Knoll Limited, Research and Development, Nottingham, UK

Correspondence to: I C Kilpatrick, Knoll Limited, St Nicholas Court, 25-27 Castle Gate, Nottingham, NG1 7AR, UK. E-mail: ian.kilpatrick@knoll.co.uk

OBJECTIVE AND DESIGN: This study examined the effects of the anti-obesity agents, phentermine and dexfenfluramine given alone or in combination, on in vitro and in vivo 5HT release from rat brain tissue.

RESULTS: In vitro, phentermine was without effect on basal [³H]5HT efflux from hypothalamic slices whereas dexfenfluramine (10 µM) evoked a 131% increase in [³H]5HT release. In combination, the two drugs did not alter [³H]5HT release beyond that caused by dexfenfluramine alone. At pharmacologically equivalent doses, phentermine (5.7 mg/kg, i.p.) caused a rapid, modest elevation, and dexfenfluramine (3 mg/kg, i.p.) a larger but equally rapid elevation of extracellular 5HT in the microdialysates from the rat anterior hypothalamus. In combination, the increase in extracellular 5HT evoked by these drugs was not significantly greater than the sum of their individual effects.

CONCLUSIONS: This study provides evidence that phentermine's actions are not restricted to catecholamine systems and indicates that combining phentermine with dexfenfluramine results in an additive increase in neuronal 5HT release.

International Journal of Obesity (2001) 25, 1450-1453

phentermine; dexfenfluramine; in vitro; in vivo; 5HT release; anterior hypothalamus

Introduction

The -phenylethylamine, phentermine, has a long history in the treatment of obesity.¹ It has been suggested that phentermine elicits its anorectic effects indirectly via activation of central catecholamine systems,² particularly noradrenergic systems.^1,3,4 Fenfluramine and its active enantiomer, dexfenfluramine, were also widely used as anti-obesity agents, acting via potent release of central 5HT.^5,6 Following the publication of Weintraub,⁷ showing that combining phentermine with fenfluramine or dexfenfluramine promoted greater weight loss in obese patients than was achieved with either drug alone, this synergistic treatment was widely used in patients. Latterly, however, it was found that the use of fenfluramines (either dexfenfluramine alone or the combination of fenfluramine with phentermine) is associated with the production of valvular heart dysfunction in man and led to fenfluramine and dexfenfluramine being withdrawn in 1997 (see Connolly and McGoon⁸ and Gardin et al⁹ for reviews).

Elevations in circulating 5HT have been linked to the incidence of heart valve damage¹⁰ and valvular 5HT receptor mechanisms are also postulated to be relevant.¹¹ However, the impact of phentermine on fenfluramine-induced 5HT release is unclear. The aim of this initial study was therefore to evaluate the influence of these drugs, both alone and in combination, on central 5HT efflux using both in vitro and in vivo techniques.

Methods

In vitro release

These experiments were performed using slices of hypothalamus preloaded with [³H]5HT from male Wistar rats (150-250 g) essentially as described by Heal et al.¹² The accumulated efflux of tritium evoked by phentermine, dexfenfluramine or the two drugs in combination was calculated as a fraction of the total radioactivity initially present in the slices. Log-transformed data were statistically analysed using Williams' test. The possibility of drug interaction was statistically examined by calculation of the ratio of the effect of the phentermine/dexfenfluramine combinations compared with either drug applied alone to the effect of either drug applied alone compared with Krebs-Henseleit buffer control. Using the multiple t-test, an interaction value not significantly different from 1 represented no interaction, ie additivity. Values significantly less than 1 represented antagonism whilst those significantly higher than 1 represented synergistic effects.

In vivo microdialysis

This study was conducted in freely moving male Wistar rats (274-310 g) using the microdialysis technique in the anterior hypothalamus.¹² Doses of drugs used were based on three times their oral ED₅₀ to inhibit food intake at 1 h. Post-injection levels of 5HT were expressed as a percentage of the mean basal level. Data were log-transformed and individual time-points were compared by two-way ANCOVA (baseline as covariate and treatment and experiment as factors) followed by post-hoc Dunnett's test. Analysis of possible drug interaction was performed as above but the comparisons were of individual time-points as well as areas under the curves.

Results

In vitro release (see Table 1)

Phentermine (1 and 10 µM) had no significant effect on basal [³H]5HT efflux from hypothalamic slices. Conversely, whilst dexfenfluramine (1 µM) did not significantly alter [³H]5HT release, a higher concentration (10 µM) elevated it by +131% (P<0.001). The phentermine/dexfenfluramine combinations did not alter the effect of dexfenfluramine alone on [³H]5HT efflux (interaction value 95% CI: for 1 µM each, 0.61-1.71; for 10 µM each, 0.39-1.09). This suggests that any drug interaction was additive.

In vivo microdialysis (see Figure 1)

Phentermine (5.7 mg/kg, i.p.) evoked a rapid increase in extracellular 5HT levels in the anterior hypothalamus. This elevation was maximal (+137% above its own baseline) and significantly greater than in saline-treated controls at 20 min post-dose (+115%, P<0.001) and remained significantly increased for up to 100 min after phentermine administration (+30% at 100 min, P<0.05). At a pharmacologically equivalent dose, dexfenfluramine (3 mg/kg, i.p.) caused an equally rapid, though larger, increase (maximum 483% above its own baseline) in dialysate 5HT levels. This elevation was significantly greater than in saline-treated controls at 20 min after dexfenfluramine injection (+430%, P<0.001) and the increase persisted for up to 3 h post-dose (+225%, P<0.001). When phentermine (5.7 mg/kg, i.p.) and dexfenfluramine (3 mg/kg, i.p.) were combined, there was a rapid, large increase in dialysate 5HT which was maximal (911% above its own baseline) 40 min after injection (+858% compared to saline-treated control, P<0.001). Levels of 5HT remained higher throughout the experiment (up to 3 h) when compared to phentermine alone (P<0.001 at each time-point) and periodically when compared to dexfenfluramine alone (40, 60, 80 and 120 min). Statistical analysis revealed that, either at individual time-points (range of interaction values 0.59-1.52, not significant) or for the post-drug duration (interaction value 1.09), there was no significant interaction between the drugs and their effects on dialysate 5HT in the hypothalamus were, therefore, likely to be additive.

Discussion

Dexfenfluramine is a potent releaser of neuronal 5HT both in vitro and in vivo and these in vivo data concur with those of others.^{5,13,14,15,16} Phentermine produces a significant increase of hypothalamic 5HT efflux in vivo at a pharmacologically relevant dose and is, therefore, not a catecholamine-selective agent.¹⁵ Whilst the rapid onset of this phentermine-induced increase in 5HT efflux is consistent with that of a releasing agent⁵ (albeit a weak one), phentermine did not evoke release of radiolabelled 5HT from in vitro slices of hypothalamus. It is therefore likely that the ability of phentermine to release 5HT in vivo is indirect and requires additional neuronal circuitry which is removed during slice preparation. The profile of 5HT release caused by phentermine does not resemble that of a 5HT uptake inhibitor which, regardless of potency, would take much longer (1-1.5 h^5,6) to maximally increase extracellular 5HT. In any event, phentermine has already been shown to be essentially inactive as a 5HT uptake inhibitor in vitro^14,17 (IC₅₀=11-14 µM). As for alternative mechanisms, although inhibition of monoamine oxidase (MAO) can yield relatively rapid elevations of extracellular 5HT,¹⁸ these are usually persistent and can continue to rise for up to 5 h.^18,19 Again, however, phentermine has been shown to be extremely weak as an inhibitor of MAO_A (IC₅₀=100-200 µM)^20,21 or MAO_B (IC₅₀=300-900 µM).^20,21 Thus, the route by which phentermine evokes increases in extracellular 5HT in vivo is unclear.

Concerning the combination of phentermine with dexfenfluramine, the present data do not provide any statistical evidence for an interaction between the two drugs on extracellular 5HT in the hypothalamus that is anything other than additive. Similar additive effects have been shown between phentermine and fenfluramine on both 5HT and dopamine levels in the nucleus accumbens.¹³ In the striatum, however, evidence has been obtained for a synergistic interaction between phentermine and fenfluramine since the minimal impact of phentermine alone on striatal 5HT levels was nevertheless reported to enhance that caused by fenfluramine when the two drugs were given in combination.¹⁶ In determining whether the interactions between phentermine and/or fenfluramine are additive, synergistic or even antagonistic, it is likely that regional differences in the sensitivity of 5HT and other systems to the drugs need to be taken into account.

Turning to fenfluramines and cardiac valve dysfunction, Hervé and colleagues linked valvulopathy to increases in circulating levels of 5HT.¹⁰ The potent 5HT-releasing action of fenfluramines noted in the hypothalamus (Gundlah et al⁵ and present data), the striatum¹⁶ and in the nucleus accumbens^13,15 has also been demonstrated using blood platelets.^22,23 This could provide a peripheral source of 5HT that is sufficient to play a role in the genesis of valvulopathy. Alternatively, the cellular accumulation of fenfluramine with or without phentermine¹⁴ or direct 5HT receptor stimulation by a fenfluramine metabolite¹¹ or otherwise could trigger events that lead to valvulopathy. In terms of assessing plasma 5HT changes after drug treatment, it should be noted that repeated fenfluramine treatment is likely to deplete platelets of their 5HT stores²⁴ and may even cause plasma 5HT levels to fall, as demonstrated both in rats treated with dexfenfluramine for 2 weeks²⁴ and in diabetic patients treated with fenfluramine and phentermine for 2 months.²⁵

Overall, the data provide the first in vivo evidence that in the hypothalamus, a brain region involved in food intake regulation,²⁶ summation but not synergy appears to exist between phentermine and dexfenfluramine and their effects on extracellular 5HT. This reinforces the idea that the reported synergy between these drugs on weight loss⁷ is not simply due to their combined actions on brain 5HT systems.

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Figure 1 Effects of phentermine or dexfenfluramine alone or in combination on extracellular 5HT levels in freely-moving rat anterior hypothalamus. Values are mean percentage basal 5HT±s.e.m. of 5-12 animals. Drugs or saline were injected at time 0. *P<0.05, **P<0.01, ***P<0.001 vs saline control.

Table 1 Effect of phentermine, dexfenfluramine or their combinations on [³H]5HT release from rat hypothalamic slices