Acute fluoxetine treatment potentiates amphetamine hyperactivity and amphetamine-induced nucleus accumbens dopamine release: possible pharmacokinetic interaction

Amphetamine stimulates locomotor activity, in large part by activating central dopaminergic systems. Serotonin shares on overlapping distribution with dopamine and has been shown to modulate dopaminergic function and dopamine-mediated behaviors. The present study examined whether increasing serotonergic function, via the selective serotonin reuptake inhibitor fluoxetine, would alter the stimulatory effects of amphetamine on locomotor activity and dopamine overflow in the nucleus accumbens. In addition, the present study determined whether fluoxetine treatment would alter the metabolism of amphetamine. Results show that 5.0 mg/kg fluoxetine potentiated the locomotor activity induced by amphetamine (0.5–1.0 mg/ kg), and enhanced the increased dopamine overflow in the nucleus accumbens induced by amphetamine. Fluoxetine treatment also resulted in a higher concentration of amphetamine in the CNS. Together, these findings indicate that acute fluoxetine treatment potentiates the locomotor stimulating and dopamine activating effects of amphetamine. Further, the results indicate that fluoxetine potentiates the effects of amphetamine by decreasing the metabolism of amphetamine, probably through inhibition of cytochrome P450 isozymes. Received: 5 May 1998/Final version: 7 July 1998     

Three-phase,liquid-phase microextraction combined with high performance liquid chromatography-fluorescence detection for the simultaneous determination of fluoxetine and norfluoxetine in human plasma

A three-phase, liquid-phase microextraction using a hollow fibre (HF-LPME) combined with high performance liquid chromatography-fluorescence detection (HPLC-FL) was developed for the analysis of fluoxetine (FLX) and its active metabolite, norfluoxetine (NFLX), in human plasma. An HF-LPME system using a disposable 7-cm polypropylene porous hollow fibre, 5 mL of alkaline plasma solution (donor phase), n-hexyl ether (extraction solvent) and 20 mM hydrochloric acid (acceptor phase) was used in the extraction. The method was validated after optimisation of several parameters that influence LPME efficiency. A reverse-phase LiChrospher 60 RP-Select B column (125 mm × 4 mm, 5 μm particle size) was used with 0.005 M sodium acetate buffer (pH 4.5) and acetonitrile at a 50:50 (v/v) as the mobile phase at a flow rate of 0.6 mL min⁻¹. In these conditions satisfactory chromatographic resolution and efficiency for the analytes were obtained. Fluorescence detection at 230 nm excitation wavelength and 290 nm emission wavelength was performed. Linearity over a range of 5–500 ng mL⁻¹, with determination coefficients (R²) of 0.9999 and 0.9962 for FLX and NFLX, respectively, was established. Venlafaxine was used as the internal standard for both analytes. Extraction recoveries from plasma samples were 70.9% for FLX and 59.7% for NFLX. The intra-day coefficients of variation (CVs) were below 5.4%, and inter-day CVs were below 13.0%, for both analytes at concentrations of 20, 80 and 160 ng mL⁻¹. HF-LPME extraction followed by HPLC-FL detection for FLX and NFLX analyses demonstrated excellent sample clean-up and selectivity. This method was simple, cheap, and easy to perform, yielding substantial analytes enrichment. The method was applied to the analysis of samples from 12 patients under fluoxetine treatment and proved suitable for routine therapeutic drug monitoring for this antidepressant.     

Influence of dose and route of administration on the kinetics of fluoxetine and its metabolite norfluoxetine in the rat

Fluoxetine (FL) is being used in neuropharmacology as a tool for studying various functional roles of serotoninergic neurons. Its kinetics was studied in rats, a species widely used in neurochemical studies, after IV (2.5–10 mg/kg) and oral (5–20 mg/kg) administration. When injected IV the drug followed apparent first-order kinetics up the 10 mg/kg dose. Its volume of distribution was large and total body clearance was relatively high compared to liver blood flow. The mean elimination half-lives (t _1/2) of FL and its active metabolite norfluoxetine (NFL) were about 5 and 15 h, respectively. The mean blood:plasma concentration ratios of FL and NFL approached unity and plasma protein binding was 85–90% for both compounds. After oral doses the kinetics of FL were complex. At the lowest dose tested (5 mg/kg) the drug was efficiently extracted by the liver (extraction ratio about 60%), resulting in bioavailability of only about 38%. Plasma areas under the curve (AUC) of the metabolite were approximately the same as after IV injection of the same dose; consequently the metabolite-to-parent drug ratio after oral administration (about 5) was approximately twice that after IV injection of FL (about 2.5). At higher doses, however, the oral bioavailability (e.g.C _max and AUC) appeared greater than expected, possibly because of transient saturation of FL first-pass metabolism in the case of the 10 mg/kg dose and concomitant saturation of elimination kinetics at the higher dose (20 mg/kg). The apparent eliminationt _1/2 of FL markedly increased and the metabolite-to-parent drug ratio declined with the higher dose, this also being consistent with saturable elimination. Brain concentrations reflected the plasma kinetics of FL and NFL and the metabolite-to-parent drug ratio varied with dose and time of administration and was modified at the highest dose tested. FL and its metabolite NFL distributed almost evenly in discrete brain areas and subcellular distribution was similar for both compounds. Neurochemical studies of FL should consider the formation of the active metabolite NFL and extrapolation of data across animal species requires consideration of dose dependence in the rat.     

Effects of norfluoxetine on the action potential and transmembrane ion currents in canine ventricular cardiomyocytes

Norfluoxetine is the most important active metabolite of the widely used antidepressant compound fluoxetine. Although the cellular electrophysiological actions of fluoxetine are well characterized in cardiac cells, little is known about the effects of its metabolite. In this study, therefore, the effects of norfluoxetine on action potential (AP) configuration and transmembrane ion currents were studied in isolated canine cardiomyocytes using the whole cell configuration of patch clamp techniques. Micromolar concentrations of norfluoxetine (1–10 M) modified AP configuration: amplitude and duration of the AP and maximum velocity of depolarization were decreased in addition to depression of the plateau and elimination of the incisura of AP. Voltage clamp experiments revealed a concentration-dependent suppression of both L-type Ca²⁺ current, I_Ca (EC₅₀=1.13±0.08 M) and transient outward K⁺ current, I_to (EC₅₀=1.19±0.17 M) having Hill coefficients close to unity. The midpoint potential of the steady-state inactivation of I_Ca was shifted from –20.9±0.75 mV to –27.7±1.35 mV by 3 M norfluoxetine (P<0.05, n=7). No such shift in the steady-state inactivation curve was observed in the case of I_to. Similarly, norfluoxetine caused no change in the steady-state current–voltage relationship of the membrane or in the density of the inward rectifier K⁺ current, I_K1. All these effects of norfluoxetine developed rapidly and were fully reversible. Comparing present results with those obtained previously with fluoxetine, it can be concluded that norfluoxetine displays stronger suppression of cardiac ion channels than fluoxetine. Consequently, the majority of the cardiac side effects observed during fluoxetine treatment are likely to be attributed to its metabolite norfluoxetine.     

Inhibition of rat brain monoamine oxidase enzymes by fluoxetine and norfluoxetine

Fluoxetine and its primary metabolite, norfluoxetine, are inhibitors of neuronal uptake of 5-hydroxytryptamine. While fluoxetine has also been reported to inhibit monoamine oxidase (MAO) in vitro at concentrations much lower than those measured in brain following chronic fluoxetine treatment, neurochemical profiles are not consistent with substantial MAO inhibition in vivo. In an attempt to explain this inconsistency, we have examined the interactions of fluoxetine and norfluoxetine with rat brain MAO-A and -B by a radiochemical assay method.Fluoxetine and norfluoxetine were competitive inhibitors of MAO-A in vitro, with K_i values of 76.3 M and 90.5 M, respectively. Both compounds were non competitive or uncompetitive inhibitors of MAO-B in vitro. Inhibition of MAO-B was time-dependent and was very slowly reversible by dialysis. IC₅₀ values versus metabolism of 50 M, -phenylethylamine were 17.8 M (fluoxetine) and 18.5 M (norfluoxetine). Analysis of the time-dependence of MAO-B inhibition by fluoxetine revealed that an initial competitive interaction between the enzyme and the inhibitor (K_i 245 M) was followed by tight-binding enzyme inactivation (k_inact 0.071 min^–1).Following administration of fluoxetine (20 mg kg^–1 day^–1]) for 7 days, the cortical concentration of fluoxetine + norfluoxetine was estimated by gas-liquid chromatography to be 700 M. Such drug treatment reduced MAO-A activity by 23% in 1:8 (w/v) cortical homogenates, but not in 1:80 homogenates. Inhibition of MAO-B in 1:8 homogenates was modest (12%) and was not significantly reduced by homogenate dilution. The concentration of 5-hydroxyindole-3-acetic acid, measured by high pressure liquid chromatography, was reduced by 47% in cortices from drug-treated rats, while concentrations of 5-hydroxytryptamine, noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid were unchanged. These results suggest that, following chronic drug administration leading to relatively high tissue concentrations of fluoxetine and norfluoxetine, inhibition of either form of MAO would be restricted by competition for the enzyme with intraneuronal amine substrates.     

Fluoxetine inhibits A-type potassium currents in primary cultured rat hippocampal neurons

The effects of fluoxetine (Prozac) on the transient A-currents (IA) in primary cultured hippocampal neurons were examined using the whole-cell patch clamp technique. Fluoxetine did not significantly decrease the peak amplitude of whole-cell K+ currents, but it accelerated the decay rate of inactivation, and thus decreased the current amplitude at the end of the pulse. For further analysis, IA and delayed rectifier K+ currents (IDR) were isolated from total K+ currents. Fluoxetine decreased IA (the integral of the outward current) in a concentration-dependent manner with an IC50 of 5.54 microM. Norfluoxetine, the major active metabolite of fluoxetine, was a more potent inhibitor of IA than was fluoxetine, with an IC50 of 0.90 microM. Fluoxetine (3 microM) inhibited IA in a voltage-dependent manner over the whole range of membrane potentials tested. Analysis of the time dependence of inhibition gave estimates of 34.72 microM(-1) s(-1) and 116.39 s(-1) for the rate constants of association and dissociation, respectively. The resulting apparent Kd was 3.35 microM, similar to the IC50 value obtained from the concentration-response curve. In current clamp configuration, fluoxetine (3 microM) induced depolarization of resting membrane potential and reduced the rate of action potential. Our results indicate that fluoxetine produces a concentration- and voltage-dependent inhibition of IA, and that this effect could affect the excitability of hippocampal neurons.     

液相色谱-串联质谱法研究盐酸氟西汀胶囊的人体药动学及生物利用度

目的:建立准确、灵敏的液相色谱-串联质谱法(LC-MS/MS)同时测定人血浆中的盐酸氟西汀和诺氟西汀,并研究健康受试者单剂量口服盐酸氟西汀胶囊试验制剂和参比制剂后的药动学和相对生物利用度.方法:20名健康男性受试者进行随机双交叉试验,分别单剂量口服 20 mg 盐酸氟西汀胶囊参比制剂和试验制剂,以盐酸舍曲林为内标,采用ESI正离子选择性反应测定盐酸氟西汀和诺氟西汀血浆浓度,计算药动学参数并进行生物等效性评价.结果:由两种制剂的AUC0-τ计算,受试胶囊盐酸氟西汀的相对生物利用度为(104.0±25.2)%.结论:建立的LC-MS/MS测定法准确、灵敏,结果可靠;统计分析表明盐酸氟西汀胶囊试验制剂和参比制剂生物等效.     

Pharmacokinetics and pharmacodynamics of norfluoxetine in rats: Increasing extracellular serotonin level in the frontal cortex

Norfluoxetine is the most important active metabolite of the widely used antidepressant fluoxetine. Although the pharmacokinetics/pharmacodynamics (PK/PD) relationship and neurochemical profile of fluoxetine is well characterized in human and in animals, little is known about the effect of its metabolite. The aim of this study was to characterize extracellular level of serotonin (5-hydroxytryptamine, 5-HT)-time profile of norfluoxetine after acute administration over 18 h post dose and to establish the relationship between this pharmacodynamic (PD) profile and its pharmacokinetic (PK) properties. Following subcutaneous administration of fluoxetine in rats, plasma and brain PK of fluoxetine and norfluoxetine were monitored respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The extracellular level of 5-HT in the frontal cortex was measured by microdialysis as a PD endpoint. Norfluoxetine when directly administrated to rats caused a significant increase in extracellular level of 5-HT in the frontal cortex and maintained for 18 h. This result is correlated well with higher plasma and brain concentration and longer plasma and brain retention time of norfluoxetine. Our results showed that norfluoxetine contributes to 5-HT transporter inhibition and extends fluoxetine efficacy.     

A simple method to monitor serum concentrations of fluoxetine and its major metabolite for pharmacokinetic studies

A rapid, selective and sensitive isocratic reversed-phase HPLC assay coupled with MS/MS detection for simultaneous quantification of fluoxetine and its major active metabolite in serum samples has been developed. Analytes were extracted with a simple three step liquid–liquid procedure and chromatographic separation was achieved on a C18 column.     

Norfluoxetine Induces Spawning and Parturition in Estuarine and Freshwater Bivalves

Fluoxetine, a commonly prescribed antidepressant (Prozac), has been detected in sewage effluent. Its active metabolite norfluoxetine is more potent and has been detected in sewage influent and in fish tissues. We tested the effects of norfluoxetine on spawning and parturition in bivalves. Norfluoxetine induced significant spawning in zebra mussels and dark false mussels at concentrations as low as 5 μM. Norfluoxetine induced significant parturition in fingernail clams at 10 μM. Fluoxetine also induced spawning in dark false mussels at concentrations as low as 100 nM. Implications for environmental impacts of norfluoxetine and fluoxetine on native and exotic bivalves are discussed.