br Materials and methods br Results

Materials and methods
Results
Discussion Based on increased tacrolimus concentrations in a patient under clementine consumption and the in vitro drug interaction potential of the compounds present in clementines that may both induce and inhibit drug metabolising enzymes and drug transporters, we conducted two pilot experiments to test the clinical relevance of the in vitro findings, which had suggested a possible beverage/food-drug interaction. In ampa receptors to the previously observed sharp rise of tacrolimus concentrations, we were unable to reproduce the effect of clementine ingestion on tacrolimus plasma concentrations in the same patient. The observed increase in tacrolimus Cmin (5.0 μg/L at −48 h, 6.1 μg/L at +48 h) and Cmax (11.9 μg/L at −48 h, 14.9 μg/L at 48 h) during clementine ingestion was within the range of normal intra-patient fluctuation (Park et al., 2007). The C/D ratio during our single case experiment ranged between 0.89 and 1.36 (Fig. 1). We neither observed changes in midazolam eCLmet indicative of significant inhibition of CYP3A4/5 during both experiments, despite the in vitro evidence for CYP3A4/5 inhibition (Theile et al., 2017). The observed increase in CYP3A dependent midazolam clearance after chronic ingestion of clementines/clementine juice in both participants is consistant with induction of CYP3A activity through increased expression of intestinal and/or hepatic CYP3A isozymes. Still the observed changes of +43% and +89% are also within the known range of intra-individual variability when repetitively phenotyping a person\'s CYP3A activity with midazolam (Halama et al., 2013). Interestingly, the LC-MS/MS analysis of the juice consumed demonstrated that, even though the flavonoid profile was very similar, the concentrations of all components were much lower than in the clementines the patient consumed in 2013 and which were used for the in vitro experiments in 2014 (Theile et al., 2017): only narirutin and hesperidin were present in quantifiable amounts confirming the batch variability as a consequence of harvesting conditions, maturity, extraction techniques, use of degreening agents and to subsequent storage conditions (Milella et al., 2011; Fujita et al., 2008; Álvarez et al., 2012) (Table 1). Overall, milligram amounts of narirutin (3.1 mg/d) and hesperidin (21.0 mg/d) were chronically ingested by the volunteer without significant inhibition of CYP3A activity. Up to date, no CYP3A inducing properties have been reported and only very weak inhibition of CYP3A4 by hesperidin has been demonstrated (Ho et al., 2001), so that subspecies appears unlikely that the amounts of hesperidin present in our clementine juice were high enough to provoke CYP3A4 induction or clinically relevant CYP3A4 inhibition. While gross changes in enzyme function would have been detected in these single case experiment, the effects on midazolam clearance (Fig. 1, Fig. 2) measured upon chronic ingestion of clementines/clementine juice albeit consistent with enyzme induction cannot be distinguished in a definitive manner from randomness in these two cases.