Identification of the metabolites of ivermectin in humans

Abstract

Mass drug administration of ivermectin has been proposed as a possible malaria elimination tool. Ivermectin exhibits a mosquito-lethal effect well beyond its biological half-life, suggesting the presence of active slowly eliminated metabolites. Human liver microsomes, primary human hepatocytes, and whole blood from healthy volunteers given oral ivermectin were used to identify ivermectin metabolites by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry. The molecular structures of metabolites were determined by mass spectrometry and verified by nuclear magnetic resonance. Pure cytochrome P450 enzyme isoforms were used to elucidate the metabolic pathways. Thirteen different metabolites (M1-M13) were identified after incubation of ivermectin with human liver microsomes. Three (M1, M3, and M6) were the major metabolites found in microsomes, hepatocytes, and blood from volunteers after oral ivermectin administration. The chemical structure, defined by LC-MS/MS and NMR, indicated that M1 is 3″-O-demethyl ivermectin, M3 is 4-hydroxymethyl ivermectin, and M6 is 3″-O-demethyl, 4-hydroxymethyl ivermectin. Metabolic pathway evaluations with characterized cytochrome P450 enzymes showed that M1, M3, and M6 were produced primarily by CYP3A4, and that M1 was also produced to a small extent by CYP3A5. Demethylated (M1) and hydroxylated (M3) ivermectin were the main human in vivo metabolites. Further studies are needed to characterize the pharmacokinetic properties and mosquito-lethal activity of these metabolites.

Keywords: LC-MS/MS; ivermectin; malaria; metabolism.

FIGURE 1

Molecular structure of ivermectin. IVM consists of a spiroketal unit (C17–C28), cyclohexene cyclic ether unit (C2–C8), and a disaccharide unit at C13. A secondary butyl side chain at C25 give rise to the major component (IVM‐B_1a) and an isopropyl side chain give rise to the minor component (<10%; IVM‐B_1b)

FIGURE 2

Metabolite chromatograms. Chromatogram (A) shows the metabolites identified using pooled human liver microsomes incubated with IVM for 60 min. The upper insert chromatogram shows a zoom of RT 8.2–12.8 min. Chromatogram (B) shows the metabolites identified in the media fraction of primary human hepatocytes exposed to IVM for 24 hours. The upper insert chromatogram shows a zoom of RT 10.3–12.8 min. Chromatogram (C) shows the metabolites identified in human volunteer blood 24 hours after IVM administration

FIGURE 3

Extracted ion chromatogram and MS/MS spectra. The figure shows (A) extracted ion chromatogram of IVM‐B_1a and IVM‐B_1b, (B) MS/MS spectrum of IVM‐B_1b, (C) MS/MS spectrum of IVM‐B_1a, and (D) major fragments of IVM

FIGURE 4

MS/MS spectra of metabolite M1 to M13. Characteristic fragment ion peaks of metabolites matched to ivermectin library peaks are shown in orange. Various characteristic neutral losses ion peaks are shown in different colors

FIGURE 5

Metabolite purification and detection for NMR analysis. Panel (A) is an extracted ion chromatogram of IVM, M1, and M3 during the preparation chromatography, and UV chromatogram at 240 nm for the trapping experiment (the dotted lines indicate the start and end of the trapping procedure, (B) is a mass spectrum from the peaks in the extracted ion chromatogram demonstrating that the molecules form stable sodium adducts

FIGURE 6

NMR spectra of ivermectin (left) compared with M1 (middle) and M3 (right). The structure of IVM was labeled in yellow at signals showing the most significant differences in the spectra of the metabolites. The cursor in HSQC spectrum indicates signals missing due to changes in the metabolites. Panel (A) the shift of the –CH group in the metabolite M1 (m/z 883) at the position labeled in yellow and (B) the missing –CH₃ group of the metabolite M3 (m/z 913) at position labeled in yellow. The changes in chemical shift values are listed in Table 3

FIGURE 7

Proposed metabolic pathway of ivermectin metabolites. The exact site of biotransformation could not be confirmed for some metabolites. In these, the potential biotransformation sites are highlighted in red shading