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.

Elevated childhood serotonergic function protects against adolescent aggression in disruptive boys.

OBJECTIVE: This longitudinal study examined whether responsiveness of the neurotransmitter serotonin (5-HT) in childhood predicts adolescent aggression. METHOD: Boys (N = 33) with disruptive behavior disorders who received assessments of central 5-HT function via the prolactin response to fenfluramine between 1990 and 1994 when they were 7 to 11 years old were re-evaluated clinically on average 6.7 years later. RESULTS: After accounting for baseline aggression, early 5-HT function accounted for a significant proportion of variance in adolescent aggression. This prospective relationship of childhood 5-HT function with adolescent aggression (r = -0.71) and antisocial behavior (r = -0.59) was found primarily in adolescents who were aggressive during childhood. Irrespective of childhood aggression, no child with high 5-HT function was particularly aggressive at follow-up. CONCLUSIONS: Low childhood 5-HT function appears important, but not sufficient, for the emergence of adolescent aggression. However, early high 5-HT function may protect against adolescent violence and aggression.

Fluorometric determination of DL-fenfluramine, DL-norfenfluramine and phentermine in plasma by achiral and chiral high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) with fluorescence detection has been developed for the simultaneous determination of sympathomimetic amines including ephedrine, norephedrine, 2-phenylethylamine, 4-bromo-2,5-dimethoxyphenylethylamine, phentermine (Phen) and DL-fenfluramine (Fen) in spiked human plasma. Furthermore, an enantioselective HPLC method for the separation of D-Fen (dexfenfluramine) and L-Fen (levofenfluramine) in addition to their active metabolites D- and L-norfenfluramine (Norf) is described. The detection was achieved at emission wavelength of 430 nm with excitation wavelength of 325 nm for both methods. The analytes were extracted from plasma (100 microl) at pH 10.6 with ethyl acetate using fluoxetine as the internal standard. The extracts were evaporated and derivatized with the fluorescence reagent 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride in the presence of carbonate buffer (pH 9.0). A gradient separation was achieved on a C18 column for the achiral separation or on a Chiralcel OD-R column for the chiral separation. The methods were fully validated, and shown to have excellent linearity, sensitivity and precision. The chiral method has been applied for the determination of D- and L-enantiomers of Fen and Norf, in addition to Phen in rat plasma after an intraperitoneal administration of DL-Fen and Phen, simultaneously.

Cigarette smoking decreases the prolactin response to serotonergic stimulation in subgroups of alcoholics and controls.

BACKGROUND: The prolactin response to serotonergic stimulation has been used as an index of central nervous system serotonin function. We evaluated the prolactin response to d,l-fenfluramine to determine whether subtypes of alcoholics differed in prolactin responsivity compared with nonalcoholics and whether cigarette smoking affected prolactin response. METHODS: One hundred ten healthy, abstinent men across four groups (controls [23% smokers]; alcoholics [72% smokers]; alcoholics with antisocial personality disorder [94% smokers]; nonalcoholic antisocials [88% smokers]) received d,l-fenfluramine (100 mg orally) in a randomized, double-blind, placebo-controlled study. Plasma prolactin levels were obtained at baseline and at half-hour intervals for 5 hr after fenfluramine/placebo administration. Plasma fenfluramine and norfenfluramine levels were obtained hourly. RESULTS: Smokers had a blunted prolactin response to fenfluramine compared with nonsmokers without any alcoholism or antisocial personality effects. Using a cutoff point of delta peak prolactin < 10 ng/ml, more smokers (41/76, 54%) had a dampened response to fenfluramine than did nonsmokers (7/34, 21%) [chi2(1) = 10.6, p < 0.003]. The percentage of low responders was greatest among smokers regardless of whether they were healthy controls, alcoholics, or antisocial. Multiple regression revealed that three variables--(1) number of pack-years of smoking, (2) actual dosage of fenfluramine received, and (3) plasma norfenfluramine level obtained--explained 43% of the variance (R2 = 0.43) in delta prolactin area under the curve. Variables that included alcoholism diagnostic status, antisocial personality diagnostic status, and impulsive aggressive personality, depressive, and suicidal traits failed to explain any additional unique variance. CONCLUSIONS: Cigarette smoking blunted the prolactin response to a pharmacological challenge with d,l-fenfluramine. Pharmacodynamic and pharmacokinetic factors related to smoking both appear to influence fenfluramine-induced prolactin secretion. Phenotypes of alcoholics did not differ in their prolactin response to this serotonergic probe.

Thermal response to serotonergic challenge and aggression in attention deficit hyperactivity disorder children.

Body temperature change in response to the serotonergic (5-HT) enhancer, d,l-fenfluramine (FEN), was examined in 27 prepubescent boys diagnosed with Attention Deficit Hyperactivity Disorder (ADHD) to determine (1) the utility of this measure as an index of central serotonergic function; and (2) if the magnitude of temperature change is associated with aggression. FEN, 1 mg/kg, produced a significant increase in body temperature, the magnitude of which was correlated with plasma levels of the FEN metabolite, norfenfluramine (NORFEN). Furthermore, a significant inverse relationship was found between temperature response to FEN and teacher ratings of aggression. Parent ratings of aggression were not significantly correlated with the hyperthermic response to FEN. Interestingly, the magnitude of the hyperthermic response was unrelated to changes in plasma levels of prolactin and cortisol, suggesting that thermal and neuroendocrine responses are mediated by distinct 5-HT mechanisms. The agreement of these finding with those of studies using other procedures to assess the relationship between 5-HT and aggression suggests that decreased central 5-HT is associated with increased aggression.

Neuropharmacological effects of low and high doses of repeated oral dexfenfluramine in rats: a comparison with fluoxetine.

The neuropharmacological effects of repeated oral doses of dexfenfluramine (DF; 1.25-10 mg/kg, twice daily for 21 days) were examined in rats and related to the drug brain levels. Results were compared with fluoxetine (FL) given at similar doses relative to its anorectic ED50. Both drugs dose-dependently slowed body weight gain and reduced brain serotonin (5-HT). However, at 1.25 mg/kg DF caused only a slight and transient decrease in cortical 5-HT. Comparable doses of FL (6.25-12.5 mg/kg) lowered 5-HT more than DF, besides slightly reducing striatal dopamine. At higher doses DF markedly reduced 5-HT in all regions, and to a lesser extent noradrenaline in hippocampus. There was a negative relationship between 5-HT and log total active drug levels and the indole was approximately halved at drug levels about 50 times lower with DF than FL. However, the ratio between drug levels causing marked 5-HT reductions and those considered anorectic was similar for DF and FL because brain levels at the anorectic ED50 were higher with FL than DF. Long-lasting reductions of 5-HT were also observed but recovery was only consistently slow beginning from 5 mg/ kg DF. Comparable doses of FL could not be used because its general toxicity leads to the death of rats after only 2-4 multiples of its anorectic ED50.

The effect of D-fenfluramine on brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in male and female rats.

Brain regional 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) concentrations were determined in freely feeding male and female rats 7 days after giving a single dose of D-fenfluramine (3.8 mg/kg, p.o.) or vehicle. Males showed negligible effects except for a significant decrease of 5-HT in the rest of the cortex, whereas females showed significant decreases of 5-HT and 5-HIAA in the frontal cortex, the rest of the cortex, hippocampus and hypothalamus; 5-HT was also decreased in female midbrain. Females had substantially higher plasma and brain concentrations of fenfluramine and moderately but significantly lower concentrations of norfenfluramine than the males. Plasma fenfluramine + norfenfluramine concentrations of the females were significantly higher than those of the males. Corresponding brain values showed smaller but significant differences. Female brain and plasma areas under the curve for fenfluramine + norfenfluramine (0-24 h after administration of D-fenfluramine) were 20 and 35% higher than male values. However, results suggest that the sex difference in the effect of D-fenfluramine on brain 5-HT metabolism is not due to differences in the metabolism of the drug.

Determination of D-fenfluramine, D-norfenfluramine and fluoxetine in plasma, brain tissue and brain microdialysate using high-performance liquid chromatography after precolumn derivatization with dansyl chloride.

A HPLC method is described for the simultaneous determination of D-fenfluramine (FEN), D-norfenfluramine (NF) and fluoxetine (FLX) using fluorometric detection after precolumn derivatization with dansyl-chloride. The method has limits of quantitation of 200 fmol for FEN and NF, 500 fmol for FLX in brain microdialysate, and 1 pmol for NF and FEN, and 2 pmol for FLX in plasma. Brain tissue standards were linear between 5 and 200 pmol/mg for all three compounds. The inter-assay variability (relative standard deviation) was 6.6%, 6.9% and 9.3% for FEN, 4.6%, 3.7% and 7.9% for NF and 10.4%, 4.9% and 12.2% for FLX, for brain microdialysate (2 pmol/microl), plasma (2 pmol/ microl) and brain tissue (50 pmol/mg), respectively. Intra-assay variability was always lower, typically several times lower than inter-assay variability. Extraction recovery was 108% and 48% for FEN, 105% and 78% for NF and 94% and 45% for FLX, in plasma (2 pmol/microl) and brain tissue (5 pmol/mg), respectively. Due to the stability of the dansyl-chloride derivatives this method is well suited for an autoinjector after manual derivatization with dansyl chloride at room temperature for 4 h.

The effect of fenfluramine dosage regimen and reduced food intake on levels of 5-HT in rat brain.

Male Wistar rats received fenfluramine in subacute (5 mg/kg b.i.d. i.p. for 4 days) or escalating (0.5, 1, 1.5, 2, 3, 4 and 5 mg/kg b.i.d. i.p., each dose given for 4 days) doses. Saline-treated controls received food ad libitum, or were pair-fed with the fenfluramine-treated animals. Rats were killed 1, 15 and 30 days after drug withdrawal. On day 1, plasma and brain fenfluramine levels were higher, and hypothalamus norfenfluramine levels were lower following escalating compared to subacute dosing, although total active drug levels were unaltered. Both treatment regimes, and pair-feeding reduced food intake and body weight. Subacute fenfluramine reduced brain 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels for up to 30 days. Brain 5-HT and 5-HIAA levels were unaltered following escalating-doses of fenfluramine. Additionally, pair-feeding transiently decreased hippocampal 5-HT levels. These data suggest that escalating-doses of fenfluramine prevent the 5-HT-depleting effect of a sub-cute challenge without altering the anorexic action of the drug.

In vitro and in vivo effects of the anorectic agent dexfenfluramine on the central serotoninergic neuronal systems of non-human primates. A comparison with the rat.

The effects of repeated subcutaneous (s.c) injections of dexfenfluramine (d-F; 10 mg/kg, twice daily, for 4 days) on the contents of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the brain were assessed in primates (cynomolgus and rhesus monkeys) and compared with the regional brain concentrations of unchanged drug and its active metabolite, dexnorfenfluramine (d-NF). This four-day, high-dose, regimen caused a large depletion of 5-HT (more than 95%) and of 5-HIAA (80-90%) in all brain areas studied (cortex, hippocampus, putamen, caudate nucleus and hypothalamus) 2 h after the last injection of d-F. Analysis of the plasma and brain contents of d-F and d-NF confirmed that both compounds were concentrated as in other species, in regions of the primate brain. However, d-NF was concentrated to a greater extent than d-F, and there were differences between the two primate species. Unlike in the rat brain, concentrations of d-NF greatly exceeded those of d-F in the primate brain suggesting that in these primates the d-NF may play a major role in the overall neurochemical response. The effects of d-F and d-NF on different in vitro parameters of serotoninergic neuronal function did not show appreciable differences between cynomolgus or rhesus monkeys when compared to rats, the ability of the two compounds to inhibit 5-HT reuptake, to enhance its release, and to affect the binding of [3H] -d-F or of [3H] -mesulergine (a ligand for 5-HT2C receptors) being similar. Kinetic differences in the disposition of d-F appear to have more relevance than biochemical effects in providing an explanation for the more marked brain depletion induced by d-F in primates than in rodents.

Demonstration in vivo of reduced serotonin responsivity in the brain of untreated depressed patients.

OBJECTIVE: For over 25 years, it has been hypothesized that major depression is due to a deficiency of available serotonin or subsensitivity of key serotonin receptors in relevant brain regions. Direct evidence supporting this hypothesis has been lacking because of the difficulty in studying regional brain serotonergic function. The authors have developed a method for visualizing in vivo regional brain responses to serotonin release by comparing regional brain glucose metabolism after administration of the serotonin-releasing drug dl-fenfluramine, relative to placebo. METHOD: Results with healthy subjects (N = 6) were compared to those obtained with drug-free inpatients with moderately severe major depression (N = 6). RESULTS: Healthy subjects had several areas of statistically significant increases in metabolism, mostly in the left prefrontal and temporoparietal cortex, and areas of decreased metabolism, such as in the right prefrontal cortex. In contrast, the depressed patients had no areas of increase or decrease in metabolism, differing significantly from healthy subjects. Results with patients resembled those with healthy subjects (N = 10) who were scanned twice without active drug on either occasion. CONCLUSIONS: This study provides the first direct visualization of blunted regional brain responses to serotonin release in the brain of patients with major depression, a finding that supports the hypothesis of impaired serotonergic transmission in depression.

New chiral high-performance liquid chromatographic methodology used for the pharmacokinetic evaluation of dexfenfluramine.

A new chiral high-performance liquid chromatographic (HPLC) method utilizing ultraviolet (UV) detection has been developed for determining plasma and urinary concentrations of d-fenfluramine and its major metabolite d-norfenfluramine, while being able to determine the possible presence of l-fenfluramine after oral administration of enantiopure d-fenfluramine hydrochloride. Sensitivity, stability, and specificity were enhanced by derivatizing the extracted analytes with 3,5-dinitrophenylisocyanate while utilizing a Pirkle-type chiral recognition approach. In vitro and in vivo statistical data are analogous. Overall plasma inter-assay precision was less than 7% with a minimum quantitation limit of 10 ng/ml. Overall urine inter-assay precision was also less than 7% with a minimum quantitation limit of 25 ng/ml.

Enhanced serotonergic responsivity following electroconvulsive therapy in patients with major depression.

Prolactin release in response to fenfluramine hydrochloride (60 mg orally) and placebo was evaluated in 18 medication-free patients with RDC major depressive disorder, endogenous subtype, before and after a series of bilateral treatments with ECT. Before ECT, fenfluramine induced a twofold increase in plasma prolactin levels. This response was significantly enhanced after the ECT series, while baseline prolactin levels and response to the placebo challenge were not altered. There was no significant difference in plasma fenfluramine and norfenfluramine levels during the pre- and post-ECT challenges. These findings suggest that ECT enhances central serotonergic responsivity and extend to depressed patients pre-clinical observations regarding the effect of electroconvulsive shock on serotonergic function.

Neuroendocrine and behavioral responses to challenge with the indirect serotonin agonist dl-fenfluramine in adults with obsessive-compulsive disorder.

Neuroendocrine and behavioral responses to a single 60-mg oral dose of the indirect serotonin agonist dl-fenfluramine were assessed in unmedicated adults with obsessive-compulsive disorder (OCD) and neuroendocrine results contrasted with those in normal control subjects. Net fenfluramine-induced prolactin release did not differ significantly between OCD patients and normal controls. Prolactin responses in the OCD group were not significantly correlated with baseline Yale-Brown Obsessive Compulsive Scale scores for either obsessions or compulsions, but were positively correlated with the baseline Hamilton Depression Scale score and Hamilton Anxiety Scale score. No clear difference in the severity of patients' obsessions or compulsions was found following challenge with fenfluramine versus placebo. Although the present study does not demonstrate a serotonergic abnormality in OCD, this may be more a reflection of limitations of the test procedures than evidence that central nervous system (CNS) serotonergic function is normal in the disorder.

Multiple hormone responses to stimulation with dl-fenfluramine in patients with major depression before and after antidepressive treatment.

The authors investigated 31 drug-free inpatients with major depression using dl-fenfluramine (dl-FEN) as a serotonin probe (fenfluramine stimulation test, FFT). The FFT was performed in the same individuals (intraindividual comparison) with and without application of dl-FEN and before and after antidepressive treatment. Blood samples were analyzed for prolactin, cortisol, thyrotropin (TSH), growth hormone (HGH), dl-FEN and its major metabolite dl-norfenfluramine. There was no difference in the conditions before and after treatment as regards peak plasma levels of dl-FEN. Compared with saline, significant (P less than 0.01) increases were detected for dl-FEN-stimulated cortisol and prolactin values. A significantly (P less than 0.01) lower increase in dl-FEN-stimulated prolactin values was found after treatment compared with the situation before treatment, a finding which was dependent on the type of medication which the patients were receiving at the time when the test procedure took place. Baseline and dl-FEN-stimulated cortisol values were significantly (P less than 0.001) lower and TSH values were significantly (P less than 0.01) higher in the condition after compared with the one before treatment. Patients' mean peak delta prolactin responses to dl-FEN before treatment were significantly (P less than 0.05) correlated with those of patients after subchronic treatment with fluvoxamine. Hormonal responses were neither associated with the severity of the illness nor with a specific psychopathological profile.

Fenfluramine treatment of twenty children with autism.

The effects of fenfluramine were examined on 20 children with autism over a 48-week period utilizing a double-blind placebo-controlled crossover design. Blood and urine samples and psychological tests (Griffith's Developmental Scales and Real Life Rating Scale) were obtained at each crossover period. The only significant improvement was a decrease in abnormal motor behavior. We did not find any significant improvement in intellectual functioning or any correlation between good clinical response and low baseline serotonin levels or high baseline IQ. Serotonin decreased 53% after fenfluramine treatment and rebounded to a level 35% higher than baseline following a placebo period. Fenfluramine and the active metabolite norfenfluramine were determined in plasma samples.

The measurement of d-fenfluramine and its metabolite, d-norfenfluramine in plasma and urine with an application of the method to pharmacokinetic studies.

1. A specific and sensitive gas chromatographic assay is described for the measurement of d-fenfluramine and its de-ethylated metabolite, d-norfenfluramine, in biological fluids, together with some data on its application to the oral pharmacokinetics of the drug. 2. The analytical method developed has advantages over the previously described methods since it uses nitrogen specific detection and, when applied routinely, enables smaller sample volumes to be used (typically 1 ml of plasma) with a shorter chromatography time and an improved sensitivity (minimum quantifiable level of 2.5 ng ml-1). 3. Peak plasma concentrations of 22 and 24 ng ml-1 of intact drug were reached at 4 h after an oral dose of 14C-d-fenfluramine hydrochloride (30 mg) given to two volunteers as part of a metabolism and disposition study. Subsequently, concentrations of intact drug declined monoexponentially with a half-life of approximately 13 h. Peak concentrations of 10 and 8 ng ml-1 of the metabolite, d-norfenfluramine, were reached after 4 and 6 h and were maintained as a plateau for a further 4-6 h. Assessment of the half-life of the metabolite could not be made because of lack of data on the terminal portion of the curves. 4. The urinary excretion of d-fenfluramine (6.0 and 10.6% of the dose) and d-norfenfluramine (5.8 and 8.8% of the dose) was low, indicating extensive metabolism of the parent drug.

Enantioselective gas chromatographic assay with electron-capture detection for dl-fenfluramine and dl-norfenfluramine in plasma.

An enantioselective gas chromatographic assay utilising electron-capture detection has been developed for the simultaneous quantitation of enantiomers of fenfluramine and nonfenfluramine in plasma. The assay involves the conversion of the enantiomers of both fenfluramine and norfenfluramine into their corresponding diastereomeric amide derivatives by an acylation reaction with n-heptafluorobutyryl-S-prolyl chloride under Schotten-Baumann conditions prior to gas chromatographic separation on an achiral polar OV-225 capillary column. Linear and reproducible standard curves were obtained over the concentration ranges 4.30-86.3 ng/ml per enantiomer and 1.25-42.25 ng/ml per enantiomer for the enantiomers of fenfluramine and norfenfluramine, respectively. The method was applied to a single-dose pharmacokinetic study in a healthy adult subject. Stereoselective differences were observed in the plasma concentration versus time profiles of the enantiomers of both fenfluramine and norfenfluramine. The area under the plasma concentration versus time curve values obtained for the l-isomers of fenfluramine or norfenfluramine were higher than the values of their corresponding d-antipodes.

Serotonergic regulation of prolactin and growth hormone secretion in man.

Controversy still exists regarding the role of serotonin in the regulation of prolactin (Prl) and growth hormone (GH) secretion in man. We gave healthy male volunteers three oral doses (0.5, 1.0 and 1.5 mg/kg) of fenfluramine, a serotonin-releasing agent and uptake inhibitor, and a corresponding placebo. There was a significant dose-response effect of fenfluramine on Prl but not on GH levels. Following the highest dose of fenfluramine, mean Prl levels increased from 9.7 ng/ml to 42.3 ng/ml. In a separate study, subjects were pre-treated with cyproheptadine, a serotonin antagonist, before the administration of fenfluramine. Cyproheptadine did not significantly affect basal Prl or GH levels, but it did blunt the response of Prl to fenfluramine. Cyproheptadine pre-treatment did not alter plasma levels of fenfluramine. Our findings support a stimulatory role for serotonin in the regulation of Prl secretion in man. They also suggest that serotonin does not have a major influence on GH secretion in man.