High-throughput microsomal stability assays

Di L, Kerns EH et al (2008) Applications of high throughput microsomal stability assay in drug discovery. Comb Chem High Throughput Screen 11 469—476... 

Di L, Kems EH, Li SQ, Petusky SL. High throughput microsomal stability assay for insoluble compounds. Int J Pharm 2006 317(1) 54—60. 

Fonsi M, Orsale MV, Monteagudo E. High-throughput microsomal stability assay for screening new chemical entities in drug discovery. J Biomol Screen 2008 13 (9) 862-869. 

Experimentally, high-throughput microsomal stability assays are conducted by incubating compounds (0.5-5 iM) at 37°C with resuspended liver microsomes in buffer along with NADPH, usually in a %-well plate format. Aliquots of the reaction mixture are taken at predetermined intervals and quenched with organic solvent to stop the reaction as well as precipitate microsomal proteins. The quenched mixture is then centrifuged and the supernatant is removed for LC-MS analysis. SRM is typically used to monitor the disappearance of the parent compounds, and the results at each time point are expressed in the form of percent of compound remaining as compared with time zero (TO). 

Di, L., Kerns, E.H., Gao, N., et al. (2004) Experimental design on single-time-point high-throughput microsomal stability assay. Journal of Pharmaceutical Sciences, 93, 1537-1544. 

The ability to obtain microsomal stability data for NCEs in ahigher throughput manner has continued to evolve. In 1999, Korfmacher et al. [14] described a microsomal stability assay based on an automated LC-MS system that was able to handle 75 compounds per week. More recently, Xu, Kassell et al. [15] have described a highly automated microsomal stability screen using an eight-channel parallel LC-MS system for the assay step this system has the capacity to provide microsomal stability data for 176 compounds per day ... 

One recent example of a high-throughput assay for metabolic stability was reported by Fonsi et al. (2008). In this report, the authors described the use of a robotic platform to prepare the compounds for a microsomal stability assay. The authors selected five time points (20, 30, 45, 60, and 90 min) so the intrinsic clearance (CL nt) could be calculated with an Excel spreadsheet. The assay was performed with a triple quadrupole tandem mass spectrometer (MS/MS) system. The system included software tools for automated MS/MS method development—an important requirement for any MS/MS system that will be used for high-throughput assays for microsomal stability assays. The authors also made use of a cassette assay system where samples were pooled after the incubation step was completed. Up to four analytes were pooled and were assayed in one HPLC—MS/MS procedure with a generic chromatographic gradient system. 

With this focus on CYP and fiver metabolism, most companies have established high throughput assays to measure compound stability in the presence of human (or preclinical species) fiver microsomes [49]. Disappearance of starting compound from an incubation with microsomes is monitored. Measurement at a single time point enables a rank-ordering of compounds for stability based on percent of parent compound remaining acquisition of data at multiple time points allows determination of half-life, intrinsic clearance, and extrapolation to a predicted in vivo clearance [50]. 

Some authors searched for common oxidative metabolites as part of metabolic stability assays. Tong et al.75 described a highly automated microsomal metabolic stability assay that achieved a throughput of 50 compounds per day with each compound tested in rats, dogs, monkeys, and humans. In addition to assaying the test compound, they monitored M+16 metabolites by using the... 

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