Introduction
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 (Gonzalez et al., 2012). IVM also has some antiviral activity including against SARS-CoV-2in vitro (Caly et al., 2020).
Malaria is a mosquito-borne disease transmitted by Anophelesmosquitoes during blood feeding. Numerous studies have reported the mosquito-lethal effect of ivermectin (Chaccour et al., 2013; Kobylinski et al., 2020; Smit et al., 2018) and the ability to inhibit sporogony ofPlasmodium in the mosquito (Kobylinski et al., 2012; Kobylinski et al., 2017; Pinilla et al., 2018). 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 Plasmodiumtransmission by mosquitoes (Alout et al., 2014) and reduce transmission to humans (Foy et al., 2019). 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 (Kobylinski et al., 2020).
Ivermectin is a semisynthetic compound derived from avermectin (B1 series), a natural fermentation product of the soil bacterium Streptomyces avermilitis. The regiospecific hydrogenation of the avermectin B1 at the 22,23-double bond produces the 22,23-single bond derivative called 22,23-dihydroavermectin B1 or ivermectin (William C. Campbell, 1989). Ivermectin is a mixture containing at least 90% of 22,23-dihydroavermectin B1a(H2B1a or ivermectin B1aor IVM-B1a) and less than 10% of 22,23-dihydroavermectin B1b(H2B1b or ivermectin B1bor IVM-B1b). Both show the same antiparasitic activity (W. C. Campbell, 1985). The chemical structures shown in Figure 1 indicate the alkyl side chain difference between IVM-B1a and IVM-B1b at C25.
There are several studies of IVM metabolites produced in non-human vertebrates (Chiu et al., 1988; Chiu et al., 1986; Chiu et al., 1984; Chiu et al., 1987; Miwa et al., 1982). The major metabolite found in rats, cattle, and sheep is the 24-hydroxymethyl derivative (Chiu et al., 1988; Miwa et al., 1982) while only trace levels are found in pigs. The 3″-O -demethyl derivative is the major metabolite present in pigs (Chiu et al., 1984). A previous in vitro study using human liver microsomes found nine IVM metabolites, mostly hydroxylated and demethylated compounds including the two listed above (Zeng et al., 1998).
In this study, we aimed to identify the common metabolites of 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 for ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) analysis. 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 . The structure of IVM metabolites were defined 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.