Low clearance HµREL^® co-culture assay

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

Data from the low clearance HµREL^® co-culture assay

 

	Ion Class	Major Drug Metabolising Enzyme	Cyprotex 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, CYP3A4	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 CL_int 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

HµRELhumanPool^TM 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 (CL_int) are calculated.

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 HµRELhumanPool^TM co-culture plate over standard plated mono-culture hepatocytes?

The HµRELhumanPool^TM 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^TM 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 HµRELhumanPool^TM 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 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).

What is the impact of using different timepoint sampling methods on CL_int 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 CL_int profile with little or no effect on the measured CL_int.

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 hepatocytes^5,8.

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

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.

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 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".