The Growing Trend of Low Clearance Compounds – How to Accurately Predict In Vivo Clearance from In Vitro Data

Low clearance compounds continue to have an increased prevalence within the pharmaceutical industry as a consequence of advances in predicting and/or optimising DMPK properties and evolution in the chemistry required to interact with new drug targets. Demand for once-a-day dosing regimens means that reducing the metabolic clearance of new chemical entities (NCEs) is paramount for improving exposure and extending half-life.

The standard approach for drug metabolism screening is to use human hepatocyte suspensions. Hepatocytes not only contain the full complement of both Phase I and Phase II metabolic enzymes, they also have an intact cell membrane to account for drug permeation including active transport.

However, using hepatocytes in suspension for accurately defining the in vitro intrinsic clearance (CLint) of slowly metabolised compounds (e.g., < 3 µL/min/106 cells) can prove challenging as the cell viability and enzymatic activity cannot be maintained long enough in this model for the detection of substrate depletion. Even plated mono-cultures of cryopreserved hepatocytes, though better suited to longer‑term metabolism studies, may not provide the sensitivity needed for these compounds, as viabilities start to decrease by 12 hr without introduction of supplementation/overlays.

The low clearance assay developed at Cyprotex utilises HµRELhumanPoolTM hepatic co-culture plates supplied by HµREL® Corporation. These plates contain a 5 donor pool of human hepatocytes and non‑parenchymal (stromal type) cells in optimal ratios to allow the hepatocytes to maintain their functionality for up to 21 days. As a result, the Cyprotex Low Clearance HµREL® Co-culture Assay can robustly define CLint for compounds which are cleared slowly over a 72 hr time course. Indeed, using this method allows for >25 fold assay sensitivity over our current hepatocyte suspension method (CLint LOQ of 0.14 µL/min/106 cells, extrapolating the half-life to 3x incubation time). Of the fourteen validation compounds assessed, thirteen had CLint values that were reproducibly and accurately determined above the LOQ compared with six compounds in suspension hepatocyte incubations (2 hr). The HµREL® CLint data were comparable to values reported in literature. In vitro CLint values were used for in vitro-in vivo scaling to predict in vivo hepatic clearance. Following regression correction, in vivo clearance for twelve of the compounds was predicted within 2-fold of the observed values.

Metabolite identification and structural elucidation are important in DMPK studies. When drug metabolism is slow, hepatocyte suspension incubations may not produce enough metabolite for detection. By increasing the incubation time, the Low Clearance HµREL® Co-Culture Assay has the scope to provide a wider range of metabolites at detectable levels for metabolite identification and structural elucidation compared with hepatocyte suspension assays.  The system also has the potential to investigate the impact of a number of time-dependent processes (e.g. CYP metabolism-based inhibition, CYP induction) which may occur in parallel.

In summary, the Low Clearance HµREL® Co-culture Assay at Cyprotex shows promise for providing meaningful predictions of human hepatic clearance for advanced NCEs designed to have enhanced metabolic stability. The assay builds on the metabolic stability services already offered at Cyprotex to provide accurate determination of CLint and prediction of in vivo human hepatic clearance.

Learn more about the assay here

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