Metabolic stability assays

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... 

One issue related to supporting a metabolic stability assay with HPLC/MS/MS is the need to set up an MS/MS method for each compound. While it may only take 10 min to infuse a compound solution and find the corresponding precursor and product ions (along with minimal optimization of the collision energy), the processes of MS/MS development would require 4 hr per day if one wanted to assay 25 compounds per day. MS vendors have responded to this need by providing software tools that can perform the MS/MS method development step in an automated fashion. Chovan et al.68 described the use of the Automaton software package supplied by PE Sciex (Toronto, Canada) as a tool for the automated MS/MS method development for a series of compounds. The Automaton software was able to select the correct precursor and product ions for the various compounds and optimize the collision energy used for the MS/MS assays of each compound. They found that the Automaton software provided similar sensitivity to methods that would have been developed by manual MS/MS procedures. Chovan et al. also reported that the MS/MS method development for 25 compounds could be performed in about an hour with the Automaton software and required minimal human intervention. 

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... 

Chovan, L.E. et al. 2004. Automatic mass spectrometry method development for drug discovery Application in metabolic stability assays. Rapid Commun. Mass Spectrom. 18 3105. 

Approximately 300 drugs were tested in aqueous solubility, log D, apparent permeability and metabolic stability assays. Compounds having low values for solubility, apparent permeability, or metabolic stability, or extreme log D values were flagged. The frequency of compounds with flags in each human bioavailability (%) bin is shown. 

O Connor, D., Mortishire-Smith, R., Morrison, D., Davies, A., and Dominguez, M. (2006). Ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry for robust, high-throughput quantitative analysis of an automated metabolic stability assay, with simultaneous determination of metabolic data. Rapid Commun. Mass Spectrom. 20 851-857. 

Until very recently, metabolic stability screening and metabolite identification (metabolite ID) have been sequential processes that is, the metabolic stability assays are typically performed hrst to identify rapidly metabolized compounds and a follow-up metabolite ID study is performed next, typically at a 10-fold higher substrate concentration to ensure generation of sufficiently high-quality MS/MS information to support structure elucidation. 

Wu X, Wang J, Tan L et al (2012) In vitro ADME profiling using high-throughput rapid-fire mass spectrometry cytochrome p450 inhibition and metabolic stability assays. J Biomol Screen 17 761-772... 

FIGURE 13.13 Sample generation for metabolic-stability assay. 

Nevertheless, 1 and 10 xM are often arbitrarily selected as the initial concentrations for metabolic stability assays (MacKenzie et al., 2002 Obach and Reed-Hagen, 2002). The effects of organic solvents on enzyme activities have been carefully investigated (Busby et al., 1999 Chauret et al., 1998 Easterbrook et al., 2001). For the purpose of data comparability for enzyme activities, the levels of organic vehicles should, in principle, be kept as low as possible, and consistent in all incubations in a given experiment. Dimethyl sulfoxide (DMSO) and methanol (or acetonitrile), the most commonly used vehicle solvents, should be kept at levels equal to, or preferably less than, 0.2 and 2% (v/v), respectively (Easterbrook et al., 2001 Hickman et al., 1998). 

The Lilly metabolic stability assay estimates the percent loss of a substrate by phase I metabolism in human hepatic microsomes over a 30 min incubation period at 37 °C. All reactions are performed using a final substrate concentration of 2 pM in 100 mM sodium phosphate buffer at pH 7.4. 

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. 

McNaney et al. (2008) described a second-tier metabolic stability assay that was used to further characterize compounds and provide a metabolic stability half-life measurement. The main change for this assay versus the MetFast assay was the measurement of the substrate depletion of a compound for up to six time points in addition to the zero time point. These additional time points allowed for the calculation of the metabolic stability half-life for each compound. 

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