Biochemical and analytical development of the CIME cocktail for drug fate assessment in humans

Abstract

Phenotyping based on drug metabolism activity appears to be informative regarding mechanism-based interactions during drug development. We report here the first steps of the development of the innovative CIME cocktail. This cocktail is designed not only for the major cytochrome P450, with caffeine, amodiaquine, tolbutamide, omeprazole, dextromethorphan and midazolam as substrates of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A, respectively, but also phase II enzymes UGT 1A1/6/9 with acetaminophen, P-gp and OATP1B1 with digoxin and rosuvastatin, and renal function with memantine. An assay combining ultra-performance liquid chromatography using a 1.7 microm particle size column with tandem mass spectrometry (UPLC/MS/MS) was set up for the simultaneous quantification of the 20 substrates and metabolites after extraction from human plasma using solid-phase extraction. The method was validated in the spirit of the FDA guidelines. Mean accuracy ranged from 87.7 to 115%, the coefficient of variance (CV%) of intra- and inter-run from 1.7 to 16.4% and from 1.6 to 14.9%, respectively, and for the limit of quantification (LOQ) with ten lots of plasma, accuracy ranged from 84 to 115% and CV% precision was <16%. Short-term stability was evaluated in eluate (4 h, room temperature), plasma (24 h, room temperature), the autosampler (24 h, 4 degrees C) and in three freeze/thaw cycles in plasma. All except three analytes were stable under these conditions. For the three others a specific process can be followed. This robust, fast and sensitive assay in human plasma provides an analytical tool for ten-probe drugs of the CIME cocktail. Clinical samples will be assayed in the near future using this new assay method.

Safety and pharmacokinetics of the CIME combination of drugs and their metabolites after a single oral dosing in healthy volunteers

“…Amodiaquine was selected for the first version of the CIME combination (Videau et al 2010;Prot et al 2011). However, the ethics committee was surprised by this use since several cases of severe toxicity have been reported with amodiaquine (Guevart and Aguemon 2009).…”

“…The CIME combination was designed to limit the risk of potential interaction between probes by: (a) selection of well-known substrates with no or few predictable interactions based on the Metabolism and Transport Drug Interaction Database (http://www.druginteractioninfo.org/) and the literature and (b) choice of low doses [previously discussed in (Videau et al 2010)], but not microdoses minimizing pharmacokinetic linearity issues. The probe doses are generally below the therapeutic range, but are used only for metabolic purposes and allow the production of metabolites for all the CYPs studied.…”

“…Blood was centrifuged and plasma was collected in two separate aliquots stored at -80°C until analysis. Probes and metabolites were assayed by means of an LC-MS/MS method previously developed and validated (Videau et al 2010 (Videau et al 2010) instead of amodiaquine, but were assayed simultaneously in the clinical samples. Lower limits of quantification (LLOQ) were 0.05 ng/mL for repaglinide, M4-hydroxy-repaglinide and digoxin and are reported elsewhere for the other compounds (Videau et al 2010).…”

“…Finally, we added memantine to the CIME combination to study the renal excretion of drugs (active tubular secretion), since the plasma pharmacokinetics of memantine correlates with urine pH and memantine is not metabolized (Freudenthaler et al 1998). After development and validation of a simultaneous LC-MS/MS bioanalytical assay for the substrates of the CIME combination and main relevant metabolites (Videau et al 2010), the CIME combination was used as an index of blood-brain barrier permeation in an in vitro model (Lacombe et al 2011), to demonstrate the drug-metabolizing capacity of a microfluidic biochip containing primary human hepatocyte cultures (Prot et al 2011), and to phenotype several CYP activities in rat (Videau et al 2012).…”

Buspirone, fexofenadine, and omeprazole: Quantification of probe drugs and their metabolites in human plasma

“…Scott et al reported an LC-MS/MS method for six probe drugs and their metabolites in both plasma and urine [24], Yin et al validated a similar approach for five drugs and their metabolites [25], Zhang et al developed an assay for four parent probe drugs in human plasma [26], and Videau et al presented a UPLC-MS/MS assay for ten probe drugs and ten metabolites in human plasma [27]. The overarching goal in each of these studies was the same as for the current work — to accelerate and simplify the processing and analysis of samples for probe drug studies, such that the efficiency of subject treatment and sampling is matched by the efficiency of the analytical process.…”