key: cord-0856754-mmy4sa95 authors: Tipthara, Phornpimon; Kobylinski, Kevin C.; Godejohann, Markus; Hanboonkunupakarn, Borimas; Roth, Alison; Adams, John H.; White, Nicholas J.; Jittamala, Podjanee; Day, Nicholas P. J.; Tarning, Joel title: Identification of the metabolites of ivermectin in humans date: 2021-01-26 journal: Pharmacol Res Perspect DOI: 10.1002/prp2.712 sha: a8b8674ddef306629d73204d2f51854131246c54 doc_id: 856754 cord_uid: mmy4sa95 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. Ivermectin (IVM) is an antiparasitic and endectocidic drug used for decades in animal health and for treating onchocerciasis, lymphatic filariasis, scabies, and strongyloidiasis in humans. 1 IVM also has some antiviral activity including against SARS-CoV-2 in vitro. 2 Malaria is a mosquito-borne disease transmitted by Anopheles mosquitoes during blood feeding. Numerous studies have reported the mosquito-lethal effect of IVM [3] [4] [5] and the ability to inhibit sporogony of Plasmodium in the mosquito. [6] [7] [8] Mass drug administration (MDA) of IVM has been suggested as a possible vector control tool to aid malaria elimination as it has been shown to reduce Plasmodium transmission by mosquitoes 9 and reduce transmission to humans. 10 A recent clinical trial in Thailand showed that mosquito-lethal effects persisted well beyond the detectable presence of the parent compound, which suggests that IVM may have active metabolites that are more slowly eliminated than the parent compound. 5 IVM is a semisynthetic compound derived from avermectin Both compounds have the same antiparasitic activity. 12 There have been several studies of IVM metabolites produced in nonhuman vertebrates. [13] [14] [15] [16] [17] The major metabolite found in rats, cattle, and sheep is the 24-hydroxymethyl derivative 13, 16 while only trace levels are found in pigs. The 3″-O-demethyl derivative is the major metabolite present in pigs. 14 A previous in vitro study using human liver microsomes found nine IVM metabolites, mostly hydroxylated and demethylated compounds including the two listed above. 18 In this study, we aimed to identify the common metabolites in humans through in vitro and in vivo experiments. Pooled human liver microsomes and primary human hepatocytes were exposed to IVM, and metabolite fractions were collected to identify metabolites produced in vitro. Human whole blood, collected from healthy volunteers after a single oral dose of IVM (400 µg/kg), was used to identify metabolites produced in vivo. While the standard dose of ivermectin is 200 µg/kg, the 400 µg/kg dose has been shown to be safe and efficacious in numerous clinical trials, is frequently used in clinical settings, and is now recommended on the package insert for lymphatic filariasis mass drug administration. Previous pharmacokinetic modeling and in vitro mosquito mortality experiments indicated that IVM 400 µg/kg is the ideal dose to target the important Anopheles malaria vectors in the Greater Mekong Subregion. 8 The structure of IVM metabolites were characterized by LC-MS/MS and verified by NMR. The metabolic pathways that generated these metabolites were characterized by incubation of IVM with purified human cytochrome P450 (CYP) enzymes, followed by LC-MS/MS analysis. (v/v)), and 5 µl of microsomes. The tube was vortexed briefly and incubated at 37°C for 5 min in a shaking water bath. The reaction was initiated by adding 10 µl of 20 mM NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) prepared in 100 mM potassium phosphate buffer pH 7.4. Total reaction volume per tube F I G U R E 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 ) was 200 µl with the final concentration of 10 µM IVM and 1.0 mM NADPH. Each tube was vortexed briefly and a baseline sample (0 min control) was collected by aliquoting 100 µl of the metabolite fraction mixture described above to a separate tube with 100 µl of pre-chilled acetonitrile, which was kept on ice until centrifugation. Two separate negative control tubes were prepared, one without NADPH (negative co-factor control) but with an extra 10 µl of buffer and a second tube without IVM substrate (negative IVM control) with an extra 2 µl of buffer. All remaining reactions, including negative co-factor control, and negative IVM control, were incubated at 37°C for 60 min with gentle shaking. After 60 min of incubation, all tubes were removed from the water bath and cold acetonitrile was added immediately to make a final 1:1 (v/v) ratio. All tubes (0-and 60-min reactions, negative co-factor, and negative IVM controls) were vortexed briefly again and centrifuged at 10,000g for 15 min at 4°C. The supernatant was IVM (IVM-B 1a > 95%, IVM-B 1b < 2%) Primary human hepatocytes were seeded on a 384-well plate as described previously to stimulate reacquisition of in vivo physiologic activity. 19 At day 3 post seed, IVM (10 µM) was added to each well. There were no media changes and no subsequent additions of IVM. Venous blood was collected from three healthy Thai volunteers given a single oral dose of IVM (400 µg/kg) (NCT02568098). The LC-MS/MS system used was an ultra-high performance liquid Mass range of TOF-MS scan was at m/z 100-1000 and product ion scan was at m/z 50-1000. IVM standard solution (100 ng/ml) was injected before and after batch analysis for validating the system performance. The analyses were performed by in-line instruments of interfacing liquid chromatography with parallel NMR and mass spectrometry. The 60 dried microsome pellets described above were each reconstituted in 300 µl of methanol and sonicated for 5 min. Supernatants were pooled into one 50 ml falcon tube. A second extraction of the microsome residue was performed by adding an additional 300 µl of acetonitrile followed by sonication for 5 min. The thirteen metabolites (M1 to M13) identified from the (Table S1 ). The relative abundance of ivermectin and metabolites, generated using specific cDNA-expressed human cytochrome P450 enzymes, is provided in the supplementary information (Table S2) . respectively) ( Figure 5B ). The NMR spectra confirmed that the demethylation of M1 occurred at C3″ (labeled in yellow, Figure 6A) and that the hydroxylation of M3 occurred at C4 (labeled in yellow, Figure 6B ). The evaluation of HSQC and HMBC spectra of IVM, M1, and M3 shows the location of biotransformation (Table 3 , Figure 6 , Supplement Appendix S2). Chemical shift values for proton and carbon resonances are shown in Table 3 . As the sensitivity of the 500 MHz instrument was not sufficient to obtain an HMBC spectrum within the period of the nitrogen refill cycle (one week), the isolated metabolites were analyzed using an 800 MHz instrument. This provided a complete set of NMR data 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 , Figure 2 ). The four most abundant of these metabolites were also present in IVM-exposed primary human hepatocytes (M1 > M3 > M5 > M6). Three of these metabolites were found also in human volunteer blood samples taken 24 h after IVM administration (M1 > M3 > M6). 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. 18 However, four additional IVM metabolites, including ketone and carboxylic derivatives, were also found in our study. Advancements 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 support the M6 structure based on the elution order. The elution order in this study is also consistent with the study of Zeng et al.. 18 Interestingly, many low abundance metabolites produced in microsomes were not detected from primary human hepatocytes in culture or from human volunteer blood after IVM administra- We report for the first time, novel IVM metabolites from human liver microsomes, primary human hepatocytes, and from human blood after oral IVM dosing. Importantly, we identified and confirmed the structure of the two major IVM metabolites in humans; 3″-Odemethylation IVM (M1) and 4-hydroxymethyl IVM (M3). The authors declare no conflict of interest. We did not purchase any of the compounds or instruments mentioned in this article from Bruker. provided with an explanation of the study and signed a written informed consent before study entry. The data that support the findings of this study are available from the corresponding author upon reasonable request. Phornpimon Tipthara Podjanee Jittamala Ivermectin in human medicine, an overview of the current status of its clinical applications The FDAapproved Drug Ivermectin inhibits the replication of SARS-CoV-2 in vitro Ivermectin to reduce malaria transmission: a research agenda for a promising new tool for elimination Safety and mosquitocidal efficacy of high-dose ivermectin when co-administered with dihydroartemisinin-piperaquine in Kenyan adults with uncomplicated malaria (IVERMAL): a randomised, double-blind, placebo-controlled trial Safety, pharmacokinetics, and mosquito-lethal effects of ivermectin in combination with dihydroartemisinin-piperaquine and primaquine in healthy adult Thai subjects Ivermectin inhibits the sporogony of Plasmodium falciparum in Anopheles gambiae Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi Ivermectin susceptibility and sporontocidal effect in Greater Mekong Subregion Anopheles Evaluation of ivermectin mass drug administration for malaria transmission control across different West African environments Efficacy and risk of harms of repeat ivermectin mass drug administrations for control of malaria (RIMDAMAL): a cluster-randomised trial Ivermectin and Abamectin, 1st edn Ivermectin: an update The metabolism of avermectins B1a, H2B1a, and H2B1b by liver microsomes The metabolism of avermectin-H2B1a and -H2B1b by pig liver microsomes Metabolic disposition of ivermectin in tissues of cattle, sheep, and rats Comparative metabolic disposition of ivermectin in fat tissues of cattle, sheep, and rats Comparative in vivo and in vitro metabolism of ivermectin in steers, sheep, swine, and rat Identification of cytochrome P4503A4 as the major enzyme responsible for the metabolism of ivermectin by human liver microsomes A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum The use of mass defect in modern mass spectrometry Mass defect filter technique and its applications to drug metabolite identification by high-resolution mass spectrometry Improving LC-MS sensitivity through increases in chromatographic performance: comparisons of UPLC-ES/MS/MS to HPLC-ES/MS/MS Liquid chromatography-mass spectrometry based global metabolite profiling: a review Current applications of high-resolution mass spectrometry in drug metabolism studies It is time for a paradigm shift in drug discovery bioanalysis: from SRM to HRMS Genetic polymorphisms in MDR1, CYP3A4 and CYP3A5 genes in a Ghanaian population: a plausible explanation for altered metabolism of ivermectin in humans Additional supporting information may be found online in the Supporting Information section. 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