W. Roger Buckett

The pharmacology of sibutramine hydrochloride (BTS 54 524), a new antidepressant which induces rapid noradrenergic down-regulation

1. Sibutramine hydrochloride (BTS 54 524) has been evaluated in a range of behavioural and biochemical models predictive of potential antidepressant activity. 2. BTS 54 524 exhibited potent activity in acute behavioural tests for detecting potential antidepressants. 3. BTS 54 524 rapidly and potently down-regulated rat cortical beta-adrenoceptors, being effective after only three days of oral administration. 4. BTS 54 524 showed very little activity in a range of experiments predictive of side-effect potential. 5. BTS 54 524 possesses a pharmacological profile which suggests that it may be a potent antidepressant with a rapid onset of action, and with a low potential for causing side-effects.

Quantification of presynaptic α2-adrenoceptors in rat brain after short-term DSP-4 lesioning

Abstract The relative numbers of pre- and postsynaptic α 2 -adrenoceptors were determined in various rat brain regions after short-term DSP-4 ( N -(2-chloroethyl)- N -ethyl-2-bromobenzylamine) lesioning. For these studies, rats pretreated with zimeldine (10 mg/kg i.p.) were injected with DSP-4 (100 mg/kg i.p.) and were killed either 3 or 15 days later. At the 3 day time-point, DSP-4 treatment produced marked reductions in the noradrenaline content of the cortex (93%), hippocampus (89%), hypothalamus (83%) and cerebellum (92%) with no change in the levels of dopamine or 5-HT. This treatment also decreased the number of α 2 -adrenoceptors labelled with [ 3 H]idazoxan in the cortex (20%), hippocampus (18%), cerebellum (24%) and hypothalamus (39%). Fifteen days after DSP-4 lesioning, the marked reductions of noradrenaline were sustained in the cortex, hippocampus and cerebellum, but there was a considerable reversal of the effect of DSP-4 in the hypothalamus. At this time-point, the decrease in α 2 -adrenoceptors was attenuated in cortex (4%) and cerebellum (0%) and their number was increased in hippocampus (8%) and hypothalamus (7%). Together, the data argue that presynaptic α 2 -adrenoceptors comprise approximately 20% of the total α 2 -adrenoceptors population in the cortex, hippocampus and cerebellum, but about 40% of it in the hypothalamus. Furthermore, they also demonstrate that although the number of presynaptic α 2 -adrenoceptors in rat brain can be determined by the reduction of radioligand-receptor binding shortly after DSP-4 lesioning, this effect is rapidly masked by receptor proliferation in response to noradrenergic denervation.

Peripheral stimulation in mice induces short-duration analgesia preventable by naloxone

Peripheral stimulation was applied to mice by mild caudal electrostimulation, by mechanical pressure or by footshock for 30 sec, before testing on a 52 degrees C hot plate. Reaction times to paw lick and to escape from the hot plate were recorded. Analgesia could be elicited and measured by these procedures. It was of short duration, declining in a minute, and was antagonized by low doses of naloxone. The analgesia measured by the escape reaction time could be elicited after multiple caudal electrostimulation as well as in morphine-tolerant mice, and it could still be reversed by naloxone. An opioid link is thus involved in this phenomenon, which also supports the notion of more than one opioid pathway existing in the brain. The short period of analgesic cover afforded in the face of noxious stimuli would permit aversive action to be taken in nature and thus might represent the prime functional role of enkephalins in the brain.

Effect on radiolabelled-monoamine uptake in vitro of plasma taken from healthy volunteers administered the antidepressant sibutramine HCl

Sibutramine HCl, a monoamine reuptake inhibitor type of antidepressant, was administered to healthy male volunteers as either a single dose (12.5 or 50 mg) or repeated treatment (5–20 mg once daily or 15 mg twice daily). Plasma, obtained at regular intervals during and after sibutramine HCl or placebo treatment, was assayed in vitro for its ability to inhibit the uptake of [3H]-noradrenaline (NA) by rat cortical synaptosomes, [3H]-5-hydroxytryptamine (5HT) by human platelets and [14C]-dopamine (DA) by rat striatal synaptosomes. After both single and repeated sibutramine HCl administration, the rank order of uptake inhibition was [3H]-NA>[3H]-5HT>[14C]-DA. The level of monoamine uptake inhibition increased on daily administration to a plateau 4–6 days after initiation of treatment, for example, approximately 60% and 40% inhibition of [3H]-NA and [3H]-5HT, respectively, following 15 mg sibutramine HCl twice daily. The pattern of monoamine uptake inhibition following sibutramine HCl administration to man is similar to that observed in sibutramine HCl-treated rats, and probably at least partly reflects inhibition of uptake by drug metabolites in both species. The inhibition of monoamine uptake following sibutramine HCl administration to man is consistent with an antidepressant effect.

Clonidine-induced hypoactivity and mydriasis in mice are respectively mediated via pre- and postsynaptic α2-adrenoceptors in the brain

Since brain alpha 2-adrenoceptors occur both pre- and postsynaptically, experiments were carried out to determine the synaptic locations of those receptors mediating clonidine-induced hypoactivity and mydriasis. Intraperitoneal (i.p.) injection of clonidine (1-3000 micrograms/kg) to mice dose dependently induced these two responses and also decreased brain concentrations of 3-methoxy-4-hydroxyphenylglycol (MHPG). The ED50 values were: 120 micrograms/kg for hypoactivity (95% confidence limits 103-140 micrograms/kg), 54 micrograms/kg for mydriasis (95% confidence limits 40-74 micrograms/kg) and 18 micrograms/kg for MHPG reduction (95% confidence limits 8-36 micrograms/kg) suggesting that these responses could all be presynaptically mediated. However, methamphetamine which increases noradrenaline turnover was found to dose dependently produce mydriasis, but not hypoactivity, after peripheral (0.1-5 mg/kg i.p.) or central (0.5-10 micrograms i.c.v.) injection. The mydriasis produced by methamphetamine (0.5 mg/kg i.p.) was abolished by i.c.v. injection of 1 micrograms idazoxan or yohimbine, but not 2.5 micrograms prazosin or pindolol, showing this effect was mediated by central alpha 2-adrenoceptors. Methamphetamine (1-10 micrograms i.c.v.) potentiated the mydriasis induced by clonidine (50 micrograms/kg i.p.) suggesting this was a postsynaptic alpha 2-adrenoceptor response. By contrast, methamphetamine (1-10 micrograms i.c.v.) dose dependently reversed clonidine (100 micrograms/kg i.p.) hypoactivity indicating this response was mediated by presynaptic alpha 2-adrenoceptors. These hypotheses were confirmed by destruction of noradrenergic neurones using DSP-4 (100 mg/kg i.p. x 2). This treatment prevented the mydriasis response to methamphetamine (0.5 mg/kg i.p.), but not clonidine (100 micrograms/kg i.p.) and markedly attenuated clonidine (100 micrograms/kg i.p.) hypoactivity.

Pharmacological studies on stimulation-produced analgesia in mice

Stimulation-produced analgesia (ESPA) was induced in mice by peripheral caudal electrostimulation and subsequently monitored on a 52 degrees C hot plate. The effects on ESPA of compounds modulating some neurotransmitter systems were studied at appropriate premedication times and at doses at which the compounds themselves did not exhibit antinociceptive actions. The manipulation of catecholaminergic, dopaminergic or GABAergic systems did not modify ESPA. It could be potentiated by an increase in serotoninergic activity following 5-hydroxy-D,L-tryptophan (80-120 mg/kg) and reduced, under certain circumstances, by serotonin depletion with p-chlorophenylalanine (3 x 132 mg/kg). However the serotonin reuptake inhibitors fluoxetine (5 mg/kg) and zimelidine (10 mg/kg) were without effect indicating a complex modulating role of the monoamine. As with morphine-tolerant mice, the induction of ESPA in mice tolerant to methadone and to meperidine has been demonstrated, while the analgesia was not enhanced by chronic naloxone treatment. Although the naloxone reversibility of ESPA has been confirmed, a second acute dose of naloxone (1 mg/kg) did not reverse the analgesia. ESPA could also be fully elicited in adrenalectomized mice. It is concluded that ESPA is a specific type of stimulation-produced analgesia, shorter in duration and pharmacologically more resistant to modulation than others, which might subserve a functional role in response to noxious stimuli.

Receptor binding and functional evidence suggest that postsynaptic α2-adrenoceptors in rat brain are of the α2D subtype

This study has determined the subtype(s) of postsynaptic α2-adrenoceptors in rat brain. This question has been addressed by using two separate approaches, i.e. ligand displacement of [3H]2-(2-methoxy)-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX 821002) from membranes prepared from rat cortex after noradrenergic denervation and, secondly, by antagonism of clonidine-induced mydriasis. After rats had been lesioned using N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4; 100 mg/kg i.p., 30 min after zimeldine 10 mg/kg i.p.), noradrenaline was undetectable in the cortex 3 days later. Displacement of [3H]RX 821002 with a range of agonists and antagonists which distinguish between the known α2-adrenoceptor subtypes (α2A–2D) yielded pKi values which correlated very well with reported values for the α2D-adrenoceptor (r = 0.929; P < 0.001), but not the α2A (r = 0.450; P = 0.192), α2B (r = 0.280, P = 0.434) or α2C (r = 0.283; P = 0.460) subtypes. Similarly, the potencies of various α2-adrenoceptor antagonists to inhibit clonidine (0.03 mg/kg i.p.)-induced mydriasis in conscious rats correlated strongly with their pKi values for α2D-adrenoceptors (r = 0.899; P = 0.015) but not α2A- (r = 0.369; P = 0.472), α2B- (r = −0.224; P = 0.670) or α2C-adrenoceptors (r = 0.253; P = 0.584). These data are, therefore, consistent and argue strongly that postsynaptic α2-adrenoceptors in the rat cortex and Edinger-Westphal nucleus are of the α2D subtype.

Clonidine produces mydriasis in conscious mice by activating central α2-adrenoceptors

Abstract Intraperitoneal (i.p.) injection of the α 2 -adrenoceptor agonist clonidine (1–3000 μg/kg) produed dose-dependent pupil dilatation in conscious C57/B1/6 mice with an ED 50 of 54 μg/kg (95% confidence limits 40–74 μg/kg). This response was rapid in onset and of approximately 30 min duration. The α 2 -adrenoceptor antagonists idazoxan (1 or 3 mg/kg i.p.) and yohimbine (1 or 3 mg/kg i.p.) both produced dose-related miosis, but the α 1 - and β -adrenoceptor antagonists prazosin (1 or 3 mg/kg i.p.) and pindolol (1 or 3 mg/kg i.p.) were without effect. These doses of idazoxan and yohimbine potently reversed the mydriasis induced by clonidine (100 μg/kg i.p.), while prazosin and pindolol were again ineffective. Clonidine-induced mydriasis was also unaltered by the 5-HT antagonists, methysergide (2.5 mg/kg i.p.) and ketanserin (0.1 mg/kg i.p.) or 0.1 mg/kg i.p. of the dopamine antagonists, haloperidol, SCH 23390 and BRL 34778. A dose of 0.25 μg clonidine, which was ineffective when administered i.p., produced marked mydriasis after intracerebroventricular (i.c.v.) injection. In addition, the mydriasis produced by i.p. injection of clonidine (100 μg/kg) was abolished by i.c.v. dosing of 2.5 μg idazoxan or yohimbine, but again not by prazosin or pindolol. Together, these data provide strong evidence to indicate that clonidine-induced mydriasis is exclusively mediated via central α 2 -adrenoceptors and that this response provides a useful model for studying the function of these receptors.

A comparison of various antidepressant drugs demonstrates rapid desensitisation of α2-adrenoceptors exclusively by sibutramine hydrochloride

The functional status of presynaptic and post-synaptic α2-adrenoceptors in murine brain was respectively monitored using the hypoactivity (sedation) and mydriasis (pupil dilatation) responses to clonidine (0.1 mg/kg IP). Both responses were attenuated 24 h after 3 days of injection of sibutramine hydrochloride (3 mg/kg IP). To ascertain whether this property was exclusive to sibutramine, the following antidepressant drugs were also tested for their ability to down-regulate α2-adrenoceptors rapidly: amitriptyline, doxepin, nomifensine, desipramine, amoxapine, fluoxetine, zimeldine, tranylcypromine and mianserin. When given for 3 or 5 days at the low dose of 3 mg/kg IP, none of the other antidepressants reduced clonidine-induced hypoactivity or mydriasis. Furthermore, increasing the dose of amitriptyline, doxepin, nomifensine, desipramine, amoxapine and tranylcypromine to 10 mg/kg IP did not enable these antidepressants to attenuate the α2-adrenoceptor-mediated responses after 3 days of treatment. An electroconvulsive shock (ECS; 200 V, 2 s) given once daily attenuated clonidine-induced mydriasis, but not hypoactivity, when administered for 3 days and both responses when administered for 5 days. In conclusion, this comparative study using antidepressant treatments with differing pharmacological modes of action demonstrated that sibutramine was the only drug which rapidly down-regulated pre- and postsynaptic α2-adrenoceptors. ECS down-regulated postsynaptic α2-adrenoceptors when given for 3 days, but required 5 days to desensitise both α2-adrenoceptor populations.

Common profile of D1 receptor antagonists and atypical antipstchotic drugs revealed by analysis of dopamine turnover

1. Selective antagonists of dopamine D1 and D2 receptors enhanced 3-methoxytyramine (3-MT) accumulation in the striata and accumbens of tranylcypromine pretreated rats. Selective D1 and D2 agonists produced opposite effects. The smaller changes produced by the D1 agonists and antagonists were probably mediated by neuronal feedback, whereas the larger effects produced by the D2 ligands predominantly reflected pharmacological actions at prejunctional dopaminergic autoreceptors. 2. Atypical antipsychotics evoked small increases in 3-MT similar to the effects of the selective D1 inhibitors, whereas the mixed D1/D2 antagonists mimicked the selective D2 inhibitors by inducing much larger elevations in 3-MT. 3. gamma-Butyrolactone, an inhibitor of dopaminergic neuronal firing, dose-dependently decreased 3-MT accumulation in both the striata and accumbens. 4. gamma-Butyrolactone pretreatment abolished the small increases in 3-MT induced by the selective D1 antagonists and the atypical antipsychotics and also the large increases produced by the mixed D1/D2 antagonists. By contrast, gamma-butyrolactone only partially reversed the marked elevation of 3-MT evoked by the selective D2 antagonists. 5. The above data suggest that in vivo the atypical antipsychotics behave predominantly as selective D1 antagonists.