Low clearance hepatocyte stability 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 hepatocytes

Reducing the metabolic clearance of new chemical entities is a common goal in drug discovery projects in order to reduce dose, improve exposure and prolong the half-life. However, accurately predicting the clearance of stable compounds is challenging using standard in vitro suspension methods.
Prolonged incubation times are restricted using suspended primary hepatocytes due to activity and viability issues. This can lead to inaccuracies in the intrinsic clearance values.
New methods are being developed to address this concern through extension of the incubation time, which, in turn, is able to provide a more accurate estimation of the intrinsic clearance.
Through its parent company, Evotec, Cyprotex are able to offer a low clearance method which utilises primary human hepatocytes, and matrix overlay to extend the time course for up to 5 days.

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

Protocol

Low clearance hepatocyte stability assay protocol

Cells	Primary human hepatocytes
Test Compound Concentration	1 µM (different concentrations available)
Overlay Matrix	Geltrex^®
Incubation Time	0, 1, 2, 4, 8, 22, 26, 30 h
Replicates	n=2
Compounds Requirements	20 μL of 10 mM solution
Analysis Method	LC-MS/MS quantification
Assay Controls	Disopyramide (low clearance) Metoprolol (moderate clearance) Sildenafil (high clearance)
Data Delivery	Intrinsic clearance Half life

Data

Data from the low clearance hepatocyte stability assay

	Ion Class	Major Drug Metabolising Enzyme	Bonn et al., 2016 PHH CL_int (µL/min/10⁶ cells)	Bonn et al., 2016 Hurel CL_int (µL/min/10⁶ cells)	Evotec CL_int (µL/min/10⁶ cells)
Bupropion	Base	CYP2B6, CYP1A2, CYP2A6, CYP3A4, CYP2E1	Not reported	Not reported	5.4
Carvedilol	Base	CYP2D6, CYP2C9	26.3	34.2	14.5
Diazepam	Neutral	CYP2C19, CYP3A4	0.8	1.3	0.7
Diclofenac	Acid	CYP2C9, UGT2B7	Not reported	Not reported	4.7
Disopyramide	Base	CYP3A4	0.2	0.4	0.1
Ethinylestradiol	Acid	UGT1A1, CYP3A4	Not reported	Not reported	3.3
Imipramine	Base	CYP1A2, CYP2C19, CYP2D6	8.6	1.7	8.5
Metoprolol	Base	CYP2D6, CYP3A4	2.2	0.8	0.9
Midazolam	Neutral	CYP3A4	Not reported	Not reported	5.1
Sildenafil	Base	CYP3A4, CYP2C9, CYP2C19	7.0	6.2	9.0
Tolbutamide	Acid	CYP2C9	Not reported	Not reported	0.8
Warfarin	Neutral	CYP2C9, CYP3A4	BLQ	0.7	0.3

Table 1
Comparison of human in vitro intrinsic clearance data generated by Evotec (Cyprotex’s parent company) and a publication by Bonn et al 2016² where plated human hepatocytes and a co-culture model were used.

Figure 1
Correlation of scaled in vitro human intrinsic clearance (using Evotec’s low clearance model) with in vivo human intrinsic clearance for a set of 12 known drugs.

The data generated by Evotec is consistent to those reported by Bonn et al., 2016 as illustrated in Table 1. Further, the scaled in vitro human intrinsic clearance data from the Evotec model demonstrates a strong correlation with in vivo human intrinsic clearance demonstrating the advantages of this approach as illustrated in Figure 1.

References

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
² Bonn 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; 527-533