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American Journal of Clinical Nutrition, Vol. 87, No. 4, 863-871, April 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Further characterization of a furanocoumarin-free grapefruit juice on drug disposition: studies with cyclosporine1,2,3

Mary F Paine, Wilbur W Widmer, Susan N Pusek, Kimberly L Beavers, Anne B Criss, Jennifer Snyder and Paul B Watkins

1 From the School of Pharmacy (MFP), the General Clinical Research Center (SNP, ABC, and PBW), and the Department of Medicine (KLB, JS, and PBW), University of North Carolina, Chapel Hill, NC; and the US Department of Agriculture, Citrus and Subtropical Products Laboratory, Winter Haven, FL (WWW)

2 Supported by the National Institutes of Health (M01RR000046 and R01GM38149). Eli Lilly donated the zosuquidar, and the Florida Department of Citrus donated the 67-dihydroxybergamottin that was used in the in vitro experiments.

3 Address reprint requests and correspondence to MF Paine, 3312 Kerr Hall, CB# 7360, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360. E-mail: mpaine{at}unc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We previously established furanocoumarins as mediators of the interaction between grapefruit juice (GFJ) and the model CYP3A4 substrate felodipine in healthy volunteers using a GFJ devoid of furanocoumarins. It remains unclear whether furanocoumarins mediate drug-GFJ interactions involving CYP3A4 substrates that are also P-glycoprotein substrates.

Objective: The effects of furanocoumarin-free GFJ on drug disposition were further characterized by using the dual CYP3A4/P-glycoprotein substrate cyclosporine.

Design: By randomized crossover design, 18 healthy volunteers received cyclosporine (5 mg/kg) with 240 mL orange juice (control), GFJ, or furanocoumarin-free GFJ. Blood was collected over 24 h. Juice treatments were separated by ≥1 wk. The effects of diluted extracts of each juice and of purified furanocoumarins on [3H]cyclosporine translocation in Caco-2 cells were then compared.

Results: The median (range) dose-corrected cyclosporine area under the curve and the maximum concentration with GFJ (P ≤ 0.007), but not with furanocoumarin-free GFJ (P ≥ 0.50), were significantly higher than those with orange juice [15.6 (6.7–33.5) compared with 11.3 (4.8–22.0) x 10–3 h/L and 3.0 (1.6–5.8) compared with 2.4 (1.1–3.1) mL–1, respectively]. The median time to reach maximum concentration and terminal elimination half-life were not significantly different between the juices (2–3 and 7–8 h, respectively; P ≥ 0.08). Relative to vehicle, the GFJ extract, orange juice extract, and purified furanocoumarins partially increased apical-to-basolateral and decreased basolateral-to-apical [3H]cyclosporine translocation in Caco-2 cells, whereas the furanocoumarin-free GFJ extract had negligible effects. Reanalysis of the clinical juices identified polymethoxyflavones as candidate P-glycoprotein inhibitors in orange juice but not in GFJ.

Conclusions: Furanocoumarins mediate, at least partially, the cyclosporine-GFJ interaction in vivo. A plausible mechanism involves the combined inhibition of enteric CYP3A4 and P-glycoprotein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Grapefruit juice (GFJ) is one of the most widely studied dietary substances shown to interact with a variety of therapeutic agents (1-4). Most of these drugs are subject to extensive first-pass metabolism mediated by cytochrome P450 3A4 (CYP3A4), an enzyme expressed predominantly in the liver and small intestine (5, 6). When consumed in usual volumes, GFJ appears to inhibit only enteric CYP3A4 (1, 7). Although multiple components in GFJ capable of inhibiting CYP3A4 have been identified (eg, flavonoids), furanocoumarins have emerged as major candidate inhibitors, at least in vitro and in vivo in some preclinical species (8-15).

Major candidate furanocoumarins suitable for human consumption (eg, bergamottin, 6,7-dihydroxybergamottin, and furanocoumarin dimers) are not yet available in most countries, including the United States. Accordingly, to elucidate the aggregate role of furanocoumarins in drug-GFJ interactions in humans in vivo, we created a GFJ that was devoid of furanocoumarins (by {approx}99%) but retained other major ingredients, including flavonoids (16). This furanocoumarin-free GFJ was then tested against orange juice (control juice) and the original GFJ on the oral pharmacokinetics of the model CYP3A4 substrate felodipine in 18 healthy volunteers (16). As anticipated, the median area under the curve (AUC) and maximum concentration (Cmax) of felodipine with GFJ were significantly greater (by 2 to 3-fold) than with orange juice, whereas the terminal half-life (t1/2) was not different between the 2 juices. In contrast, no difference was detected in any pharmacokinetic outcome between furanocoumarin-free GFJ and orange juice, as the median felodipine concentration-time profile with furanocoumarin-free GFJ was virtually superimposable with that of orange juice.

Although the aforementioned in vivo study established furanocoumarins as mediators of the felodipine-GFJ interaction, this may not hold true for all CYP3A4 substrates. Unlike felodipine, several CYP3A4 substrates reported to interact with GFJ are also substrates for P-glycoprotein (P-gp), an ATP-dependent transmembrane export pump that is expressed, among other cell types, in the enterocytes (17, 18). Because of its location on the apical (lumenal) membrane, P-gp functions to extrude its substrates back into the intestinal lumen. Thus, inhibition of enteric P-gp would be expected to enhance drug systemic exposure.

The widely used immunosuppressant cyclosporine is a dual CYP3A4/P-gp substrate shown to interact with GFJ (19-23). Taken together, an additional mechanism underlying the GFJ effect may involve inhibition of enteric P-gp. To further characterize our furanocoumarin-free GFJ as a potential alternative to GFJ, the effects of this juice on the pharmacokinetics of oral cyclosporine were compared with those of orange juice and the original GFJ in healthy volunteers. Experiments with Caco-2 cell monolayers and organic extracts of the clinical test juices and of purified furanocoumarins were then undertaken to gain mechanistic insight into the effects of GFJ and its components on the intestinal translocation of cyclosporine.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and chemicals
[3H]Cyclosporine, with a specific content of 9 Ci/mmol, was purchased from Amersham Biosciences (Piscataway, NJ). Bergamottin was purchased from Indofine Chemical Co (Hillsborough, NJ). 67-Dihydroxybergamottin (DHB) was a kind gift from the Florida Department of Citrus (Lakeland, FL). Zosuquidar (otherwise known as LY335979), a selective P-gp inhibitor (24), was a kind gift from Eli Lilly (Indianapolis, IN). Caco-2 cell culture materials (uncoated polyethylene terephthalate culture inserts and murine laminin) and media ingredients (DMEM, fetal bovine serum, nonessential amino acids, gentamicin, dl-{alpha}-tocopherol, zinc sulfate, and sodium selenite) were purchased from sources as described previously (25). The Caco-2 cell clone P27.7 was characterized previously (26). All other chemicals were of tissue culture or analytic grade where appropriate.

Preparation of a furanocoumarin-free grapefruit juice
A GFJ devoid of furanocoumarins ({approx}99%) that retained other major components (flavonoids) was prepared by using a series of food-grade solvents and absorption resins as described in detail in our previous publication (16).

Subjects
Healthy nonsmoking volunteers (9 women, 9 men), ranging in age from 19 to 64 y and weighing from 83 to 153 kg, were enrolled. Most of the participants took no chronic prescription medications; one man took sertraline and one woman took bupropion and alendronate. Except for one man who was taking baby aspirin, none of the participants were taking nonprescription medications, including vitamin and mineral supplements and herbal products. Before enrollment, each participant underwent a screening procedure that consisted of a medical history, physical examination, evaluation of vital signs, and laboratory tests that included a complete blood count and blood chemistries (blood urea nitrogen, serum creatinine, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and total bilirubin). All of the women underwent a serum pregnancy test. All participants were instructed to abstain from grapefruit-containing products beginning ≥1 wk before and during the course of the study and to abstain from caffeinated and alcoholic beverages beginning the evening before each admission. Each participant was randomly assigned to 1 of 6 possible treatment sequences: ABC, ACB, BAC, BCA, CAB, or CBA (A = GFJ, B = orange juice, and C = furanocoumarin-free GFJ).

The University of North Carolina Institutional Review Board and Clinical Research Advisory Committee reviewed and approved the clinical protocol and consent form. All volunteers provided written informed consent before participating in the study.

Study design
Each participant was admitted to the General Clinical Research Center on the evening before the study day on 3 separate occasions. The next morning, after the subjects fasted overnight, an indwelling venous catheter was placed into an antecubital vein for blood collection. The participants were then administered a single dose (5 mg/kg) of cyclosporine (Sandimmune capsules; Novartis Pharmaceuticals, East Hanover, NJ) by mouth with 240 mL whole GFJ, furanocoumarin-free GFJ, or orange juice (Thirster, 100% orange juice from concentrate; Vitality Foodservice Inc, Tampa, FL). Because it had been reported previously that orange juice does not produce an interaction with cyclosporine (19), orange juice was used as the reference juice to control for potential physiologic effects of the treatment juices, such as carbohydrate and calorie load. Blood (10 mL) was drawn into EDTA-containing Vacutainer tubes (Becton-Dickinson, Rutherford, NJ) just before cyclosporine and juice administration and 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, and 24 h thereafter. Whole-blood samples were stored at –80 °C until analyzed for cyclosporine. Meals and snacks, devoid of grapefruit-containing products and caffeinated beverages, were provided after the 4-h blood collection. Vital signs (blood pressure, pulse, respirations, and temperature) were obtained just before cyclosporine and juice administration, every 2 h for the first 8 h, and then every 4 h until discharge the next morning. Each admission was separated by ≥1 wk. On the evening of the second and third admissions, all participants underwent a complete blood count to evaluate hematocrit, hemoglobin, and blood chemistries (blood urea nitrogen and serum creatinine) to evaluate for any effects of previous doses of cyclosporine. On the evening of all admissions, all of the women underwent a repeat serum pregnancy test.

Analysis of whole blood for cyclosporine
Thawed whole blood (100 µL), or quality-control material (Iris Technologies, Lawrence, KS), was added to the wells of a deep 96-well microtiter plate, followed by 0.1 mol ZnSO4/L (100 µL) and acetonitrile (500 µL) containing internal standard (cyclosporine D). After vortex mixing (1 min) and centrifugation (5 min), the supernatant (20 µL) was injected onto a Quattro Micro liquid chromatography/mass spectrometry/mass spectrometry system (Waters, Milford, MA). The short chromatography step used a C18 guard cartridge (4.0 x 3.0 mm; Phenomenex, Torrance, CA) and a methanol gradient (load: 0% methanol, 100% 2 mmol/L ammonium acetate, and 0.1% formic acid; wash: 50% methanol, 50% 2 mmol/L ammonium acetate, and 0.1% formic acid; elute: 100% methanol) at a flow rate of 0.6 mL/min. The sample was introduced to the mass spectrometry/mass spectrometry system via the electrospray interface, and ion fragmentation was measured in positive ion mode with multiple reaction monitoring. The ion transitions monitored had mass-to-charge ratios of 1220->1203 (cyclosporine) and 1234->1217 (internal standard). The concentrations of cyclosporine in the blood collections were measured from a 3-point calibration curve generated by Mass Lynx software (Waters).

Pharmacokinetic analysis
The oral pharmacokinetics of cyclosporine were assessed by standard noncompartmental methods with the use of WINNONLIN (version 4.1; Pharsight Corp, Mountain View, CA). The terminal elimination rate constant ({lambda}z) was determined by log-linear regression of at least the last 3 data points of the blood concentration-versus-time curve. The terminal t1/2 was calculated as ln2/{lambda}z. The AUC-versus-time curve was calculated by using the mixed log-linear trapezoidal method from time 0 to the time corresponding to the last measured concentration (Clast) and extrapolation to infinite time (Clast/{lambda}z). The apparent oral clearance (CL/F) was calculated as dose/AUC. Cmax and the time to reach Cmax (tmax) were obtained by visual inspection of the concentration-versus-time curve.

Evaluation of citrus juice extracts and of purified furanocoumarins as inhibitors of [3H]cyclosporine translocation in Caco-2 cells
Concentrated (200-fold) extracts of each citrus juice administered to the subjects were prepared previously as described (16). The fold-concentration of each extract, dissolved in methanol, was verified by comparing the concentrations of the marker furanocoumarins bergamottin and DHB in the extracts with those in the starting juices by HPLC, as described previously (27). The concentrations of bergamottin and DHB in each extract were, respectively, as follows: 3.8 and 5.4 mmol/L in GFJ, below the limit of quantification (BLQ) and 40 µmol/L in furanocoumarin-free GFJ, and BLQ for both in orange juice. The limit of quantification was 0.5 µmol/L.

The inhibitory effect of each test substance on the translocation of [3H]cyclosporine was evaluated in the human intestinal cell line Caco-2. The Caco-2 cell clone P27.7 was seeded onto laminin-coated culture inserts, grown to confluence, and treated with differentiation medium as described previously (25). Monolayer integrity was assessed periodically, including on the day of the experiment, by measuring the transepithelial electrical resistance, which was always ≥250 {Omega} cm2. All test substances were initially dissolved in methanol to yield 200-fold concentrated stock solutions, which were then diluted 1:200 in incubation medium (25). For apical-to-basolateral (A->B) translocation, incubation medium (1.5 mL) containing [3H]cyclosporine (0.5 µmol/L, 1 µCi) and juice extract or vehicle (0.5% methanol) was added to the apical compartment, followed by plain incubation medium (1.5 mL) to the basolateral compartment, of culture inserts. The final concentrations of bergamottin and DHB in each diluted juice extract were calculated, respectively, as 19 and 27 µmol/L in GFJ, 0 and 0.2 µmol/L in furanocoumarin-free GFJ, and 0 and 0 µmol/L in orange juice. To determine the inhibitory effect of individual furanocoumarins on [3H]cyclosporine translocation, purified bergamottin or DHB (30 µmol/L each) was used in place of the juice extract. As a positive control for P-gp inhibition, the selective P-gp inhibitor zosuquidar (0.5 µmol/L) was used in place of the juice extract (24). After 1, 2, 3, and 4 h at 37 °C, a 40-µL aliquot was collected from each compartment and added to 10 mL of scintillation cocktail; radioactivity was then counted. The percentage of [3H]cyclosporine translocated to the receiver compartment was calculated as the ratio of the amount of radioactivity collected from the receiver compartment to the sum of the amounts of radioactivity collected from the apical and basolateral compartments.

Analysis of clinical test juices for polymethoxylated flavone content
Polymethoxyflavone content in the 3 juices used for the clinical study was measured by combining 2 procedures. Because polymethoxyflavones exhibit retention characteristics similar to those of limonin on a C18 column (28), samples were prepared by the direct injection and in-line sample concentration and clean-up procedure described previously for limonin analysis (29). The same solid-phase extraction and trace enrichment column (3.0 x 10 mm; Upchurch, Oak Harbor, WA) was used with 20% aqueous acetonitrile as the wash solvent. An injection volume of 100 µL and 0.5 mL/min column wash flow rate for 1.1 min was used for sample clean-up and gave 100% retention of the polymethoxyflavones while allowing the major flavonoids, hesperidin, and naringin to be washed off the column. A valve was then switched that directed flow using a gradient of acetonitrile (20–40% over 10 min, held at 40% for 10 min, 40–50% over 10 min, 50–90% over 10 min, and held at 20% for 15 min) from a second pump through the trace enrichment column and eluted the polymethoxyflavones onto a YMC J'sphere M80 C18 analytic column (4 µm, 3.0 x 250 mm; Waters). The YMC column was selected because it provided good resolution of the polymethoxyflavones and furanocoumarins contained in citrus (30). This direct injection and in-line sample clean-up procedure greatly simplified sample preparation and eliminated the need for solvent extraction (31) or solid-phase extraction followed by concentration (32). Sample preparation for the direct injection technique consisted of the addition of 1 mL acetonitrile to a 4-mL aliquot of juice, sonication for 10 min, filtration through a 0.45-µm nylon filter, and then injection. Quantification of the polymethoxyflavones was accomplished by averaging duplicate injections of each juice and was based on response factors determined for tangeretin and nobiletin as external standards by ultraviolet light at 335 nm. The response factors for nobiletin at 335 nm were used to quantify all polymethoxyflavones except tangeretin. The response factor for tangeretin was used to quantify tangeretin. The limit of detection was 0.005 ppm ({approx}0.01 µmol/L).

Statistical analysis
Statistical analyses were performed by using STATVIEW (version 5.0.1; SAS Institute Inc, Cary, NC). For the human volunteer study, results are presented as medians and ranges with 95% CIs. Comparisons of median pharmacokinetic outcomes of cyclosporine among the 3 juices were made by pairwise comparisons with the Wilcoxon's signed-rank test and a Bonferroni-corrected level of significance (ie, 0.05/3 = 0.017). Comparisons of median pharmacokinetic outcomes between men and women were made by using the Mann-Whitney U test, with a significance level of 0.05. For the Caco-2 cell experiments, results are presented means ± SDs of 3–6 incubations unless indicated otherwise. Comparisons between the A->B and B->A translocation of [3H]cyclosporine for a given treatment were made by using the unpaired Student's t test (P < 0.05). Comparisons between vehicle and the juice extracts and zosuquidar were made by using one-way analysis of variance, followed by Dunnett's post hoc test when an overall significant difference ensued (P < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of the effects of citrus juices on the pharmacokinetics of oral cyclosporine in healthy volunteers
All of the cyclosporine and juice treatments were well tolerated. No adverse effects were reported by any of the participants. As with our previous study (16), none of the participants commented on the taste of the furanocoumarin-free GFJ, which, of the opinion of one of the investigators (MFP), was sweeter and less bitter than the original juice.

Representative individual (Figure 1Go, A and B) and median (plus upper extreme of the 95% CI; Figure 1CGo) cyclosporine concentration-time profiles show the effects of the 3 juices on cyclosporine disposition. Because the dose of cyclosporine was based on body weight (5 mg/kg), AUC and Cmax were dose-corrected to compare the various treatments among the 18 participants. In all but 3 subjects, the AUC of cyclosporine with GFJ was greater than that with orange juice (Figure 2Go A). The percentage difference among the 18 individuals ranged from –32% to 140%. The median AUC with GFJ was significantly greater (by 38%) than that with orange juice (P ≤ 0.004) (Table 1Go). Similarly, in all but 4 subjects, the Cmax with GFJ was higher than that with orange juice (Figure 2BGo). The percentage difference among the 18 subjects ranged from –29% to 160%, and the median Cmax with GFJ was significantly greater (by 25%) than that with orange juice (P ≤ 0.007) (Table 1Go). The median Cl/F with GFJ was significantly lower (by 28%) than that with orange juice (P = 0.001) (Table 1Go). The median tmax and terminal t1/2 were not different between orange juice and GFJ (P ≥ 0.30) (Table 1Go).


Figure 1
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  		FIGURE 1.. Concentration-time profile for cyclosporine (5 mg/kg) after coadministration of a single glass (240 mL) of orange juice (OJ), furanocoumarin-free (FC-free) grapefruit juice (GFJ), and GFJ in a subject with one of the smallest (A) and largest (B) GFJ-mediated increases in the area under the concentration-time curve and the maximum concentration (relative to orange juice) and the median profile (C) for all 18 subjects. Error bars represent the upper extremes of the 95% CI.

 

Figure 2
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  		FIGURE 2.. Effects of a single glass (240 mL) of orange juice (OJ), grapefruit juice (GFJ), and furanocoumarin-free (FC-free) GFJ on the dose-corrected area under the concentration-time curve (AUC) (A) and the maximum concentration (B) of cyclosporine (5 mg/kg) in 18 healthy subjects. Open symbols and solid lines denote individual values. Closed symbols and dashed lines denote the median values.

 

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  		TABLE 1. Pharmacokinetics of cyclosporine after oral administration (5 mg/kg) with a single glass (240 mL) of orange juice (OJ), grapefruit juice (GFJ), or furanocoumarin-free (FC-free) GFJ to 18 healthy subjects1

 
Relative to orange juice, furanocoumarin-free GFJ had no consistent effect on cyclosporine AUC and Cmax (Figure 2Go), with the percentage difference ranging from –56% to 58% and from –49% to 124%, respectively. The corresponding median differences were –0.2% and –3.0%, respectively. The median AUC and Cmax values with furanocoumarin-free GFJ were not different from those with orange juice (P ≥ 0.71) (Table 1Go). The median Cl/F, tmax, and terminal t1/2 were not different between orange juice and furanocoumarin-free GFJ (P ≥ 0.08) (Table 1Go). The median concentration-time profile for cyclosporine with furanocoumarin-free GFJ was nearly superimposable with that with orange juice (Figure 1CGo).

In all but 2 individuals, the AUC of cyclosporine with GFJ was greater than that with furanocoumarin-free GFJ (Figure 2AGo). The percentage difference ranged from –30% to 99% among the 18 subjects. The median AUC with GFJ was significantly greater (by 36%) than that with furanocoumarin-free GFJ (P ≤ 0.001) (Table 1Go). Likewise, in all but 2 individuals, the Cmax with GFJ was higher than that with furanocoumarin-free GFJ (Figure 2BGo). The percentage difference ranged from –6% to 92% in the 18 subjects. The median Cmax with GFJ was significantly greater (by 50%) than that with furanocoumarin-free GFJ (P ≤ 0.003) (Table 1Go). The median Cl/F with GFJ was significantly lower (by 27%) than that with furanocoumarin-free GFJ (P = 0.002). The median tmax and t1/2 were not different between GFJ and furanocoumarin-free GFJ (P ≥ 0.23) (Table 1Go). For all 3 juices, a sex difference was not detected in any of the pharmacokinetic outcomes of cyclosporine (P ≥ 0.19).

Comparison of the effects of citrus juice extracts and purified furanocoumarins on [3H]cyclosporine translocation in Caco-2 cells
To gain mechanistic insight into the cyclosporine-GFJ interaction, diluted extracts of each clinical test juice, at concentrations equivalent to those given to the human volunteers, were compared on the translocation of [3H]cyclosporine through Caco-2 cell monolayers. In the presence of vehicle, the translocation of [3H]cyclosporine in the B->A direction was significantly greater than that in the A->B direction (Figure 3Go A), by a factor of {approx}40 (Table 2Go). In the presence of the selective P-gp inhibitor zosuquidar, A->B translocation was significantly higher, and B->A translocation significantly lower, than in the presence of vehicle (Figure 3BGo), leading to an efflux ratio that was near unity (Table 2Go). In the presence of the GFJ extract, A->B and B->A translocations were also significantly higher and lower, respectively, (Figure 3CGo), but the effect was less pronounced than that with zosuquidar in the A->B direction; thus, the efflux ratio was lowered to a lesser extent than with zosuquidar (Table 2Go). The furanocoumarin-free GFJ extract had much less of an effect than did the GFJ extract. Relative to vehicle-treated cells, A->B translocation was 3-fold higher and B->A translocation was slightly higher (Figure 3DGo), which yielded an efflux ratio that was approximately half that with the vehicle (Table 2Go). The orange juice extract (Figure 3EGo) had a greater effect than did the furanocoumarin-free GFJ extract, but less of an effect than did the GFJ extract, on the translocation of [3H]cyclosporine. Relative to vehicle, the orange juice extract had a greater effect on A->B than on B->A translocation (Figure 3EGo). Because the changes in directional translocation with orange juice were proportional to those with GFJ, the efflux ratio with orange juice was similar to that with GFJ (Table 2Go).


Figure 3
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  		FIGURE 3.. Effects of vehicle (0.5% methanol) (A); the selective P-glycoprotein inhibitor zosuquidar (0.5 µmol/L) (B); diluted extracts (1:200) of grapefruit juice (GFJ) (C), furanocoumarin-free (FC-free) GFJ (D), and orange juice (OJ) (E); and purified bergamottin (BG; 30 µmol/L) (F) and 6,7-dihydroxybergamottin (DHB; 30 µmol/L) (G) on the apical-to-basolateral (A->B) and basolateral-to-apical (B->A) translocation of [3H]cyclosporine (0.5 µmol/L, 1 µCi) in Caco-2 cell monolayers. For A->B translocation, [3H]cyclosporine was added simultaneously with each test compound (1.5 mL total volume) to the apical compartment, followed by 1.5 mL plain incubation medium to the basolateral compartment; for B->A translocation, plain incubation medium was added to the apical compartment, followed by [3H]cyclosporine + test compound to the basolateral compartment. At the indicated times, a 40-µL aliquot was collected from each compartment and added to 10 mL of scintillation cocktail; radioactivity was then counted. The percentage of [3H]cyclosporine translocated to the receiver compartment was calculated as ratio of the amount of radioactivity measured in the receiver compartment to the sum of the amounts of radioactivity measured in the donor and receiver compartments. The concentrations of components in the diluted extracts were equivalent to those in the clinical juices. The concentrations of BG and DHB approximated those measured in GFJ. Symbols denote the means of 3–6 (A, B, C, D, and E) or duplicate (F and G) culture inserts. Error bars represent the SDs.

 

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  		TABLE 2. Effects of citrus juice extracts and of purified furanocoumarins on the apical-to-basolateral (A->B) and basolateral-to-apical (B->A) translocation of [3H]cyclosporine (CsA) in Caco-2 cell monolayers

 
To determine whether furanocoumarins could contribute to the effects of GFJ on [3H]cyclosporine translocation, purified forms of bergamottin and DHB, at concentrations approximating those in the original GFJ, were evaluated. Both furanocoumarins had similar effects on [3H]cyclosporine translocation (Figures 3Go, F and G); A->B translocation was slightly higher, and B->A translocation slightly lower, than that in vehicle-treated cells (Table 2Go).

Polymethoxylated flavone content in the clinical test juices
Polymethoxyflavones, compounds contained in some citrus fruit, including oranges, have been identified as inhibitors of P-gp (but not of CYP3A) activity in vitro (33-35). To determine whether these compounds might account for the inhibitory effect of the orange juice extract toward [3H]cyclosporine translocation in Caco-2 cells, the 3 juices used in the clinical study were analyzed for polymethoxyflavone content. Of the 9 compounds examined, all were readily detected in orange juice, with concentrations ranging from {approx}1 µmol/L (hexamethyl-O-gossypetin) to {approx}8 µmol/L (nobiletin) (Table 3Go). In contrast, only 3 and 2 compounds were detected, respectively, in GFJ and furanocoumarin-free GFJ and were <1/10th the corresponding concentrations in orange juice. The total polymethoxyflavone content in orange juice was 24-fold and >700-fold greater than that in GFJ and furanocoumarin-free GFJ, respectively. The total polymethoxyflavone content in GFJ was 33-fold greater than that in furanocoumarin-free GFJ, which converted to a percentage reduction of 97%.


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  		TABLE 3. Polymethoxylated flavone content in orange juice (OJ), grapefruit juice (GFJ), and furanocoumarin-free (FC-free) GFJ1

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a GFJ that was devoid of furanocoumarins, we recently established furanocoumarins as the mediators of the interaction between GFJ and the antihypertensive agent and model CYP3A4 substrate, felodipine (16). However, this observation cannot be assumed to apply to CYP3A4 substrates that are also substrates for the efflux transporter P-gp. Cyclosporine, a widely used immunosuppressant with a narrow therapeutic window, is such a dual CYP3A4/P-gp substrate that has been shown to interact with GFJ, potentially leading to serious adverse events (eg, nephrotoxicity) (3, 36). As a consequence, patients are advised to avoid GFJ while taking this medication (Neoral package insert; Novartis Pharmaceuticals, East Hanover, NJ). Therefore, to further characterize our furanocoumarin-free GFJ as a potential alternative to GFJ, its effects on the oral pharmacokinetics of cyclosporine were compared with those of the original GFJ and orange juice in healthy volunteers.

As anticipated, the median dose-corrected AUC and Cmax with GFJ were significantly higher than corresponding values with orange juice, which substantiated previous reports involving either formulation of cyclosporine (Sandimmune or Neoral) (19, 22, 23, 37). In contrast, relative to orange juice, furanocoumarin-free GFJ had no consistent effect on cyclosporine systemic exposure, with a median concentration-time profile that was indistinguishable from that with orange juice. These observations, coupled with those from the felodipine study (16), argue that the cyclosporine-GFJ interaction involves inhibition of enteric CYP3A4 by furanocoumarins.

Because the mechanism underlying the cyclosporine-GFJ interaction may also involve inhibition of enteric P-gp by furanocoumarins, the effects of diluted extracts of the clinical test juices on the translocation of [3H]cyclosporine in Caco-2 cell monolayers were then compared. Relative to vehicle, the selective P-gp inhibitor zosuquidar significantly decreased translocation in the B->A direction and increased translocation in the A->B direction, which confirmed that cyclosporine is a P-gp substrate. The GFJ extract partially decreased B->A and increased A->B translocation, consistent with previous reports involving the dual CYP3A4/P-gp substrate [3H]vinblastine (38) and the nonmetabolized P-gp substrate talinolol (39), and supported that components in GFJ are inhibitors of P-gp. Moreover, the furanocoumarin-free GFJ extract had negligible effects on [3H]cyclosporine translocation in both directions, which indicated that P-gp inhibitors were removed from the original GFJ. Candidate P-gp inhibitors include bergamottin and DHB, because both furanocoumarins partially altered [3H]cyclosporine translocation in both directions at concentrations comparable with those measured in the original juice. Bergamottin and DHB have also been shown to partially alter the translocation of [3H]vinblastine and another dual CYP3A4/P-gp substrate, [14C]saquinavir, in Caco-2 cells (38). In contrast, bergamottin and DHB had no effect on [3H]digoxin translocation in our Caco-2 cell system (MF Paine, AB Criss, PB Watkins, unpublished observations, 2005), and DHB had no effect on [3H]vinblastine translocation in MDR1-transfected LLC-PK1 cells (40). Collectively, these observations suggest that furanocoumarins contribute to the cyclosporine-GFJ interaction by inhibiting P-gp, and that the effects of furanocoumarins on P-gp are substrate- and cell system–dependent.

The effect of the orange juice extract on [3H]cyclosporine translocation indicated that citrus juice components other than furanocoumarins can inhibit P-gp. Other investigators have reported similar findings, with an orange juice extract, on [3H]vinblastine and [14C]saquinavir translocation in Caco-2 cells and identified the polymethoxylated flavones tangeretin, heptamethoxyflavone, and nobiletin as candidate P-gp inhibitors (34, 38). Each of these compounds, at 20 µmol/L, increased the A->B translocation (by {approx}2-fold) and decreased the B->A translocation (by {approx}30%) of both drugs, which resulted in a reduction of {approx}~70% in the B->A/A->B ratio (38). More recently, these compounds, as well as sinensetin, were shown to inhibit the B->A translocation of talinolol in Caco-2 cells, with half maximal inhibitory concentrations of 3–4 µmol/L (35). Reanalysis of our clinical test juices showed that orange juice contained these and other polymethoxyflavones at an aggregate concentration of 24 µmol/L, which was {approx}25-fold higher than that in the original GFJ. These observations support polymethoxyflavones as candidate inhibitors of cyclosporine translocation by orange juice, but not by GFJ, at least in Caco-2 cells.

The {approx}97% reduction in polymethoxyflavone content in the furanocoumarin-free GFJ does not necessarily implicate an important role for polymethoxyflavones in drug-GFJ interactions. This contention is based on observations that an orange juice extract (which would be abundant in polymethoxyflavones and devoid of furanocoumarins), and/or individual polymethoxyflavones, had no effect on CYP3A4 activity in human liver (34) and intestinal (16) microsomes. The lack of effect of orange juice components on CYP3A4 activity, combined with results from an earlier healthy volunteer study (n = 14) demonstrating no interaction between orange juice and cyclosporine (19), further suggest that these components play a negligible role in cyclosporine-GFJ interactions. Although it is possible that additional unknown bioactive, nonflavonoid components were removed during preparation of the furanocoumarin-free GFJ, furanocoumarins must at least partially mediate the cyclosporine-GFJ interaction. Whereas polymethoxyflavones likely do not contribute to drug-GFJ interactions, these components could contribute to drug–orange juice interactions. The mechanism underlying the latter appears to involve inhibition of an apically located enteric uptake transporter, organic anion transporting polypeptide (OATP), rather than P-gp, at least with fexofenadine, of which the systemic exposure with orange juice was significantly lower, rather than higher, than that with water (2, 41). Indeed, diluted orange juice and purified tangeretin and nobiletin have been shown to inhibit uptake of the OATP probe substrate [3H]estrone-3-sulfate in OATP2B1-transfected human embryonic kidney cells (42). In addition, the major orange juice–containing flavonoid, hesperidin, was recently shown to inhibit [3H]fexofenadine uptake in OATP1A2-transfected HeLa cells (43). Diluted GFJ and purified bergamottin, DHB, and/or the major GFJ-containing flavonoid, naringin, have also been shown to inhibit [3H]estrone-3-sulfate or [3H]fexofenadine uptake (42, 43), which could account for the apparent paradoxical lower systemic exposure of fexofenadine when given with GFJ compared with that when given with water (2, 41, 43).

Our assertion that furanocoumarins at least partially mediate the cyclosporine-GFJ interaction is incongruent with the observation that juice prepared from the related citrus fruit, the Seville (sour) orange, which contained furanocoumarins at concentrations comparable with those in GFJ (11, 44), had no interaction with cyclosporine (40). Opposing effects between OATP and CYP3A4/P-gp are unlikely, because cyclosporine is not known to be an OATP substrate. In view of these observations, it seems prudent to repeat the cyclosporine-Seville orange juice interaction study with a larger sample size. The small number of subjects included in the one published study (n = 7) (40), along with the large inter- and intraindividual variation in cyclosporine pharmacokinetics (45), could have masked a GFJ-type interaction. In support of this notion, Seville orange juice was shown to significantly increase the AUC of saquinavir by {approx}40% (46). As with cyclosporine, the interaction between saquinavir and GFJ is modest ({approx}50% increase in average AUC) (47); thus, a large sample size (n = 20) was required to detect an interaction between saquinavir and Seville orange juice.

In summary, the current in vivo and in vitro observations collectively indicate that furanocoumarins represent important mediators of the cyclosporine-GFJ interaction. The most plausible mechanism involves the combined inhibition of enteric CYP3A4 and P-gp. Whether the furanocoumarin-free GFJ can be used as a safe alternative to GFJ requires further testing on other drugs (eg, those that are substrates for OATP) and in different patient populations receiving chronic drug therapies.


   ACKNOWLEDGMENTS
 
We thank Dorothene Thompson of the General Clinical Research Center nutrition staff for her careful monitoring of the different juices used throughout the course of the clinical study.

The authors' responsibilities were as follows—MFP: responsible for the conduct, analysis, and data interpretation of the in vitro and clinical studies and compiled the manuscript; WWW: created the furanocoumarin-free GFJ and analyzed the clinical test juices for furanocoumarin and polymethoxyflavone contents; SNP: participated in the design and execution of the clinical study; KLB: participated in the design of the clinical study and acted as the study physician; ABC: participated in the design and execution of the in vitro experiments; JS: analyzed the whole-blood samples for cyclosporine; and PBW (principal investigator): acted as the attending physician. None of the authors had any conflicts of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication August 27, 2007. Accepted for publication October 12, 2007.





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