Nobiletin, sinensetin, and tangeretin are the main perpetrators in clementines provoking food-drug interactions in vitro

Highlights

• Flavonoid fractions of clementines juice were assessed for food-drug interactions.

• Polymethoxyflavones were the main perpetrators identified.

• CYP1A2 was profoundly inhibited by nobiletin, sinensetin, and tangeretin.

• Nobiletin and sinensetin activated AhR and thus induced CYP1A1/CYP1A2 expression.

• Tangeretin was a PXR activator and thus induced CYP3A4 and ABCB1 expression.

Abstract

For clementine juice, previous data indicate a possible food-drug interaction with substrates of key enzymes responsible for drug metabolism (i.e. cytochrome P450 [CYP] 3A4, CYP1A2). However, which compounds in clementine juice are responsible for these effects are unknown. Therefore, we aimed to identify the compounds in clementine juice provoking metabolic enzyme inhibition or induction. The results demonstrated that the flavonoid fraction of clementine juice provoked induction of several genes and inhibition of both CYP3A4 and CYP1A2, matching effects observed with whole clementine juice. CYP1A2 inhibition and induction can most likely be attributed to nobiletin, sinensetin, and tangeretin. Tangeretin was the only compound causing CYP3A4 induction while CYP3A4 inhibition was most likely the result of additive or synergistic effects caused by several compounds. Thus, whenever evaluating the clinical relevance of clementine interactions, flavonoid contents should be reported because these might explain differences between cultivars and harvests.

Introduction

Food components such as flavonoids can alter the pharmacokinetics of drugs by inhibiting or increasing activities of drug-metabolising enzymes and drug transporters that have important roles in absorption, distribution, metabolism, and excretion (Rodríguez-Fragoso et al., 2011). Inhibition of drug-metabolising enzymes, or efflux transporters in the intestine or the liver, can lead to increased bioavailability and, thus, increased plasma concentrations and, consequently, increased risk for potentially severe adverse effects (Guengerich, 1997, Lund et al., 2017). In contrast, induction of these proteins might provoke treatment failure (Guengerich, 1997, Lund et al., 2017). Induction is mediated mostly by transcription factors, pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR), which increase transcription of their target genes when activated by drug or food compounds behaving as ligands. Adverse food-drug interactions due to citrus juices, especially grapefruit juice, are well known and are included in patient informations (Seden, Dickinson, Khoo, & Back, 2010).

For clementines/clementine juice, we were the first to report a possible interaction (Theile et al., 2017). Based on the observation of increased tacrolimus troughs in a renal transplant patient with high consumption of clementines (>1 kg/d), we analysed the food-drug interaction potential of clementines in vitro using clementine juice from fruits of the same lot as those consumed by the patient (Theile et al., 2017). In our earlier study, strong inducing and/or inhibiting effects were revealed for several genes regulated by PXR, such as cytochrome P450 (CYP) CYP3A4 and ABCB1 (encoding for P-glycoprotein, P-gp). These proteins are important for the distribution of a large number of drugs to their sites of action (P-gp) as well as drug metabolism and clearance (CYP3A). Thus, they have a crucial role concerning the exposure of patients to the active compound(s). Alterations in expression or activities of these genes are a major cause of clinically relevant drug-drug and food-drug interactions (Guengerich, 1997, Lund et al., 2017, Rodríguez-Fragoso et al., 2011). Moreover, recently, we demonstrated in vitro that clementine juice induces strongly AhR regulated genes CYP1A1 and CYP1A2 and inhibits CYP1A2 (Theile et al., 2017), which can lead to interactions with co-administered drugs in vivo. Given that clementines do not contain furanocoumarins (bergamottin, dihydroxybergamottin, or epoxybergamottin), which are established CYP3A4 inhibitors responsible for most clinically relevant food-drug interactions with grapefruit juice (Seden et al., 2010) and the primary clementine compounds, hesperidin and narirutin, are not known to be strong inducers or inhibitors of, for example, CYP3A4 and CYP1A2 (Ho et al., 2001, Pan et al., 2011), identification of the compounds responsible is necessary.

The present study, therefore, aimed to identify compounds in clementine juice that provoked the observed effects. To do this, we isolated the total flavonoid fraction and, after verifying induction/inhibition effects were preserved, we analysed sub-fractions and single flavonoids not only for inhibition of CYP1A1 and CYP3A4, but also induction of genes driven by AhR (CYP1A1 and CYP1A2) and PXR (CYP3A4 and ABCB1).