Fenfluramine modulates the anti-amnesic effects induced by sigma-1 receptor agonists and neuro(active)steroids in vivo.

Fenfluramine (N-ethyl-α-methl-3-(trifluoromethyl)phenethylamine) is an anti-seizure medication (ASM) particularly effective in patients with Dravet syndrome, a severe treatment-resistant epileptic encephalopathy. Fenfluramine acts not only as neuronal serotonin (5-HT) releaser but also as a positive modulator of the sigma-1 receptor (S1R). We here examined the modulatory activity of Fenfluramine on the S1R-mediated anti-amnesic response in mice using combination analyses. Fenfluramine and Norfenfluramine, racemate and isomers, were combined with either the S1R agonist (PRE-084) or the S1R-acting neuro(active)steroids, pregnenolone sulfate (PREGS), Dehydroepiandrosterone sulfate (DHEAS), or progesterone. We report that Fenfluramine racemate or (+)-Fenfluramine, in the 0.1-1 mg/kg dose range, attenuated the dizocilpine-induced learning deficits in spontaneous alternation and passive avoidance, and showed low-dose synergies in combination with PRE-084. These effects were blocked by the S1R antagonist NE-100. Dehydroepiandrosterone sulfate or PREGS attenuated dizocilpine-induced learning deficits in the 5-20 mg/kg dose range. Co-treatments at low dose between steroids and Fenfluramine or (+)-Fenfluramine were synergistic. Progesterone blocked Fenfluramine effect. Finally, Fenfluramine and (+)-Fenfluramine effects were prevented by the 5-HT1A receptor antagonist WAY-100635 or 5-HT2A antagonist RS-127445, but not by the 5-HT1B/1D antagonist GR 127935 or the 5-HT2C antagonist SB 242084, confirming a 5-HT1A and 5-HT2A receptor involvement in the drug effect on memory. We therefore confirmed the positive modulation of Fenfluramine racemate or dextroisomer on S1R and showed that, in physiological conditions, the drug potentiated the low dose effects of neuro(active)steroids, endogenous S1R modulators. The latter are potent modulators of the excitatory/inhibitory balance in the brain, and their levels must be considered in the antiepileptic action of Fenfluramine.

An Emerging Role for Sigma-1 Receptors in the Treatment of Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathies (DEEs) are complex conditions characterized primarily by seizures associated with neurodevelopmental and motor deficits. Recent evidence supports sigma-1 receptor modulation in both neuroprotection and antiseizure activity, suggesting that sigma-1 receptors may play a role in the pathogenesis of DEEs, and that targeting this receptor has the potential to positively impact both seizures and non-seizure outcomes in these disorders. Recent studies have demonstrated that the antiseizure medication fenfluramine, a serotonin-releasing drug that also acts as a positive modulator of sigma-1 receptors, reduces seizures and improves everyday executive functions (behavior, emotions, cognition) in patients with Dravet syndrome and Lennox-Gastaut syndrome. Here, we review the evidence for sigma-1 activity in reducing seizure frequency and promoting neuroprotection in the context of DEE pathophysiology and clinical presentation, using fenfluramine as a case example. Challenges and opportunities for future research include developing appropriate models for evaluating sigma-1 receptors in these syndromic epileptic conditions with multisystem involvement and complex clinical presentation.

Fenfluramine acts as a positive modulator of sigma-1 receptors.

OBJECTIVE: Adjunctive fenfluramine hydrochloride, classically described as acting pharmacologically through a serotonergic mechanism, has demonstrated a unique and robust clinical response profile with regard to its magnitude, consistency, and durability of effect on seizure activity in patients with pharmacoresistant Dravet syndrome. Recent findings also support long-term improvements in executive functions (behavior, emotion, cognition) in these patients. The observed clinical profile is inconsistent with serotonergic activity alone, as other serotonergic medications have not been demonstrated to have these clinical effects. This study investigated a potential role for σ1 receptor activity in complementing fenfluramine's serotonergic pharmacology. METHODS: Radioligand binding assays tested the affinity of fenfluramine for 47 receptors associated with seizures in the literature, including σ receptors. Cellular function assays tested fenfluramine and norfenfluramine (its major metabolite) activity at various receptors, including adrenergic, muscarinic, and serotonergic receptors. The σ1 receptor activity was assessed by the mouse vas deferens isometric twitch and by an assay of dissociation of the σ1 receptor from the endoplasmic reticulum stress protein binding immunoglobulin protein (BiP). In vivo mouse models assessed fenfluramine activity at σ1 receptors in ameliorating dizocilpine-induced learning deficits in spatial and nonspatial memory tasks, alone or in combination with the reference σ1 receptor agonist PRE-084. RESULTS: Fenfluramine and norfenfluramine bound ≥30% to ß2-adrenergic, muscarinic M1, serotonergic 5-HT1A, and σ receptors, as well as sodium channels, with a Ki between 266 nM (σ receptors) and 17.5 µM (ß-adrenergic receptors). However, only σ1 receptor isometric twitch assays showed a positive functional response, with weak stimulation by fenfluramine and inhibition by norfenfluramine. Fenfluramine, but not the 5-HT2C agonist lorcaserin, showed a positive modulation of the PRE-084-induced dissociation of σ1 protein from BiP. Fenfluramine also showed dose-dependent antiamnesic effects against dizocilpine-induced learning deficits in spontaneous alternation and passive avoidance responses, which are models of σ1 activation. Moreover, low doses of fenfluramine synergistically potentiated the low-dose effect of PRE-084, confirming a positive modulatory effect at the σ1 receptor. Finally, all in vivo effects were blocked by the σ1 receptor antagonist NE-100. SIGNIFICANCE: Fenfluramine demonstrated modulatory activity at σ1 receptors in vitro and in vivo in addition to its known serotonergic activity. These studies identify a possible new σ1 receptor mechanism underpinning fenfluramine's central nervous system effects, which may contribute to its antiseizure activity in Dravet syndrome and positive effects observed on executive functions in clinical studies.

In vitro evaluation suggests fenfluramine and norfenfluramine are unlikely to act as perpetrators of drug interactions.

Studies support the safety and efficacy of fenfluramine (FFA) as an antiseizure medication (ASM) in Dravet syndrome, Lennox-Gastaut syndrome, or CDKL5 deficiency disorder, all pharmacoresistant developmental and epileptic encephalopathies. However, drug-drug interactions with FFA in multi-ASM regimens have not been fully investigated. We characterized the perpetrator potential of FFA and its active metabolite, norfenfluramine (nFFA), in vitro by assessing cytochrome P450 (CYP450) inhibition in human liver microsomes, CYP450 induction in cultured human hepatocytes, and drug transporter inhibition potential in permeability or cellular uptake assays. Mean plasma unbound fraction was ~50% for both FFA and nFFA, with no apparent concentration dependence. FFA and nFFA were direct in vitro inhibitors of CYP2D6 (IC50 , 4.7 and 16 µM, respectively) but did not substantially inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5. No time- or metabolism-dependent CYP450 inhibition occurred. FFA and nFFA did not induce CYP1A2; both induced CYP2B6 (up to 2.8-fold and up to 2.0-fold, respectively) and CYP3A4 (1.9- to 3.0-fold and 3.6- to 4.8-fold, respectively). Mechanistic static pharmacokinetic models predicted that neither CYP450 inhibition nor induction was likely to be clinically relevant at doses typically used for seizure reduction (ratio of area under curve [AUCR] for inhibition <1.25; AUCR for induction >0.8). Transporters OCT2 and MATE1 were inhibited by FFA (IC50 , 19.8 and 9.0 µM) and nFFA (IC50 , 5.2 and 4.6 µM) at concentrations higher than clinically achievable; remaining transporters were not inhibited. Results suggest that FFA and nFFA are unlikely drug-drug interaction perpetrators at clinically relevant doses of FFA (0.2-0.7 mg/kg/day).

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 MAP Kinase Cascade Is Activated prior to the Induction of Gliosis in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Model of Dopaminergic Neurotoxicity.

Injury to the central nervous system (CNS) provokes microglial activation and astrocytic hypertrophy at the site of damage. The signaling events that underlie these cellular responses remain unknown. Recent evidence has implicated tyrosine phosphorylation systems, in general, and the mitogen-activated protein kinase (MAP kinase) cascade, in particular, in the mediation of growth-associated events linked to neural degeneration, such as glial activation. 1 Moreover, an increase in the mRNA coding for the 14.3.3 protein, a known regulator of the MAP kinase pathway, 2 appears to be involved in methamphetamine neurotoxicity. 3 To examine the potential role of these protein kinase pathways in drug-induced damage to the CNS, we used the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to damage nerve terminals in the mouse neostriatum and elicit a glial reaction. The onset of reactive gliosis then was verified by Northern blot analysis of glial fibrillary acidic protein (GFAP) mRNA and qualified by enzyme-linked immunosorbent assay (ELISA) of GFAP (protein). A single administration of MPTP (12.5 mg/kg, subcutaneously (s.c.)) to the C57B1/6J mouse resulted in a 10-fold increase in GFAP mRNA by 1 day and a 4-fold increase in GFAP (protein) by 2 days. To determine the potential role of protein tyrosine phosphorylation and MAP kinase activation in these events, blots of striatal homogenates were probed with antibodies directed against phospho-tyr 204 and phospho-thr 202, residues corresponding to the active sites of p42/44 MAP kinase. After mice were sacrificed by focused microwave irradiation to preserve steady-state phosphorylation, proteins from striatal homogenates were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots of these samples showed a number of phosphotyrosine-labeled bands, but there were no apparent differences between control and MPTP groups. In contrast, phospho-MAP kinase was elevated over 1.5-fold, 3-6hours post MPTP. These findings are suggestive of a role of the MAP kinase cascade in the early phase of injury-induced glial activation.