Microsomal incubation assay

Mutagenicity studies conducted using the Salmonel-/a/microsome incubation assays gave negative results for furan. The same results were observed when Salmonella typhimurium strains were incubated in the presence or absence of rat and hamster liver S-9 fractions. 

Many factors may confound the assessment of the D DI potential of early discovery compounds [93], Limited or no solubility data exist to understand the likelihood that the compound will precipitate out of an in vitro incubation. The compounds have generally not been analyzed from a spectroscopic perspective their characteristics may interfere with a fluorogenic DDI assay. Metabolism data are typically not available. The binding of a compound to plasma proteins or microsomal incubation constituents is not well understood, which may lead to underprediction of its inhibitory potential. The compounds are typically delivered in DMSO, which may cause solvent-related inhibition of the enzymatic assay. Also, since little is known about in vivo concentrations or projected dose, framing the consequences of an early DDI in vitro experiment may be difficult. With these factors in mind, general experimental paradigms have been developed to help minimize their potential impact. 

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. 

Rourick and co-workers endeavored to test the feasibility of nanoscale LC-MS on metabolite analysis with an in vitro assay based on human liver microsomal incubation [100]. Buspirone, an anxiolytic drug with a well-characterized metabolic profile [101], was chosen as a test compound. At an initial concentration of 4 pM, buspirone was incubated with microsomal protein (1 mg/mL) and 4 mM NADPH in 50 mM sodium phosphate... 

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. 

Figure 11.28 (a) Six compounds and their metabolites chosen as specific probes for activity of six CYP450 isozymes mainiy responsible for metabolism of xenobiotic compounds, (b) An example of the ESI-MS and ESI-MS/MS spectra used to select MRM transitions used in the LC-MS/MS assays for inhibition/induction of a specific isozyme, (c) HPLC-MRM chromatograms for the internal standard (propronanol) and for the metabolites of the six probe compounds obtained for a microsomal incubation. Reproduced from Peng, Rapid Commun. Mass Spectrom. 17, 509 (2003), with permission of John Wiley Sons Ltd. 

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). 

NADPH total clearance (oxidative cuid hydrolytic) of parent chemical from microsomal assay, —NADPH NADPH-independent hydrolytic clearance of parent chemical from microsomal incubation, ND. no detectable elimination... 

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]. 

An elegant study of lycopene uptake in LNCaP, PC-3, and DU-145 cells using beadlet-delivered 1.48 J.M all-trans lycopene (a maximal level in human plasma) found that all three cell lines rapidly took up lycopene during the first 10 h of incubation. Cells continued to accrue lycopene, but more slowly, over the next 48h. The uptake by the LNCaP cells was 2.5-fold higher than PC-3 cells and 4.5-fold higher than DU-145 cells at 24h of incubation but lycopene showed no affinity for the AR receptor, which is expressed in the LNCaP cells (Liu et al. 2006). LNCaP uptake followed Michaelis-Menten kinetics with a V m i, of 66.3pmol/106 cells/h and a Km of 7.72 pM lycopene. Because of the sensitivity of their LC-MS-MS lycopene assay, Liu et al. were also able to investigate the subcellular lycopene distribution. The nuclear membrane contained 55%, the nuclear matrix 26%, and the microsomal fraction 19% of the intracellular fraction. The cytosol contained no lycopene(Liu et al. 2006). 

Forti, G.C., Paolini, M., Hrelia, P. et al. (1984) NADPH-generating system influence on microsomal monooxygenase stability during incubation for the liver microsomal assay with rat and mouse S9 fractions. Mutation Research, 129, 291-297. 

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... 

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