Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots

Abstract

The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session.

Trial registration: ClinicalTrials.gov NCT01731067.

Figure 1

Concentration–time profiles for CYP probe substrates (circles) and their metabolites (triangles) obtained in 10 µl of capillary DBS (dashed lines) and venous plasma samples (continuous lines) after oral administration of cocktail alone (left column), in the presence of inhibitor(s) (middle), or in the presence of an inducer (right) in 10 healthy volunteers. CYP1A2 profile is presented only for volunteers who received coffee (n = 6); CYP2D6 profiles are presented only for EMs and UMs (n = 7). Error bars represent SD. CYP, cytochrome P450; DBS, dried blood spot; EM, extensive metabolizer; UM, ultrarapid metabolizer.

Figure 2

Concentration–time profile of fexofenadine in capillary DBS (dashed lines) and venous plasma samples (continuous lines) after administration of the cocktail capsule alone (circles), with a P-gp inhibitor (squares), or with a P-gp inducer (triangles) in 10 healthy volunteers. Error bars represent SD. DBS, dried blood spot; P-gp, P-glycoprotein.

Figure 3

Metabolic ratio profiles after oral administration of cocktail capsule alone (diamonds), with an inhibitor (squares), or with an inducer (triangles) in dried blood spots. *P < 0.05; **P < 0.01. dor/dem: continuous lines are used for extensive metabolizers (EMs) and dashed lines for intermediate metabolizers (IMs). bup, bupropion; caf, caffeine; dem, dextromethorphan; dor, dextrorphan; mdz, midazolam; OH-bup, 4-hydroxybupropion; OH-flb, 4-hydroxyflurbiprofen; flb, flurbiprofen; OH-mdz, 1-hydroxymidazolam; OH-opz, 5-hydroxyomeprazole; opz, omeprazole; par, paraxanthine.