Accurately determine the intrinsic clearance for metabolically stable compounds for which a traditional suspension assay fails to quantify.

The low clearance hepatocyte stability assay is in our portfolio of in vitro ADME services. We deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

Accurate measurement of low intrinsic clearance values using the HµREL^® co-culture assay:

• Low clearance compounds are increasingly prevalent in drug discovery², with the emphasis on reducing the metabolic clearance of new chemical entities (NCEs) in order to minimize dose, improve exposure and prolong the half-life.
• Determining an accurate in vitro measurement for clearance prediction of such compounds in human hepatocyte suspensions may not be possible due to the short incubation times required to maintain viability/activity. Although longer term studies using simple plated mono-cultures of cryopreserved human hepatocytes can be used for a more accurate determination of low CL_int, drug metabolizing enzyme activities start to decline by 12 hr³, thus leading to inaccuracies in the intrinsic clearance values.
• Hepatocytes contain the full complement of hepatic drug metabolizing enzymes (both Phase I and Phase II), making them the 'gold standard' for use in metabolic studies. HµREL (part of Viskol^®) provide a co-culture of primary hepatocytes (5 donor pool) and non-parenchymal stromal cells, which have been designed to maintain their cellular function for use in long term culture⁴.
• The low clearance method developed utilizes HµRELhumanPool^TM co-culture models over a 72 hr incubation period, allowing for more accurate assessment of CL_int for low clearance NCEs.

HµRELhumanPool 96-well hepatic co-culture plates (5 donor pool) are purchased from Visikol^®. Following 6 days co-culture and shipment of cells, media is replaced and cells allowed to acclimatize. Test compounds are prepared in serum-free incubation medium provided by HµREL (1 µM incubation concentration, 0.1 % DMSO) and added to relevant wells. Plates are incubated at 37°C, 5 % CO₂ over a 72 hr time course (separate well per time point). Samples are taken at 6 time points (0, 2, 6, 24, 48 and 72 hr) and quenched by addition to acetonitrile containing 1% formic acid. Plates are centrifuged at 2500 rpm and internal standard is added to supernatants prior to analysis using Cyprotex generic LC-MS/MS methods.

The disappearance of test compound is monitored over the 72 hr time period. From a plot of ln peak area ratio against time, the gradient of the line is determined. Subsequently, half-life (t½) and intrinsic clearance (CL_int) are calculated.

The liver is the main organ of drug metabolism in the body. Hepatocytes contain both Phase I and Phase II drug metabolizing enzymes, which are present in the intact cell, and provide a valuable in vitro model for predicting in vivo hepatic clearance.

The HµRELhumanPool contains an optimal mixture of pooled primary cryopreserved hepatocytes and non-parenchymal (stromal type) cells. The presence of stromal cells allows the hepatocytes to remain functional for weeks, without the requirement for additional supplements or overlay matrices which are vital to maintain long-term functionality for mono-cultures of hepatocytes. Consequently, the HµRELhumanPool plates are ideal in providing a simple and reproducible system for longer term metabolism studies.

Cyprotex's low clearance assay utilises HµRELhumanPool co-culture, containing a pool of five different individual donors, both male and female. This reduces the problems associated with inter individual variation in drug metabolism.

Clients tend to use the suspension hepatocyte stability as an initial screening assay, however this assay is not sensitive enough to discriminate between low clearance compounds due to the short incubation times required to maintain viability of hepatocytes. Theoretically, the lower limit of quantification for a 2 hr suspension assay incubated at 0.5 million cells/mL is a CL_int of 3.85 µL/min/10⁶ cells (assuming maximum measurable t_1/2 of 3 times incubation time).

The low clearance assay can be used as a secondary screen for specific compounds of interest where a CL_int can not reliably be generated in suspension hepatocyte incubations and an increased level of assay sensitivity is required to assist with more accurate predictions of in vivo clearance values. Cyprotex’s low clearance assay using HµREL co-culture provides > 25 fold increased assay sensitivity over the suspension hepatocyte assay with the lower limit of quantification being a CL_int of 0.143 µL/min/10⁶ cells (assuming maximum measurable t_1/2 of 3 times incubation time).

In the original low clearance assay protocol, media was sampled from a well at each timepoint and diluted into 3 volumes of acetonitrile (media sampling method). From January 2022 the protocol has been changed to a ‘whole well crash’ approach where 3 volumes of acetonitrile containing 1 % formic acid is added to wells at the required timepoint. This has been found to improve recovery of highly bound compounds and reduce the variability in the CL_int profile with little or no effect on the measured CL_int.

Cyprotex's low clearance assay can be extended to profile the metabolites that are formed. Cyprotex's biotransformation services are supported by high resolution, accurate mass spectrometry. Structural elucidation can also be performed on the potential metabolites' MS/MS fragmentation data. All biotransformation studies are performed by a dedicated team of experts.

For slowly metabolized compounds, the suspension hepatocyte assay may not provide an adequate incubation time to generate sufficient levels of metabolites for detection. Validation data generated at Cyprotex has supported literature findings that the HµREL model can robustly produce a wider range of metabolites over the incubation period, in comparison to the use of suspension hepatocytes^5,8.

In vitro CL_int can be scaled to predict human pharmacokinetics (PK). From in vitro CL_int, the predicted in vivo CL_int can be obtained by applying physiological human scaling factors and taking incubational binding into account:

Human liver weight = 25.7g liver/kg⁹
Hepatocellularity = 120 x 10⁶ cells/g liver³

A regression line correction method can be used for the prediction of in vivo CL_int⁷. Using a validation set of compounds, the slope and intercept from a plot of log predicted in vivo CL_int and log derived CL_int provides a regression correction for a particular hepatocyte donor to more accurately predict in vivo CL_int.

It is well reported that there is a systematic under-prediction of in vivo clearance when using in vitro CL_int data generated using plated hepatocytes, potentially due to reduced surface area for drug diffusion in comparison to suspension. The regression line correction is an effective method to remove bias from the various systems available to determine CL_int⁷.

At Cyprotex, the regression correction has been established for the validated method and cell batch using 13 literature compounds spanning various routes of metabolism and physiochemical properties. Details of the correlations obtained are described in the posters "Evaluation of HµREL Co-culture for Low Clearance Assessment", and "Time point sampling methodology has minimal impact on HμREL IVIVe".

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