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ADME PK

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 ClassMajor Drug Metabolising EnzymeBonn et al., 2016 PHH CLint (µL/min/106 cells)Bonn et al., 2016 Hurel CLint (µL/min/106 cells)Evotec
CLint (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

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