Paul F. Cancalon

The effect of grapefruit juice on drug disposition

Introduction: Since their initial discovery in 1989, grapefruit juice (GFJ)–drug interactions have received extensive interest from the scientific, medical, regulatory and lay communities. Although knowledge regarding the effects of GFJ on drug disposition continues to expand, the list of drugs studied in the clinical setting remains relatively limited. Areas covered: This article reviews the in vitro effects of GFJ and its constituents on the activity of CYP enzymes, organic anion-transporting polypeptides (OATPs), P-glycoprotein, esterases and sulfotransferases. The translational applicability of the in vitro findings to the clinical setting is discussed for each drug metabolizing enzyme and transporter. Reported AUC ratios for available GFJ–drug interaction studies are also provided. Relevant investigations were identified by searching the PubMed electronic database from 1989 to 2010. Expert opinion: GFJ increases the bioavailability of some orally administered drugs that are metabolized by CYP3A and normally undergo extensive presystemic extraction. In addition, GFJ can decrease the oral absorption of a few drugs that rely on OATPs in the gastrointestinal tract for their uptake. The number of drugs shown to interact with GFJ in vitro is far greater than the number of clinically relevant GFJ–drug interactions. For the majority of patients, complete avoidance of GFJ is unwarranted.

Methods for determining the adulteration of citrus juices

The adulteration of fruit juices and juice-based beverages is a serious economic problem. Juice adulteration has progressed from simple dilution with water and the substitution of cheap ingredients to highly sophisticated manipulations designed to mask the adulteration process. Major adulteration problems currently encountered by regulatory agencies include undeclared addition of sugar and pulp wash, the admixture of juices, and the addition of colorants, amino acids and organic acids. Also of regulatory concern is ascertaining the geographical origin of a juice. While numerous methods have been developed to detect juice adulteration, only a limited number have proved workable in deterring sophisticated adulteration. Methodologies critically evaluated in this report include isotopic analyses, SNIF-NMR, HPLC, tracer addition, UV/VIS spectrophotometry, ICP-AES, electrochemical detection and pattern recognition.

Mechanism-based inhibition of human cytochrome P450-3A activity by grapefruit hybrids having low furanocoumarin content.

A citrus breeding program aimed at developing low furanocoumarin (FC) grapefruit cultivars provided 40 grapefruit juice (GFJ) samples containing variable concentrations of FC derivatives, established as being mechanism-based (irreversible) inhibitors of human CYP3A isoforms. The principal inhibitory FCs were identified as 6′,7′-dihydroxybergamottin, along with a series of dimeric compounds (spiroesters) having high inhibitory potency. A random subset of the GFJ samples (n = 25) were tested as CYP3A inhibitors using an in vitro model based on human liver microsomal metabolism of the index substrate triazolam. The reciprocal values of in vitro 50% inhibitory concentrations (IC50) were highly correlated with concentrations of inhibitory FCs in the GFJ samples (r2 = 0.96). However the correlations were driven mainly by a few samples having high FC content and high reciprocal IC50 (corresponding to low IC50). Among the rest of the samples, the relationship was less robust. Further study is needed to determine how low the FC content needs to be (or how high the IC50 needs to be) to assure minimal risk of clinical interactions involving GFJ and CYP3A substrate drugs.

New furanocoumarins detected from grapefruit juice retentate

Grapefruit (Citrus paradisi Macf.) furanocoumarins and related compounds have been shown to interact with the enterocyte cytochrome P450, CYP3A4, and as a result they affect the bioavailibility of certain drugs. Only a few grapefruit furanocoumarins have been identified so far. In this study, grapefruit juice retentate, rich in furanocoumarins, was extracted and then separated by flash chromatography for the examination of new compounds. Finally, nine new furanocoumarins were detected in different fractions according to their UV spectra and mass spectrometric properties by LC-MS (liquid chromatography mass spectrometry) and tentatively designated as FC 338, FC 420, FC 524, FC 530, FC 540, FC 546, FC 552, FC 570 and FC 614.

Effect of blueberry juice on clearance of buspirone and flurbiprofen in human volunteers

AIM The present study evaluated the possibility of drug interactions involving blueberry juice (BBJ) and substrate drugs whose clearance is dependent on cytochromes P4503A (CYP3A) and P4502C9 (CYP2C9). METHODS A 50:50 mixture of lowbush and highbush BBJ was evaluated in vitro as an inhibitor of CYP3A activity (hydroxylation of triazolam and dealkylation of buspirone) and of CYP2C9 activity (flurbiprofen hydroxylation) using human liver microsomes. In clinical studies, clearance of oral buspirone and oral flurbiprofen was studied in healthy volunteers with and without co-treatment with BBJ. RESULTS BBJ inhibited CYP3A and CYP2C9 activity in vitro, with 50% inhibitory concentrations (IC50 ) of less than 2%, but without evidence of mechanism-based (irreversible) inhibition. Grapefruit juice (GFJ) also inhibited CYP3A activity, but inhibitory potency was increased by pre-incubation, consistent with mechanism-based inhibition. In clinical studies, GFJ significantly increased area under the plasma concentration-time curve (AUC) for the CYP3A substrate buspirone. The geometric mean ratio (GMR = AUC with GFJ divided by AUC with water) was 2.12. In contrast, the effect of BBJ (GMR = 1.39) was not significant. In the study of flurbiprofen (CYP2C9 substrate), the positive control inhibitor fluconazole significantly increased flurbiprofen AUC (GMR = 1.71), but BBJ had no significant effect (GMR = 1.03). CONCLUSION The increased buspirone AUC associated with BBJ is quantitatively small and could have occurred by chance. BBJ has no effect on flurbiprofen AUC. The studies provide no evidence for concern about clinically important pharmacokinetic drug interactions of BBJ with substrate drugs metabolized by CYP3A or CYP2C9.

Identification of genes associated with low furanocoumarin content in grapefruit

Some furanocoumarins in grapefruit (Citrus paradisi) are associated with the so-called grapefruit juice effect. Previous phytochemical quantification and genetic analysis suggested that the synthesis of these furanocoumarins may be controlled by a single gene in the pathway. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of fruit tissues was performed to identify the candidate gene(s) likely associated with low furanocoumarin content in grapefruit. Fifteen tentative differentially expressed fragments were cloned through the cDNA-AFLP analysis of the grapefruit variety Foster and its spontaneous low-furanocoumarin mutant Low Acid Foster. Sequence analysis revealed a cDNA-AFLP fragment, Contig 6, was homologous to a substrate-proved psoralen synthase gene, CYP71A22, and was part of citrus unigenes Cit.3003 and Csi.1332, and predicted genes Ciclev10004717m in mandarin and orange1.1g041507m in sweet orange. The two predicted genes contained the highly conserved motifs at one of the substrate recognition sites of CYP71A22. Digital gene expression profile showed the unigenes were expressed only in fruit and seed. Quantitative real-time PCR also proved Contig 6 was down-regulated in Low Acid Foster. These results showed the differentially expressed Contig 6 was related to the reduced furanocoumarin levels in the mutant. The identified fragment, homologs, unigenes, and genes may facilitate further furanocoumarin genetic study and grapefruit variety improvement.

Effect of Low‐Furanocoumarin Hybrid Grapefruit Juice Consumption on Midazolam Pharmacokinetics

The objectives of this study were to investigate the effect of grapefruit juice low in furanocoumarins on CYP3A activity and to summarize previous findings of enzyme inhibition measured by the metabolism of midazolam after intake of grapefruit juice. Twelve healthy volunteers participated in a prospective, randomized, double‐blinded, 3‐way crossover clinical study to determine the effect of regular grapefruit juice (RGJ) and a novel, low‐furanocoumarin hybrid grapefruit juice (HGJ) on the metabolism of oral midazolam, used as a probe for in vivo CYP3A activity, compared with water as a control. The RGJ was 100% hand‐squeezed “Hudson” grapefruit juice, and the HGJ contained low amounts of furanocoumarin constituents. The point estimates (90% confidence intervals) for the RGJ/water midazolam AUC geometric mean ratio was 122% (107‐140). The point estimate for the HGJ/water midazolam AUC ratio was within the 80% to 125% bioequivalence range, indicating an absence of interaction. This finding also prompted a systematic review of available evidence on the pharmacokinetic alteration of midazolam by grapefruit juice. Although most studies demonstrated alteration in midazolam pharmacokinetics supporting inhibition of CYP3A activity as a likely mechanism, the cohorts included in these studies and the extent of the pharmacokinetic interaction varied widely. The current study indicated grapefruit juice‐drug interaction varies substantially based on patient characteristics and/or grapefruit juice product‐related factors, including the amount of furanocoumarin constituents present in the juice.