Metabolic stability
| Service | Cell Line | Transporter | Technology | Membrane Type | Probe Substrate | Read Out | Reference Inhibitor | Positive Control | Dynamic Range |
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Description
Metabolic clearance using HepatoPac®
SOLVO offers HepatoPac® in collaboration with Ascendance, to predict metabolic stability and provide reliable CLint parameters for all your molecules. With HepatoPac®, the proprietary patterning of hepatocyte “islands” in a “sea” of stromal cells fully replicates the physiological microenvironment of the liver, promoting hepatocyte health and, most importantly, enabling stable enzyme activity for weeks, not hours or days, as is the case for other primary isolated hepatocyte models. In addition, the HepatoPac® system maintains functional uptake and efflux transporter systems as well as a bile formation capability.
When compared to industry-standard suspension or monolayer cultures, HepatoPac® provides superior estimates of low and medium CLint molecules, and improved in vivo clearance predictions (Table 1). Multiple species are available for test, allowing cross-species comparisons.
Solvo’s team of highly trained bioanalysts, with access to state of the art analytical techniques can provide data for all your molecules.
The HepatoPac® system can be used in 24 or 96-well formats, and using human, monkey, rat and dog hepatocytes, as best suited to your requirements. A typical study outline might be as follows:
In vitro Metabolic Stability study:
• 96 well plate format
• Appropriate controls: stromal and acellular; +/- Imipramine
• Single concentration of test article
• 6 collection time points up to 7 days
• Deliverables: parent compound depletion, predicted (human) clearance
Superior Clearance Predictions, Especially Low Clearance Molecules
Cell viability and loss of metabolism (and transporter) function with time restricts use of traditional plated or suspension hepatocytes, especially for low clearance or slowly metabolised molecules (Table 1).
Twenty four molecules classified as low(<5ml/min/kg) or intermediate(>5mL/min/kg) in vivo clearance were tested using human HepatoPac® and in vivo clearance (CL) predictions compared (where available) with values from standard systems (CLh). Green-filled cells indicate predictions of clearance that are within a three-fold cut off the observed in vivo clearance. Using these criteria, HepatoPac® predicts both moderate and low clearance molecules well (green filled cells, approximately 75% success rate, n=24). , Performance for low clearance molecules is even more impressive (10 of 12 molecules were well predicted, approximately 83%)1,2 and is superior to traditional plated or suspension hepatocyte approaches.
Table 1: Predicted versus actual clearance of molecules tested in human HepatoPac® and traditional methods
| Mean Predicted CLh | |||||
| Compound | In Vivo CL* | HepatoPac®** | Monolayer | Suspension | |
| Low in vivo clearance | Warfarin | 0.081 | 0.036 | - | - |
| Meloxicam | 0.12 | 0.027 | - | - | |
| Naproxen | 0.19 | 0.18 | NC | NC | |
| Tolbutamide | 0.31 | 0.29 | - | - | |
| Tolbutamide | 0.38 | 0.21 | 0.06 | NC | |
| Diazepam | 0.53 | 0.18 | - | - | |
| Diazepam | 0.53 | 0.21 | 0.03 | NC | |
| Alprazolam | 0.61 | 1.1 | - | - | |
| Lorazepam | 1 | 0.46 | 0.11 | NC | |
| Theophylline | 1.1 | 0.34 | 0.43 | NC | |
| Glimepriride | 1.12 | 0.4 | - | - | |
| Theophylline | 1.13 | 1.25 | - | - | |
| Prednisolone | 1.44 | 1.69 | - | - | |
| Riluzole | 2.05 | 2.35 | - | - | |
| Voriconazole | 3.8 | 7.16 | - | - | |
| Moderate in vivo clearance | Risperidone | 5.16 | 3.65 | - | - |
| Erythromycin | 5.6 | 0.71 | 0.73 | NC | |
| Flecainide | 5.82 | 0.96 | - | - | |
| Methylprednisolone | 6.1 | 4.67 | 0.91 | NC | |
| Atomoxetine | 7.05 | 1.29 | - | - | |
| Diclofenac | 7.67 | 1.4 | - | - | |
| Atazanavir | 8.13 | 10.5 | - | - | |
| Midazolam | 8.59 | 4.52 | - | - | |
| Timolol | 10.2 | 5.39 | 0.95 | 4.66 | |
| Lidocaine | 10.7 | 6.21 | - | - | |
| Naloxone | 18 | 14.45 | 11.98 | 11.12 | |
| Verapamil | 19.5 | 6.79 | 0.8 | 1.23 | |
*In Vivo clearance values were sourced from literature sources and represent either total or non-renal clearance
** Values are based on an average of 3 separate donors (Chen et al), or a single donor (Lin et al)
- Not tested
NC - Not Calculated as insufficient turnover observed to allow calculation of clearance terms
Green coloured cells indicate predicted clearance is within 3-fold of observed
Low Clearance < 5 mL/min/kg, Intermediate Clearance >5 ml/min/kg.
Combined dataset from Chan et al, DMD 41: 2024-2032, 2013 and Lin et al, DMD 44: 127-136, 2016
References
1. Chan, T. S., Yu, H., Moore, A., Khetani, S. R. & Tweedie, D. Meeting the Challenge of Predicting Hepatic Clearance of Compounds Slowly Metabolized by Cytochrome P450 Using a Novel Hepatocyte Model, HepatoPac. Drug Metab. Dispos. 41, 2024–2032 (2013).
2. Lin, C., Shi, J., Moore, A. & Khetani, S. R. Prediction of Drug Clearance and Drug-Drug Interactions in Microscale Cultures of Human Hepatocytes. Drug Metab. Dispos. 44, 127–36 (2016).
