Metabolic stability process

The development of synthetic methods for the selective introduction of short-chain perfluoroalkyl groups into organic molecules is of interest in drug development [464]. Fluoromodifications often confer unique properties on a molecule, for example in terms of increased metabolic stability and lipophilicity and, as a consequence, the pharmacokinetic profiles are often improved [465]. Burger and coworkers developed a domino process consisting of a SN reaction combined with a Claisen and a Cope rearrangement which allows the transformation of simple fluorinated compounds into more complex molecules with fluoro atoms [466]. Treatment of furan 2-917 with 2-hydroxymethyl thiophene (2-918) in the presence... 

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. 

The are several clearance and toxicological aspects that have to be considered in the drug discovery process such as metabolic stability, enzyme selectivity, CYP inhibition and type of inhibition. Among these factors, the prediction of the site of metabolism has become one of the most successful parameters for prediction. The knowledge of the site of metabolism enhances the opportunity to chemically modify the molecule to improve the metabolic stability. There are several approaches based on database mining, chemical reactivity, protein interaction or both that have been developed for the prediction of this property, with different degree of success and applicability. 

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... 

Rourick, R. A., Jenkins, K. M., Walsh, J., Xu, R., Cai, Z. and Kassel, D. B., Integration of Custom LC/MS Automated Data Processing Strategies for the Rapid Assessment of Metabolic Stability and Metabolic Identification in Drug Discovery, American Society for Mass Spectrometry 2002 Conference Abstract, Orlando, FL, USA, 2002. 

Whether a toxin is naturally reactive to biological macromolecules or receptors, or requires metabolic activation to produce such a species, e.g. the enzyme-mediated transformation of tremorine (9) to the active parasympathomimetic agent oxotremorine (10), it will usually be subject to chemical or enzymic inactivation in vivo. Interruption of the latter process via appropriate substitution may thus lead to an increase in biological activity or toxicity over that of the parent compound. Perhaps the most striking example of this is provided by the extreme metabolic stability and toxicity of TCDD and the nontoxicity of its de- chloro analogue dibenzodioxin (Table 3). 

The LTQ-FT mass spectrometer was introduced in late 2003 and, as expected, the main application discussed in the literature is for the analysis of proteins and peptides (Johnson et al., 2004 Syka et al., 2004). A recent book chapter (van der Greef et al., 2004) and a review article (Brown et al., 2005) discussed the application of the LTQ-FT to metabolomics. FTMS applications to dmg metabolism are still very new and dmg discovery research laboratories which have recently purchased the instmment are still in the process of developing and validating methods and approaches. A recent publication describes the depth and flexibility of the experimental setup utilizing accurate mass data-dependent exclusion MS" measurements with a LTQ-FT (Tozuka et al., 2005). We have reported several integrated approaches for determination of metabolic stability, characterization of metabolites and metabolic... 

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. 

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