In vitro 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... 

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... 

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. 

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... 

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. 

Metabolism Metabolic stability (see Section 6.3.2.1) Metabolic soft-spot identification (see Table 6.10) Reactive metabolites (see Table 6.10) Assessing in vitro metabolic liabilities by drug-metabohsm enzymes such as CYP Predicting in vivo clearance Assay ° Liver microsomes from various species ° NADPH as co-factor ° 0.5-5 i,M incubation concentration Analysis ° PPT followed by LC-MS/MS or onhne SPE-MS/MS... 

Metabolic Stability Metabolic stability assays are the most widely used in vitro ADME screening assay. Metabolism is the body s major mechanism of detoxification of foreign compounds (xenobiotics) such as drug molecules. Common metabolic reactions can be categorized as (1) phase I reactions, which are direct modifications such as oxidation and (2) phase II reactions, which are conjugations such as glucuronidation. As a result of these metabolic conversions, xenobiotics become polar metabolites which can be cleared more... 

Drug candidates that are intended for oral dosing need to have good ADME properties so that they can be dosed once or twice daily. The drug should be well absorbed, survive first pass metabolism, and have sufficiently low clearance. At the lead identification stage, the primary in vitro ADME assays employed are those that assess permeability and metabolic stability. There are a variety of assays available for both parameters, as described in the previous chapter. 

In addition, compound 15 also had good metabolic stability in human liver microsome in vitro assay (hLM ti/2 = 39min) and in rat in vivo pharmacokinetic studies (ty2 = 3.3 h, po), with a rat oral bioavailability of 15%, showing a significant improvement in these PK parameters over the lead compound 1. The observed improvement in PK during the optimization was another validation of the strategy discussed above. This part of the optimization process is summarized in Scheme 19.2. 

Bioavailability can be assessed early in the discovery process by combining data from a number of independent in vitro assays run as part of a pharmaceutical properties profile. These assays generally include some measurement of aqueous solubility, lipophilicity, cell or membrane permeability, and metabolic stability. In most cases, proper interpretation of... 

Cyprotex, using Cloe Screen , evaluates pharmacokinetic properties in vitro and establishes a broad portfolio of in vitro assays that allows researchers to investigate the metabolism parameters for drug discovery and development. This company supplies the following data and assays (276) microsomal stability,... 

In theory, an isosteric/isoelectronic aromatic ring replacement within a toxic or therapeutic molecule should lead to a retention of biological activity. The fact that this is sometimes not observed may mean either that the activity is heterocycle-specific or that unforseen changes in metabolism, biological distribution and excretion, partition coefficient or stability have accompanied the molecular change. In such cases, activity may be observed in in vitro assays while not being observed in vivo (cf. the possible case of the dithiin analogue of TCDD in Table 3). 

---