Effects of grapefruit juice on the pharmacokinetics of pitavastatin and atorvastatin

Abstract

Aims

To compare the effects of grapefruit juice (GFJ) on the pharmacokinetics of pitavastatin and atorvastatin.

Methods

In a randomized, four-phase crossover study, eight healthy subjects consumed either GFJ or water t.i.d. for 4 days in each trial. On each final day, a single dose of 4 mg pitavastatin or 20 mg atorvastatin was administered.

Results

GFJ increased the mean AUC_0–24 of atorvastatin acid by 83% (95% CI 23–144%) and that of pitavastatin acid by 13% (−3 to 29%).

Conclusions

Pitavastatin, unlike atorvastatin, appears to be scarcely affected by the CYP3A4-mediated metabolism.

Introduction

Most of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) except pravastatin are metabolized by the cytochrome P450 (CYP) enzymes [1]. Interactions involving CYP are therefore possible. For example, itraconazole has been shown to increase the area under the plasma concentration-time curve (AUC) of atorvastatin acid and lactone by 3-fold and 4-fold, respectively [1].

Pitavastatin is a novel synthetic statin, which has a highly potent cholesterol-lowering action similar to that of atorvastatin [2]. The long-term lipid-modifying effects and safety profiles of pitavastatin have already been confirmed in hypercholesterolaemic patients [2]. Pitavastatin acid, which is the major active form, and the inactive lactone form are metabolized to some extent by CYP2C9 and CYP3A4, respectively, in human hepatic microsomes, but the clearance of pitavastatin is very low and only one-thirteenth of atorvastatin clearance [3]. The bioavailabilities of pitavastatin and atorvastatin are 60 and 12%, respectively [2]. Moreover, after the repeated administration of pitavastatin in healthy subjects, its metabolites other than pitavastatin lactone were scarcely detected in plasma [2]. The pharmacokinetics of pitavastatin therefore appear to be hardly affected by the CYP-system modulators, but no direct evidence has been reported clinically. To address this issue, we investigated the effects of a repeated intake of grapefruit juice (GFJ), a potent CYP3A4 inhibitor [1, 4], on the pharmacokinetics of pitavastatin and atorvasatin in healthy subjects.

Methods

This study was performed in an open, randomized, four-phase crossover design with the intervals of 2 weeks. The protocol was approved by the Ethics Committee of Jichi Medical School (Tochigi, Japan).

Eight healthy Japanese men (age, 23–34 years; weight, 60–72 kg) participated after written informed consent. The subjects were instructed not to take any medications, herbal supplements and tea, and any foods or drinks containing component(s) of grapefruits, pomelos, sweet or sour oranges, throughout the study period. Caffeine-containing beverages and smoking were avoided from one night before each study day until after the final blood sampling on the next morning. The subjects consumed 250 mL of single-strength GFJ (Tropicana, Kirin Beverage Co., Tokyo, Japan) or water t.i.d. for 3 days just before each trial according to a randomized schedule. On each study day, they took a single oral dose of 4 mg pitavastatin (two 2-mg Livalo tablets, Kowa Co., Nagoya, Japan) or 20 mg atorvastatin (two 10-mg Lipitor tablets, Yamanouchi Pharmaceutical Co., Tokyo, Japan) with 250 mL of GFJ or water at 08.00 h. The subjects also consumed 250 mL of GFJ or water at 12.00 h and 20.00 h. They had fasted overnight, had a standardized meal at 12.00 h, and were allowed to take supper 12 h after dosing.

On each study day, venous blood was taken immediately before and at 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h after the administration of pitavastatin or atorvastatin. To prevent photolysis, the plasma samples for the measurement of pitavastatin were harvested into the light-protected tubes, immediately flash-frozen in the dry ice/methanol bath, and then stored at −80 °C until analysis. The samples for the measurement of atorvastatin were stored at −20 °C until use.

Plasma concentrations of pitavastatin acid and lactone, atorvastatin acid and lactone, and 2-hydroxy atorvastatin acid were quantified by the liquid chromatography-mass spectrometry methods. Briefly, 150 µL of internal standard solution containing racemic iprolact (for pitavastatin acid), 2-hydroxy atorvastatin-D5 (for pitavastatin lactone and 2-hydroxy atorvastatin acid), atorvastatin-D5 (for atorvastatin acid), or atorvastatin lactone-D5 (for atorvastatin lactone) was added to 50 µL of plasma. After centrifugation, aliquots were injected onto the HPLC column. Zorbax Extend-C18 columns (50 × 2.1 mm, 3.5 µm) (Agilent Technologies, Palo Alto, CA, USA) were used for analytical separation. The mobile phase consisted of acetonitrile and either 0.1% ammonium hydroxide in water (for pitavastatin compounds) or 10 m m ammonium acetate with 0.4% ammonia hydroxide in water (for atorvastatin compounds). The effluent was delivered to a Sciex 3000 quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Canada). The ion transitions monitored were m/z 422.3/290.0 for pitavastatin acid, m/z 404.0/290.2 for pitavastatin lactone, m/z 559.2/440.6 for atorvastatin acid, m/z 541.2/448.5 for atorvastatin lactone, and m/z 575.3/440.6 for 2-hydroxy atorvastatin acid. The lower quantification limits for pitavastatin and atorvastatin compounds were 0.2 and 0.4 ng mL⁻¹, respectively. The interassay coefficients of variation and accuracy were all ≤ 14.0% and 91.8–110.0%.

The pharmacokinetics were characterized by maximum concentration in plasma (C_max), time to maximum plasma concentration (t_max), elimination half-life (t_½), and AUC between time zero and 24 h after dosing (AUC_0–24). The elimination t_½ was calculated as ln2/k_e. The AUC_0–24 was calculated by the trapezoidal rule.

Furanocoumarins were extracted with ethyl acetate and isolated using the HPLC systems as described previously [4]. We measured in triplicate the following four compounds: bergamottin, 6’,7’-dihydroxybergamottin (DHB), 4-[[6-hydroxy-7[[1-[(1-hydroxy-1-methyl) ethyl]-4-methyl-6-(7-oxo-7H-furo[3,2-g][1] benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl-2-octenyl]oxy]-7H-furo[3,2-g][1]benzopyran-7-one (GF-I-1), and 4-[[6-hydroxy-7[[4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo[3,2-g][1]benzopyran-4-yl)-4-hexenyl] oxy]-3,7-dimethyl-2-octenyl]oxy]-7H-furo[3,2-g][1] benzopyran-7-one (GF-I-4).

Data were analysed by the Wilcoxon signed rank test. Values are presented as the means and their 95% confidence intervals (CI), and a P-value of less than 0.05 was considered significant.

Results

Because the amounts of furanocoumarins might depend on the lots of GFJ, their concentrations in each lot used in this study (n = 3) were measured. The mean values (ranges) of bergamottin, DHB, GF-I-1, and GF-I-4 concentrations were 21.0 (16.2–24.4), 10.4 (5.2–13.8), 0.50 (0.36–0.59), and 0.59 (0.45–0.70) µM, respectively. These concentrations of GF-I-1 and GF-I-4 can completely inhibit the CYP3A4-mediated oxidations in vitro [4]. The differences of furanocoumarin concentrations among the lots were small.

To investigate the effect of GFJ on the pharmacokinetics of atorvastatin, we measured the concentrations of its active acid form, inactive lactone form, and 2-hydroxy atorvastatin acid, one of the major active metabolites, after a single dosing of 20 mg atorvastatin. The formation of atorvastatin metabolites, including 2-hydroxy atorvastatin, is shown to be catalysed by CYP3A4 [5]. GFJ significantly increased the mean AUC_0–24 of atorvastatin acid by 83% (Table 1), from 21.3 to 39.0 ng mL⁻¹ h (95% CI for difference, 4.8–30.7), as reported previously [5, 6]. The AUC_0–24 were increased in seven of the eight subjects, and the increase ranged from 1.5-fold to 7.1-fold. The increase of C_max did not reach to a statistical significance (P = 0.09), probably due to small sample size. The AUC_0–24 and C_max of atorvastatin lactone and of 2-hydroxy atorvastatin acid were not significantly altered, whereas the mean t_max of 2-hydroxy atorvastatin acid was prolonged from 3.2 to 9.4 h by GFJ.

Table 1.

The pharmacokinetic parameters of atorvastatin acid, atorvastatin lactone, and 2-hydroxy atorvastatin acid after a single dosing of 20 mg atorvastatin with water or grapefruit juice (mean and 95% confidence interval)

Parameter	Water	GFJ	Difference between water and GFJ	P-value
Atorvastatin acid
Cmax (ng mL−1)	4.2 (2.2, 6.3)	7.2 (2.6, 11.7)	2.9 (−0.2, 6.1)	0.09
tmax (h)	1.4 (0.4, 2.4)	1.4 (0.8, 2.0)	−0.1 (−1.0, 0.9)	0.92
AUC0–24 (ng mL−1 h)	21.3 (10.6, 32.0)	39.0 (24.4, 53.7)	17.7 (4.8, 30.7)	<0.05
Atorvastatin lactone
Cmax (ng mL−1)	2.1 (0.8, 3.5)	3.5 (1.7, 5.2)	1.3 (−0.3, 3.0)	0.12
tmax (h)	6.1 (2.4, 9.9)	3.3 (1.2, 5.3)	−2.9 (−6.8, 1.0)	0.13
AUC0–24 (ng mL−1 h)	25.6 (3.9, 47.2)	41.1 (17.8, 64.5)	15.6 (−2.3, 33.4)	0.07
2-hydroxy atorvastatin acid
Cmax (ng mL−1)	3.6 (1.1, 6.1)	2.0 (1.1, 2.9)	−1.6 (−4.1, 0.8	0.12
tmax (h)	3.2 (0.0, 6.4)	9.4 (6.2, 12.5)	6.2 ( 2.5, 9.9)	<0.05
AUC0–24 (ng mL−1 h)	28.6 (18.6, 38.7)	29.5 (18.3, 40.5)	0.8 (−7.4, 9.0)	0.78

GFJ, Grapefruit juice; C_max, maximum concentration in plasma; t_max, time to maximum plasma concentration; AUC_0–24, area under the plasma concentration – time curve between time zero and 24 h after dosing.

GFJ increased the mean AUC_0–24 of pitavastatin acid only by 13%, from 194.2 to 220.1 ng mL⁻¹ h (95% CI for difference, −5.0 to 56.9; Table 2). Moreover, the C_max remained unchanged. The t_½ and AUC_0–24 of pitavastatin lactone were significantly increased.

Table 2.

The pharmacokinetic parameters of pitavastatin acid and pitavastatin lactone after a single dosing of 4 mg pitavastatin with water or grapefruit juice (mean and 95% confidence interval)

Parameter	Water	GFJ	Difference between water and GFJ	P-value
Pitavastatin acid
Cmax (ng mL−1)	81.4 (22.2, 140.5)	86.0 (42.7, 129.2)	4.6 (−16.9, 26.0)	0.48
tmax (h)	0.9 (0.4, 1.3)	1.0 (0.6, 1.4)	0.1 (−0.4, 0.6)	0.58
t1/2 (h)	9.0 (7.7, 10.4)	10.1 (7.6, 12.6)	1.1 (−0.8, 2.9)	0.12
AUC0–24 (ng mL−1 h)	194.2 (104.3, 284.0)	220.1 (149.0, 291.3)	26.0 (−5.0, 56.9)	<0.05
Pitavastatin lactone
Cmax (ng mL−1)	49.5 (28.9, 70.1)	64.4 (30.6, 98.2)	14.9 (−3.3, 33.1)	0.07
tmax (h)	1.6 (1.2, 2.1)	1.6 (0.9, 2.2)	−0.1 (−0.9, 0.7)	0.72
t1/2 (h)	−6.1 (4.5, 7.7)	−9.8 (7.2, 12.3)	−3.5 (0.2, 6.8)	<0.05
AUC0–24 (ng mL−1 h)	269.3 (169.6, 369.1)	351.0 (196.6, 505.5)	81.7 (−4.2, 167.7)	<0.05

GFJ, Grapefruit juice; C_max, maximum concentration in plasma; t_max, time to maximum plasma concentration; t_1/2, elimination half-life; AUC_0–24, area under the plasma concentration – time curve between time zero and 24 h after dosing.

Discussion

GFJ had only a minimal effect on pitavastatin pharmacokinetics, while the expected increased concentrations of atorvastatin were confirmed.

Interestingly, the pharmacokinetic parameters of both atorvastatin and pitavastatin varied widely in the subjects even in the trial with water. For example, the AUC_0–24 of atorvastatin acid ranged from 4.5 to 43.3 ng mL⁻¹ h, and the C_max of pitavastatin acid varied from 25.6 to 246.0 ng mL⁻¹ in the trial with water. The AUC_0–24 of atorvastatin acid in the subject who had the highest value in the trial with water did not increase any more in the trial with GFJ. It can thus be speculated that there are great differences in the inherent ability to metabolize the statins among the subjects. Recent studies have revealed that polymorphisms of both CYP enzymes and transporters may influence the cholesterol-lowering effect of the statins in hypercholesterolaemic patients [7], and both pathways must be taken into account to avoid potential drug–statin interactions.

In conclusion, repeated intake of GFJ affected the pharmacokinetics of atorvastatin, but had minimal effect on pitavastatin acid. Pitavastatin may be clinically preferable for preventing pharmacokinetic drug-interactions.