The influence of phase II enzymes on in vitro half-life of pirydo[1,2-c]pirymidine derivatives as structural analogues of arylpiperazine

Abstract Metabolic stability plays a crucial role in assessing the safety of potential drug candidates. Assays to designate in vitro half-life are well established and are focused on phase I metabolism using human liver microsomes. However, such an assay can easily be modified to include phase II metabolism. Yet, to authors best knowledge, no attempts at designating in vitro half-life values that includes phase II enzymes were performed. That means, that all current metabolic stability estimations reflect only partial information – as only phase I of metabolism takes place in currently used assays. The following study uses originally modified metabolic stability assay, coupled with LC-MS analysis to provide in vitro half-life values for four pirydo[1,2-c]pirymidine derivatives. Testosterone was used as a positive control. The assay was modified using alamethicin (a pore-forming agent which enhances membrane transport, allowing for phase II reactions) and UDPGA (Uridine 5′-diphosphoglucuronic acid) – co-factor for most common phase II biotransformation reaction – glucuronidation. In vitro half-life values estimated using two assays (a standard one comprising sole phase I enzymes and modified one – providing phase II reactions) have been compared using U Mann-Whitney’s test for statistical significance. Values for phases I+II were slightly higher, however, the difference was not statistically significant. Such results lead to the conclusion, that in vitro half-life values are not strongly influenced by phase II biotransformation in the case of the considered compounds.