| Explain the benefits of using liver microsomes for drug metabolism studies? The liver is the main organ of drug metabolism in the body. Subcellular fractions such as liver microsomes are useful in vitro models of hepatic clearance as they contain many of the drug metabolising enzymes found in the liver. Microsomes are easy to prepare and can be stored for long periods of time. They are easily adaptable to high throughput screens which enable large numbers of compounds to be screened rapidly and inexpensively.
Please provide an overview of the Cloe Screen Microsomal Stability assay. The microsomes are incubated with the test compound at 37°C in the presence of the co-factor, NADPH, which initiates the reaction. The reaction is terminated by the addition of methanol containing internal standard. Following centrifugation, the supernatant is analysed on the LC-MS/MS. The disappearance of test compound is monitored over a 45 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 liver microsomes. 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 microsomal stability assay? There are several ways in which the data can be used. Firstly, the 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. The CLint classification bands for each species in Table 1 are calculated from a rearrangement of the well stirred model4 detailed in the following equation assuming an extraction ratio (E) of 0.3 and 0.7 for the low and high boundaries, respectively. This can then be scaled to intrinsic clearance (µL/min/mg protein) using the relevant liver weights5 and microsomal protein concentration6, 7 obtained from the literature. Due to lack of literature information the monkey and mouse microsomal protein concentration was assumed at 50mg microsomal protein/g liver.  Where CLH = E x QH QH = liver blood flow (mL/min/kg)5
E = Extraction Ratio
CLH = Hepatic Clearance (mL/min/kg)
fu = fraction unbound in plasma (assumed at 1) Table 1: Classification bands typically used for categorising compounds into low, medium or high clearance. Clearance Category | Intrinsic Clearance (µL/min/mg protein) | Human | Monkey | Dog | Rat | Mouse | Low | < 8.6 | < 12.5 | < 5.3 | < 13.2 | < 8.8 | High | > 47.0 | > 67.8 | > 28.9 | > 71.9 | > 48.0 | Secondly, the data can be used in conjunction with other in vitro parameters to predict the pharmacokinetics of a compound in vivo using the simulation software Cloe PK. Thirdly, species specific differences in drug metabolism can be investigated. This may be useful in identifying an appropriate species for pre-clinical development. Why would I screen my compounds in the microsomal stability assay rather than the hepatocyte stability assay? Microsomes are adaptable to high throughput screening and enable large numbers of compounds to be screened inexpensively. Our clients tend to use this assay as an initial screen to rank order compounds of interest in terms of their metabolic stability, and then perform a secondary screen on a smaller number of selected compounds using hepatocytes. What controls are used in the assay? • Two positive control compounds are included for each species. These compounds are known to be metabolised by liver microsomes. Species | Control Compounds | Human | Dextromethorphan Verapamil | Rat | Diazepam Diphenhydramine | Mouse | Diazepam Diphenhydramine | Monkey | Dextromethorphan Verapamil | Dog | Dextromethorphan Verapamil | • A control incubation is performed in the absence of NADPH to reveal any chemical instability or non-NADPH dependent enzymatic degradation. • A control is included that contains all reaction components with the exception of the test compound. This control identifies any potential interfering component which may affect the analysis. Can I investigate Phase II metabolism in liver microsomes? The microsomal stability assay is primarily used to investigate Phase I metabolism using NADPH as the enzyme co-factor. However liver microsomes can also be used to study Phase II metabolism if the correct incubation conditions are used. We have recently validated this using the pore forming agent alamethicin in conjunction with appropriate Phase II cofactors for example UDPGA. This allows you to gain an understanding as to the contribution Phase II metabolism has on the overall metabolism of a test compound. It is also possible to study coupled Phase I and Phase II metabolism by using all of the relevant co-factors in the incubation. back to top  | 
