Microsomal stability assays

In a third example, when a compound was initially tested in a microsomal stability assay, no metabolism was observed, with 100% of the compound remaining after a 15-min incubation. When it was retested under the same conditions, only 50% remained after 15 minutes. The results were irreproducible and erratic. The reason was that the compound had poor aqueous solubility. It precipitated during the first dilution into the aqueous media (Dilution is essential to reduce the DMSO content, because it inhibits Cytochrome P450 activity). The solid precipitate was not metabolized, but it was quantitated in the assay when acetonitrile was added to the aqueous reaction mixture. The compound, thus, appeared artificially to be more stable. Precipitation is quite a variable process and depends on many different factors, such as temperature, incubation time, seeding conditions and agitation. The completeness of precipitation affects the final results, thus, assay results tend to be more variable for insoluble compounds. 

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 ... 

Examples of how this integrated approach is applied to assays in the profiling suite are illustrated for determining permeability via the parallel artificial-membrane permeabilty assay (PAMPA) [45] and metabolic stability via a microsomal stability assay. 

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... 

Bujfers Tris-HCl and potassium phosphate buffer (or phosphate-buffered saline, PBS) are used for the microsomal stability assays, while PBS is often the choice for the metabolism in hepatocyte suspensions. 

Giuliano C, Jairaj M, Zafiu CM, Laufer R. Direct determination of unbound intrinsic drug clearance in the microsomal stability assay. Drug Metab Dispos 2005 33 1319-1324. 

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. 

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. 

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. 

---