Metabolic stability incubation

An important DMPK property of a NCE is oral bioavailability (F) of the compound in various pre-clinical species.3 The oral bioavailability of a compound is dependent on several factors including intestinal permeability (estimated by the Caco-2 assay) and hepatic clearance (estimated with an in vitro metabolic stability assay).3 30 The metabolic stability assay is typically performed by incubating test compounds in liver microsomes or hepatocytes. The results can provide estimates of in vivo stability in terms of metabolic liabilities.3 8 59 62 Several authors described this assay as an important tool for the rapid assessment of the DMPK properties of NCEs.3 6 8111819 26 44 59 62-65... 

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. 

When chemical inhibition experiments are conducted with a relatively metabolically stable drug candidate (one that must be incubated with relatively high concentrations of human liver microsomes for a relatively long time in order to generate quantifiable levels of metabolite), it is important to take into account the metabolic stability of the inhibitors themselves. Lack of metabolic stability makes some compounds poor choices as chemical inhibitors despite their selectivity. For example, coumarin is a selective substrate of CYP2A6 (Km 0.25 to 0.5 pM) (111) and it would be a good selective competitive inhibitor of CYP2A6 if it were not metabolized so rapidly by human liver microsomes. 

Samples obtained are analyzed by chromatographic means, e.g. LC-MS/MS, HLPC using UV detection or radiodetection, if radiolabeled substrates are applied. Metabolic stability as final result is given as percent remaining after incubation by dividing peak area of parent compound in the sample by the area of the time 0 sample and multiplication by 100. Also evaluation of parent and metabolites detected is possible for generat-... 

Figure 11-13. Time-course human liver microsomal incubation profiles for a number of positive and negative controls, as well as project compounds. Metabolic stability profiles are represented both in tabular format and graphically above.

Figure 11-15. Flow diagram for performing intelligent metabolite ID studies. Compounds are incubated with liver microsomes or hepatocytes at a test concentration of 1 xM over a limited time course. The percent of substrate remaining after 30-min incubation is determined on-the-fly. Compounds exhibiting poor metabolic stability are automatically queued for detailed metabolite ID studies. This approach enables metabolic stability and metabolite ID data to be generated in a completely automated, independent manner.

Metabolic stability in human CYP3A4 cDNA-expressed microsomal preparation offers a suitable approach to predict the metabolic stability of external compounds. From a dataset (n= 1507) from Pharmacia Corporation, each compound was incubated at a fixed concentration for 60 min with a fixed concentration of protein at 37 °C. The reaction was stopped by adding acetonitrile to the solution and, after centrifugation to remove the protein, the supernatant was analyzed using LC/MS and MS. Compounds with a final concentration > 90% of the corre-... 

Predictive models can be better produced when recombinant cytochrome data are available, an experimental technique which may increase the probability of obtaining consistent and predictive models, since one protein is involved in the metabolic reaction. The most accurate data to describe the rate and affinity of the ligand towards an enzyme are the kinetic parameters Vmax and Km. Nevertheless, the calculation of these parameters is time consuming. A less precise parameter is the determination of the compound percentage remaining in a cytochrome incubation after a certain period of time. These metabolic data are less accurate and can only be used to classify the compounds in a metabolic system as stable or unstable. This type of data was the basis for a predictive model of metabolic stability towards CYP3A4 [35]. 

Several TRK-850 derivatives were synthesized based on the general strategies mentioned above, and their metabolic stabilities were evaluated in the presence of human liver microsomes. Metabolic rates were determined by incubating the compounds with human liver microsomal enzymes and then quantifying the remaining parent peak by HPLC analysis. The data were expressed as the elimination rate constant (Kel) and the relative metabolic rate, or the /C , [ of the compound compared with the /C, [ of TRK-850 (ATF, ratio). 

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%). 

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