Page - (000119) - in Biomedical Chemistry: Current Trends and Developments

is often not compatible with the amount of drug present in the samples. At this point, some companies are opting to develop LC-MS techniques or automated LC-MS/MS approaches to circumvent this limitation (Table 2.1.6). Nevertheless, the major application of LC-MS and LC-MS/MS during in vitro ADME analysis regards drug metabolism. In fact, due to the publication of the FDA guidelines on metabolites in safety testing (MIST) and DDI, metabolite profiling and screening for metabolites in plasma samples have been recently included as part of new drug discovery stages, implying the development of bioanalytical techniques that quickly identify and quantify the parent drug and its metabolites. Information generated from these studies could therefore enable the scientist to predict earlier the major metabolite exposure in animals and humans, allowing a better strategy to be developed. For instance, for compounds with extensive metabolism, identification of metabolites may help medicinal chemists to modify the NCE to block the sites of metabolism. On the other hand, active metabolites may have better pharmacokinetic profiles than the dosed NCE and may become a new lead compound. Moreover, the DDI guidance of EMA recommends the identification of the enzymes involved in the formation and elimination pathway of metabolites that contribute to more than 50% of the in vivo pharmacological effect (EMA, 2012). Since CYP enzymes are the most frequently involved in drugs metabolism, the identification of the specific isoforms involved in the metabolism of new compounds at early in vitro stages become crucial, specifically whether the drug candidate is metabolized by single or multiple enzyme isoforms and whether highly polymorphic enzymes, such as CYP2D6 and CYP2C19, contribute to their metabolic clearance. In addition, these in vitro techniques (mainly employing microsomes and hepatocyte cell lines) can also be useful in identifying drug candidates that are inhibitors or inducers of CYP isoforms, and can therefore predict their DDI potential. For these investigations, CYP probe substrates must be used and their respective metabolites must be quantified in the presence and absence of the drug candidate. Thus, it is not surprising that significant efforts have been devoted to develop rapid chromatographic techniques that