Human Liver Microsomes Immobilized on Magnetizable Beads: A Novel Approach to Study In Vitro Drug Metabolism

Abstract

Human liver microsomes (HLM) are a commonly used tool to study drug metabolism in vitro. Typical experiments conducted using suspensions of HLM can be challenging to separate from the incubation solution without lengthy ultracentrifugation steps. Magnetizable beads coated with silica (MGBS) were found to bind strongly to HLM, which could then be isolated and purified using a magnet. Binding of HLM to the MGBS (HLM-MGBS) was demonstrated to be mediated by strong interactions between microsomal phospholipids and MGBS, as artificially prepared phosphatidylcholine (PC) liposomes could be more efficiently captured by the MGBS. HLM-MGBS complexes retained functional cytochrome P450 and uridine-diphosphate-glucuronosyltransferase (UGT) activity as indicated by CYP2C8-mediated amodiaquine de-ethylation, CYP3A4mediated midazolam 1'hydroxylation, UGT1A1-mediated glucuronidation of estradiol, UGT1A9-mediated glucuronidation of propofol, and UGT2B7-mediated glucuronidation of zidovudine. When comparing suspension HLM alone with HLM-MGBS complexes containing equivalent amounts of HLM, the intrinsic clearance (CL int ) of CYP450 substrates was comparable; however, CL int of UGT1A1, UGT1A9, and UGT2B7 was increased in the HLM-MGBS system between 1.5-and 6-fold. HLM-MGBS used in an incubation could also be readily replaced with fresh HLM-MGBS to maintain the presence of active enzymes. Thus, HLM-MGBS demonstrate increased in vitro metabolic efficiency and manipulability, providing a new platform for determination of accurate metabolic parameters. SIGNIFICANCE STATEMENTThe following work describes the strong binding of HLM to magnetizable beads. In addition, the preservation of enzyme activity on the bound HLM provides a novel means to conduct preclinical metabolism studies.

HLM-beads: Rapid Assessment of Nonspecific Binding to Human Liver Microsomes Using Magnetizable Beads

“…Moreover, our studies show that the extent of compound adsorption to beads in the absence of HLM is negligible (data not shown). Furthermore, we previously showed that phospholipids can block HLM from binding to the beads, which indicates that it is possible to block the binding of material to the beads (Horspool et al, 2020). Thus, we hypothesized that the HLM would have the same effect of blocking other compounds from binding to the beads.…”

“…Previously, a 30 min incubation time was used to ensure that the binding of HLM to the beads reached equilibrium (Horspool et al, 2020). To optimize the time required for the binding, the time course of HLM binding to the beads was investigated.…”

“…HLM are vesicular subcellular fractions composed of endoplasmic reticulum and are commonly used to study hepatic metabolism in support of small-molecule drug development. Previously, we demonstrated that HLM can bind strongly to silica-coated magnetizable beads and that these HLM-beads retain functional drug metabolizing enzymes (Horspool et al, 2020).…”

“…Previously, we have demonstrated that HLM are able to bind to silica-coated magnetizable beads (Horspool et al, 2020). The HLM could not be released from the beads by multiple washing steps, indicating strong binding.…”

“…Unfortunately, the mechanism by which HLMs bind to the magnetizable beads (via phospholipid interactions with silica coating) is not amenable to non-membrane proteins. Our previous work showed that binding of human serum albumin to silica coated beads is not as complete as the binding of phospholipids to the beads (Horspool et al, 2020). It is however worth noting that at least one marketed product exists which utilizes binding of plasma proteins to non-magnetizable beads (Schuhmacher et al, 2004).…”