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

Abstract

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 (t_1/2 > 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).

Keywords: antiepileptics; cytochrome P450; drug transport; drug-drug interactions.

FIGURE 1

Metabolic stability (percentage substrate loss) of 1 µM Fenfluramine (FFA) in (A) liver S9 fractions or (B) intestinal S9 fractions from rat, dog, or human over time in the presence or absence of NADPH‐containing cofactor mix. Loss of 10 µM FFA substrate in liver (C) and intestinal (D) S9 fractions from rat, dog, and human in the presence or absence of NADPH/cofactors. FFA, fenfluramine; NADPH, nicotinamide adenine dinucleotide phosphate (hydrogen)

FIGURE 2

Identification of Fenfluramine (FFA) and norfenfluramine (nFFA) in 120‐min incubations of human liver S9 fraction (representative spectra). (A) Extracted ion chromatogram of FFA in the presence or absence of NADPH‐generating system (120 min). (B) CID MS/MS spectrum of 2 µM FFA reference standard (m/z 232; t_R = 9.9 min). (C) Representative extracted ion chromatogram of nFFA in the presence or absence of NADPH‐generating system. (D) CID MS/MS spectrum of 2 µM nFFA reference standard (m/z 204; t_R = 9.12 min). CID, collision‐induced dissociation; FFA, fenfluramine; MS/MS, tandem mass spectrometry; m/z, mass‐to‐charge ratio; NADPH, nicotinamide adenine dinucleotide phosphate (hydrogen); nFFA, norfenfluramine; t_R, retention time

FIGURE 3

Identification of C2 in 120‐min incubations of Fenfluramine (FFA) (10 µM) with rat, dog, or human liver S9 fractions (2 mg protein/mL). (A) Extracted ion chromatogram of C2 from rat, dog, or human liver S9 fractions in the presence or absence of NADPH‐generating system. (B) CID MS/MS spectrum of C2 (m/z 220; t_R = 10.8 min). CID, collision‐induced dissociation; FFA, fenfluramine; MS/MS, tandem mass spectrometry; m/z, mass‐to‐charge ratio; NADPH, nicotinamide adenine dinucleotide phosphate (hydrogen); t_R, retention time. In the absence of NADPH, no C2 peak was observed in any species.

FIGURE 4

Identification of C1 and C6 in 120‐min incubations of Fenfluramine (FFA) (10 µM) with rat liver S9 fraction (2 mg protein/mL). (A) Extracted ion chromatogram of FFA metabolites formed in the presence or absence of NADPH‐generating system. (B) CID MS/MS spectrum of FFA (m/z 424; t_R = 4.0 min) in C1. (C) CID MS/MS spectrum of FFA (m/z 424; t_R = 12.7 min) in C6. CID, collision‐induced dissociation; FFA, fenfluramine; MS/MS, tandem mass spectrometry; m/z, mass‐to‐charge ratio; NADPH, nicotinamide adenine dinucleotide phosphate (hydrogen); t_R, retention time

FIGURE 5

Identification of C3, C4, and C5 in 120‐min incubations of Fenfluramine (FFA) (10 µM) with rat liver S9 fraction (2 mg protein/mL). (A) Extracted ion chromatogram of FFA metabolites formed in the presence or absence of NADPH‐generating system. (B) CID MS/MS spectrum of C3 (m/z 394; t_R = 11.7 min). (C) CID MS/MS spectrum of C4 (m/z 218; t_R = 12.5 min). (D) CID MS/MS spectrum of C5 (m/z 218; t_R = 12.6 min). CID MS/MS spectrum of norfenfluramine (nFFA) (m/z 204; t_R = 9.1 min). CID, collision‐induced dissociation; FFA, fenfluramine; MS/MS, tandem mass spectrometry; m/z, mass‐to‐charge ratio; NADPH, nicotinamide adenine dinucleotide phosphate (hydrogen); nFFA, norfenfluramine; t_R, retention time

FIGURE 6

Fenfluramine (FFA) substrate loss, norfenfluramine (nFFA) formation, and nFFA substrate loss in human liver microsomes (1 mg/mL protein). (A) NADPH‐dependent FFA loss in incubations of 1, 10, or 100 µM substrate. (B) NAPDH‐dependent nFFA formation in incubations of 1, 10, or 100 µM FFA. (C) nFFA loss in incubations of 0.1, 1, or 10 µM substrate. Points represent the mean of n=2 replicates per condition. When substrate was incubated for 30 min without the NADPH‐generating system, all values were BLQ or near the lower limits of quantitation. BLQ, below limits of quantitation; FFA, fenfluramine; NADPH, nicotinamide adenine dinucleotide phosphate (hydrogen); nFFA, norfenfluramine

FIGURE 7

Fenfluramine (FFA) and norfenfluramine (nFFA) substrate loss and nFFA formation by rCYPs. (A) FFA substrate loss (%), (B) nFFA metabolite formation (pmol/incubation), and (C) nFFA substrate loss (%) were determined in recombinant human CYP enzymes expressed in E. coli. Enzymes were incubated with 1 or 10 µM FFA (A, B) and 0.1 or 1 µM nFFA (C). Controls: membranes from E. coli. transfected with empty‐expression plasmid (control bactosome) or with plasmid expressing human NADPH‐cytochrome P450 oxidoreductase but no human CYP enzyme (reductase control). rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C19, and rCYP3A4 were co‐incubated with cytochrome b₅ reductase. Hashed line indicates 20% threshold. Values are the mean of duplicate determination. BLQ, below the limits of quantification (0.01 μM, which is equivalent to 2 pmol per incubation); FFA, fenfluramine; NADPH, nicotinamide adenine dinucleotide phosphate (hydrogen); nFFA, norfenfluramine; ND, not detected; rCYP, recombinant cytochrome P450

FIGURE 8

Metabolic pathway of fenfluramine. Adapted from Brownsill 1991