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Perspective Journal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4343parent compound. It was discovered later that thehigher activity of the ex vivo plasma samples, as shownby the leftward shift in the graph (Figure 3), was dueto the presence of an active metabolite in the samplesfrom the in vivo study. The metabolite was thenisolated, and partial structural identification led to thesynthesis of a hydroxylated analogue that became thelead compound for the chemotype.An additional clue that could point toward the presenceof an active metabolite is the observation of agreater pharmacological effect upon extravascular administrationof a compound relative to parenteraladministration. In most cases, the systemic concentrationsare lower when the compound is administeredorally because of the first-pass effect, especially duringthe first few hours after dose. As a result, greater invivo activity is expected to be observed when thecompound is administered intravenously compared towhen it is administered orally at a similar nominal dose,provided the pharmacological site of action is not a firstpassorgan. If the converse is observed, then an activemetabolite(s) may be playing a role. This was exactlywhat was observed when enzyme activity was measuredex vivo upon administration of the BMS-A drug candidatediscussed above. 34 In these studies, the concentrationof the parent compound as well as the enzymaticactivity was measured in ex vivo plasma samples. It wasfound that in samples where 80% inhibition of theenzyme activity was measured, the concentration of theparent compound was 46 nM after oral administrationand 132 nM after intravenous administration. Thisdifference in apparent potency after oral and intravenousadministration was evident at all levels of enzymeinhibition. The data clearly showed that the enzymeactivity was inhibited more significantly upon oraladministration and was consistent with the presenceof an active metabolite.An additional trigger point for the search for activemetabolites based on PK/PD disconnects is the observationof a prolonged PD effect for a drug candidate despitea short pharmacokinetic profile. 3 In particular, if acompound shows a relatively high in vivo activity anda prolonged pharmacodynamic effect compared to othercompounds of the same class, while demonstratingsimilar pharmacokinetic profiles, then there is a strongindication of the presence of an active metabolite(s).Recently, on the basis of this type of observation, wewere able to identify a number of active metaboliteswith superior “developability” characteristics comparedto the parent compounds. 35 In one such case, severaldrug candidates (BMS-B, -C, -D, and -E) from the samechemical class were observed to have essentially similarPK (similar plasma concentrations, similar proteinbinding, and target tissue concentrations) and similarin vitro activity profiles. However, what was intriguingwas the dissimilarity in the apparent in vivo activitydata of these compounds, with some showing many-foldmore activity than others. For example, BMS-B wasabout 20- to >40-fold more active in vivo than BMS-C,-D, and -E. Since the result was suggestive of thepresence of active metabolites, a rapid bioassay-guidedmethod was designed to generate and detect the activemetabolites (vide infra). In this method, compoundswere incubated with rat liver microsomes for a specifiedperiod of time, after which the incubation was terminated.The biological activity was determined in theinitial and final incubation mixture, without isolation,using an in vitro cell-based assay. The IC 50 (concentrationvalue resulting in 50% inhibition) values weredetermined to be 12, 11, 41, and 60 nM in the initialincubation mixtures and 19, 51, 585, and 490 nM in thefinal incubation mixture for BMS-B, -C, -D, and -E,respectively. The amount of the parent compoundsremaining in the final incubation mixture was determinedto be