Human microsomal metabolic stability

1) Use a large data set containing experimental property values, here human microsomal metabolic stability measurements. 

3) Find MMPs and extract contexts and corresponding transformations. 

5) Group the transformations and calculate the difference in property value AP for each MMP. Bin the AP values and compute their distribution. 

7) Evaluate the effect of the context on the behavior of the transformation. Identify cases where the effect of the context leads to a AP distribution that differs from the general trend in a statistically significant way. 

Metabolic stability is the extent of susceptibility to enzymatic modifications encountered by a compound in the body. It is an important ADM E property as it is an in vivo determinant of oral bioavailability, clearance, and half-life. 

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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... 

Pfizer has recently evaluated open source descriptors and model building algorithms using a training set of approximately 50 000 molecules and a test set of approximately 25 000 molecules with human liver microsomal metabolic stability data. A C5.0 decision tree model demonstrated that the Chemistry Development Kit descriptors together with a set of SMARTS keys had good statistics (Kappa = 0.43, sensitivity = 0.57, specificity 0.91, positive predicted value (PPV) = 0.64) equivalent to models built with commercial MOE2D and the same set of SMARTS keys (Kappa = 0.43, sensitivity = 0.58, specificity... 

This chapter follows the large-scale MMP analysis paradigm and studies the relationship between molecular transformations and the resulting changes in experimental property, particularly human liver microsomal metabolic stability. More specifically, the ultimate aim has been the identification of metabolic stability isosteres, that is, transformations and replacements with zero or positive effect on metabolic stability. Furthermore, in line with the recent study of Papadatos et al. [18], the possibility of context-dependent isosteric replacements is explored. 

The work presented here follows and builds upon the context-dependent MMP analysis approach described by Papadatos et al. [18]. Specifically, the said approach is employed here in order to detect context-sensitive bioisosteric replacements in a large data set of human liver microsomal metabolic stability data. In what follows, the methodology of this work will be thoroughly described and the results will be discussed in detail. 

The aim of the present example was to investigate whether the assessment of an in silico model of metabolic stability from a training set of several hundred drugs or drug-like compounds in human CYP3A4 cDNA-expressed microsomal preparations, would offer a suitable approach to predict the metabolic stability of external compounds. 

This model integrates existing in vitro data, such as Caco-2 permeability (Papp) and metabolic stability in liver S9 or microsomes, to estimate bioavailability as being either low, medium, or high. Oral bioavailability predictions for not only humans but also other species can be made by using the metabolic stability values of drugs in liver microsomal enzyme preparations from that species. A premise of this model is that metabolic clearance is more important than renal or biliary clearance in determining bioavailability. However, despite the lack of in vitro renal... 

Fig. 19.10. Oral bioavailability estimates of drugs in rat, guinea pig, dog and human from their respective Caco-2 Papp and metabolic stability in liver microsomes or S9. (Reproduced with permission from Ref. [26], 2002 American Chemical Society.)...

Several kinetic parameters can be measured on different experimental systems to account for the interaction of a compound with CYPs. For example when studying the metabolic stability of a compound, it could be measured in a recombinant CYP system, in human liver microsomes, in hepatocytes and so on. Each system increases in biological complexity. Although in the recombinant CYP system only the cytochrome under consideration is studied, in the case of the human liver microsomes, there is a pool of enzyme present that includes several CYPs, and finally in the hepatocyte cell system, metabolizing enzymes play an important role in the metabolic compound stability. In addition, transport systems are also present that could involve recirculation or other transport phenomena. The more complex the experimental system, the more difficult it is to extract information on the protein/ligand interaction, albeit it is closer to the in vivo real situation and therefore to the mechanism that is actually working in the body. 

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. 

Using human physiology and in vitro data and clinically relevant input parameters (i.e., metabolic stability in human hepatocytes and microsomes as well as prothrombin time measured in various batches of human plasma) the model was extended to human. This human PK/PD model was then used to investigate the impact of the various physicochemical, pharmacokinetic and pharmacodynamic properties on the anticoagulant profile (i.e., prothrombin time) expected in man. 

Kantharaj, E., Ehmer, P. B., Tuytelaars, A., Van Vlaslaer, A., Mackie, C., and Gilissen, R. A. (2005b). Simultaneous measurement of metabolic stability and metabolite identification of 7-methoxymethylthiazolo[3,2-a]pyrimidin-5-one derivatives in human liver microsomes using liquid chromatography/ion-trap mass spectrometry. Rapid Commun. Mass Spectrom. 19 1069-1074. 

When chemical inhibition experiments are conducted with a relatively metabolically stable drug candidate (one that must be incubated with relatively high concentrations of human liver microsomes for a relatively long time in order to generate quantifiable levels of metabolite), it is important to take into account the metabolic stability of the inhibitors themselves. Lack of metabolic stability makes some compounds poor choices as chemical inhibitors despite their selectivity. For example, coumarin is a selective substrate of CYP2A6 (Km 0.25 to 0.5 pM) (111) and it would be a good selective competitive inhibitor of CYP2A6 if it were not metabolized so rapidly by human liver microsomes. 

Lee SH, Slattery JT (1997) Cytochrome P450 isozymes involved in lisofylline metabolism to pentoxifylline in human liver microsomes. Drug Metab Dispos 25 1354-1358 Li AP (1999) Cryopreserved human hepatocytes characterization of DME activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability and drug-drug interaction potential. Chem Biol Interact 121 17-35... 

Busby WF, Ackermann JM, Crespi CL (1999) Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450. Drug Metab Dispos 27 246-249 Clohs L, Wong J (2002) Validation of a capillary electrophoresis assay for assessing the metabolic stability of verapamil in human liver microsomes. J Cap Elec Microchip Tech 7 113 Coughtrie MWH, Fisher MB (2003) The role of sulfotransferase and UDP-glucuronosyltransferases. In Lee JS, Obach RS, Fisher MB (eds) Drug Metabolizing Enzymes. Marcel Dekker, New York pp 541-575... 

Linget JM, du Vignaud P (1999) Automation of metabolic stability studies in microsomes, cytosol and plasma using a 215 Gilson liquid handler. J Pharm Biomed Anal 19 893-901 Long L, Dolan RC, Dolan ME (2001) Debenzylation of 06-benzyl-8-oxoguanine in human liver implications for 06-benzylguanine metabolism. Biochem Pharmacol 61 721-726... 

Figure 11-13. Time-course human liver microsomal incubation profiles for a number of positive and negative controls, as well as project compounds. Metabolic stability profiles are represented both in tabular format and graphically above.

Because clearance at the whole-body level often is determined by metabolism at the cellular level, it is possible to use a variety of human-derived in vitro systems to determine rates of metabolism. These systems include pure human enzymes (such as cytochrome P450 enzymes) (13) and human liver subcellular fractions (S9 and microsomes) (14). However, with enzymes and subcellular fractions, some information is lost because the whole-cell integration of subcellular processes has been disrupted. The use of cultured human hepatocytes retains the whole-cell integration at the expense of greater experimental complexity (15). Each system provides a different window on the metabolic processes, is relatively easy to use, and can be obtained from commercial sources. Rates and pathways of metabolism may be compared with a series of discovery compounds to identify those with the greatest relative metabolic stability or with a benchmark compound of known human PK characteristics to provide a more absolute estimate of hepatic metabolic clearance. 

Several TRK-850 derivatives were synthesized based on the general strategies mentioned above, and their metabolic stabilities were evaluated in the presence of human liver microsomes. Metabolic rates were determined by incubating the compounds with human liver microsomal enzymes and then quantifying the remaining parent peak by HPLC analysis. The data were expressed as the elimination rate constant (Kel) and the relative metabolic rate, or the /C , [ of the compound compared with the /C, [ of TRK-850 (ATF, ratio). 

Another approach to human bioavailability estimation is based on in vitro data using Caco-2 as a measure of permeability and human liver microsomes for metabolism estimates. These data are combined in a graphical method to get a rough estimate of human oral bioavailability [22]. In principle, but not yet proven, this method could also be applied by using calculated permeability and metabolic stability. 

The use of fast gradient elution LC-MS techniques for metabolic screening was first described by Ackermann and coworkers in 1998 [91] and Korfmacher and coworkers in 1999 [69], In the method developed by Ackermann et al., a HPLC column-switching apparatus is used to desalt and analyze lead candidates incubated with human liver microsomes. The resulting data can be quickly resolved into specific categories of metabolic stability high (>60%) moderate (>30%-59%) low (>10%-29%) and very low (<10%). 

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