Grapefruit juice-felodipine interaction: Effect of naringin and 6’,7’- dihydroxybergamottin in humans

Objective: To test whether naringin or 6’,7’-dihydroxybergamottin is a major active substance in grape- fruit juic+felodipine interaction in humans. Methods: Grapefruit juice was separated by means of centrifugation and filtration into supernatant and particulate fractions, which were then assayed for naringin and 6’,7’-dihydroxybergamottin. The effect of these fractions, grapefruit juice (containing comparable amounts of both fractions), and water on the pharmacokinetics of oral felodipine were assessed in 12 healthy men in a randomized, 4-way crossover study. Results: The amounts of naringin and 6’,7’-dihydroxybergamottin in the supernatant fraction (148 mg and 1.85 mg) were greater than in the particulate fraction (7 mg and 0.60 mg). The area under the plasma concentration-time curve (AUC) and the peak concentration (C,) of felodipine were higher with super- natant fraction (81 nmol . h/L and 20 nmol/L), particulate fraction (117 nmol . h/L and 24 nmol/L), and grapefruit juice (130 mu01 . h/L and 33 nmol/L) compared with water (53 mnol . h/L and 11 nmol/L). However, the supernatant fraction had a lower AUC for felodipine and a similar C,, of felodi- pine relative to the particulate fraction. The supernatant fraction neither augmented the AUC of the pri- mary metabolite dehydrofelodipine nor decreased the AUC ratio of dehydrofelodipine to felodipine com- pared with water. IndividuaIly the supernatant fraction consistently produced lower felodipine AUC and C compared with grapefruit juice. In contrast, the particulate fraction had values ranging from more thZ grapefruit juice to less than supernatant fraction. Conclusions: Naringin and 6’,7’-dihydroxybergamottin are not the major active ingredients, although they may contribute to the grapefruit juic+felodipine interaction. The variable effect with the particulate frac- tion may result from erratic bioavailability of unidentified primary active substances. The findings show the importance of in vivo testing to determine the ingredients in grapefruit juice responsible for inhibi- tion of cytochrome I?450 3A4 in humans. (Clin Pharmacol Ther 1998;64:248-56.)

David G. Bailey, PhD, John H. Kreefk, MD, Claudio Munoz, MD, David J. Freeman, PliD, and John R Bend, PhD London, Ontario, Canada

Grapefruit juice is known to interact with a broad range of medications.172 In our initial report, it produced marked elevation of plasma felodipine concentration

From the Department of Medicine, London Health Sciences Centre, and Department of Pharmacology and Toxicology, University of Western Ontario.

Supported by grant MT-13750 from the Medical Research Council of Canada (Ottawa, Ontario, Canada) and by grant 94014 from the Department of Citrus, State of Florida.

Received for publication Feb. 24, 1998; accepted May 11, 1998. Reprint requests: David G. Bailey, PhD, Department of Medicine,

London Health Sciences Centre, Victoria Campus, 375 South St, London, Ontario, N6A 4G5 Canada. E-mail: David.Bailey@ LHSC.ON.CA

Copyright 0 1998 by Mosby, Inc. 0009-9236/98/$5.00+0 13/l/91730

when this dihydropyridine calcium channel antagonist was administered to patients with borderline hyperten- sion.3 Reduction in blood pressure, increase in heart rate, and frequency of vasodilatation-related adverse events also were greater, supporting the clinical rele- vance of the interaction. Felodipine is completely absorbed from the gastrointestinal tract after oral admin- istration, but it normally has only 15% absolute bioavail- ability because of high presystemic metabolism.4 Grape- fruit juice caused a severalfold increase in the area under the plasma concentration-time curve (AUC) and peak concentration (Cm,) of felodipine and dehydrofelodi- pine, its single primary metabolite, and a concomitant decrease in the AUC ratio of dehydrofelodipine to felodipine.t,s The half-life (tt/,) values of felodipine and

248

CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 64, NUMBER 3 Bailey et al. 249

dehydrofelodipine did not change. These pharmacoki- netic results indicated that grapefruit juice reduced the presystemic metabolism of felodipine and dehydro- felodipine. Both are substrates for cytochrome P4503A4 (CYP3A4), which is present in both the intestinal wall and liver.5-7 However, grapefruit juice appears to inhibit only the intestinal metabolism of this drug.s-to Grape- fruit juice acted by decreasing intestinal CYP3A4 pro- tein expression through a post-transcriptional mecha- nism, most likely accelerated CYP3A4 degradation after mechanism-based enzyme inactivation.9

Identification of the active ingredient or ingredients in grapefruit juice would enable prediction and testing of this type of interaction with other foods.1 The appar- ently nontoxic substance might also be used commer- cially to make orally active drugs that are currently effective only through the intravenous route because of complete presystemic metabolism involving intestinal CYP3A4 monooxygenase activity or to produce higher and more dependable drug bioavailability and clinical response among and within individuals for medications with high but incomplete first-pass elimination caused by this enzyme.1 Grapefruit juice does not appear to inhibit liver CYP3A4 activity. This suggests that an important mechanism for systemic drug elimination is not jeopardized. However, the continuance of liver CYP3A4 activity indicates that it would likely not be possible to make oral drug bioavailability complete. There also is concern for interaction with other drugs that are extensively metabolized by enteric CYP3A4 but do not have active ingredients included in their for- mulations.

The flavonoid naringin and the furanocoumarin 6’,7’- dihydroxybergamottin have been suggested to be important ingredients in grapefruit juice-drug interac- tion (see Structure).lJt These substances were initially identified in studies of in vitro activity and quantitative prominence in the juice.“-l3 However, oral administra- tion of commercially available pure naringin to humans did not increase the plasma concentration of the dihy- dropyridines, felodipine, or nisoldipine compared with the same amount of naringin found in grapefruit juice.14J5 Although the reason for this lack of effect is unknown, it is evident that in vitro results were not pre- dictive of in vivo activity in this case. To our knowl- edge, the effect of 6’,7’-dihydroxybergamottin as an in vivo inhibitor of CYP3A4 in humans has not been reported.

The purpose of this study was to evaluate further the role of naringin and 6’,7’-dihydroxybergamottin in grapefruit juice-drug interactions by studying in vivo effects on humans subjects. The approach was to sepa-

rhamnoglucose - 0

OH 0

NARINGIN

t5-C~~ CH -C-CH2 -CH2 -CH-k-CH3 bH3 bH 6H

6’, 7’ - DIHYDROXYBERGAMOTTIN Chemical structures of naringin and 6’, 7’-dihydroxyberg- amottin. *Indicates position of the asymmetric carbon atom in the naringin molecule.

rate grapefruit juice into fractions that contained dif- ferent amounts of the 2 proposed active ingredients. The effects of these fractions, grapefruit juice contain- ing a comparable amount of both fractions, and water on the pharmacokinetics of oral felodipine were com- pared for healthy male volunteers.

METHODS Study popuhtion. Twelve white men (age range, 18

to 40 years) participated in the study. An examination before the study showed that subjects had normal phys- ical findings and laboratory test results, including results of routine hematologic tests, serum chemistry studies, and urinalysis. Individuals provided written informed consent for the study, which had been approved by the Health Sciences Standing Committee on Human Research at the University of Western Ontario (London, Ontario).

Experimental protocol. Subjects received 10 mg racemic felodipine extended release formulation (Plendil; Astra Pharma Inc., Mississauga, Ontario) with supematant fraction (240 mL from 250 mL grapefruit juice), particulate fraction (9 g from 250 mL grapefruit juice resuspended in 250 mL water), whole grapefruit juice (250 mL) or water (250 mL) in a balanced ran- domized crossover study. The interval between each test day was 1 week. The same brand and lot number of white grapefruit juice (Equality brand lot 96 OCO8/D; The Great Atlantic and Pacific Company of Canada, Toronto, Ontario) was used throughout the

250 Bailey et al. CLINICAL PHARMACOLOGY & THERAPEUTICS

SEPTEMBER 1998

study. Subjects abstained from alcohol for 48 hours and fasted for 10 hours before testing. Blood was sampled just before dosing and at %, 1, l%, 2, 2%, 3, 4, 5, 6, 8, 10, and 12 hours after administration. Subjects con- sumed a standardized lunch 4 hours (noon) and supper 9 hours (5 PM) after drug administration. Smoking and consumption of beverages that contained caffeine were not allowed, but water was allowed from 3 hours after administration.

Preparation of grapefruit juice supernatant and particulate fractions. Grapefruit juice (250 mL) was centrifuged at 9000g for 30 minutes. The supernatant fraction was removed, centrifuged a second time at 20,OOOg for 1 hour and filtered sequentially (5 pm, 1.2 pm, 0.8 pm x 47 mm membrane filters) under vacuum. The material sedimented by means of centrifugation (particulate fraction) was weighed and combined. The total wet weight of the particulate fraction was 9 g. The filters used for preparing the supernatant fraction were combined and extracted by means of passing 5 mL absolute ethanol through the filters under vacuum. The ethanolic extract was reduced in volume to 1 to 2 mL under nitrogen and added to the particulate fraction. Supernatant and particulate fractions were prepared the week before each study day and stored frozen (-30°C). Just before use, the fractions and whole grapefruit juice were thawed. The particulate fraction was resuspended in 250 mL water.

Assay of naringin in supernatant and particulate fractions. An aliquot (250 p.L) of supematant fraction (previously diluted to one-tenth concentration with water) and aqueous internal standard solution of 150 pg/mL rutin (INN, rutoside; 250 pL; Sigma Chemical Co, St. Louis, MO) were applied to a lOO-mg Cts preparatory solid-phase extraction column (Burdick and Jackson, Muskegon, Mich).14 The sample was washed with water (1 x 250 pL) and extracted with methanol (6 x 500 pL>. This procedure has been shown to produce complete recovery of naringin from aque- ous solution at concentrations encountered in this assay.14 The extract was evaporated to dryness under a gentle stream of nitrogen.

The wet particulate fraction (9 g) was resuspended in water to a total volume of 10 mL. An aliquot (5 mL) was extracted twice with 30 mL methanol by means of shaking for 2 hours followed by centrifugation at room temperature. Separate analysis of each extract showed that the first extract contained nearly all of the naringin. Further extraction with 30 mL hot (65°C) methanol for 1 hour of incubation showed it contained less than 2% of the total in the first 2 extracts. The first and second methanolic extracts were combined, and an aliquot (250

pL) was mixed with rutin (internal standard) in 250 pL methanol and evaporated to dryness under nitrogen.

The residues from the supematant and particulate fractions were dissolved in HPLC mobile phase (250 pL), which consisted of methanol/water (25:75 vol/vol) at pH 3 with phosphoric acid. An aliquot (25 pL) was injected onto a 10 cm x 3.2 mm Spherisorb Cs 5-pm column (Phase Separation, Deeside, Clwyd, England) at a flow rate of 0.7 mL/min. Ultraviolet absorbance was monitored at 285 nm. The retention time of naringin was 8.5 minutes and of rutin was 14.4 min- utes. The standard curve was linear within the range tested (0 to 100 pg/mL), and the coefficient of varia- tion was less than 5% at 50 pg/mL (n = 5).

The concentration of naringin in the supematant frac- tion was 61.5 pg/mL and in the particulate methanolic extract was 56.0 pg/mL. Because 250 mL grapefruit juice weighed 256 g and because the amount of super- natant fraction administered to volunteers weighed 247 g, the amount of naringin in the supernatant fraction was 148 mg (247 g + 256 g x 250 mL x 61.5 pg/mL x lo-fold dilution = 148 mg). The amount of naringin in the particulate fraction was 7 mg (10 mL + 5 mL x 65 mL x 56 yg/mL = 7 mg).

Assay of naringenin in supernatant and particulate fractions. Sample preparation of the supematant frac- tion was the same as that for the assay of naringin. Recovery of naringenin was determined. Naringenin, 10 pg/rnL, in 10% methanolic aqueous solution (n = 5) was subjected to solid-phase extraction, using the same method as that for naringin. HPLC results were com- pared with those for 10 pg/mL naringenin in mobile phase (n = 5) analyzed directly by means of HPLC. The solid-phase extraction procedure produced complete naringenin recovery. Naringenin content in the particu- late fraction was determined in the methanolic extract used for the naringin assay. Additional extraction with hot (65°C) methanol for 1 hour did not result in detectable amounts of naringenin. The internal standard was 150 pg/mL quercetin (Sigma Chemical Co) in methanol; an aliquot (250 pL) was added after extrac- tion. The extracts from supematant and particulate frac- tions were evaporated to dryness under nitrogen.

The residues were redissolved in HPLC mobile phase, which consisted of methanol/water (35:65 vol/vol) at pH 3 with phosphoric acid. Other HPLC chromatographic conditions were the same as those for the naringin assay. The retention time of naringin was 8.2 minutes and that of quercetin was 10.6 minutes. The limit of detection of naringenin was 1 pg/mL. Narin- genin was not detected in extracts from the supematant or particulate fractions. Thus the supematant fraction

CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 64, NUMBER 3 Bailey et al. 25 1

contained less than 0.25 mg (250 mL x 1 pg/mL), and the particulate fraction contained less than 0.13 mg (10 mL + 5 mL x 65 mL x 1 yg/mL).

Assay of 6; 7’-dihydroxybergamottin in supematant andparticulutefractioions. An aliquot (1 .O mL) of super- natant, particulate fraction (from 1 mL grapefruit juice resuspended in 1 mL water) or 6’,7’-dihydroxybergamot- tin standard (pure substance generously supplied by Department of Citrus, Lake Alfred, Fla) in orange juice, which does not normally contain 6’,7’-dihydroxyberg- amottin, were extracted with dichloromethane (1 .O mL) by means of shaking for 1 hour. After centrifugation, a portion of the dichloromethane (500 pL) was evaporated to dryness under nitrogen and the residue was redis- solved in HPLC mobile phase (100 pL) that consisted of methanol/water (45:55 vol/vol) at pH 3 with phos- phoric acid. The solution was filtered (0.45 pm), and a sample (25 pL) was injected on to a 15 cm x 4.6 mm Spherisorb Cs 3 pm column at a flow rate of 0.8 r&/mm. Ultraviolet absorbance was measured at 310 nm. The retention time of 6’,7’-dihydroxybergamottin was 12.5 minutes. The standard curve was linear over the tested range (0 to 20 yg/mL) with a coefficient of variation of 5.8% at 10 pg/rnL (n = 5). The absolute recovery of 6’,7’- dihydroxybergamottin was not directly measured. Instead, standards and unknowns were assayed together on the same day. The high linearity of the standard curve and the low coefficient of variation showed that recov- ery of 6’,7’-dihydroxybergamottin among samples was consistent and undoubtedly high.

The concentration of 6’,7’-dihydroxybergamottin in supernatant was 7.4 pg/mL and that in resuspended par- ticulate fractions was 2.4 pg/mL. This meant that the supernatant fraction contained 1.85 mg (250 mL x 7.4 pg/mL = 1.85 mg) and the particulate fraction 0.60 mg (250 n-L x 2.4 pg/mL = 0.60 mg).

Assay of felodipine and dehydrofelodipine. The plasma concentrations of felodipine and its primary metabolite dehydrofelodipine were quantified with a modification of an earlier method.16 In brief, plasma (200 pL) was extracted with toluene (200 pL) that contained the internal standard (H165/04; AB Haessle, Gothenburg, Sweden) by means of gentle oscillation of the mixture overnight. After centrifugation, a sample of the toluene extract (1 pL) was introduced by means of splitless injec- tion into a dual tapered deactivated glass insert (Hewlett- Packard Canada, Toronto, Ontario) to prevent chemical oxidation of felodipine in the injector port. Chromatog- raphy was performed with a Hewlett-Packard 5890 Series II gas chromatograph equipped with a 63Ni electron cap- ture detector and a 25 m x 0.32 mm internal diameter fused silica capillary column coated with stationary-phase

35

30

25

20

15

10

5

0 0 2 4 6 6 10 12

HOURS

*If** 30 TT

HOURS

Figure 1. Mean plasma felodipine (top panel) and dehy- drofelodipine (bottom panel) concentration-time profiles for persons (n = 12) given 10 mg felodipine with either 250 mL grapefruit juice (GFJ), particulate fraction (PF; from 250 mL grapefruit juice suspended in 250 mL water), supernatant fraction (SF; from 250 mL grapefruit juice) or 250 mL water. Bars represent SEM. Comparisons were made at each mea- surement time between the 3 treatments and water: **P < .Ol; ***p< .OOl.

methyl silicone 0.52 pm (HP- 1; Hewlett-Packard). After purge at 1 minute, the initial oven temperature of 90°C was increased 30°C per minute to 180” C, 5°C per minute to 260°C for 3 minutes, and 30°C per minute to a final temperature of 280°C for 5 minutes. The injector port temperature was maintained at 260°C and the detector at 300°C. The carrier gas was ultrapure helium (column inlet pressure of 100 kPa), and the make-up gas was ultrapure nitrogen (60 ml/mm). The retention time of felodipine was 20.1 minutes, the retention time of dehydrofelodip- ine was 14.5 minutes, and the retention time of internal standard was 21.7 minutes. The interday coefficient of variation for plasma felodipine at 5.0 nmol/L was 3.5%; for dehydrofelodipine it was 9.6% (n = 8). The limit of detection was 0.5 nmol/L.

252 Bailey et al. CLINICAL PHARMACOLOGY &THERAPEUTICS

SEMEMBER 1998

Table I. Pharmacokinetics of felodipine with water, grapefruit juice (GFJ), supematant (SF), and particulate (PF) fractions

AUC(O-12) (nmol . h/L.) c,, mow $,m @) tx @)

Subject No. Water GFJ SF PF Water GFJ SF PF Water GFJ SF PF Water GFJ SF PF

1 33 2 29 3 34 4 41 5 52 6 93 7 76 8 24 9 71 10 78 11 81 12 27 Mean value 53 SEM 7 P Value *

158 92 93 7 48 26 16 2.5 2.0 2.5 2.0 6.0 5.1 1.8 4.7 98 54 98 6 26 15 23 4.0 3.0 4.0 4.0 2.1 2.8 4.3 4.5

129 70 54 9 35 14 10 5.0 2.5 3.0 8.0 3.3 7.6 3.5 3.6 37 29 37 7

3: 2: 9 1.0 1.5 2.5 3.0 - - - -

143 79 171 9 35 4.0 3.0 3.0 3.0 9.9 9.2 2.5 7.3 181 136 216 14 48 36 43 2.5 3.0 3.0 2.5 6.3 5.5 6.0 4.7 136 101 202 17 38 24 31 2.5 2.5 2.5 2.0 - - - -

96 86 96 6 21 15 22 5.0 3.0 3.0 2.5 3.4 6.7 4.2 7.6 238 140 186 13 63 37 43 3.0 2.5 4.0 3.0 3.6 7.5 2.6 4.2 164 88 48 19 33 26 12 2.5 3.0 2.0 2.0 4.7 4.2 3.2 0.7 109 70 111 20 25 16 26 2.5 4.0 2.5 3.0 3.5 5.0 5.4 4.2 72 29 96 5 25 9 21 3.0 1.5 2.0 1.0 2.2 5.7 6.8 3.1

130 81 117 11 33 20 24 3.1 2.6 2.8 3.0 4.5 5.9 4.0 4.5 15 10 18 2 4 3 3 0.3 0.2 0.2 0.5 0.8 0.6 0.5 0.6

<.OOl c.01 <.Ol * <.OOl <.Ol c.01 * NS NS NS * NS NS NS t <.Ol t NS t NS t NS

AUC(O-12). Area under the curve from 0 to 12 hours; C,, peak concentration; t-3 time to C,; t q eliiation half-life; NS, not significant. *Comparisons between water and other treatments. tComparisons between supematant and particulate fractions.

Table II. Pharmacokinetics of dehydrofelodipine with water, grapefruit juice (GFJ), supematant (SF), and particulate (PF) fractions

AUC(O-12) (nmol ’ hn) c,, wwfi) &,a Vd

Subject No. Water GFJ SF PF Water GFJ SF PF Water GFJ SF PF

1 93 190 146 149 21 49 43 24 1.0 2.0 2.5 3.0 2 72 113 81 130 16 29 27 29 1.5 4.0 4.0 5.0 3 56 112 82 76 14 29 18 14 5.0 2.5 3.0 8.0 4 67 68 63 73 18 16 17 18 1.0 1.5 2.5 1.5 5 94 138 102 182 19 33 29 42 2.5 3.0 3.0 3.0 6 132 174 160 191 28 46 42 39 2.5 3.0 3.0 2.5 7 95 104 117 164 28 31 30 26 2.5 2.5 2.5 2.0 8 66 108 147 100 16 23 26 22 3.0 3.0 2.5 2.5 9 111 208 170 168 26 53 46 37 3.0 3.0 3.0 3.0 10 103 150 114 64 27 33 30 18 2.5 3.0 2.5 1.0 11 92 111 72 100 22 25 21 24 4.0 4.0 2.5 3.0 12 75 100 79 151 14 33 23 32 3.0 2.0 2.0 1.0 Mean value 88 131 111 129 21 33 29 27 2.6 2.8 2.8 3.0 SEM 6 12 11 13 2 3 3 3 0.3 0.2 0.1 0.6 P Value * <.OOl NS <.Ol * <.OOl <.Ol NS * NS NS NS

AUC(O-12), Area under the curve from 0 to 12 hours; C,, *Comparisons between water and other treatments.

peak concentration; t,-, time to C,& tv elimination half-life; NS, not significant.

tx fh)

Water GFJ SF PF

3.5 5.8 2.2 6.1 2.3 4.3 3.9 3.8 3.5 5.8 3.8 2.4 - - - - 6.8 15.4 2.9 5.2 6.9 5.5 5.8 5.4 - - - - 6.7 7.4 5.7 5.6 4.0 9.0 3.5 10.0 7.9 3.8 4.5 1.6 4.3 4.7 4.0 4.1 2.2 5.3 7.9 3.5 4.8 6.7 4.4 4.8 0.7 1.1 0.5 0.7 * NS NS NS

Data analysis. Plasma felodipine and dehydrofelodi- pine concentrations were analyzed with a noncompart- mental method. The terminal elimination rate constant (k& was determined by means of log-linear regression of the final data points (at least 3). The apparent elimination half- life of the log-linear phase (t%) was calculated as 0.693& Because of fluctuations in plasma felodipine and dehy-

drofelodipine concentrations during the terminal phase, tK could not be calculated for 2 subjects. The AUC was cal- culated from 0 to 12 hours by means of the linear trape- zoidal method. Plasma C,, and the time to reach C,, (&) were obtained directly from the experimental data.

Statistical comparisons among the 4 groups initially were performed with ANOVA for repeated measures.

CLINICAL I’ HARMACOLOGY & THERAPEUTICS VOLUME 64, NUMBER 3 Bailey et al. 2 5 3

For analyses with P < .05, subsequent comparisons were performed between groups with a paired t test cor- rected for multiple comparisons by means of the Bon- ferroni method. In the case of absolute felodipine phar- macokinetics, a total of 4 comparisons were performed; that is, 3 treatments compared with water and super- natant fraction compared with particulate fraction, with P c .013 (2-tailed test) considered to be significant. For all other comparisons, a total of 3 contrasts were per- formed; that is, 3 treatments compared with water, with P < .017 (2-tailed test) considered to be significant. Data are presented as mean values f SE.

RESULTS Naringin and 6 “, 7 ‘-dihydroxybergamottin dose. The

amounts of naringin and 6’,7’-dihydroxybergamottin were different in the 2 fractions. The supernatant frac- tion from 250 mL grapefruit juice contained 148 mg naringin and 1.85 mg 6’,7’-dihydroxybergamottin. The particulate fraction from the same amount of juice con- tained only 7 mg naringin and 0.60 mg 6’,7’-dihydroxy- bergamottin. Naringenin was not detected in either frac- tion, meaning that the supernatant fraction contained less than 0.25 mg and the particulate fraction contained less than 0.13 mg.

Effect of grapefruit juice. Whole grapefruit juice augmented plasma felodipine concentrations, AUC, and C,,,, for 11 of 12 volunteers compared with water (Fig- ure 1 and Table I). The AUC of felodipine increased to 274% i 34% (range, 90% to 479%; P < .OOl) and the C max to 346% f 49% (range, 100% to 686%; P < .OOl) of that of water. Plasma dehydrofelodipine concentra- tion, AUC, and C,,, also were greater (Table II), whereas the dehydrofelodipine/felodipine AUC ratio (1.9 f 0.2 versus 1.1 f 0.1; P < .OOl) decreased. Grape- fruit juice did not alter felodipine or dehydrofelodipine tmax or tt/,.

Effect of supernatantfraction. The supernatant frac- tion enhanced plasma felodipine concentrations and pharmacokinetics. The AUC of felodipine was 170% * 24% (range, 71% to 358%; P = .015) and the C,, was 202% f 25% (range, 80% to 371%; P < .Ol) compared with water. Although the C,,, of dehydrofelodipine increased, the AUC and dehydrofelodipine/felodipine AUC ratio (1.5 f 0.2) did not change. The t,r,ax and tX of felodipine and dehydrofelodipine were not different. Individually the supernatant fraction produced lower felodipine AUC and C,, than grapefruit juice among all 11 responders to grapefruit juice (Figure 2).

Eflect of particulate fraction. The particulate frac- tion augmented plasma felodipine concentrations, AUC, and C,,,. The AUC of felodipine increased to

PF

FELODIPINE AUC (nmol.h/L)

PF

FELODIPINE Cmax (nmol/L) Figure 2. Individual felodipine area under the plasma con- centration-time curve (AUC; top panel) and the felodipine peak concentration (Cm=; bottom panel) with grapefruit juice (GFJ) plotted against supernatant (SF) and particulate (PF) fractions. The diagona2 in each quadrant represents the line of unity. The one subject who did not have an increase in felodipine AUC with grapefruit juice compared with water is identified with the solid square.

243% * 31% (range, 90% to 400%; P c .OOl) and the C,, to 253% f 37% (range, 63% to 420%; P < .Ol) of those of water. The dehydrofelodipine AUC was greater, but the dehydrofelodipine/felodipine AUC ratio (1.2 f 0.1; P c .Ol) decreased. Felodipine and dehydro- felodipine & and tK were not altered. Felodipine AUC with the particulate fraction was greater than that with the supernatant fraction. Felodipine C,,, was not dif- ferent between these 2 treatments. There was consider-

254 Bailey et al. CLINICAL PHARMACOLOGY & THERAPEUTICS

SEPTEMBER 1998

able individual variability in felodipine AUC and C,, with the particulate fraction, which ranged from being greater than grapefruit juice to less than the supematant fraction among the 11 responders to grapefruit juice.

DISCUSSION Flavonoids have been known for some time to inhibit

in vitro oxidative drug metabolism and were the initial group of substances considered to be the active ingredi- ents in grapefruit juice.17 Naringin is the most prevalent flavonoid in grapefruit juice and is not present in orange juice,18 which does not increase plasma felodipine con- centration among humans.3 Naringin inhibits felodipine and nifedipine oxidation by human liver microsomes.l* However, it has low potency compared with its aglycone naringenin. Although naringenin is not normally present in grapefruit juice, it can be formed in vivo, probably in the small intestine, after consumption of the juice.‘9 Because naringenin was not detected in plasma after administration of juice and because only a small frac- tion of the naringin in grapefruit juice was recovered in urine as naringenin conjugates, naringenin may have low oral bioavailability, consistent with the observed selective inhibition of intestinal CYP3A4 by grapefruit juice.s-‘0 Nevertheless, commercially available pure naringin administered in an aqueous solution or a gelatin capsule (dissolution and bioavailability characteristics not determined) did not augment plasma concentrations of felodipine or nisoldipine, respectively, compared with grapefruit juice that contained the same amount of naringin administered to healthy volunteers.14,l5 Sug- gested possibilities for the lack of in vivo effect of pure naringin in humans include the following: (1) a change in optical isomer composition of naringin with accom- panying loss of activity during purification from grape- fruit juice, (2) insufficient naringenin formation, (3) the requirement for other components in the juice to facili- tate the effect of naringin-naringenin, or (4) the prospect that naringin is not an important constituent in grape- fruit juice-drug interactions.’

Furanocoumarins inactivate cytochromes P450.20 6’,7’- Dihydroxybergamottin belongs to this class of com- pounds.21 It also is present in grapefruit juice, but not orange juice, and has been shown to inhibit CYP3A-medi- ated testosterone oxidation by rat liver microsomes.ll 6’,7’-Dihydroxybergamottin was also shown to cause a dose-dependent decrease in CYP3A4 activity and immunoreactive CYP3A4 protein concentration in a Caco-2 cell culture model of human intestinal epithe- lium.22 The concentration in grapefruit juice exceeded the 50% inhibitory concentration for loss of CYP3A4 activ- ity. 6’,7’-Dihydroxybergamottin acted initially by means

of competitive inhibition, followed by mechanism-based inactivation of recombinant CYP3A4. Because of these in vitro findings, 6’,7’-dihydroxybergamottin was pro- posed as the main active ingredient in grapefruit juice.**

The hypothesis tested in this study was that naringin or 6’,7’-dihydroxybergamottin or both in grapefruit juice are primarily responsible for the interaction with felodi- pine in humans. Because pure 6’,7’-dihydroxybergamot- tin was not available for human administration and because other substances in the juice may contribute indi- rectly to their activity in the interaction with felodipine, the approach of this study was to separate grapefruit juice by methods suitable for human testing into 2 partially purified fractions that contained different amounts of naringin and 6’,7’-dihydroxybergamottin. It was expected that the water-soluble components would be found in the supematant fraction and the lipid-soluble substances would be bound to the particulate fraction. Because the supematant fraction had nearly all of the naringin and 300% higher 6’,7’-dihydroxybergamottin content com- pared with the particulate fraction, it was postulated that the activity of the supematant fraction would range from being greater than that of the particulate fraction and equivalent to that of the grapefruit juice. However, this was not the case. Although the supematant fraction was active, it produced a felodipine AUC that was 30% less than and a felodipine C,,, that was not different from values obtained with the particulate fraction. Further- more, the supematant fraction did not alter the dehydro- felodipine AUC or the dehydrofelodipine/felodipine AUC ratio, as was observed with grapefruit juice and the par- ticulate fraction. Thus the hypothesis was not supported, and naringin and 6’,7’-dihydroxybergamottin are not the main active substances in grapefruit juice.

If there is only one main active ingredient in grape- fruit juice that would be mainly distributed to the par- ticulate portion of grapefruit juice, our findings suggest that neither naringin nor 6’,7’-dihydroxybergamottin has any substantial activity in humans. Under the sup- position that there are 2 or more major active ingredi- ents, it can be inferred that naringin or 6’,7’-dihydroxy- bergamottin may possess some in vivo activity. How- ever, it still must be concluded there is at least one other component in grapefruit juice that makes a greater con- tribution to the interaction.

What is the main active component? Because the aglycone naringenin, which is normally present in only trace amounts in grapefruit juice, was not measured in either supematant or particulate fractions at concentra- tions known to inhibit in vitro CYP3A4-mediated drug metabolism,t*,13 it cannot be this substance. Other flavonoids, such as quercetin and kaempferol, which

CLINICAL P HARMACOLOGY & THERAPEUTICS VOLUME 64, NUMBER 3 Bailey et al. 2 5 5

have been shown to be active inhibitors in vitro,12J3 are known to be present in grapefruit juice in only trace amounts’s and thus are not likely involved in the inter- action. Preliminary data from our laboratory indicated that bergamottin, a more lipophilic analog of 6’,7’-dihy- droxybergamottin, is found in high concentrations in grapefruit juice (10 yg/mL) and is measurable only in the particulate fraction. Because bergamottin has been shown to be a mechanism-based in vitro inhibitor of CYP3A4, it may be a main active ingredient in grape- fruit juice.22 Other furanocoumarin dimers isolated from grapefruit juice also have been reported to inhibit human CYP3A in vitro.23,24 However, a final decision on their importance in the interaction must await results from testing with human subjects.

The reason for the inconsistent effect of the particu- late fraction among individuals seems unlikely to be a variable amount of active ingredient administered. Because the effect of whole grapefruit juice has been shown to be reasonably reproducible within a month of rechallenge, individual responsiveness normally does not appear to change greatly from that during the period of testing. Although the particulate fraction was prepared on 4 occasions before each of the study days, it seemed unlikely that day-to-day variability in the procedure was a cause, because the same brand and lot number of grape- fruit juice was processed by means of a standardized pro- tocol by the same person over the short (l-month) period needed to conduct the clinical aspects of the study. Because the active ingredient was bound to the particu- late material, it seems more plausible that erratic bioavailability of the active ingredient from the particu- late fraction might explain the observed inconsistency of effect. Delayed bioavailability of the active component may account for the fact that felodipine C,, was not different between the 2 fractions despite the greater felodipine AUC with the particulate fraction.

The results of this study show that 2 previously indi- cated substances, naringin and 6’,7’-dihydroxyberg- amottin, are not likely to be the main intestinal CYP3A4 inhibitory ingredients, although they may contribute to the interaction. The findings also support the necessity for in vivo human testing in attempts to identify the active substances in grapefruit juice.

We thank Dr. Anthonio Montanari of the Florida Department of Citrus for his donation of 6’,7’-dihydroxybergamottin.

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