Chloroquine (CQ) and hydroxychloroquine (HCQ) are established 4-aminoquinoline antimalarials with therapeutic applications extending to autoimmune and viral diseases (Mubagwa, 2020). Research indicates that HCQ exhibits a safer profile compared to CQ. The primary contributors to HCQ's toxic side effects are believed to be its broad dosage range and substantial volume of distribution (Vd) (Al-Bari, 2015). In clinical practice, HCQ administration often relies on empirical judgment, which can lead to misuse and potential adverse outcomes (Tett et al., 1988; Shi et al., 2020; Abbas et al., 2021). HCQ is notably characterized by its propensity to accumulate in acidic environments and distribute within tissue cells. This feature, along with its absorption, distribution, metabolism, and excretion (ADME) properties, makes it a significant therapeutic option for inflammatory conditions such as malaria-induced red blood cell disorders and rheumatoid arthritis (RA) (Rainsford et al., 2015). Nonetheless, its high tissue distribution can contribute to drug resistance, as well as adverse effects including gastrointestinal reactions, ocular toxicity, cardiotoxicity, and other toxic side effects (Pers and Padern, 2020; Doyno et al., 2021). Drug-drug interactions (DDIs) are a leading cause of adverse effects in clinical medication use. Research indicates that the concurrent administration of HCQ with other pharmaceuticals can precipitate adverse reactions, despite potential improvements in therapeutic efficacy (Paniri et al., 2020). In the context of drug metabolism, DDIs involving HCQ can impede the metabolism of metoprolol by competing for the CYP2D6 enzyme, leading to increased plasma concentrations and bioavailability of metoprolol (Somer et al., 2000). These DDIs, often resulting from changes in metabolic enzyme activity, are a significant contributor to adverse drug reactions. They are critical for drug metabolism and clearance, making the comprehension and management of these interactions essential for enhancing patient safety and therapeutic outcomes (Lawrence et al., 2014). It is imperative to elucidate HCQ's metabolic profiles and to quantify the formation rates of its major metabolites across different species.

It is well accepted that choosing a suitable animal models with similar metabolic profile or pharmacokinetic behaviors to human is crucial for in vivo pharmacological and pharmacokinetic tests of a given drug candidate, due to the species-dependent variations in drug metabolism and disposition always lead to species-specific effects or toxicity (Graham and Lake, 2008). As an ideal or a suitable animal model for preclinical studies, it should exhibit similar metabolic behaviors to humans, which means the metabolic profiles and the major enzymes involved in these biotransformations should be identical, while the metabolic clearances between human and the surrogate animal model are also similar (Baillie and Rettie, 2011). Therefore, understanding the metabolic pathways and the key enzymes responsible for the formation of major metabolites across the species will be very helpful for choosing the animal models and for the animal-to-human extrapolations in pharmacokinetics, pharmacology and toxicity.

Although numerous studies have suggested that HCQ is metabolized into three active metabolites—desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and didesethylchloroquine (BDCQ), with DHCQ being the major circulating metabolite—these claims are primarily based on chemical reaction speculation rather than definitive identification using high-resolution mass spectrometry. Following an intravenous injection of HCQ at 5 mg/kg in mice, the half-life (t1/2) was found to be 12.7 ± 1.1 h, whereas in humans, after an oral dose of 155 mg, the t1/2 was 50 ± 16 days (Tett et al., 1989; Chhonker et al., 2018). In addition, pharmacokinetic studies of artemisinin-HCQ combination therapy have demonstrated significant interspecies disparities between rats and dogs. In rat models, HCQ exhibited delayed absorption (Tmax 4–6 h), higher systemic exposure (AUC), shorter elimination half-life (t1/2 10–15 h), and marked accumulation (1.5–1.9-fold AUC increase) versus dogs' faster absorption (Tmax 1.4–2.9 h), lower AUC, prolonged elimination (t1/2 21–25 h), and milder accumulation (1.3–1.4-fold). Importantly, at equipotent doses, rats manifested markedly enhanced toxicological responses compared to dogs (Li et al., 2021). This indicates that the ADME characteristics of HCQ are quite different between humans and animals. HCQ is widely distributed in tissues and varies significantly across species (Solitro and MacKeigan, 2018; Liu et al., 2021). Consequently, the effects of HCQ and its ADME properties may vary greatly among different species of animals. However, research investigating species differences in HCQ metabolism remains notably lacking.

In clinical settings, directly measuring drug concentrations in human tissues is challenging, so PBPK models rely on experimental physiological and biochemical data. Initially validated in preclinical animals, these models are adapted through allometric scaling and species-specific adjustments to predict human tissue exposure (Collins et al., 2018; Zhang et al., 2021). Without clear insights into species-specific HCQ metabolism, accurately predicting its pharmacokinetics in humans remains difficult. Therefore, this study aimed to investigate the interspecies differences in phase I metabolism of HCQ in liver microsomes from human and five common experimental animals, including minipig, mouse, rat and dog. The metabolic profiles, the involved enzymes and the related enzyme kinetics for the major metabolic pathways in liver microsomes from the above-mentioned species, were well characterized for the first time.