Discussion and conclusions
The peak area of metabolites, relative to IVM, was used to estimate the
relative abundance of each metabolite. In 60-min microsomes reactions,
the five most abundant metabolites were M3 > M1
> M5 > M6 > M9 (Table
1). Metabolites M1, M3, and M6 were found after 24-hour exposure to
primary human hepatocytes and in volunteer blood samples taken 24 hours
post IVM administration. This is the first report of IVM metabolites
identified from human hepatocytes and clinical blood samples.
The IVM demethylation, oxidation, and monosaccharide metabolites
identified from microsomes in this study were consistent with those
reported previously from human microsomes (Zeng et al., 1998). However,
four additional IVM metabolites, including ketone and carboxylic
derivatives, were also found in our study. Advancements in UHPLC
technology (Churchwell et al., 2005) and state-of-the-art high
resolution mass spectrometry (Meyer & Maurer, 2012; Ramanathan et al.,
2011; Theodoridis et al., 2012) used here improved the sensitivity for
metabolite detection. By using pure CYP enzymes, we observed evidence
for two CYP metabolism pathways for IVM; CYP3A5 produced demethylated
IVM (M1) and CYP2C8 produced hydroxylated IVM (M13).
The chemical structures of the most abundant metabolites were obtained
by NMR for M1 and M3 but not for M6. The metabolic pathway data suggest
that M1, M3, and M6 were all produced by CYP3A4, and that M6 is a
combination of oxidation and demethylation. Thus, we propose that M6 is
3″-O-demethyl, 4-hydroxymethyl-ivermectin, a further (common) metabolite
product of M1 and M3. With two sites of transformation occurring in M6
(demethylation and oxidation), it is more polar and elutes earlier than
M1 and M3. Additional reversed phase chromatography data supports the M6
structure based on the elution order. The elution order in this study is
also consistent with Zeng et al. 1998.
Interestingly, many low abundance metabolites produced in microsomes
were not detected from primary human hepatocytes in culture nor from
human volunteer blood after IVM administration. Several factors that
could influence the metabolic function of hepatocytes in vitro ,
including initial cell suspension, confluence density of adherent cells,
and drug concentration. The lower number of metabolites found in
volunteer blood samples compared to microsomes could be because of
phospholipids in blood samples. In vitro systems are also more
efficient in producing metabolites and do not have elimination pathways
(such as renal elimination) compared to in vivo systems, which
could have an impact on detection results. In future studies, we will
characterize IVM metabolites produced at later time points from
hepatocytes and human blood, when metabolite abundance may possibly be
altered compared to the 24-hour time point thus allowing
characterization of in vivo metabolism over time. To support
these efforts, we have performed a clinical trial (NCT03690453) to asses
the pharmacokinetic profile of key IVM metabolites in orally treated
volunteers over several weeks, characterized and synthesized these
metabolites, and evaluated their potential mosquito-lethal and
antimalarial effects. A better understanding of IVM metabolite
pharmacokinetics can provide further insight into pharmacodynamics and
efficacy for NTDs, especially ones requiring multiple administrations
such as scabies and strongyloidiasis. Furthermore, these IVM metabolites
may inhibit viral replication and should be evaluated against
SARS-CoV-2, the causative agent of COVID-19.
In conclusion, we report for the first time, novel IVM metabolites from
human liver microsomes and IVM metabolites from primary human
hepatocytes and from human blood after oral IVM dosing. Importantly, we
identify that the two major IVM metabolites in humans are
3″-O -demethylation IVM and 4-hydroxymethyl IVM.