RESUMO
Plasma concentration data points (n = 2,640) from 16 healthy adults were used to develop and validate limited sampling strategies (LSS) for estimation of phenotypic metrics for CYP enzymes and the ABCB1 transporter, using a cocktail of subtherapeutic doses of the selective probes caffeine (CYP1A2), metoprolol (CYP2D6), midazolam (CYP3A), losartan (CYP2C9), omeprazole (CYP2C19), and fexofenadine (ABCB1). All-subsets linear regression modelling was applied to estimate the AUC0-12h for caffeine, fexofenadine, and midazolam, and the AUC0-12h ratio of metoprolol: α-OH metoprolol and omeprazole:5-OH omeprazole. LSS-derived metrics were compared with the parameters' 'best estimates' obtained by non-compartmental analysis using all plasma concentration data points. The correlation coefficient (R 2) was used to identify the LSS equations that provided the best fit for n timed plasma samples, and the jack-knife statistics was used as an additional validation procedure for the LSS models. Single time-point LSS models provided R 2 values greater than 0.95 (R 2 > 0.95) for the AUC0-12h ratio of metoprolol:α-OH metoprolol and omeprazole:5-OH omeprazole, whereas 2 time-point models were required for R 2 > 0.95 for the AUC0-12h of caffeine, fexofenadine, and midazolam. Increasing the number of sampling points to three led to minor increases in R 2 and/or the bias or prediction of the estimates. In conclusion, the LSS models provided accurate prediction of phenotypic indices for CYP1A2, CYP2C19, CYP2D6, CYP3A, and ABCB1, when using subtherapeutic doses of selective probes for these enzymes and transporter.
RESUMO
Drug transporters and CYP enzymes are important sources of pharmacokinetics (PK) variability in drug responses and can cause various pharmacological and toxicological consequences, leading to either toxicity or an insufficient pharmacological effect. In recent years, the cocktail approach was developed to determine in vivo CYP and transporters activities, but these approaches are somewhat limited. We described the development and validation of three sensitive and specific LC-MS/MS assays for the determination of P-gp and major human CYP isoenzyme activities following oral administration of a drug cocktail of subtherapeutic doses (lower than 10 times) of caffeine (CAF), omeprazole (OME), losartan (LOS), midazolam (MDZ), metoprolol (METO) and fexofenadine (FEX) in healthy volunteers. The three validated methods were selective for all tested analytes. No interference or matrix effect was observed for the mass transition and retention times for all compounds monitored. Additionally, assays were linear over a wide range, and limits of quantification varied between 0.01-5 ng/mL plasma. The coefficients of variation obtained in the precision studies and the inter- and intra-assay accuracies were less than 15%, guaranteeing the reproducibility and repeatability of the results. All substrates and metabolites were stable in plasma during freeze-thaw cycles. Three healthy volunteers were selected based on genotyping for CYP2C9, CYP2C19 and CYP2D6. One volunteer was genotyped as an extensive metabolizer (EM) for all tested CYP isoforms, one volunteer was genotyped as a poor metabolizer (PM) for the CYP2C9 isoform (CYP2C9*3/*3), and one volunteer was genotyped as a PM for the CYP2D6 isoform (CYP2D6*4/*4). The methods allowed the quantification of all analytes over the entire sampling period (12 h) in all studied genotypes. Thus, the analytical methods described here were sufficiently sensitive for use in low-dose pharmacokinetic studies.