Thomas P. Blackburn

The medical benefit of 5-HT research.

5-HT research is now more than 50 years old, and it has generated a wealth of therapeutic agents, some of which have had a major impact on disease management. The 5-HT reuptake inhibitors (SSRIs) are among the most widely prescribed drugs for treating depression and a variety of other disorders including anxiety, social phobia and premenstrual dysphoria (PMD). The other major success stories of 5-HT research are the discovery of 5-HT1B/D receptor agonists for treating migraine and 5-HT3 receptor antagonists for chemotherapy and radiation-induced emesis. The role of 5-HT in the mechanism of action of antipsychotic agents remains a topic of intense research, which promises better treatments for schizophrenia in the future. Compounds interacting with 5-HT1F, 5-HT2C, 5-HT6 and 5-HT7 receptors are currently under investigation and may prove to have important therapeutic applications in the future.

Anxiolytic-like effect of paroxetine in a rat social interaction test

The effects of short- and long-term administration of the selective serotonin reuptake inhibitor paroxetine were investigated in a rat social interaction test. A single administration of paroxetine at oral doses of 0.3, 1, 3 and 10 mg/kg had no effect on social interaction between pairs of male rats under bright light (high anxiety) conditions. After 21 days of daily administration, paroxetine given orally at 3 mg/kg significantly (p < 0.01) increased the time spent in social interaction by pairs of rats tested under the same conditions, with no effect on locomotor activity, indicating an anxiolytic-like effect. The magnitude of increase (+97%) was comparable to that seen after a single dose of chlordiazepoxide (4 mg/kg orally). Although there was also an increase in time spent in social interaction after 21 days of repeated oral administration of paroxetine at 0.3, 1, and 10 mg/kg (+44, +56, and +54% increases, respectively), statistical significance was not achieved. These results indicate that in the long term paroxetine has an anxiolytic action, and thus support the clinical evidence for its therapeutic use in the treatment of anxiety disorders in addition to its established role as an antidepressant.

Evidence that 5-HT2C receptor antagonists are anxiolytic in the rat Geller-Seifter model of anxiety

Four non-selective 5-HT2C/5-HT2A receptor antagonists, mianserin (2–8 mg/kg), 1-naphthyl piperazine (1-NP) (0.5–1 mg/kg), ICI 169,369 (20 mg/kg) and LY 53857 (5 mg/kg), increased punished responding for a food reward in the rat Geller-Seifter test 30 min after subcutaneous (SC) administration. This property was shared by the benzodiazepine anxiolytic chlordiazepoxide (5 mg/kg SC). However, the selective 5-HT2A receptor antagonists ketanserin (0.2–1 mg/kg SC) and altanserin (0.5, 1 mg/kg SC) had little effect. The 5-HT1A, 5-HT1B andβ-adrenergic receptor antagonists pindolol and cyanopindolol (6 mg/kg SC) did not affect punished responding either, nor did the 5-HT1D receptor partial agonist andα2 adrenergic receptor antagonist yohimbine (2.5 mg/kg SC) or the histamine H1 receptor antagonist mepyramine (1 mg/kg SC). Unpunished responding was also modestly increased after some doses of the 5-HT2C/5-HT2A receptor antagonists. However, this effect was inconsistent and was also seen after chlordiazepoxide. Furthermore, it was not associated with the increase in punished responding observed in rats orally treated with mianserin (10, 20 mg/kg), 1-NP (10, 20 mg/kg) or ICI 169,369 (50 mg/kg). The action of the 5-HT2C/5-HT2A receptor antagonists tested is therefore consistent with anxiolysis. The results also strongly suggest that this effect is mediated by blockade of the 5-HT2C receptor, although the possibility of 5-HT2B receptor mediation is discussed.

BRL 46470A: a highly potent, selective and long acting 5-HT3 receptor antagonist with anxiolytic-like properties.

The novel 5-HT3 antagonist, BRL 46470A (endo-N-(8-methyl-8-azabicyclo [3.2.1]oct-3-yl)2,3-dihydro-3,3 dimethyl-indole-1-carboxamide, hydrochloride), has been investigated in a series of in vitro and in vivo tests, including the effect of the drug in models of anxiolysis. In classical tests for 5-HT3 receptor antagonism, BRL 46470A was shown to antagonise 5-HT3 mediated responses in the guinea-pig ileum [pA2 8.3±0.5, slope 0.98±0.20, mean±SEM (5)], the rabbit isolated heart (pA2 10.1±0.1, slope 1.2±0.2,n=5) and the rat Bezold-Jarisch model (ID50 0.7 µg/kg IV±0.1,n=8), with a long duration of action (>3 h). BRL 46470A selectively displaced [3H]-BRL 43694 from 5-HT3 binding sites in rat brain membranes (Ki 0.32 nM±0.04,n=4) without displacing (at concentrations greater than 1 µM) a wide variety of ligands binding to other neurotransmitter receptors, opioid receptors and to neurotransmitter gated ion channel complexes. In vivo, BRL 46470A showed anxiolytic-like activity in two animal models predictive of antianxiety effects-elevated X-maze and social interaction in rats. In both models, BRL 46470A showed significant activity over a wide dose-range following both oral (0.0001–0.1 mg/kg PO) and systemic administration. The unique level of potency of BRL 46470A was apparent in the rat social interaction test and was shown to be 100 fold more potent than the 5-HT3 receptor antagonist ondansetron, with no evidence of a bell-shaped dose response curve over 4 orders of magnitude. BRL 46470A (0.0001 and 0.01 mg/kg SC) also reduced the anxiogenic effects of m-CPP (1-(3-chlorophenyl) piperazine) in the rat elevated X-maze. BRL 46470A is therefore a chemically novel potent and selective 5-HT3 receptor antagonist with anxiolytic potential and a long duration of action in animal studies.

Effect of SB 200646A, a 5-HT2C/5-HT2B receptor antagonist, in two conflict models of anxiety

SB 200646A is the first selective 5-HT2C/5-HT2B receptor antagonist and has previously been observed to have anxiolytic-like properties in the rat social interaction test. In the present study the effects of the compound in two conflict models of anxiety, the rat Geller-Seifter and marmoset conflict test, were examined. In the rat Geller-Seifter test, suppressed responding was increased by all doses of SB 200646A between 5 and 40 mg/kg PO when given 1 h pretest. Unsuppressed responding was slightly increased only at 10 mg/kg PO. Suppressed responding was also increased by the benzodiazepine anxiolytic, chlordiazepoxide, at 1, 2.5 and 5 mg/kg PO 1 h pretest. Unsuppressed responding was modestly increased by chlordiazepoxide only at 5 mg/kg PO. In the marmoset conflict test marmosets were trained to lever press for a palatable food reward. Lever pressing was subsequently suppressed by air puffs. In this procedure suppressed responding was increased by both the benzodiazepine anxiolytic diazepam at 2 and 5 mg/kg PO and SB 200646A after 10 and 20 mg/kg PO. Both treatments caused small increases in unsuppressed responding at 2 and 20 mg/kg PO respectively. Taken together with the previous effects of SB 200646A in the rat social interaction test, this is compelling evidence that 5-HT2C/2B receptor antagonists may possess anxiolytic properties.

Dextromethorphan attenuates post-ischemic hypoperfusion following incomplete global ischemia in the anesthetized rat

The effects of dextromethorphan (DM) were tested in an in vivo model of incomplete global cerebral ischemia. Anesthetized rats were divided into 4 groups: Group 1 (saline); Group 2 (DM pre-treatment, 20 mg/kg i.v. bolus followed by 10 mg/kg/h DM infusion); Group 3 (DM post-treatment, 2 mg/kg i.v. bolus followed by 10 mg/kg/h DM infusion at the onset of post-ischemic hypoperfusion); and Group 4 (sham-operated, drug-treated). Groups 1-3 underwent 15 min of 4-vessel occlusion followed by 3 h of reperfusion. Administration of DM in sham-operated animals (Group 4) had no effect on cerebral blood flow or electroencephalographic (EEG) activity. In contrast, when compared to the Group 1 saline controls, significant attenuation of post-ischemic hypoperfusion and EEG dysfunction was demonstrated in ischemic rats treated with DM (both pre- and post-treatment), suggesting an ability of DM to improve cerebral blood flow (CBF) and brain function in cerebral ischemia.

An investigation of the mechanism responsible for fluoxetine-induced hypophagia in rats

The effect of fluoxetine on feeding in p-chlorophenylalanine (PCPA) pretreated rats and the nature of its interaction with 5-HT2C receptors have been investigated. Animals that received 3 days PCPA (150 mg/kg i.p.) pretreatment and vehicle on the test day consumed a similar amount as control, saline pretreated animals under the test paradigm used in this study. Fluoxetine (20 and 30 mg/kg p.o.) significantly reduced food intake in PCPA and control pretreated animals to a similar extent, despite an approximately 90% reduction in the levels of brain 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in the PCPA-pretreated animals. Thus, hypophagia is unlikely to be caused by inhibition of 5-HT reuptake. In the pig choroid plexus in vitro, fluoxetine and norfluoxetine inhibited specific [3H] mesulergine binding with pKIs (+/- S.E.M.) of 6.45 +/- 0.09 (n = 4) and 6.05 +/- 0.05 (n = 3), and slope factors (+/- S.E.M.) of 1.06 +/- 0.14 and 0.99 +/- 0.13, respectively. In slices of piglet choroid plexus fluoxetine (1, 10 and 33 microM) caused a rightward shift in the dose-response curve produced by 5-HT with no effect on the maximal response, and a mean pKB of 5.94 +/- 0.09. Norfluoxetine (10 microM) also produced a rightward shift in the 5-HT dose-response curve with no effect on the maximal response, and a pKB of 6.20. Thus, both compounds acted as surmountable antagonists with no agonist efficacy at 5-HT2C receptors present in choroid plexus. The hypophagic effect of fluoxetine (20 mg/kg p.o.) was also unaffected by the non-specific 5-HT2C receptor antagonist metergoline (2 and 5 mg/kg i.p.). These findings suggest that the hypophagic effect of fluoxetine is not likely to be dependent upon intact brain 5-hydroxytryptaminergic presynaptic function, nor is it mediated by an agonist action at the 5-HT2C receptor, but by an additional, unknown mechanism.

Suppressant effects of selective 5-HT2 antagonists on rapid eye movement sleep in rats

The effects of the novel, highly selective serotonin-2 (5-HT2) antagonists, ICI 169,369 and ICI 170,809, on 24 h EEG sleep-wake activity were studied in the rat. Both compounds caused a dose-related increase in the latency to rapid eye movement sleep (REMS) and significantly suppressed cumulative REMS time up to 12 h postinjection. In contrast, neither drug disrupted slow-wave sleep continuity in as much as the latency to non-REMS (NREMS) and cumulative NREMS time were unchanged. However, at the highest dose tested (20 mg/kg) ICI 170,809 did produce a significant increase in total NREMS time during the second half of the sleep-awake cycle. These results demonstrate effects of selective 5-HT2 antagonists on sleep in rats which appear to be specific for REMS behavior, suggesting that the priming influence of serotonin on REMS may involve 5-HT2 receptor subtypes. The relationship between the REMS suppressant actions of these compounds and their consideration as therapeutic agents in depression is discussed.

Supersensitivity of nigral serotonin receptors and rat rotational behaviour

Direct injections of 5-hydroxytryptamine (5-HT) into the corpus striatum (CS) and substantia nigra (SN) after unilateral 5,7-dihydroxytryptamine (5,7-DHT) of the dorsal raphe nucleus (DRN), resulted in a significant dose related contralateral turning behaviour in SN-injected but not in CS-injected animals. Receptor binding studies in these animals revealed a 3 fold increase in [3H]5-HT binding sites (Bmax) and a similar increase in affinity constant (KD) in the SN from the lesioned side when compared with the unlesioned side. There was no change in binding characteristics in the CS of DRN-lesioned animals. The data presented would confirm our previous findings that nigral 5-HT receptors become supersensitive after denervation of the DRN-SN pathway.

1-[2-[(Heteroarylmethoxy)aryl]carbamoyl]indolines are selective and orally active 5-HT2C receptor inverse agonists.

Bisarylmethoxyethers have been identified with nanomolar 5-HT2C affinity and selectivity over both 5-HT2A and 5-HT2B receptors. Compounds such as 1, 2, 8, 12, 14 and 18 have potent oral activity in a centrally mediated pharmacodynamic model of 5-HT2C function and their therapeutic potential is currently under further investigation.