Effects of repeated ingestion of grapefruit juice on the single and multiple oral-dose pharmacokinetics and pharmacodynamics of alprazolam

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1 Psychopharmacology (2000) 150: Digital Object Identifier (DOI) /s ORIGINAL INVESTIGATION Norio Yasui Tsuyoshi Kondo Hanako Furukori Sunao Kaneko Tadashi Ohkubo Tsukasa Uno Takako Osanai Kazunobu Sugawara Koichi Otani Effects of repeated ingestion of grapefruit juice on the single and multiple oral-dose pharmacokinetics and pharmacodynamics of alprazolam Received: 27 October 1999 / Accepted: 8 March 2000 / Published online: 14 April 2000 Springer-Verlag 2000 Abstract The effects of repeated ingestion of grapefruit juice, an inhibitor of cytochrome P 450 3A4 (CYP3A4), on the pharmacokinetics and pharmacodynamics of both single and multiple oral doses of alprazolam, a substrate of CYP3A4, were examined. In study 1, eight healthy volunteers ingesting 600 ml/day water or grapefruit juice for 10 days took a single oral 0.8-mg dose of alprazolam on the eighth day. Plasma drug concentrations were monitored up to 48 h after alprazolam dosing together with evaluation of psychomotor function. Grapefruit juice altered neither the plasma concentrations of alprazolam at any time points, any pharmacokinetic parameters, nor the majority of psychomotor function parameters in subjects. In study 2, 11 patients with anxiety disorders receiving alprazolam ( mg/day) ingested grapefruit juice (600 ml/day) for 7 days. Blood samples were collected before and during grapefruit juice ingestion and 1 week after its discontinuation together with an assessment of clinical status. Grapefruit juice altered neither the steady-state plasma concentration of alprazolam nor the clinical status in patients. The present study shows that grapefruit juice is unlikely to affect pharmacokinetics or pharmacodynamics of alprazolam due to its high bioavailability. Key words Alprazolam Grapefruit juice Cytochrome P 450 3a4 Bioavailability N. Yasui T. Kondo ( ) H. Furukori S. Kaneko Department of Neuropsychiatry, Hirosaki University School of Medicine, Hirosaki , Japan Tel.: , Fax: T. Ohkubo T. Uno T. Osanai K. Sugawara Department of Pharmacy, Hirosaki University Hospital, Hirosaki, Japan K. Otani Department of Neuropsychiatry, Yamagata University School of Medicine, Yamagata, Japan Introduction The triazolobenzodiazepine alprazolam is extensively used in the treatment of anxiety and panic disorders (Greenblatt and Wright 1993). A significant concentration effect relationship for alprazolam has been suggested in the treatment of panic disorder: optimal reduction of anxiety occurs in the plasma concentration range of ng/ml, while the central nervous system depressant side effects increase progressively at higher plasma concentrations (Greenblatt et al. 1993). Thus, information on kinetic disposition of alprazolam affected by environmental factors is of clinical importance. Alprazolam is metabolized primarily by hepatic microsomal oxidation, yielding 4- and α-hydroxyalprazolam as its principal metabolites (Greenblatt and Wright 1993). An in vitro study (von Moltke et al. 1994) with human liver microsomes has shown that ketoconazole, an inhibitor of cytochrome P 450 (CYP) 3A4 (Watkins 1994; Wilkinson 1996), inhibits the 4- and α-hydroxylation of alprazolam, suggesting that alprazolam is metabolized by CYP3A4. Our recent studies have suggested that erythromycin (Yasui et al. 1996), itraconazole (Yasui et al. 1998), and carbamazepine (Furukori et al. 1998), inhibitors and an inducer of CYP3A4 (Watkins 1994; Wilkinson 1996), respectively, alter the metabolism of alprazolam in healthy volunteers, providing in vivo evidence of the involvement of CYP3A4. Grapefruit juice is known to be an inhibitor of CYP3A4 (Wilkinson 1996) based on several interaction studies between grapefruit juice and substrates for CYP3A4, such as cyclosporin, terfenadine, and several calcium antagonists (Ameer and Weintraub 1997). Moreover, several in vivo studies have shown that grapefruit juice significantly changes pharmacokinetic dispositions of midazolam (Kupferschmidt et al. 1995; Vanakoski et al. 1996) and triazolam (Hukkinen et al. 1995), both of which are also substrates for CYP3A4 (Watkins 1994; Wilkinson 1996). However, to our knowledge, there are no available data indicating any interactions between alprazolam and grapefruit juice.

2 186 To clarify the drug interaction between alprazolam and grapefruit juice, we studied the effects of repeated ingestion of grapefruit juice on single oral-dose pharmacokinetics and pharmacodynamics of alprazolam in healthy volunteers using randomized two-way crossover study design. We also examined the effects of co-administration of grapefruit juice on the steady-state plasma concentration and clinical effects of alprazolam in patients with anxiety disorders. Methods Two studies were separately conducted to examine the effects of grapefruit juice on single and multiple oral doses of alprazolam. The protocols of both studies were approved by the ethics committee of Hirosaki University Hospital, and all subjects in both studies gave their written informed consent before the studies. Study 1 Eight healthy male volunteers participated in study 1. Their normal health statuses were confirmed by a medical history, a physical examination, and hematology and blood chemistry tests. The mean±sd of age was 31.1±6.3 years, and that of body weight 61.5±4.7 kg. Six subjects were smokers ( 10 cigarettes/day), and the others were non-smokers. The study was conducted in a randomized two-way crossover study design, with an interval of 4 weeks. Regular-strength grapefruit juice (200 ml) (Dole, Snow Brand Milk Products Co., Ltd., Tokyo, Japan) or water (200 ml) was given three times per day (0900 hours, 1700 hours, and 2300 hours) for 10 days. Four volunteers each as a group were randomly allocated to either of the different sequences: water grapefruit juice or grapefruit juice water. After an overnight fast, at 0900 hours on the eighth day, a single oral 0.8-mg dose of alprazolam (Solanax, Pharmacia and Upjohn, Tokyo, Japan) as a tablet formulation was given with 200 ml grapefruit juice or water. No food was allowed until 3 h after alprazolam dosing. The blood samples (10 ml each) were collected into heparinized tubes from an antecubital vein before and 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 36, and 48 h after alprazolam dosing. At the same time points, psychomotor function status was evaluated using the digit symbol substitution test (DSST) adapted from the Wechsler Adult Intelligence Scale, the item thinking speed of visual analog scale (VAS) of mood and subjective states (Greenblatt et al. 1977), and the item sleepiness of the Udvalg for kliniske undersøgelser (UKU) side-effect rating scale (Lingjærde et al. 1987). Study 2 Eleven physically healthy patients (one male, ten females), who fulfilled the criteria for anxiety disorders (panic disorder without agoraphobia, two cases; generalized anxiety disorder, five cases; social phobia, one case; and anxiety disorder not otherwise specified, three cases) according to the diagnostic and statistical manual (DSM)-IV of the American Psychiatric Association (1994), participated in study 2. Ten cases were in-patients and the remaining one was an out-patient. Their normal health status was also confirmed by a medical history, a physical examination, and hematology and blood chemistry tests. The mean±sd of age was 47.2±10.0 years and that of body weight 50.3±9.9 kg. Five subjects were smokers ( 10 cigarettes/day), and the others were non-smokers. The subjects had been treated with alprazolam (Solanax, Pharmacia and Upjohn, Tokyo, Japan) 0.4 mg in four patients, 0.8 mg in four patients, and 1.2 mg in three patients twice a day (0800 hours and 2000 hours) for 2 10 weeks. The elimination half-life of alprazolam was reported as about h (Greenblatt and Wright 1993). Therefore, it was considered that steady-state plasma concentration of alprazolam was achieved before the study. No other drugs were co-administered except flunitrazepam (2 6 mg/day) in seven patients and sennoside (12 36 mg/day) in five patients. The doses of these drugs were fixed throughout the study. For each patient, regular-strength grapefruit juice (200 ml) (Dole) was given three times a day (0800 hours, 1300 hours, and 2000 hours) for 7 days. Blood samples (20 ml) were collected into heparinized tubes from an antecubital vein at 0800 hours, 12 h after the last night dosing, just before and during co-administration of grapefruit juice and 1 week after its discontinuation. At the same time points, clinical status was assessed using clinical global impressions (CGI) (Guy 1976), global assessment of functioning (GAF) (American Psychiatric Association 1994) scale, and UKU side-effect rating scale. Assay and statistics Plasma alprazolam concentrations were measured in duplicate using the high-performance liquid chromatography method developed in our laboratory (Nagasaki et al. 1997). The lowest limit of detection was 0.5 ng/ml, and the coefficient of variation (both intraassay and interassay) was less than 7.8%. The elimination rate constant (k) of alprazolam was estimated from the linear regression analysis of the terminal log linear concentration time data, and the elimination half-life (t 1/2 ) was calculated from 0.693/k. The area under the plasma concentration time curve from 0 h to 48 h [AUC(0 48)] was calculated using the trapezoidal rule. The area under the plasma concentration time curve from 0 h to infinity [AUC(0 )] or total AUC was calculated from AUC(0-48)+C48/k, in which C48 is the plasma concentration of alprazolam at 48 h after the dosing. The apparent oral clearance (CL oral ) was calculated from dose/total AUC. This clearance term is influenced by absolute oral bioavailability, which was not determined in this study. The peak plasma concentration (C max ) and the time to C max (t max ) were determined graphically. Statistical analyses were performed using a paired t-test and Wilcoxon singed-rank test and Friedman rank test followed by Tukey test using SPSS J for Windows (SPSS Japan Inc., Tokyo, Japan). A P value of <0.05 was regarded as statistically significant. Results Study 1 There was no difference in plasma alprazolam concentrations at any time points (Fig. 1) or such pharmacokinetic parameters as C max (water vs grapefruit juice: 12.4±2.3 ng/ml vs 13.3±3.1 ng/ml), the AUC(0 ) (243±48 ng h/ml vs 272±62 ng h/ml), and the elimination t 1/2 (15.6±4.6 h vs 18.9±3.6 h) between the treatment with water and grapefruit juice in all subjects (Table 1 ). In six smokers, an increased AUC (288± 62 ng h/ml, P<0.05) and prolonged elimination t 1/2 (19.5±2.9 h, P<0.05) were found during the grapefruit juice phase compared with the values during the water phase (AUC 245±56 ng h/ml and elimination t 1/2 14.1±4.1 h), while such a tendency was not observed in two non-smokers (Table 1 ). The number of cigarettes per day was not associated with the impact of these pharmacokinetic changes. Psychomotor function after alprazolam dosing was almost unchanged during grapefruit juice treatment in smokers, non-smokers, and all subjects except for a delayed thinking speed assessed using the VAS from 1 h to 2 h after alprazolam dosing during

3 187 Table 1 Single-dose pharmacokinetic parameters of alprazolam (0.8 mg) after the treatment with 600 ml/day of water or grapefruit juice in eight subjects. GFJ grapefruit juice, C max peak plasma concentration, t max time to C max, AUC (0 ) area under the plasma concentration time curve from 0 h to infinity, t 1/2 half-life Case no. C max (ng/ml) t max (h) AUC (0 ) (ng h/ml) Elimination t 1/2 (h) Water GFJ Water GFJ Water GFJ Water GFJ Smokers Mean±SD 13.0± ± ± ± ±56 288±62 * 14.1± ±2.9 * Non-smokers All subjects 12.4± ± ± ± ±48 272± ± ±3.6 (mean±sd) *P<0.05, significantly different from the values after the treatment with water (Wilcoxon signed-rank test) (17.8±9.8 ng/ml) or 1 week after its discontinuation (14.9±11.5 ng/ml), although no significant difference was found in the mean scores of CGI, GAF, or UKU throughout the study (Table 2 ). Discussion Fig. 1 Mean plasma alprazolam concentration time data after a single oral dose of 0.8 mg alprazolam during repeated ingestion of water or grapefruit juice in eight subjects. Open and solid circles indicate the mean data with water and grapefruit juice, respectively. Bracketed bars indicate the values of standard deviations grapefruit juice treatment compared with those during water co-administration in all subjects. Study 2 There was no significant difference in the plasma alprazolam concentrations (before and during co-administration of grapefruit juice and one week after its discontinuation: 14.6±9.9, 16.9±11.8, and 13.4±10.4 ng/ml, respectively) and the mean scores of CGI (4.4±1.4, 4.0±1.4, and 4.4±1.7), GAF (49.1±19.2, 50.9±20.7, and 50.0±21.9) or UKU (1.6±1.0, 1.5±1.3, and 1.5±1.0) in all patients (Table 2 ). However, in five smokers, the mean plasma concentration during co-administration of grapefruit juice (22.7±13.6 ng/ml) was significantly (P<0.05) higher than that before co-administration of grapefruit juice Grapefruit contains several components, e.g., flavanone glycosides, naringin and narirutin, and flavone glycoside quercetin (Ameer and Weintraub 1997). Naringin has been shown to inhibit the oxidative metabolism of several drugs in human liver microsomes (Guengerich and Kim 1990). Also, stronger inhibition has been demonstrated with the aglycon of naringin, naringenin, which is hydrolyzed in the body from naringin (Guengerich and Kim 1990). Meanwhile, naringin and naringenin have been suggested not to be predominant factors for inhibitory effects of grapefruit on CYP3A activity (Edwards and Bernier 1996). Recently, some components including furocoumarins have been clarified as causative agents of grapefruit juice-mediated drug interaction (Fukuda et al. 1997; Schmiedlin-Ren et al. 1997). Bergamottin, in particular, has been reported to be the primary furanocoumarin extracted from grapefruit juice which inhibits not only the activity of CYP3A4 but also those of CYP1A2, 2A6, 2C19, 2C19, 2D6, and 2E1 (He et al. 1998). Grapefruit juice significantly increases the bioavailability and the AUC of midazolam (Kupferschmidt et al. 1995) and also increases the C max and the AUC of triazolam (Hukkinen et al. 1995). However, no changes have been found in the elimination half-life of midazolam (Kupferschmidt et al. 1995) or that of triazolam (Hukkinen et al. 1995). Relatively low bioavailability of midazolam (33%; Garzone and Kroboth 1989; Kupferschmidt et al. 1995) and triazolam (44%; Garzone and Kroboth 1989) suggests that these drugs undergo exten-

4 188 Table 2 Steady-state plasma concentrations of alprazolam and total scores of each clinical assessment before and during co-administration of grapefruit juice and 1 week after its discontinuation in patients with anxiety disorder. CGI clinical global impression, GAF global assessment of functioning, UKU UKU side-effect rating scale Case no. Co-administered drugs Plasma concentrations (ng/ml) CGI score GAF score UKU score Flunitra- Sennoside Before During After Before During After Before During After Before During After zepam Smokers 1 4 mg/day mg/day mg/day 24 mg/day mg/day mg/day Mean±SD 17.8± ±13.6 * 14.9± ± ± ± ± ± ± ± ± ±0.8 Non-smokers 6 8 mg/day mg/day mg/day mg/day mg/day 36 mg/day Mean±SD 12.0± ± ± ± ± ± ± ± ± ± ± ±1.2 All patients 14.6± ± ± ± ± ± ± ± ± ± ± ±1.0 (mean±sd) *P<0.05 compared with before co-administration of grapefruit juice or 1 week after its discontinuation (Friedman rank test followed by Tukey test)

5 189 sive first-pass metabolism. Furthermore, it is known that CYP3A4 is present not only in the liver (Shimada et al. 1994) but also in the small intestine (Kolars et al. 1994; Lown et al. 1994). A recent study (Lown et al. 1997a) whose design was similar to ours, i.e., about 600 ml/day ingestion of grapefruit juice for 7 days, has shown that grapefruit juice significantly decreases the CYP3A4 protein expression in the small intestine, but does not influence the CYP3A4 activity in the liver. These facts suggest that midazolam and triazolam undergo extensive intestinal metabolism catalyzed by CYP3A4, which plays a major role in the first-pass metabolism of both drugs. Therefore, it should be also determined whether grapefruit juice inhibits the CYP3A4-catalyzed metabolism of alprazolam. In the present two studies, however, the pharmacokinetics of alprazolam in all subjects were unchanged even after repeated ingestion of grapefruit juice, although CYP3A4 is involved to a major extent in the metabolism of alprazolam (von Moltke et al. 1994; Yasui et al. 1996; Furukori et al. 1998). Grapefruit juice might not increase the bioavailability of alprazolam due to its high bioavailability (92%; Greenblatt and Wright 1993). In addition, the high bioavailability of alprazolam, in contrast to those of midazolam and triazolam, suggests that alprazolam is unlikely to undergo extensive intestinal first-pass metabolism. Therefore, few inhibitory effects of grapefruit juice on the pharmacokinetic dispositions of alprazolam in all subjects may be ascribable to less of a contribution of presystemic intestinal metabolism to the CYP3A4-catalyzed metabolism of this drug. Bailey et al. (1996) have shown that erythromycin, but not grapefruit juice, prolonged the elimination half-life of a calcium antagonist, felodipine, suggesting some differences between the mechanisms of grapefruit juice- and erythromycin-mediated drug interaction. Grapefruit juice may only alter presystemic clearance via inhibited intestinal metabolism, while erythromycin additionally alters hepatic clearance. In fact, our previous study (Yasui et al. 1996) showed that erythromycin altered pharmacokinetics of alprazolam, although grapefruit juice did not in this study. These findings may also support the difference in the inhibitory mechanism of CYP3A4 activity between erythromycin and grapefruit juice. Lown et al. (1997b) reported that intestinal P-glycoprotein, which is a transmembrane protein involved in drug efflux from the enterocyto back into the intestinal lumen, is a more important determinant of the oral bioavailability of cyclosporin than intestinal CYP3A4. Edwards et al. (1999) suggested that compounds other than 6,7 -dihydroxybergamottin in grapefruit juice change cyclosporin disposition through inhibition of P-glycoprotein activity. On the contrary, Lown et al. (1997a) showed that the mechanism for the effect of grapefruit juice on oral felodipine kinetics is its selective downregulation of CYP3A4, but not P-glycoprotein, in the small intestine. In addition, it has been suggested that grapefruit juice components inhibit the CYP3A4- mediated metabolism of saquinavir, a human immunodeficiency virus (HIV)-1 protease inhibitor, and also modulate, to a minor extent, P-glycoprotein-mediated transport of this drug (Eagling et al. 1999). Thus, the contribution of P-glycoprotein to drug disposition altered by grapefruit juice has not yet fully been proven. Although the further study is required to determine whether or not alprazolam is a substrate of P-glycoprotein, it is possible to conclude that neither CYP3A4 in small intestine nor P-glycoprotein play a clinically significant role in alprazolam disposition due to no interaction between grapefruit juice and alprazolam in this study. Surprisingly, significant pharmacokinetic changes for alprazolam in smokers were found during the grapefruit juice phase in both studies 1 and 2, which were not predicted before the studies. Smoking is known to be an inducing factor to CYP1A2 activity (Bock et al. 1994). Meanwhile, there has been no direct in vivo evidence for the involvement of CYP1A2 in the metabolism of alprazolam. An in vitro study has also shown no activity of 4- or α-hydroxylation for alprazolam catalyzed by CYP1A2 (Venkatakrishnan et al. 1998). Accordingly, significant pharmacokinetic interaction with alprazolam through the change of CYP1A2 activity is unlikely to occur. However, the possibility that grapefruit juice inhibits the metabolism of alprazolam catalyzed by some smoking inducible enzymes cannot be excluded entirely, as our previous study (Otani et al. 1997) has suggested different pharmacokinetics for alprazolam between smokers and non-smokers. It is also noted that the present results should preferably be reconfirmed by a larger study because the number of the subjects (both smokers and non-smokers) may be too small to obtain a definitive clinical relevance of the interaction between alprazolam and grapefruit juice in smokers. In accordance with negative results regarding the pharmacokinetics of alprazolam in the volunteers study, no significant difference was found in the majority of psychomotor function parameters between the treatments with grapefruit juice and water. Also, in patients with anxiety disorders, clinical effects of alprazolam were unchanged throughout the study. Recently, it has been reported that the effect of grapefruit juice on drug disposition reaches a maximum after three glasses (600 ml) of grapefruit juice ingestion daily (Takanaga et al. 2000), but this rapidly and fully develops after the first glass (Lundahl et al. 1998). Despite repeated (7 days) and a large amount (600 ml/day) of grapefruit juice consumption, no changes in pharmacokinetics and pharmacodynamics of alprazolam in these studies suggest that grapefruit juice is probably safer for patients treated with alprazolam than other substrates of CYP3A4. In particular, more attention should be paid to the combination of grapefruit juice and triazolam and midazolam. In conclusion, repeated ingestion of grapefruit juice does not affect pharmacokinetic disposition or clinical effects of alprazolam, though it slightly alters only the pharmacokinetics of alprazolam in smokers. This finding suggests that an interaction between grapefruit juice and alprazolam does not need to be considered in the clinical situation. Acknowledgement This study was supported by a grant from the Hirosaki Research Institute for Neurosciences.

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