Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

Abstract

Objectives:

An extended release formulation of dalfampridine (4-aminopyridine; 4-AP), a potassium channel blocker is available in the USA to improve walking in patients with multiple sclerosis. This study investigated the human metabolites of 4-AP and the cytochrome P450 (CYP450) pathways responsible for 4-AP metabolism.

Methods:

Metabolites were identified, using thin layer chromatography, high performance liquid chromatography, and gas chromatography/mass spectroscopy, in plasma and urine samples obtained during an excretion balance study of four subjects who were administered a single oral 15-mg dose of ¹⁴C-4-AP. Samples were compared with authentic standards of 4-AP, 2-hydroxy-4AP, 3-hydroxy-4AP, and 4-AP-N-oxide. Reaction phenotyping was performed in vitro using human liver microsomes and recombinant CYP450 enzymes with incubation in the presence of direct and time-dependent inhibitors to determine the CYP450 pathways involved in metabolite formation.

Results:

While most (∼70%) of the radioactivity detected in plasma at each time point corresponded to unchanged 4-AP, two major metabolites were recovered. One metabolite co-localized with the authentic reference standard of 3-hydroxy-4-AP, and the other metabolite was identified as the sulfate conjugate of 3-hydroxy-4-AP. Two minor components were observed, one accounting for 2% of radioactivity and the other below the level of quantitation. Reaction phenotyping showed moderate correlations for conversion of 4-AP to 3-hydroxy-4AP with both CYP2E1 (r = 0.596; p < 0.001) and CYP2C8 (r = 0.608; p < 0.001). Use of a CYP2E1 metabolism-dependent inhibitor inhibited formation of 3-hydroxy-4-AP with and without pre-incubation (higher inhibition with pre-incubation), further supporting the likelihood of CYP2E1 as a metabolic pathway. The main limitation of this study was the inability to identify the CYP enzymes responsible for the 3-hydroxylation of 4-AP, although this conversion represents only a minor metabolic pathway.

Conclusion:

There is limited metabolism of 4-AP in humans. The two major metabolites were 3-hydroxy-4-AP and 3-hydroxy-4-AP sulfate, likely through CYP2E1 pathways; the possibility of other CYP enzymes playing a minor role in 4-AP metabolism could not be established unequivocally. Overall, these data suggest that there is a low risk for drug–drug interactions via an impact on 4-AP metabolism through cytochrome pathways.

Keywords::

• Dalfampridine
• 4-aminopyridine
• Metabolism
• Reaction phenotyping
• Cytochrome P450

Transparency

Declaration of funding

This study was funded by Acorda Therapeutics, Inc., Ardsley, NY, USA.

Declaration of financial/other relationship

AC and AB are employees and stockholders of Acorda Therapeutics, Inc., Ardsley, NY, USA.

Acknowledgments

The authors wish to thank E.J. Bienen, PhD, of The Curry Rockefeller Group, LLC, Tarrytown, New York, for medical editorial assistance with this report. Editorial support was funded by Acorda Therapeutics, Inc.