Stereoselective Analysis of the Antiseizure Activity of Fenfluramine and Norfenfluramine in Mice: Is l-Norfenfluramine a Better Follow-Up Compound to Racemic-Fenfluramine?

The aim of this study was to investigate the comparative antiseizure activity of the l-enantiomers of d,l-fenfluramine and d,l-norfenfluramine and to evaluate the relationship between their concentration in plasma and brain and anticonvulsant activity. d,l-Fenfluramine, d,l-norfenfluramine and their individual enantiomers were evaluated in the mouse maximal electroshock seizure (MES) test. d,l-Fenfluramine, d,l-norfenfluramine and their individual l-enantiomers were also assessed in the DBA/2 mouse audiogenic seizure model. All compounds were administered intraperitoneally. Brain and plasma concentrations of the test compounds in DBA/2 mice were quantified and correlated with anticonvulsant activity. In the MES test, fenfluramine, norfenfluramine and their enantiomers showed comparable anticonvulsant activity, with ED50 values between 5.1 and 14.8 mg/kg. In the audiogenic seizure model, l-norfenfluramine was 9 times more potent than d,l-fenfluramine and 15 times more potent than l-fenfluramine based on ED50 (1.2 vs. 10.2 and 17.7 mg/kg, respectively). Brain concentrations of all compounds were about 20-fold higher than in plasma. Based on brain EC50 values, l-norfenfluramine was 7 times more potent than d,l-fenfluramine and 13 times more potent than l-fenfluramine (1940 vs. 13,200 and 25,400 ng/g, respectively). EC50 values for metabolically formed d,l-norfenfluramine and l-norfenfluramine were similar to brain EC50 values of the same compounds administered as such, suggesting that, in the audiogenic seizure model, the metabolites were responsible for the antiseizure activity of the parent compounds. Because of the evidence linking d-norfenfluramine to d,l-fenfluramine to cardiovascular and metabolic adverse effects, their l-enantiomers could potentially be safer follow-up compounds to d,l-fenfluramine. We found that, in the models tested, the activity of l-fenfluramine and l-norfenfluramine was comparable to that of the corresponding racemates. Based on the results in DBA/2 mice and other considerations, l-norfenfluramine appears to be a particularly attractive candidate for further evaluation as a novel, enantiomerically pure antiseizure medication.

Pharmacokinetics of d- and l-norfenfluramine following their administration as individual enantiomers in rats.

The effect of fenfluramine and norfenfluramine enantiomers in rodent seizure models and their correlation with the pharmacokinetics of d- and l-fenfluramine in rats have been reported recently. To complement these findings, we investigated the pharmacokinetics of d- and l- norfenfluramine in rat plasma and brain. Sprague-Dawley rats were injected intraperitoneally with 20 mg/kg and 1 mg/kg l- norfenfluramine. A 1 mg/kg dose of d-norfenfluramine was used because higher doses caused severe toxicity. The concentration of each enantiomer in plasma and brain was determined at different time points by liquid chromatography/mass spectrometry. Pharmacokinetic parameters were compared between norfenfluramine enantiomers, and with those reported previously for fenfluramine enantiomers after a 20 mg/kg, i.p., dose. All enantiomers were absorbed rapidly and eliminated, with half-lives ranging from 0.9 h (l-fenfluramine) to 6.1 h (l- norfenfluramine, 20 mg/kg) in plasma, and from 3.6 h (d-fenfluramine) to 8.0 h (l-fenfluramine) in brain. Brain-to-plasma concentration ratios ranged from 15.4 (d-fenfluramine) to 27.6 (d-norfenfluramine), indicating extensive brain penetration. The fraction of d- and l-fenfluramine metabolized to norfenfluramine was estimated to be close to unity. This work is part of ongoing investigations to determine the potential value of developing enantiomerically pure l-fenfluramine or l-norfenfluramine as follow-up compounds to the marketed racemic fenfluramine.

Placental disposition of cannabidiol: An ex vivo perfusion study.

OBJECTIVE: In the absence of safety data in humans, the use of cannabidiol (CBD) is not recommended during pregnancy. Yet >50% of pregnancies in women with epilepsy are unintended, making fetal exposure to CBD possible. As a small-molecule, highly lipid-soluble drug, CBD is likely to be distributed into the placenta and cross it. To estimate the placental distribution profile of CBD and its potential short-term placental effects, we conducted an ex vivo perfusion study in human placentas. METHODS: Placentas were obtained from healthy women undergoing cesarean deliveries. Selected cotyledons were cannulated and perfused for 180 min with a CBD-containing medium (250 ng/mL, .796 µmol·L-1 ; representative of a low therapeutic concentration; n = 8). CBD concentrations were determined at 180 min in the medium and placental tissue using liquid chromatography-tandem mass spectrometry. A customized gene panel array was used to analyze the expression of selected genes in the perfused placental cotyledons as well as in placentas perfused with 1000 ng/mL CBD (3.18 µmol·L-1 ; high therapeutic concentration; n = 8) and in those exposed to the vehicle. RESULTS: CBD was sequestered in the placental tissue, exhibiting significant variability across samples (median = 5342 ng/g tissue, range = 1066-9351 ng/g tissue). CBD concentrations in the fetal compartment were one fifth of those measured in the maternal compartment (median = 59 ng/mL, range = 48-72 ng/mL vs. 280 = ng/mL, range = 159-388 ng/mL, respectively; p < .01). Placental gene expression was not significantly altered by CBD. SIGNIFICANCE: The placenta acts as a depot compartment for CBD, slowing down its distribution to the fetus. This phenomenon might yield flatter but prolonged fetal CBD levels in vivo. The attenuated transplacental CBD transfer does not imply that its use by pregnant women is safe for the fetus. Only pregnancy registries and neurocognitive assessments would establish the risk of being antenatally exposed to CBD.

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.

Synthesis and Enantioselective Pharmacokinetic/Pharmacodynamic Analysis of New CNS-Active Sulfamoylphenyl Carbamate Derivatives.

We recently reported a new class of carbamate derivatives as anticonvulsants. Among these, 3-methylpentyl(4-sulfamoylphenyl)carbamate (MSPC) stood out as the most potent compound with ED50 values of 13 mg/kg (i.p.) and 28 mg/kg (p.o.) in the rat maximal electroshock test (MES). 3-Methylpropyl(4-sulfamoylphenyl)carbamate (MBPC), reported and characterized here, is an MSPC analogous compound with two less aliphatic carbon atoms in its structure. As both MSPC and MBPC are chiral compounds, here, we studied the carbonic anhydrase inhibitory and anticonvulsant action of both MBPC enantiomers in comparison to those of MSPC as well as their pharmacokinetic properties. Racemic-MBPC and its enantiomers showed anticonvulsant activity in the rat maximal electroshock (MES) test with ED50 values in the range of 19-39 mg/kg. (R)-MBPC had a 65% higher clearance than its enantiomer and, consequently, a lower plasma exposure (AUC) than (S)-MSBC and racemic-MSBC. Nevertheless, (S)-MBPC had a slightly better brain permeability than (R)-MBPC with a brain-to-plasma (AUC) ratio of 1.32 (S-enantiomer), 1.49 (racemate), and 1.27 (R-enantiomer). This may contribute to its better anticonvulsant-ED50 value. The clearance of MBPC enantiomers was more enantioselective than the brain permeability and MES-ED50 values, suggesting that their anticonvulsant activity might be due to multiple mechanisms of action.