Metabolic Stability and Metabolite Identification of CYP450 Probe Substrates in Ferret Hepatocytes
Authors: Pu Jiang , Feng Wanyong 
Published: September 2024

Background:
Ferrets exhibit similar lung physiology to humans and display similar clinical signs
following influenza infection, making them a valuable model for studying high susceptibility and infection
patterns. However, the metabolic fate of several common human CYP450 probe substrates in ferrets is still
unknown and has not been studied.

Objective:
The purpose of this study was to investigate the metabolism of nine human CYP450 probe substrates
in ferret hepatocytes and explore their metabolic rate differences between ferrets and other species.

Method:
Nine substrates were individually incubated in ferret hepatocytes for up to 120 min. At each time
point, 30 μL mixtures were extracted for stability analysis using LC-MS/MS methods. After a 120-minute
incubation period, 400 μL of the mixtures were extracted for metabolite identification using UHPLC-QExactive
Plus.

Results:
The metabolic clearance was determined as follows: testosterone > phenacetin > bupropion > omeprazole
> midazolam > dextromethorphan > chlorzoxazone > taxol > diclofenac. Seven metabolites were identified
from phenacetin. Deethylation was found to be the major pathway, and the major metabolite was matched
with acetaminophen as probed with the CYP1A2 enzyme. Six metabolites were identified from diclofenac.
Glucuronidation was the primary pathway, and a metabolite was found to match 4-OH-diclofenac as probed
with the CYP2C9 enzyme. Twenty-two metabolites were identified from omeprazole. The major metabolic
pathways included mono-oxygenation and sulfoxide to thioether conversion. No metabolite was found to
match with 5-OH-omeprazole as probed with the CYP2C19 enzyme. Twenty-two metabolites were identified
from dextromethorphan. Demethylation was found to be the major metabolic pathway, and one demethylation
metabolite was matched with dextrorphan as probed with the CYP2D6 enzyme. Fourteen metabolites were
identified from midazolam. Mono-oxygenation was found to be the primary metabolic pathway, and one of
the mono-oxygenation metabolites was matched with 1-OH-midazolam as probed with the CYP3A4 enzyme.
Eight metabolites were identified from testosterone. Mono-oxygenation and glucuronidation were identified
as the major metabolic pathways. One mono-oxygenation was matched with 6-β-testosterone as probed with
the CYP3A4 enzyme. Six metabolites were identified from taxol. Hydrolysis and mono-oxygenation were the
top two metabolic pathways. No metabolite was matched with 6-α-OH-taxol as probed with the CYP2C8
enzyme. Ten metabolites were identified from bupropion. Mono-oxygenation and hydrogenation were identified
as the top two metabolic pathways. No mono-oxygenation metabolite was matched with hydroxy-bupropion
as probed with the CYP2B6 enzyme. Nine metabolites were identified from chlorzoxazone. Monooxygenation
and sulfation were the top two metabolic pathways. One mono-oxygenation metabolite was
matched with 6-OH-chlorzoxazone as probed with the CYP2E1 enzyme.

Conclusion:
Nine human CYP probe substrates were clearly metabolized in ferret hepatocytes, demonstrating
substrate-dependent metabolic rates in ferret hepatocytes and species-dependent metabolic rates in mouse, rat,
dog, monkey, and human hepatocytes. Except for 6-a-5-OH-omeprazole, 6-α-OH-taxol, and hydroxy-bupropion,
specific metabolites of other six probe substrates in ferret hepatocytes were detected and identified
as probed with six human CYP enzymes, respectively.