|Service||Cell Line||Transporter||Technology||Membrane Type||Probe Substrate||Read Out||Reference Inhibitor||Positive Control||Dynamic Range|
Metabolite generation, profiling and identification
Our HepatoPac® co-cultured micropatterned culture (MPCC) primary hepatocytes system provides a superior method for generation, profiling and identification of primary and secondary metabolites for your molecule, particularly if it is only slowly metabolized in other in vitro systems such as primary hepatocytes or microsomes. MPCC offer the following advantages over other methods:
• Stable expression of DMEs (Phase 1 and Phase 2) and uptake and efflux transporters for an extended period (>7 days), compared to plated or suspension hepatocytes.
• Greater range and extent of metabolism, particularly for slowly metabolized molecules.
• Adaptable to multiple species.
• Intact biliary excretion mechanism.
HepatoPac® MPCC have been shown to maintain important DME expression levels over extended time periods (24–42 days).1 This is reflected in their ability to generate a greater range of metabolites, more accurately reflecting that observed in vivo (Table 1).2 A typical outline study design for metabolic identification study is given below:
• 24 well plate format
• Controls: stromal cells; +/- enzyme inhibitors (as appropriate)
• Single concentration of test article
• 4 sample time points taken over a period of up to 7 days
Our highly trained bioanalysts can provide a comprehensive metabolite profiling and identification service. Alternatively, our scientists can generate samples for shipment to your bioanalysts to analyse in-house.
Table 1: Comparison of human HepatoPac® MPCC generation of major human in vivo metabolites for 27 compounds with traditional in vitro systems
|Metabolites Identified (Percentage of Total Metabolites Observed In Vivo)|
|Types of Metabolite and Matrix Observed||No. Metabolites Observed In Vivo||Microsomes*||S9*||Hepatocyte Suspension*||HepatoPac® **|
|48 h||7 days|
|Excretory metabolites >10% of dose|
|All excretory metabolites||39||49%||56%||64%||69%||82%|
|Phase 1 reactions||29||59%||66%||66%||69%||83%|
|Phase 2 reactions||10||20%||30%||60%||70%||80%|
|Secondary or more metabolites||23||30%||48%||57%||61%||74%|
|Circulatory metabolites >10% of total drug-related material|
|All circulating metabolites||40||43%||48%||53%||70%||75%|
|Phase 1 reactions||31||45%||52%||45%||71%||74%|
|Phase 2 reactions||9||33%||33%||78%||67%||78%|
|Secondary or more metabolites||24||25%||33%||38%||67%||67%|
Adapted from Wang et al, 20103
* Data used was adapted from Dalvie et al, 20092
** Data adapted from Wang et al, 20103
Phase 1 - Metabolites arising by phase 1 reactions only (e.g. via CYPs)
Phase 2 - Metabolites arising by a phase 2 reaction (e.g. via UGTs)
Primary metabolites - Metabolites that are produced directly from parent (e.g. hydroxylation)
Secondary or more metabolites - Metabolites of metabolites (e.g. OH-glucuronide)
1. Khetani, S. R. & Bhatia, S. N. Microscale culture of human liver cells for drug development. Nat. Biotechnol. 26, 120–6 (2008).
2. Dalvie, D. et al. Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites. Chem. Res. Toxicol. 22, 357–68 (2009).
3. Wang, W. W., Khetani, S. R., Krzyzewski, S., Duignan, D. B. & Obach, R. S. Assessment of a Micropatterned Hepatocyte Coculture System to Generate Major Human Excretory and Circulating Drug Metabolites. DMD 38, 1900–1905 (2010).