Metabolic stability screens

Advances in techniques for chemical synthesis allow medicinal chemists to synthesize hundreds to thousands of compounds per month. Metabolic stability screening in liver microsomes is used extensively in early discovery to select the analogs or compounds most likely to have favorable pharmacokinetic parameters. This provides information on the relation of structure to stability, thus guiding synthesis strategies. 

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. 

Halladay JS, Wong S, Jaffer SM, Sinhababu AK, Khojasteh-Bakht SC. Metabolic stability screen for drug discovery using cassette analysis and column switching. Drug Metabol. Lett. 2007 1 67-72. 

For metabolic stability screens to be most effective, the screens must be tightly linked to some means of gathering information on metabolite structure. Methods... 

The LC-MS methods introduced by Ackermann et al. and Korfmacher et al. paved the way for more powerful platforms for high-throughput metabolic stability screening that featured the use of LC-MS/MS [80,92], These powerful methods relied on robotic sample preparation systems that were integrated with an automated LC-MS/MS system. 

Zhong, F. et al., High throughput metabolic stability screening by MALDI triple quadra-pole analysis, The Application Note Book, September, 24, 2007. 

For metabolic stability screens to be most effective, the screens must be tightly linked to some means of gathering information on metabolite structure. Methods for rapidly determining metabolite molecular weight and limited structural information have improved dramatically and allow this approach to be routinely employed (Anari and Baillie, 2005 Watt et al., 2003). The goal of this type of approach is to allow the identification of metabolic soft spots which can then be altered to produce compounds with improved metabolic stability. The literature of successful structural modification to increase stability has recently been reviewed (Thompson, 2001). 

A standard model for higher-throughput oral screening is shown in Fig. 12.5. As shown in Fig. 12.5, a common scenario for an oral bioavailability screen would be to combine the results of an in vitro absorption screen with the results on an in vitro metabolic stability screen to select compounds to go into an in vivo oral PK screen. The NCEs that still appeared to be promising lead compounds after these various screens could then be selected for full PK studies (oral and intravenous dosing in three to four animals for each dose route). In this way, only those compounds that were likely to exhibit good oral bioavailability would be tested in the full PK studies. 

FIGURE 12.5 Schematic representation of a typical process for oral bioavaUability screening in a drug discovery setting. The results of both an in vitro absorption screen as well as an in vitro metabolic stability screen are combined to select compounds for an in vivo oral PK screen. The compounds that survive these various screens can then be assayed in the standard in vivo full PK study. 

A different approach to the analysis of the large numbers of samples generated in metabolic stability screens (Zhang 2000a O Connor 2006) used a combination of HPLC (in one case with an ultra-small particle stationary... 

The RapidFire method is well-suited to smdies such as CYP inhibition screening and for monitoring any enzymatic reaction, because the same substrate/product pair is monitored for each incubation. Thus, it is possible to optimize the on-line SPE/MS/MS method for each specific product formed from an enzymatic assay. For studies requiring evaluation of the appearance/disappearance of the test compound (e.g., metabolic stability screening), an optimized SPE/MS/MS method for each compound must be developed, which may present challenges if the analytes being evaluated have substantially different physicochemical properties. 

In vitro metabolic stability assays are a valued first-pass assessment of potential metabolic liabilities. However, detailed information about the location of metabolic soft-spots is particularly useful in understanding whether the observed liabilities are specific to a molecule s core or introduced as part of a side chain in the lead optimization process. Whether the metabolic liability is associated with the core or side chain has clear implications for the degree to which the liability can be engineered out of a chemical series. Up until recently, metabolic stability screening and metabolite detection have been decoupled processes, that is, the metabolic stability assays are typically performed at a... 

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