Microsomal metabolic stability screens

Advances in techniques for chemical synthesis allow medicinal chemists to synthesize hundreds to thousands of compounds per month. Metabolic stability screening in liver microsomes is used extensively in early discovery to select the analogs or compounds most likely to have favorable pharmacokinetic parameters. This provides information on the relation of structure to stability, thus guiding synthesis strategies. 

Korfmacher, W. A. et al. 1999. Development of an automated mass spectrometry system for the quantitative analysis of liver microsomal incubation samples A tool for rapid screening of new compounds for metabolic stability. Rapid Commun. Mass Spectrom. 13 901. 

Lee SH, Slattery JT (1997) Cytochrome P450 isozymes involved in lisofylline metabolism to pentoxifylline in human liver microsomes. Drug Metab Dispos 25 1354-1358 Li AP (1999) Cryopreserved human hepatocytes characterization of DME activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability and drug-drug interaction potential. Chem Biol Interact 121 17-35... 

CRITICAL ASSESSMENT OF THE METHOD The high batch-to-batch variability of enzyme activities affords usage of pooled microsomes if metabolic stability has to be screened. Individual donors are used only for specific applications like correlation analysis. Since no cytosolic enzymes are present, the screening in liver microsomes might sometimes lead to misleading results if compared to in vivo data. On the other hand, the enzyme activities are higher in microsomal... 

K. Lin, C-C. Elicone, C. Liu, C. Duchoslav, E. Development of an Automated Mass Spectrometry System for the Quantitative Analysis of Liver Microsomal Incubation Samples A Tool for Rapid Metabolite Screening of New Compounds for Metabolic Stability, Rapid Commun. Mass Spectrom. 13, 901-907 (1999). 

The use of fast gradient elution LC-MS techniques for metabolic screening was first described by Ackermann and coworkers in 1998 [91] and Korfmacher and coworkers in 1999 [69], In the method developed by Ackermann et al., a HPLC column-switching apparatus is used to desalt and analyze lead candidates incubated with human liver microsomes. The resulting data can be quickly resolved into specific categories of metabolic stability high (>60%) moderate (>30%-59%) low (>10%-29%) and very low (<10%). 

As usual, though, the devil is in the details. The conditions used to run a particular microsome stability screen can radically affect the results. Microsomes are very sensitive to the presence of co-solvents, so DMSO concentrations normally need to be kept at no more than 0.2% and ACN at no more than 1%. Insoluble compounds, then, can be particularly problematic. Compound precipitation can mean that most of the material is unavailable for metabolism, but may dissolve when later diluted with ACN for analysis, resulting in a falsely high stability estimate. Ways of optimizing assays to minimize such effects have recently appeared in the literature.The concentration of compound used in the assay can make for more than a 70-fold difference in the percent remaining at the measured time point, while the concentration of the microsomes is also... 

One of the early in vitro assays that is used as part of the new drug discovery paradigm is the metabolic stability assay. This assay is also referred to as the microsomal stability assay or the hepatocyte stability assay or sometimes simply the in vitro stability assay (Thompson, 2000, 2001 Xu et ah, 2002 Jenkins et ah, 2004 Baranczewski et ah, 2006). As these names suggest, there are various ways to perform the in-life part of the assay some departments prefer to screen with liver microsomes, while others find that hepatocytes provide more meaningful data (Lau et ah, 2002). Regardless of the in-life part of the in vitro stability assay, the analytical part represents a significant challenge because the analyst must have compound-specific methods in order to properly analyze the samples. 

Due to the high cost of cell culture, Caco-2 assays are usually used as a follow-up to PAMPA in ADME screening [78], and as a result, the sample burden for bioanalysis is not as heavy as for some first-hne assays, such as metabolic stability. There have been a number of reports in the literature that use automated optimization and single LC-MS/MS for sample analysis for Caco-2 assay support [46,79-81]. Nevertheless, Caco-2 samples pose a unique bioanalytical challenge. Unlike plasma or microsomal samples rich in proteins that help solubilize compounds and prevent adsorptive loss, Caco-2 samples are essentially aqueous buffer samples with very little protein. As a result, compounds with low solubility and/ or adsorption problems tend to exhibit poor recoveries in the assay due to precipitation and adsorptive losses [82,83]. An effective solution to this problem is the use of organic solvent to catch compounds immediately after incubation, but prior to analysis, in order to maintain solubility and prevent adsorptive loss to container surfaces. Another approach involves the addition of some protein such as bovine serum albumin (BSA) to the assay buffer system, thus reducing compound loss/ precipitation and improving recoveries [84]. 

Figure 21.5. T1/2 and intrin c clearance measmements are made taking into account milligrams of microsomal protein used in the metabolic stability assay and the species used (e.g. human, rat). Compounds are generally binned according to their metabolic stability, as either stable, moderately stable, moderately unstable, or unstable light, medium, and dark shading, respectively. This information is used, in conjunction with other information (e.g. PAMPA, biological data, selectivity data, etc.) to prioritize compounds for pharmacokinetic screening.

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