Comparative activity of the enantiomers of fenfluramine and norfenfluramine in rodent seizure models, and relationship with their concentrations in plasma and brain.

OBJECTIVES: To investigate the comparative antiseizure activity of the individual enantiomers of fenfluramine and its major active primary metabolite norfenfluramine in rodent seizure models, and its relationship with the pharmacokinetics of these compounds in plasma and brain. METHODS: The antiseizure potency of d,l-fenfluramine (racemic fenfluramine) was compared with the respective potencies of its individual enantiomers and the individual enantiomers of norfenfluramine using the maximal electroshock (MES) test in rats and mice, and the 6-Hz 44 mA test in mice. Minimal motor impairment was assessed simultaneously. The time course of seizure protection in rats was compared with the concentration profiles of d-fenfluramine, l-fenfluramine, and their primary active metabolites in plasma and brain. RESULTS: All compounds tested were active against MES-induced seizures in rats and mice after acute (single-dose) administration, but no activity against 6-Hz seizures was found even at doses up to 30 mg/kg. Estimates of median effective doses (ED50 ) in the rat-MES test were obtained for all compounds except for d-norfenfluramine, which caused dose-limiting neurotoxicity. Racemic fenfluramine had approximately the same antiseizure potency as its individual enantiomers. Both d- and l-fenfluramine were absorbed and distributed rapidly to the brain, suggesting that seizure protection at early time points (≤2 h) was related mainly to the parent compound. Concentrations of all enantiomers in brain tissue were >15-fold higher than those in plasma. SIGNIFICANCE: Although there are differences in antiseizure activity and pharmacokinetics among the enantiomers of fenfluramine and norfenfluramine, all compounds tested are effective in protecting against MES-induced seizures in rodents. In light of the evidence linking the d-enantiomers to cardiovascular and metabolic adverse effects, these data suggest that l-fenfluramine and l-norfenfluramine are potentially attractive candidates for a chiral switch approach leading to development of a novel, enantiomerically-pure antiseizure medication.

In vitro evaluation of fenfluramine and norfenfluramine as victims of drug interactions.

Fenfluramine (FFA) has potent antiseizure activity in severe, pharmacoresistant childhood-onset developmental and epileptic encephalopathies (e.g., Dravet syndrome). To assess risk of drug interaction affecting pharmacokinetics of FFA and its major metabolite, norfenfluramine (nFFA), we conducted in vitro metabolite characterization, reaction phenotyping, and drug transporter-mediated cellular uptake studies. FFA showed low in vitro clearance in human liver S9 fractions and in intestinal S9 fractions in all three species tested (t1/2  > 120 min). Two metabolites (nFFA and an N-oxide or a hydroxylamine) were detected in human liver microsomes versus six in dog and seven in rat liver microsomes; no metabolite was unique to humans. Selective CYP inhibitor studies showed FFA metabolism partially inhibited by quinidine (CYP2D6, 48%), phencyclidine (CYP2B6, 42%), and furafylline (CYP1A2, 32%) and, to a lesser extent (<15%), by tienilic acid (CYP2C9), esomeprazole (CYP2C19), and troleandomycin (CYP3A4/5). Incubation of nFFA with rCYP1A2, rCYP2B6, rCYP2C19, and rCYP2D6 resulted in 10%-20% metabolism and no clear inhibition of nFFA metabolism by any CYP-selective inhibitor. Reaction phenotyping showed metabolism of FFA by recombinant human cytochrome P450 (rCYP) enzymes rCYP2B6 (10%-21% disappearance for 1 and 10 µM FFA, respectively), rCYP1A2 (22%-23%), rCYP2C19 (49%-50%), and rCYP2D6 (59%-97%). Neither FFA nor nFFA was a drug transporter substrate. Results show FFA metabolism to nFFA occurs through multiple pathways of elimination. FFA dose adjustments may be needed when administered with strong inhibitors or inducers of multiple enzymes involved in FFA metabolism (e.g., stiripentol).

The role of 5-HT2B receptors in mitral valvulopathy: bone marrow mobilization of endothelial progenitors.

BACKGROUND AND PURPOSE: Valvular heart disease (VHD) is highly prevalent in industrialized countries. Chronic use of anorexigens, amphetamine or ergot derivatives targeting the 5-HT system is associated with VHD. Here, we investigated the contribution of 5-HT receptors in a model of valve degeneration induced by nordexfenfluramine, the main metabolite of the anorexigens, dexfenfluramine and benfluorex. EXPERIMENTAL APPROACH: Nordexfenfluramine was infused chronically (28 days) in mice ((WT and transgenic Htr2B -/- , Htr2A -/- , and Htr2B/2A -/- ) to induce mitral valve lesions. Bone marrow transplantation was also carried out. Haemodynamics were measured with echocardiography; tissues and cells were analysed by histology, immunocytochemistry, flow cytometry and RT -qPCR. Samples of human prolapsed mitral valves were also analysed. KEY RESULTS: Chronic treatment of mice with nordexfenfluramine activated 5-HT2B receptors and increased valve thickness and cell density in a thick extracellular matrix, mimicking early steps of mitral valve remodelling. Lesions were prevented by 5-HT2A or 5-HT2B receptor antagonists and in transgenic Htr2B -/- or Htr2A/2B -/- mice. Surprisingly, valve lesions were mainly formed by numerous non-proliferative CD34+ endothelial progenitors. These progenitors originated from bone marrow (BM) as revealed by BM transplantation. The initial steps of mitral valve remodelling involved mobilization of BM-derived CD34+ CD31+ cells by 5-HT2B receptor stimulation. Analysis of human prolapsed mitral valves showing spontaneous degenerative lesions, demonstrated the presence of non-proliferating CD34+ /CD309+ /NOS3+ endothelial progenitors expressing 5-HT2B receptors. CONCLUSIONS AND IMPLICATIONS: BM-derived endothelial progenitor cells make a crucial contribution to the remodelling of mitral valve tissue. Our data describe a new and important mechanism underlying human VHD.

Action of a serotonergic anorectic in meal-fed mice working for food.

The aim of this study was to examine the effects of a serotonergic anorectic agent, dexnorfenfluramine (DNOR), on food intake in mice whose meals were constrained to specified periods each day and by effort. Mice were forced to adopt a human-like pattern of regular meals by making food available for four periods of 40 min/24-h period, mostly at night. They lived in behavior test chambers with a closed economy for food and were required to emit a fixed unit price (FUP) of either 2 or 25 nose pokes (FUP2, FUP25) to receive a 20 mg pellet of food. Once responding and intake were stable, mice were injected with a vehicle or DNOR (3 or 6 mg/kg) 1 h before a specified feeding opportunity. Food intake was dose-dependently suppressed at the next meal and to a greater extent when the cost of food was high (FUP25). Within a meal, the effect of the drug was the greatest in the first half of the available time. Therefore, the anorectic effect of DNOR was modified by the concurrent cost of food.

Organ culture as a tool to identify early mechanisms of serotonergic valve disease.

BACKGROUND AND AIM OF THE STUDY: Although the late effects of serotonergic valve disease are known, the early mechanisms of the characteristic plaque formation are poorly understood. METHODS: To model conditions leading to plaque formation on mitral valves, samples (n = 6-8 per treatment) cultured in a splashing bioreactor were exposed to serotonin (5HT) and norfenfluramine (NF). In order to assess the role of 5HT2B receptor activation, the effects of these drugs were also tested with a 5HT2B receptor antagonist. After two weeks, tissue samples were stained immunohistochemically to localize changes in multiple extracellular matrix (ECM) components and synthesis mediators. RESULTS: Decorin and versican expression tended to increase with 5HT treatment compared to NF or baseline controls, regardless of the presence of the receptor antagonist. Samples treated with 5HT or with the receptor antagonist tended to express less collagen (types I and III) and biglycan than NF or the baseline controls. Heat shock protein 47, prolyl-4-hydroxylase, matrix metalloproteinase 9 (MMP9) and MMP13 tended to be down-regulated with 5HT or NF exposure, although some samples treated with the antagonist displayed normal levels of these mediators. Superficial plaques grew on a subgroup of the NF-treated organ cultures, but on none of the 5HT and control valves. CONCLUSION: Although both serotonin agents lead to plaque formation in a clinical setting, the early effects of exposure to the different drugs were found to be quite different. Additionally, the different drug responses suggest that a mechanism other than 5HT2B receptor activation might contribute to plaque formation.

Serotonergic responses in depressed patients with or without a history of alcohol use disorders and healthy controls.

Dysfunction of serotonergic neurotransmission has been implicated in the etiopathogenesis of major depression (MDD) and alcohol use disorders (AUD). To compare serotonin function in MDD with co-occurring AUD (MDD/AUD), MDD without co-occurring AUD (MDD only) and healthy controls (HC) we sought to study differences in prolactin responses to fenfluramine administration in patients with MDD/AUD, patients with MDD only and HC. In all, 169 subjects (62 MDD/AUD, 75 MDD only, and 32 HC) were entered into the study. Controlling for gender, prolactin responses were lower in the MDD/AUD group compared to the MDD only or the HC group. Controlling for gender and aggression, prolactin responses in the MDD/AUD group remained significantly lower compared to the HC group but the difference between the MDD/AUD and the MDD only groups disappeared. The difference in prolactin responses between MDD/AUD and MDD only could be attributed to higher aggression scores in the MDD/AUD group compared to the MDD group.

The synthesis and biological activity of pentafluorosulfanyl analogs of fluoxetine, fenfluramine, and norfenfluramine.

The trifluoromethyl group of fluoxetine 1 and fenfluramine and norfenfluramine, 2 and 3, was substituted by the pentafluorosulfanyl group. On examination of the efficacy of the pentafluorosulfanyl containing compounds as inhibitors of 5-hydroxytryptamine receptors, it was found that substitution could lead to enhanced selectivity and in the case of the pentafluorosulfanyl analog of fenfluramine, 18, it significantly enhanced potency against the 5-HT(2b), 5-HT(2c), and 5-HT(6) receptors.

The 5-hydroxytryptamine2A receptor is involved in (+)-norfenfluramine-induced arterial contraction and blood pressure increase in deoxycorticosterone acetate-salt hypertension.

The highly effective anorexigen (+)-fenfluramine was widely used to control body weight until the association with primary pulmonary hypertension and valvular heart disease. (+)-Norfenfluramine is the major hepatic metabolite of (+)-fenfluramine and is primarily responsible for the anorexic effect as well as side effects. We reported that (+)-norfenfluramine causes vasoconstriction and a blood pressure increase in rats with normal blood pressure via the 5-hydroxytryptamine (5-HT)2A receptor. With the knowledge that (+)-norfenfluramine also has affinity for 5-HT2B receptors and that arterial 5-HT2B receptor expression is up-regulated in deoxycorticosterone acetate (DOCA)-salt hypertension, we tested the hypothesis that (+)-norfenfluramine-induced vasoconstriction and pressor effects are potentiated in DOCA-salt hypertensive rats in a 5-HT2 receptor-dependent manner. Contractions of arteries were measured using an isolated tissue bath system or myograph. Mean arterial blood pressure was measured in chronically instrumented conscious rats. Effects of (+)-norfenfluramine in stimulating arterial contraction (leftward shift versus SHAM, aorta, 5.13-fold; renal artery, 1.95-fold; mesenteric resistance artery, 1.77-fold) and raising blood pressure were significantly enhanced in hypertension. In arteries from both normotensive and hypertensive rats, (+)-norfenfluramine-induced contraction in aorta was inhibited by 5-HT2A receptor antagonists, ketanserin and LY53857 (4-isopropyl-7-methyl-9-(2-hydroxy-1-meth ylpropoxycarbonyl)4,6,6a,7,8,9,10,10a-octahydroindolo[4,3-fg]quinoline), but not by the 5-HT2B receptor antagonist, LY272015 [6-chloro-5-methyl-N-(5-quinolinyl)-2,3-dihydro-1H-indole-1-carboxamide]. Ketanserin (3 mg/kg) reduced (+)-norfenfluramine-induced pressor response in both SHAM and DOCA rats. Our results demonstrate that (+)-norfenfluramine-induced arterial contraction and blood pressure increases are potentiated in DOCA-salt hypertensive rats. However, it is the 5-HT2A receptor and not the 5-HT2B receptor that participates in these effects.

(+)-Norfenfluramine-induced arterial contraction is not dependent on endogenous 5-hydroxytryptamine or 5-hydroxytryptamine transporter.

(+)-Norfenfluramine, the major metabolite of fenfluramine, causes vasoconstriction dependence on the 5-hydroxytryptamine (5-HT)(2A) receptor in rat. (+)-Norfenfluramine was reported as a 5-hydroxytryptamine transporter (5-HTT) substrate and 5-HT releaser. Because the arterial 5-HTT exists and is functional in the rat, we hypothesized that (+)-norfenfluramine causes vasoconstriction by releasing 5-HT from vascular smooth muscle via 5-HTT. The released 5-HT, in turn, activates the 5-HT(2A) receptor. Isometric contractility experiments showed that (+)-norfenfluramine-induced mouse aortic contraction was reduced by the 5-HTT inhibitor fluoxetine (1 micriM) but not by fluvoxamine (1 microM). Tryptophan hydroxylase (TPH)-deficient (Tph1-/-) mice lack peripheral 5-HT. (+)-Norfenfluramine (10 nM-100 microM)-contracted aorta from wild-type and Tph1-/- mice with equivalent potency (-log EC(50) [M], wild type = 5.73 +/- 0.02, Tph1-/- = 5.62 +/- 0.09), and these contractions were inhibited by the 5-HT(2A) receptor antagonist ketanserin (3 nM) by a similar magnitude in aorta from wild-type and Tph1-/- mice (wild type = 19.4, Tph1-/- = 15.4-fold rightward shift versus control), as did fluoxetine (1 microM) (wild type = 22.4, Tph1-/- = 28.8-fold rightward shift versus control). To further test the role of 5-HTT in (+)-norfenfluramine-induced aortic contraction, the 5-HTT-targeted mutation mouse was used. (+)-Norfenfluramine induced similar aortic contraction in wild-type and 5-HTT-targeted mutation mice, and these contractions were inhibited by fluoxetine (1 microM). Thus, (+)-norfenfluramine vasoconstriction is not dependent on 5-HTT-mediated release of endogenous 5-HT but by activating membrane 5-HT(2A) receptors directly. Understanding of the mechanism by which (+)-norfenfluramine induces vasoconstriction is important to characterize and understand the function of the serotonergic system in peripheral arterial vasculature.

Relationship between anorexia and loss of serotonin uptake sites in brain of mice and rats receiving d-norfenfluramine or d-fenfluramine.

Previously, we have shown that repeated administration of d-fenfluramine (D-F) to rats is associated with development of tolerance to the initial anorexia, but that in mice no such tolerance seems to occur. In the first study, we show that chronic administration of neither d-norfenfluramine (D-NF; the principal metabolite of D-F) nor the serotonin (5-HT) 2C receptor agonist m-chlorophenyl-piperazine (mCPP) is associated with the development of anorectic tolerance tested using a dessert protocol. However, compared with mice receiving these drugs for the first time, both of these chronic treatments were associated with a significant attenuation of Fos immunoreactivity (Fos-ir) in several brain regions, including bed nucleus of the stria terminalis, paraventricular hypothalamus, and central nucleus of amygdala. This attenuation is similar to that described previously in rats. Because loss of efficacy of these agents could be related to loss of 5-HT transporter (5-HTT) sites, their presumptive primary mode of action, in the final study we determined the effect of various, low-dose regimens of D-F and D-NF on 5-HT uptake in frontal cortex of mice and rats. We show in mice that D-F causes a greater loss of 5-HT uptake than D-NF, and that at the lowest dose regimen used uptake was unaffected in rats but was reduced in mice. The data are discussed in terms of the species difference in behavioral tolerance and differences in neurochemical profile of D-F and D-NF.

The fenfluramine metabolite (+)-norfenfluramine is vasoactive.

The anorexigen (+)-fenfluramine was used for treatment of obesity until the association of use with valvular heart disease and primary pulmonary hypertension. (+)-Fenfluramine has been found in Chinese and Korean slimming pills. The hepatic metabolite of (+)-fenfluramine, (+)-norfenfluramine, has affinity for 5-hydroxytryptamine (5-HT)(2A) and 5-HT(2B) receptors. We tested the hypothesis that (+)-norfenfluramine contracts arterial smooth muscle in a 5-HT receptor-dependent manner and acts as a pressor in the conscious rat. Isometric contraction experiments showed that (+)-norfenfluramine (10 nM, 100 microM) but not (+)-fenfluramine nor the isomer (-)-norfenfluramine caused concentration-dependent contraction in arteries [-log EC(50) (moles per liter), thoracic aorta = 5.77 +/- 0.09; renal artery = 6.29 +/- 0.02; mesenteric resistance artery = 5.70 +/- 0.06]. Contraction was dependent on the 5-HT(2A) receptor because ketanserin (10 nM) rightward shifted (+)-norfenfluramine response curves (aorta = 16-fold, renal artery = 26-fold, and resistance artery = >100-fold). Dependence on activation of 5-HT(2A) receptors and independence of (+)-norfenfluramine-induced contraction from stimulation of alpha-adrenergic receptors and the sympathetic nervous system was validated by demonstrating 1) unchanged contraction to (+)-norfenfluramine in arteries from chemically denervated rats; 2) a minimal effect of the alpha(1)-adrenergic receptor antagonist prazosin (100 nM) on contraction; and 3) antagonism by [6-methyl-l-(1-methylethy)ergoline-8beta-carboxylic acid 2-hydroxy-1 methylpropyl ester maleate] LY53857 [6-methyl-1-(1-methylethy)-ergoline-8beta-carboxylic acid 2-hydroxy-1 methylpropyl ester maleate], a 5-HT(2) receptor antagonist without alpha-receptor affinity. (+)-Norfenfluramine (10-300 microg/kg i.v.) caused a dose-dependent increase in mean arterial blood pressure in conscious rats, the maximum of which could be virtually abolished by ketanserin (3 mg/kg i.v.) but not prazosin (0.2 mg/kg i.v.). Our findings demonstrate for the first time that (+)-norfenfluramine is vasoactive and has the potential to increase blood pressure.

Nordexfenfluramine causes more severe pulmonary vasoconstriction than dexfenfluramine.

The anorectic agent dexfenfluramine (dex) causes the development of primary pulmonary hypertension in susceptible patients by an unknown mechanism. We compared the effects of dex with those of its major metabolite, nordexfenfluamine (nordex), in the isolated perfused rat lung and in isolated rings of resistance pulmonary arteries. Nordex caused a dose-dependent and more intense vasoconstriction, which can be inhibited by the nonspecific 5-hydroxytryptamine type 2 (5-HT(2)) blocker ketanserin. Similarly a rise in cytosolic calcium concentration ([Ca(2+)](i)) in dispersed pulmonary artery smooth muscle cells (PASMCs) induced by nordex could be prevented by ketanserin. Unlike prior observations with dex, nordex did not inhibit K(+) current or cause depolarization in PASMCs. Removal of Ca(2+) from the tissue bath or addition of nifedipine (1 microM) reduced ring contraction to nordex by 60 +/- 9 and 63 +/- 4%, respectively. The addition of 2-aminoethoxydiphenyl borate (2-APB), a blocker of store-operated channels and the inositol 1,4,5-trisphosphate receptor, caused a dose-dependent decrease in the ring contraction elicited by nordex. The combination of 2-APB (10 microM) and nifedipine (1 microM) completely ablated the nordex contraction. Likewise the release of Ca(2+) from the sarcoplasmic reticulum by cyclopiazonic acid markedly reduced the nordex contraction while leaving the KCl contraction unchanged. We conclude that nordex may be responsible for much of the vasoconstriction stimulated by dex, through the activation of 5-HT(2) receptors and that the [Ca(2+)](i) increase in rat PASMCs caused by dex/nordex is due to both influx of extracellular Ca(2+) and release of Ca(2+) from the sarcoplasmic reticulum.

The effect of the anorectic agent, d-fenfluramine, and its primary metabolite, d-norfenfluramine, on intact human platelet serotonin uptake and efflux.

Dexfenfluramine, a drug formerly prescribed for treatment of obesity, caused heart valve damage and pulmonary hypertension in some people. The cause of the toxicity has not been defined, but 5-HT has been implicated. The objective of this study was to evaluate the effect of the anorectic agent, d-fenfluramine, and its major metabolite, d-norfenfluramine, on intact human platelet serotonin (5-HT) transport in vitro. The effects of d-fenfluramine and d-norfenfluramine on platelet uptake and efflux of 3H-5-HT were measured in buffer at pH 6.7, to optimize serotonin transporter (SERT) function, and at pH 7.4. Uptake of 3H-5-HT at pH 6.7 and 7.4 was inhibited by both agents at micro m concentrations (IC50, d-fenfluramine approximately 3 microM; d-norfenfluramine approximately 10 microM). However, no efflux of 3H-5-HT from labeled platelets at either pH 6.7 or 7.4 occurred at similar concentrations of d-fenfluramine or d-norfenfluramine. With inhibition of platelet dense granule 3H-5-HT uptake by reserpine, efflux of 3H-5-HT was observed at pH 6, but not at pH 7.4. Fluoxetine, a SERT inhibitor, was a more potent inhibitor of uptake (IC50 0.05 microM) than d-fenfluramine, but the anorectic agent, phentermine, had no effect. Therefore, d-fenfluramine and d-norfenfluramine inhibit human platelet uptake of 5-HT in vitro at tissue concentrations attainable in vivo, but they do not stimulate 5-HT efflux due to dense granule sequestration. Inhibition of platelet 5-HT uptake may play a role in the cardiopulmonary toxicity of d-fenfluramine, but other factors probably contribute, since similar toxicity has not been observed with fluoxetine.

3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") induces fenfluramine-like proliferative actions on human cardiac valvular interstitial cells in vitro.

Recent findings have implicated the 5-hydroxytryptamine 2B (5-HT2B) serotonin receptor in mediating the heart valve fibroplasia [valvular heart disease (VHD)] and primary pulmonary hypertension observed in patients taking the now-banned appetite suppressant fenfluramine (Pondimin, Redux). Via large-scale, random screening of a portion of the receptorome, we have discovered that the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") and its N-demethylated metabolite 3,4-methylenedioxyamphetamine (MDA) each preferentially bind to and activate human recombinant 5-HT2B receptors. We also demonstrate that MDMA and MDA, like fenfluramine and its N-deethylated metabolite norfenfluramine, elicit prolonged mitogenic responses in human valvular interstitial cells via activation of 5-HT2B receptors. We also report that pergolide and dihydroergotamine, two drugs recently demonstrated to induce VHD in humans, potently activate 5-HT2B receptors, thus validating this assay system for its ability to predict medications that might induce VHD. Our discovery that MDMA and a major metabolite, MDA, induce prolonged mitogenic responses in vitro similar to those induced by fenfluramine and norfenfluramine in vivo (i.e., valvular interstitial cell fibroplasia) predict that long-term MDMA use could lead to the development of fenfluramine-like VHD. Because of the widespread abuse of MDMA, these findings have major public health implications. These findings also underscore the necessity of screening current and future drugs at h5-HT2B receptors for agonist actions before their use in humans.

(+)-Fenfluramine and its major metabolite, (+)-norfenfluramine, are potent substrates for norepinephrine transporters.

(+/-)-Fenfluramine is an amphetamine analog that was once widely prescribed as an appetite suppressant. Although (+/-)-fenfluramine is no longer clinically available, the mechanisms underlying its anorectic properties are still of interest. Upon peripheral administration, stereoisomers of (+/-)-fenfluramine are N-deethylated to form the metabolites, (+)- and (-)-norfenfluramine. It is well accepted that isomers of (+/-)-fenfluramine and (+/-)-norfenfluramine interact with 5-hydroxytryptamine (serotonin, 5-HT) transporters to release 5-HT from neurons. However, the effects of these drugs on other monoamine transporters are not well characterized. In this study, we examined the interaction of stereoisomers of (+/-)-fenfluramine and (+/-)-norfenfluramine with transporters for 5-HT, norepinephrine (NE), and dopamine (DA). Results from in vitro assays confirmed these drugs are potent substrates for 5-HT transporters: (+)-fenfluramine, (-)-fenfluramine, (+)-norfenfluramine, and (-)-norfenfluramine released [3H]5-HT from synaptosomes with EC50 values of 52, 147, 59, and 287 nM, respectively. Importantly, (+)-fenfluramine and (+)-norfenfluramine released [3H]NE with EC50 values of 302 and 73 nM. Results from in vivo microdialysis experiments showed that intravenous injection of (+)-norfenfluramine elevates extracellular levels of 5-HT, NE, and DA in rat frontal cortex. The effects of (+)-norfenfluramine on NE and DA were antagonized by pretreatment with the NE uptake blocker nisoxetine. In summary, administration of fenfluramines can increase synaptic levels of 5-HT, NE, and DA in the cortex, and (+)-norfenfluramine likely contributes to these effects. Release of NE and DA evoked by (+)-norfenfluramine is at least partly mediated via NE transporters. Our results emphasize the potential involvement of noradrenergic mechanisms in the actions of fenfluramines.

Effect of a 5-HT(2C) serotonin agonist, dexnorfenfluramine, on amyloid precursor protein metabolism in guinea pigs.

Stimulation of serotonin receptor subtypes 5-HT(2A) or 5-HT(2C) in stably transfected 3T3 cells by dexnorfenfluramine (DEXNOR) or serotonin increases secretion of the APP metabolite APP(s). It is not known whether activation of these receptors can also affect APP metabolism in vivo. We examined the effects of a single intraperitoneal (i.p.) injection of DEXNOR on APP(s) levels in cerebrospinal fluid (CSF) of guinea pigs. These levels were significantly (P<0.05) increased by a single dose of DEXNOR (1-4 mg/kg); those of the APP metabolites Abeta(1-40) and Abeta(1-42) were unaffected. The DEXNOR-induced (1 mg/kg) increases in CSF APP(s) were suppressed by ritanserin (1 mg/kg) but not by ketanserin (2 mg/kg). When given alone, ritanserin did not affect CSF levels of APP(s), Abeta(1-40), or Abeta(1-42). Chronic treatment with DEXNOR for 9 days (1 mg/kg bid, i.p.) increased CSF APP(s) levels, measured 2 h after the last injection (P<0.05), and decreased those of CSF Abeta(1-42) (P<0.05). Neither hippocampal nor cortical levels of the APP holoprotein (APP(h)), nor body weight, were affected by DEXNOR. Chronic administration of mCPP (1-(m-chlorophenyl)piperazine) (2 mg/kg bid, i.p.), a 5-HT(2B/2C) agonist, for 9 days also increased CSF APP(s) levels (P<0.5) when measured 2 h after the drug's last administration; hippocampal and cortical APP(h) levels were unaffected. However, mCPP also caused a significant decrease in body weight gain. These data indicate that the pharmacological activation of 5-HT(2C) receptors can stimulate CSF APP(s) secretion and reduce Abeta production in vivo. Hence 5-HT(2C) receptors, which apparently are localized to the brain, may represent useful targets for the development of treatments for Alzheimer's disease.

Evidence that hypophagia induced by d-fenfluramine and d-norfenfluramine in the rat is mediated by 5-HT2C receptors.

The present series of studies is the first to investigate the pharmacological mechanisms underlying d-fenfluramine- and d-norfenfluramine-induced hypophagia in the rat using highly selective serotonin 5-HT2 receptor antagonists. Administration of d-fenfluramine, and its major metabolite d-norfenfluramine, suppresses food intake in animals. Both compounds stimulate the release of serotonin and are potent inhibitors of the re-uptake of 5-HT into nerve terminals. In addition, d-norfenfluramine also acts as a direct 5-HT(2B/2C) receptor agonist. Pre-treatment with the selective 5-HT2C receptor antagonist, SB-242084 (0.3-3 mg/kg), dose-dependently inhibited both d-fenfluramine- (3 mg/kg) and d-norfenfluramine-induced (2 mg/kg) hypophagia. In contrast, the hypophagic effect of d-fenfluramine and d-norfenfluramine was unaffected by prior treatment with the highly selective 5-HT2B receptor antagonists, SB-215505 (0.3-3 mg/kg) and RS-127445 (1-3 mg/kg) or the 5-HT2A receptor antagonists MDL 100,907 (0.003-0.03 mg/kg) and ketanserin (0.2, 0.5 mg/kg). In addition, the 5-HT1A receptor antagonist WAY-100635 (0.3, 1 mg/kg) and the 5-HT1B receptor antagonists GR-127935 (1, 2 mg/kg) and SB-224289 (2-10 mg/kg) did not affect d-fenfluramine-induced hypophagia. These data provide unequivocal evidence for an important role of the 5-HT2C receptor in the mediation of d-fenfluramine and d-norfenfluramine-induced hypophagia in the rat and do not support the involvement of 5-HT1A/1B/2A/2B receptors.

An investigation of the serotonergic effects of fenfluramine, dexfenfluramine and dexnorfenfluramine using platelets as neuronal models.

The appetite-suppressant, fenfluramine (d,l-F) has been used for several decades to treat obesity. Dexfenfluramine (d-F), the d-enantiomer of d,l-F, was approved in 1996 for long-term administration. Subsequently, these drugs were voluntarily withdrawn due to reports of adverse effects on heart valves. So far, the evidence regarding the serotonergic action of d,l-F and d-F has relied on animal-based experiments. We used human platelets as neuronal models to assess the serotonergic action of both d,l-F, d-F and the main metabolite of d-F, d-norfenfluramine (d-norF). This was evaluated using a sensitive method that assesses platelet shape change (PSC) as expressed by an increase in median platelet volume (MPV). Human platelets increased their MPV in response to d-norF. This action was probably mediated via serotonin (subtype 2) receptors because a specific antagonist blocked it. This is the first demonstration, using human tissue, of the serotonergic action associated with the administration of d-F.

Dexfenfluramine and norfenfluramine: comparison of mechanism of action in feeding and brain Fos-ir studies.

Dexfenfluramine (dF) and dexnorfenfluramine (dNF), its metabolite, are anorectic agents that release serotonin (5-HT) and may have a direct postsynaptic action. The effects on the anorectic effects of dF and dNF of either acute (p-chlorophenylalanine, PCPA) or chronic (5,7-dihydroxytryptamine, 5,7-DHT) brain 5-HT depletions were studied in rats and compared with the actions of a 5-HT uptake inhibitor (fluoxetine) and 5-HT(1B/2C) receptor agonists [1-(3-trifluoromethyl-phenyl)-piperazine and 1-(3-chlorophenyl) piperazine]. The anorexia caused by these agonists was enhanced in rats with 5,7-DHT lesions, possibly a result of receptor supersensitivity. In contrast, fluoxetine anorexia was somewhat reduced in one study and was unchanged in a second. Both dF and dNF anorexias were enhanced in rats with 5,7-DHT lesions. In contrast, the anorectic effects of either dF or dNF were unchanged in PCPA-treated rats relative to controls. Compared with controls, 5, 7-DHT-lesion rats showed greatly increased dF- and dNF-induced Fos-like immunoreactivity (ir) in the paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei, and in the median preoptic area (MnPO), but were similar to controls in most other areas. PCPA pretreatment increased dF- and dNF-induced Fos-ir in the PVN, SON, and MnPO. In controls, equianorectic doses of dF and dNF induced Fos-ir in similar brain regions, but dNF produced relatively larger effects than dF in SON, PVN, and MnPO. The data are discussed in terms of multiple pathways in the anorectic actions of dF and dNF.

Possible role of valvular serotonin 5-HT(2B) receptors in the cardiopathy associated with fenfluramine.

Dexfenfluramine was approved in the United States for long-term use as an appetite suppressant until it was reported to be associated with valvular heart disease. The valvular changes (myofibroblast proliferation) are histopathologically indistinguishable from those observed in carcinoid disease or after long-term exposure to 5-hydroxytryptamine (5-HT)(2)-preferring ergot drugs (ergotamine, methysergide). 5-HT(2) receptor stimulation is known to cause fibroblast mitogenesis, which could contribute to this lesion. To elucidate the mechanism of "fen-phen"-associated valvular lesions, we examined the interaction of fenfluramine and its metabolite norfenfluramine with 5-HT(2) receptor subtypes and examined the expression of these receptors in human and porcine heart valves. Fenfluramine binds weakly to 5-HT(2A), 5-HT(2B), and 5-HT(2C) receptors. In contrast, norfenfluramine exhibited high affinity for 5-HT(2B) and 5-HT(2C) receptors and more moderate affinity for 5-HT(2A) receptors. In cells expressing recombinant 5-HT(2B) receptors, norfenfluramine potently stimulated the hydrolysis of inositol phosphates, increased intracellular Ca(2+), and activated the mitogen-activated protein kinase cascade, the latter of which has been linked to mitogenic actions of the 5-HT(2B) receptor. The level of 5-HT(2B) and 5-HT(2A) receptor transcripts in heart valves was at least 300-fold higher than the levels of 5-HT(2C) receptor transcript, which were barely detectable. We propose that preferential stimulation of valvular 5-HT(2B) receptors by norfenfluramine, ergot drugs, or 5-HT released from carcinoid tumors (with or without accompanying 5-HT(2A) receptor activation) may contribute to valvular fibroplasia in humans.