Advanced In Vitro Tools for Evaluating Low-Clearance Compounds in Drug Discovery

In modern drug discovery, a critical factor in early-stage compound evaluation is how efficiently a candidate drug is metabolized in the liver as well as in other organs. Hepatic clearance plays a central role in determining a drug’s pharmacokinetics, safety, and efficacy.

Based on their rate of hepatic elimination, xenobiotics are commonly classified into two categories: high-clearance and low-clearance compounds (LCCs). High-clearance compounds are metabolized rapidly, often at rates approaching hepatic blood flow. Understanding the metabolic fate of these compounds is crucial because LCCs often exhibit prolonged half-lives, leading to drug accumulation, delayed toxicity, or unforeseen drug–drug interactions in clinical settings. In such cases, metabolite identification becomes particularly important, as low-turnover compounds tend to form metabolites slowly and sometimes only after extended exposure. These metabolites—especially when reactive, lipophilic, or conjugated—may be pharmacologically active or toxic, contributing to idiosyncratic adverse effects that are not detectable in early high-clearance-focused assays.

A major challenge with LCCs is their slow interaction with hepatic enzymes, making them difficult to evaluate using conventional in vitro assays such as hepatocyte suspensions or liver microsomes. Despite this, reliable characterization of a compound’s metabolic route and outcome is essential during drug development. Early clearance assays can generally distinguish between high- and low-clearance compounds, but more advanced approaches are required for detailed metabolic identification.

To address these limitations, several advanced in vitro systems have been developed. These platforms are generally grouped into the following categories: relay assays, plateable hepatocyte-based models, spheroid-based systems, and microphysiological systems (MPS).

Relay assays involve the repeated incubation of test compounds with fresh suspensions of human or animal hepatocytes over multiple time points (1). Because hepatocytes in suspension remain viable for only a few hours, this approach extends metabolic incubation by transferring the compound to new cell preparations several times, usually 4 or 5. While effective in capturing slowly formed metabolites, the method requires multiple steps and careful coordination.

Plateable hepatocytes can be used either as monocultures or co-cultured with feeder cells to support hepatic functionality (2, 3, 4). These cells can be cryopreserved and pooled, offering improved reproducibility and operational convenience. Their longer-term stability in culture provides valuable insight into compound clearance over extended periods. However, traditional plating methods were proven to lead to decreased cytochrome P450 (CYP) enzyme levels, prompting the development of co-culture techniques aimed at maintaining enzyme activity (2). While much progress has been made, many commercial co-culture systems—particularly those based on feeder layers—require multi-day establishment periods or rely on freshly plated (non-cryopreserved) hepatocytes. As a result, these platforms are difficult to integrate into routine workflows, demanding additional setup time, logistics coordination, and specialized handling. This significantly limits their scalability and practicality for early-stage screening or high-throughput applications, where consistency and operational efficiency are essential.

Spheroid cultures represent a 3D model that helps retain hepatocyte function, including CYP activity, over time (5, 6, 7). These models are typically formed from spheroid-qualified hepatocytes that can be cryopreserved. Upon culture, the cells often maintain enzyme activity levels similar to those in suspension cultures, making them useful for studying LCCs. However, the relatively small number of cells in each spheroid may limit the scale and throughput of standard metabolic assays.

MPS platforms, often referred to as "organ-on-a-chip" systems, replicate organ-level complexity by incorporating perfusion and tissue-mimicking structures (8). These models simulate the dynamic environment of the liver, enabling the study of metabolism and pharmacokinetics in a more physiologically relevant context. MPS systems have demonstrated utility in LCC metabolism studies but may require more specialized equipment and expertise, and are generally better suited for low- to medium-throughput applications.

In Vitro System	Advantages	Limitations
Relay Hepatocyte Assays	Captures slow metabolite formation; extends incubation time without losing enzyme activity	Labor-intensive; requires precise timing and multiple cell preparations
Plateable Hepatocytes	Cryopreserved; reproducible; supports longer-term metabolism studies; co-culture options preserve CYP activity	Enzyme activity may decline in monocultures; co-cultures can be complex; variability in cryopreservation
Spheroid Hepatocytes	3D structure enhances cell functionality; retains CYP activity over time; physiologically relevant	Limited cell number per spheroid; lower throughput for quantitative assays
Microphysiological Systems (MPS)	Highly physiologically accurate; supports dynamic flow; ideal for long-term metabolism and PK studies	Requires specialized equipment and expertise; low to medium throughput; costly setup

Preci’s in vitro clearance system delivers the metabolic performance of a relay assay within a simplified suspension format. Utilizing extended-clearance human hepatocytes, our platform retains CYP enzyme activity and supports phase I metabolism for up to 12 hours in a single incubation—eliminating the need for repeated cell transfers. This enables robust evaluation of low-clearance compounds with minimal workflow complexity. In addition, the system is based on cryopreserved hepatocytes, allowing convenient storage and batch-to-batch consistency for long-term studies and scalable screening programs.

Preci’s extended-clearance hepatocyte system is designed to reliably assess both high- and low-clearance compounds within a single, streamlined suspension assay. The high initial enzymatic activity of our pre-qualified and gradient-purified hepatocytes enables rapid metabolism of high-clearance compounds, while the sustained CYP function—retained for up to 12 hours—allows accurate profiling of slow-turnover, low-clearance compounds. This dual capability eliminates the need to switch between assay platforms during early-stage drug development, offering a unified solution for comprehensive hepatic clearance evaluation.suspension assays, including those targeting low-clearance compound metabolism.

Upon long-term incubation suspension hepatocytes do not use their drug-metabolizing capacity and predict the precise values of Clint for LCC.

Preci’s hepatocyte system is engineered to achieve metabolic stabilization at the cellular level, ensuring both accuracy and reproducibility in drug clearance studies. As shown in our reference compound data, the hepatocytes consistently maintain clearance rates for high-turnover compounds over a 10-hour incubation, reflecting sustained CYP activity without decline. This long-term stability also enables the reliable detection of low-clearance compounds, producing clearance values that are not only measurable but also highly translatable to in vivo pharmacokinetics. By preserving enzyme function across extended timeframes in a suspension format, Preci’s platform overcomes a key limitation of conventional assays—delivering a unified, stable, and predictive model for hepatic metabolism.