An evaluation of metabolite profiling of six drugs using dried blood spot.

Dry blood spot (DBS) analysis has been extensively used for the quantitative analysis of drugs by mass spectrometry; however, utilization of DBS for qualitative metabolite profiling has been very limited. In the present study, we investigated the use of DBS for metabolite profiling of genistein, carbamazepine, losartan, sunitinib, sildenafil and zoniporide representing a range of Phase I and Phase II biotransformations following oral and intravenous dosing to rats. Plasma and DBS were collected for PK and metabolite profiling. Analyte extraction recovery from DBS was optimized using the parent compound and metabolite standard. Rat DBS metabolite profiles from all six compounds were similar to plasma metabolite profiles, however Phase II metabolites appeared to be extracted less efficiently from DBS compared to plasma, and compounds that were unstable in blood showed different metabolite profiles. In summary, this study showed that in addition to PK bioanalytical analysis, DBS samples may also be utilized for metabolite profiling and a comparison of plasma and DBS metabolite profiling can also provide partitioning/association of major circulating metabolites compared to the parent drug even in the absence of a metabolite standard.

In Vitro and In Vivo Correlation of Hepatic Fraction of Metabolism by P450 in Dogs.

1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases-mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUCABT/AUCveh ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.