We neither observed changes in midazolam eCLmet
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 scr 11 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 it 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.
This is the first clinical evidence demonstrating that clementine juice lacking substantial amounts of flavonoids other than narirutin and hesperidin does not inhibit CYP3A substrates. These data also indicate that the effects observed in vivo and in vitro with the clementine batch from 2013 can most likely be attributed to one or more of the other flavonoids like nobiletin, sinensetin, or tangeretin. In addition, these data underline that different harvesting seasons (November versus February), as well as different harvests in general may lead to profoundly different composition of fruits (de Castro et al., 2007). For beverage-drug interactions variability between batches is a known phenomenon, as shown for instance when two different kinds (labels) of grapefruit juice were tested for their effect on tacrolimus pharmacokinetics, with one increasing tacrolimus trough concentrations 2.1-fold and the other having no effect (Liu et al., 2009).
Other underlying reasons for the discrepancy between in vivo and in vitro findings and between the initial clinical observation in 2013 and the single case experiments in early 2015 and early 2016 might be:
Hence, juice for future investigations (e.g. a clinical trial) should be analysed for their content of constituents with drug interaction potential such as tangeretin beforehand. From a clinical perspective, patients treated with narrow therapeutic index CYP3A substrates should be cautious when consuming clementines, because depending on the constituents high variability of CYP3A substrates levels may occur due to enzyme inhibition and possibly enzyme induction.
Conclusion Taken together, while ample data and warnings are available for adverse health effects of grapefruit juice in patients on drugs that are CYP3A substrates, clementines were not under scrutiny, yet. We have recently provided in vitro evidence for drug interaction potential (Theile et al., 2017) and now report in vivo data that clementines or clementine juice do not necessarily lead to relevant inhibition in CYP3A activity. We also provide in vivo data indicative for potential induction of CYP3A activity. Constituents and hence effects on CYP3A-dependent drug metabolism vary from batch to batch, hence more clinical data such as an adequately powered clinical food-drug interaction trial to further quantify this effect are necessary for a clear recommendation. For now patients taking drugs with a narrow therapeutic index that are CYP3A substrates should be cautious when consuming clementines or clementine juice. The clinical and analytical data may serve as basis for such a trial.