| Explain 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.
Please provide an overview of the Cloe Screen Hepatocyte Stability assay. The hepatocytes are incubated with the test compound at 37°C. Samples are removed at the appropriate time points into methanol containing internal standard to terminate the reaction. Following centrifugation, the supernatant is analysed by LC-MS/MS. The disappearance of test compound is monitored over a 60 minute time period. An example of a typical depletion profile is shown in Figure 3. Figure 3 Graph shows test compound disappearance with time in the presence of hepatocytes. The ln peak area ratio (compound peak area/ internal standard peak area) is plotted against time and the gradient of the line determined. 
How do I interpret the data from the hepatocyte stability assay? One of the main uses is that compounds can be ranked in terms of their intrinsic clearance values. Unless the compound is a pro-drug, very highly cleared compounds are generally considered to be unfavourable as they are likely to be rapidly cleared in vivo, resulting in a short duration of action. Classification bands can be used to categorise compounds into low, medium or high clearance. These classification bands are calculated from a rearrangement of the well stirred model2 detailed in the following equation assuming extraction ratios of 0.3 and 0.7 (the fraction of drug which is eliminated from the blood by an organ) for the low and high boundaries respectively. This can be scaled to intrinsic clearance (µL/min/106 cells) using the relevant liver weights3 and hepatocellularity4,5. Due to lack of literature information the monkey hepatocellularity was assumed to be 120x106 cell/g liver. CLint =  Where CLH = E x QH QH = liver blood flow (mL/min/kg)2
E = Extraction Ratio
CLH = Hepatic Clearance (mL/min/kg)
fu = fraction unbound in plasma (assumed at 1) | Clearance Category | Intrinsic Clearance (µL/min/106 cells) | | Human | Monkey | Dog | Rat | Mouse | | Low | <3.5 | <5.2 | <1.9 | <5.1 | <3.3 | | High | >19.0 | >28.3 | >10.5 | >27.5 | >17.8 | Table 1 Classification bands typically used for categorising compounds into low, medium or high clearance. What are the benefits of using cryopreserved hepatocytes, and how does the activity of freshly isolated hepatocytes compare with cryopreserved hepatocytes? Cryopreservation of hepatocytes enables the cells to be stored for long periods of time and ensures no supply problems or delays in screening. With advances in cryopreservation techniques, cell viability and activity have improved dramatically, and cryopreserved cells now provide a viable alternative to freshly isolated cells (Figure 4). Cryopreserved hepatocytes provide a convenient way of investigating interspecies differences in drug metabolism. Figure 4 Comparison of the metabolic stability of ethoxycoumarin, 7-hydroxycoumarin, and testosterone in the presence of freshly isolated and cryopreserved human hepatocytes. What control compounds are included in the Cloe Screen hepatocyte stability assay? Two compounds are included as controls. These include testosterone (phase I metabolism) and 7-hydroxycoumarin (phase II metabolism). How do you overcome the problems with inter-individual variability in humans? The Cloe Screen Hepatocyte Stability assay uses cells pooled from a minimum of three 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 hepatocyte stability assay tend to be used? Clients tend to use the microsomal stability assay as a primary screen early in the drug discovery process. The hepatocyte stability assay is then used as a secondary screen for the more favourable compounds from the primary screening. What may cause differences in the rates of clearance obtained from hepatocyte and microsomal assays? Compounds that do not readily permeate cell membranes or are subject to efflux may appear to be more stable in hepatocyte incubations than in microsomal incubations. Compounds that undergo Phase II metabolism may appear less stable in hepatocyte compared to microsomal incubations. back to top  | 
