ki = 5.6 nM) than for the 5-HT2C receptor (Ki = 42.8 nM). The potential implications of this receptor binding profile are discussed in comparison with data for other antipsychotic compounds.

Animal models with potential applications for screening compounds for the treatment of obsessive-compulsive disorder

The availability of an animal model for obsessive-compulsive disorder (OCD) is necessary for the development of novel pharmacological treatments. To be useful, the model must be predictive of clinical performance, possess characteristic criteria and distinguish anti-OCD from antidepressant compounds. Due to the lack of OCD models useful for drug discovery, all compounds currently used for OCD were developed first as antidepressants. In this article, we discuss the relative merits of: stereotypic behaviours (canine acral lick, feather picking, amphetamine- and 5-HT-induced stereotypy); adjunctive and displacement behaviours (schedule-induced polydipsia, wheel running, resident-intruder grooming); anxiolytic tests (separation and shock-induced ultrasonic vocalisation and marble burying); and depression tests (inescapable shock-induced escape and immobility in forced swim) as potential OCD models. We conclude that adjunctive and displacement behaviours, and in particular schedule-induced polydipsia, may prove to be the best models for compulsive behaviour in animals that can be used for the discovery of novel anti-OCD agents.

Effectiveness of GABAB antagonists in inhibiting baclofen‐induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat

1 The purpose of the present experiments was to assess the activities of GABAB receptor antagonists in mammalian isolated melanotrophs. 2 Cytosolic free Ca concentration ([Ca2+]i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura‐2. 3 (−)‐Baclofen lowered [Ca2+]i in a concentration‐dependent manner with an EC50 of 0.96 μm. The reduction in [Ca2+]i produced by (−)‐baclofen at a maximally effective concentration (100 μm) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nm), or by perifusion with a nominally Ca‐free solution. 4 The GABAB receptor antagonists, 3‐aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2‐hydroxy saclofen, phaclofen and 4‐amino‐3‐(5‐methoxybenzo[b]furan‐2‐yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca2+]i produced by (−)‐baclofen (3 μm). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 μm, compared to 120 to 400 μm for the others. CGP 35348 acted competitively. 5 CGP 35348 alone had no effect on basal [Ca2+]i, or on the changes in [Ca2+]i produced by dopamine (10 nm) or the specific GABAA receptor agonist, muscimol (10 μm). 6 The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABAB receptors in melanotrophs.

Why Are Several Inhibitory Transmitters Present in the Innervation of Pituitary Melanotrophs

The aim of the present experiments was to determine whether some rationale for the presence of the several inhibitory neurotransmitters in the innervation to the toad melanotroph might be found in differences in their individual effects or in possible cooperative interactions affecting secretion. Measurements of peptide release from isolated, perifused neurointermediate lobes of the toad Xenopus laevis showed that each of the three identified inhibitory transmitters, dopamine, GABA and NPY, was able to inhibit secretion profoundly and no less effectively than omission of Ca. Moreover, the inhibitory effects were rather similar in onset, duration and recovery. Furthermore, there was no evidence of any cooperative interactions when the transmitters were given in various combinations. And finally, the inhibitory response to each of the transmitters was abolished by pretreatment with pertussis toxin. While not excluding differential postsynaptic effects on other parameters of melanotroph function, the similarities observed have encouraged alternative speculations on the significance of the apparent redundancy of inhibitory transmitters.

Effects of besipirdine at the voltage‐dependent sodium channel

1 Besipirdine (HP 749) is a compound undergoing clinical trials for efficacy in treating Alzheimers disease. Among other pharmacological effects, besipirdine inhibits voltage‐dependent sodium and potassium channels. This paper presents a pharmacological study of the interaction of besipirdine with voltage‐dependent sodium channels. 2 Besipirdine inhibited [3H]‐batrachotoxin binding (IC50 = 5.5±0.2 μm) in a rat brain vesicular preparation and concentration‐dependently inhibited veratridine (25 μm)‐stimulated increases in intracellular free sodium ([Na+]i) and calcium ([Ca2+]i) in primary cultured cortical neurones of rat. 3 Besipirdine (30–100 μm) concentration‐dependently inhibited (up to 100%) veratridine‐stimulated release of [3H]‐noradrenaline (NA) from rat cortical slices. 4 When examined in greater detail, besipirdine was found to inhibit [3H]‐batrachotoxin binding in vesicular membranes competitively. However, when examined in rat brain synaptosomes, we found that the antagonism by besipirdine was not competitive; that is, the maximal stimulation of [Ca2+]i induced by veratridine decreased with increasing concentrations of besipirdine. 5 These results show that besipirdine is an inhibitor of voltage‐sensitive sodium channels and appears to bind to a site close to the batrachotoxin/veratridine binding site.

Studies on pituitary melanotrophs reveal the novel GABAB antagonist CGP 35-348 to be the first such compound effective on endocrine cells

One obstacle to understanding the action and physiological significance of the responsiveness of various endocrine cells to γ-aminobutyric acid (GABA) has been that previously available substances, all active as GABAB antagonists in the nervous system, are ineffective on endocrine cells. The introduction of a potent new member of this class, CGP 35-348, of very different chemical structure, encouraged us to examine its effect on endocrine cells. For this purpose, we studied melanotroph secretion from pituitary neurointermediate lobes. We found that CGP 35-348, in contrast to previously available members of this class, suppressed completely, in rat and toad, secretory responses to baclofen, the classic GABAB agonist. Analysis, in toad, showed CGP 35-348 did not affect responses to GABAA agonists (muscimol; isoguvacine), dopamine, or neuropeptide Y. When tested against GABA, the physiological ligand present in the innervation of melanotrophs (along with dopamine and neuropeptide Y), CGP 35-348 completely suppressed the secretory response, which, in toad, is purely inhibitory and unaffected by bicuculline, the specific GABAA antagonist. In addition, CGP 35-348 unmasked a stimulant effect that bicuculline blocked. In CGP 35-348, we thus have a new tool with which to analyse responses to GABA and their physiological involvement in endocrine cells.

Both GABAA and GABAB receptors participate in suppression of [Ca2+]i pulsing in toad melanotrophs

The receptor mechanisms involved in the inhibitory effect of gamma-aminobutyric acid (GABA) in suppressing spontaneous [Ca2+]i pulsing in melanotrophs of Xenopus laevis were investigated. The selective GABAB receptor agonist, baclofen reversibly arrested [Ca2+]i pulsing. This inhibition was unaffected by the selective GABAA receptor antagonist, bicuculline methiodide, but was blocked by the selective GABAB receptor antagonist, CGP 35348 (3-aminopropyl diethyoxymethyl phosphinic acid). The selective GABAA receptor agonist, muscimol, also arrested [Ca2+]i pulsing after causing a transient rise in [Ca2+]i. This biphasic response to muscimol was unaffected by CGP 35348, but was blocked by bicuculline. The inhibitory effect of GABA was unaffected by either CGP 35348 or bicuculline when given alone, but was blocked by both antagonists given together. In cells pretreated with pertussis toxin, the response to baclofen was completely lost, whereas responses to GABA and muscimol persisted; the response to GABA was blocked by bicuculline alone. Thus, both GABAA and GABAB receptors are involved in the inhibitory effect of GABA in suppressing spontaneous [Ca2+]i pulsing in Xenopus melanotrophs.

Changes in paroxetine binding in the cerebral cortex of polydipsic rats.

Schedule-induced polydipsia was induced when food-deprived rats were subjected to a fixed-time (60 s) feeding schedule for 150 min daily for 3 weeks (training period). Subsequent chronic administration of the serotonin reuptake inhibitor fluoxetine reduces schedule-induced polydipsia over 2-4 weeks. We asked whether changes in the serotonin reuptake carrier occur following the development of schedule-induced polydipsia and its reduction by fluoxetine. Using [3H]paroxetine binding, we found a 40% increase in Kd and a 50% decrease in Bmax in polydipsic rats; both were reversed by fluoxetine. Food deprivation alone did not affect these parameters. These observations suggest that changes in the serotonin reuptake carrier correlate with the development and reversal of schedule-induced polydipsia.

In Vitro electrophysiological activity of nerispirdine, a novel 4-aminopyridine derivative

1 The non‐selective K+ channel blocker 4‐aminopyridine (4‐AP) has shown clinical efficacy in the treatment of neurological disorders such as multiple sclerosis. The clinical usefulness of 4‐AP is hampered by its ability to produce seizures. Nerispirdine, an analogue of 4‐AP, is currently under clinical investigation for the treatment of multiple sclerosis. In contrast with 4‐AP, nerispirdine is not proconvulsant, suggesting mechanistic differences between the two drugs. 2 Using whole‐cell patch‐clamp electrophysiology, we compared the effects of 4‐AP and nerispirdine on the cloned human K+ channels Kv1.1 and Kv1.2, expressed in Chinese hamster ovary cells, and on voltage‐dependent Na+ channels recorded from human SH‐SY5Y cells. 3 Nerispirdine inhibited Kv1.1 and Kv1.2 with IC50 values of 3.6 and 3.7 μmol/L, respectively. 4‐Aminopyridine was approximately 50‐fold less potent at blocking these channels. Nerispirdine also inhibited voltage‐dependent Na+ channel currents recorded from human SH‐SY5Y cells with an IC50 of 11.9 μmol/L when measured from a –70 mV holding potential. In contrast, 4‐AP had no effect on Na+ channel currents. 4 The results demonstrate that nerispirdine, like 4‐AP, can inhibit axonal K+ channels and that this mechanism may underlie the ability of the drug to enhance neuronal conduction. Unlike 4‐AP, nerispirdine can also inhibit neuronal Na+ channels, a mechanism that may explain why nerispirdine lacks proconvulsant activity.

Serotonergic activity of HP 184: does spontaneous release have a role?

Examination of HP 184, [N-n-propyl)-N-(3-fluoro-4-pyridinyl)-1H-3-methylindodel-1-amine hydrochloride], in a variety of tests for serotonergic activity revealed some unique properties of this compound. We report here that 100 μM HP 184 enhanced spontaneous release of [3H]serotonin (5-HT) from rat hippocampal slices. This release was independent of the uptake carrier. In vivo assays confirmed that HP 184 (20 mg/kg, i.p.) lacked significant interactions at the norepinephrine (NE) or 5-HT uptake carrier itself. Notably, HP 184 (15 mg/kg, i.p.) reduced drinking behavior in schedule-induced polydipsic (SIP) rats. We previously reported that some selective 5-HT reuptake inhibitors decrease SIP 30–40% after a 14–21 day treatment. In the current study, HP 184 decreased SIP beginning with the first treatment, and this reduction (30%) was maintained for 28 days. We further investigated HP 184 and serotonin metabolite levels. One hour after i.p. administration of 30 mg/kg HP 184, the ratio of whole brain 5-hydroxyindolacetic acid (5-HIAA) to 5-HT was increased, suggesting serotonergic activation. Under these conditions, the brain: plasma ratio of HP 184 was approximately 2∶1, with brain concentrations of 1.6 μg/gram. We speculate that the spontaneous release effects of HP 184 may be responsible for the behavioral effects observed.