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Low clearance HµREL® co-culture assay

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 minimise 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 CLint, drug metabolising 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 metabolising 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 utilises HµRELhumanPoolTM co-culture models over a 72 hr incubation period, allowing for more accurate assessment of CLint for low clearance NCEs.
Optimization of clearance is one of the more significant challenges for a drug discovery project. Identification of the rate in preclinical species and optimization in human are major goals in most projects

1Grime KH, Barton P & McGinnity DF (2013) Mol Pharm 10; 1191-1206


Low clearance assay protocol (using HµREL® co-culture)

Cells HµRELhumanPoolTM (5 donor)
Species Human
Test Article Concentration 1µM (different concentrations available)
Incubation Times 0, 2, 6, 24, 48 and 72 hr
Test Article Requirements 50 μL of 10 mM DMSO solution
Analysis Method LC-MS/MS quantification
Assay Controls Prednisolone
Data Delivery Intrinsic clearance
Standard error of intrinsic clearance
Half life


Data from the low clearance HµREL® co-culture assay

 Ion ClassMajor Drug Metabolising EnzymeCyprotex HµREL® CLint 72 hr (µL/min/106 cells)Hultman HµREL® CLint 70 hr (µL/min/106 cells)Bonn HµREL® CLint
72 hr (µL/min/106 cells)
Theophylline Base CYP1A2 BLQ Not reported BLQ
Disopyramide Base CYP3A4 0.22 0.3 0.4
Warfarin Neutral CYP2C9, CYP3A4 0.54 0.6 0.71
Diazepam Neutral CYP2C19, CYP3A4 0.54 1.20 1.35
Metoprolol Base CYP2D6,
0.73 1.00 0.78
Tolbutamide Acid CYP2C9 1.04 Not reported Not reported
Prednisolone Neutral CYP3A4 0.35 Not reported Not reported
Ketoprofen Acid UGT 4.36 5.90 4.3
Verapamil Base CYP3A4, CYP1A2, CYP2C9 14.5 16.8* Not reported
Imipramine Base CYP2C9, CYP2D6, CYP3A4, CYP1A2 1.24 19.6* 1.7
Diclofenac Acid CYP2C9, UGT2B7 30.6 Not reported Not reported
Carvedilol Base CYP2D6, CYP2C9 54.2 Not reported 34.2
Quinidine Base CYP3A4 0.36 0.60 Not reported
Bupropion Base CYP2B6, CYP1A2, CYP2A6, CYP3A4, CYP2E1 6.42 Not reported Not reported
Table 1
Comparison of human mean in vitro intrinsic clearance data generated using the HµREL® plated co-culture model by Cyprotex (media sampling method) alongside publications by Hultman et al.(media sampling method), 20165 and Bonn et al 20166 (media sampling method).

BLQ = below level of quantification

* measured over 3 hr rather than 70 hr
Figure 1
Comparison of CLint values generated using Cyprotex's HµREL® low clearance assay (media sampling method) in 3 separate assays, based on n=1 per assay.
Figure 2
In vitro-in vivo scaling of HµREL® data for donor HU1021 following application of regression line correction, as described previously (Sohlenius-Sternbeck et al., 20127).


Please provide an overview of Cyprotex's low clearance assay

HµRELhumanPoolTM 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 acclimatise for approximately 20 hr. 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. 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 (CLint) are calculated.

Figure 3
Representative in vitro clearance data for the control compound prednisolone generated in Cyprotex's low clearance assay (media sampling method) using the HµREL® co-culture model.
Figure 4
Representative in vitro clearance data for the control compound ketoprofen generated in Cyprotex's low clearance assay (media sampling method) using the HµREL® co-culture model.

What are the benefits of using hepatocytes for drug metabolism studies?

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

What are the benefits of using a HµRELhumanPoolTM co-culture plate over standard plated mono-culture hepatocytes?

The HµRELhumanPoolTM 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 HµRELhumanPoolTM plates are ideal in providing a simple and reproducible system for longer term metabolism studies.

How do you overcome the problems with inter-individual variability in humans?

Cyprotex's low clearance assay utilises HµRELhumanPoolTM 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.

What stage in the drug discovery process does the low clearance assay tend to be used?

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 CLint of 3.85 µL/min/106 cells (assuming maximum measurable t1/2 of 3 times incubation time).

The low clearance assay can be used as a secondary screen for specific compounds of interest where a CLint 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 CLint of 0.143 µL/min/106 cells (assuming maximum measurable t1/2 of 3 times incubation time).

What is the impact of using different timepoint sampling methods on CLint values?

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 CLint profile with little or no effect on the measured CLint.

What are the benefits of completing follow-on metabolite profiling and identification studies in the low clearance assay over the suspension hepatocyte assay?

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 metabolised 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 hepatocytes5,8.

Figure 5
Metabolite profiling comparison across assay types for diazepam. Metabolites displayed as a % of parent peak area ratio.

How do I interpret the data from the low clearance assay?

In vitro CLint can be scaled to predict human pharmacokinetics (PK). From in vitro CLint, the predicted in vivo CLint 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 CLint7. Using a validation set of compounds, the slope and intercept from a plot of log predicted in vivo CLint and log derived CLint provides a regression correction for a particular hepatocyte donor to more accurately predict in vivo CLint.

What is the importance of regression correction?

It is well reported that there is a systematic under-prediction of in vivo clearance when using in vitro CLint 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 CLint7. 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".


1Grime KH et al., (2013) Application of in silico, in vitro and preclinical pharmacokinetic data for the effective and efficient prediction of human pharmacokinetics. Mol Pharm 10(4); 1191-1206
2Di L et al., (2012) A novel relay method for determining low-clearance values. Drug Metab Dispos 40(9); 1860-1865
3Hutzler JM et al., (2015) Low-turnover drug molecules: A current challenge for drug metabolism scientists. Drug Metab Dispos 43; 1917-1928
4Novik E et al., (2010) A microfluidic hepatic coculture platform for cell-based drug metabolism studies. Biochem Pharmacol 79(7); 1036-1044
5Hultman I et al., (2016) Use of HμREL® human coculture system for prediction of intrinsic clearance and metabolite formation for slowly metabolized compounds. Mol Pharmaceutics 13(8); 2796-2807
6Bonn B et al., (2016) Determination of human hepatocyte intrinsic clearance for slowly metabolised compounds: Comparison of a primary hepatocyte/stromal cell co-culture with plated primary hepatocytes and HepaRG. Drug Metab Dispos 44(4); 527-533
7Sohlenius-Sternbeck A-K et al., (2012) Practical use of the regression offset approach for the prediction of in vivo intrinsic clearance from hepatocytes. Xenobiotica 42(9); 841-853
8Burton RD et al., (2018) Assessment of the biotransformation of low-turnover drugs in the HµREL® human hepatocyte coculture model. Drug Metab Dispos 46(11); 1617-1625
9Davies B & Morris T (1993) Physiological parameters in laboratory animals and humans. Pharmaceut Res 10(7); 1093-1095

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Learn more about drug metabolism in Chapter 3 of our popular Everything you need to know about ADME guide. 

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