Abstract
Aims
Grapefruit juice inhibits CYP3A4 in the intestinal wall leading to a reduced intestinal first pass metabolism and thereby an increased oral bioavailability of certain drugs. For example, it has been shown that the oral bioavailability of midazolam, a CYP3A4 substrate, increased by 52% in healthy subjects after ingestion of grapefruit juice. However, this interaction has not been studied in patients with impaired liver function. Accordingly, the effect of grapefruit juice on the AUC of midazolam and the metabolite α-hydroxymidazolam was studied in patients with cirrhosis of the liver.
Methods
An open randomized two-way crossover study was performed. Ten patients (3 females, 7 males) with liver cirrhosis based on biopsy or clinical criteria participated. Six patients had a Child-Pugh score of A, one B and three C. Tap water (200 ml) or grapefruit juice were consumed 60 and 15 min before midazolam (15 mg) was administered orally. Plasma samples were analysed for midazolam and α-hydroxymidazolam.
Results
Grapefruit juice increased the AUC of midazolam by 106% (16, 197%) (mean (95% confidence interval)) and the AUC of the metabolite α-hydroxymidazolam decreased to 25% (12, 37%) (P < 0.05 for both). The ratio of the AUCs of the metabolite α-hydroxymidazolam to midazolam decreased from 0.77 (0.46, 1.07) to 0.11 (0.05, 0.19) (P<0.05). t1/2 remained unaltered for both drug and metabolite. Midazolam Cmax, tmax, and α-hydroxymidazolam tmax increased, but these changes were not statistically significant, whereas Cmax of the metabolite decreased to 30% (14, 47%) (P<0.05).
Conclusions
A marked interaction between oral midazolam and grapefruit juice was found and the data are consistent with a reduced first-pass metabolism of midazolam. This is likely to occur at the intestinal wall inhibition of CYP3A4 activity by grapefruit juice. These results indicate that patients with liver cirrhosis are more dependent on the intestine for metabolism of CYP3A4 substrates than subjects with normal liver function.
Keywords: grapefruit juice, intestinal drug metabolism, liver cirrhosis, midazolam
Introduction
The intestine plays a significant role in the metabolism and transport of drugs [1, 2], which may lead to significant intestinal first pass effect and clinically important changes in bioavailability [1, 3].
The most important intestinal drug metabolizing enzyme is CYP3A4 [4, 5]. CYP3A4 is also abundant in the liver [4, 6]. Midazolam is metabolized by the CYP3A subfamily to α-hydroxymidazolam, and has been used as a model compound for studying CYP3A activity [7–11].
Grapefruit juice inhibits intestinal but not liver CYP3A4 or colon levels of CYP3A5 [12–16]. Consequently, intestinal CYP3A4 first pass metabolism can be reduced by inhibition with grapefruit juice. Kupferschmidt [17] has shown that the bioavailability of midazolam increased by 50% in healthy subjects after ingestion of grapefruit juice. Studies on the effect of clarithromycin and ketoconazole on intravenous and oral midazolam pharmacokinetics have indicated that midazolam intestinal first pass metabolism in healthy subjects accounts for 50% of the dose [18, 19]. During liver transplantation an intestinal first pass effect of 40% was found [20].
The hepatic drug metabolism is generally reduced in patients with cirrhosis of the liver leading to increased drug availability [21, 22]. Furthermore, increased sensitivity to certain drugs is seen in these patients [21]. Drug absorption is believed not to be significantly influenced by cirrhosis, but whether or not the intestinal metabolism of drugs is affected is not known. The aim of this study was to investigate the effect of grapefruit juice on the intestinal first pass metabolism of midazolam in patients with liver cirrhosis.
Methods
Subjects
Ten patients with liver cirrhosis took part in the study. Diagnosis was based on liver biopsy and/or clinical criteria (prior or actual varices of the oesophagus and/or ascites) together with ultrasonic evaluation of the liver showing typical nodular changes. All of the patients continued their medication (diuretics, β-adrenoceptor blockers, paracetamol, vitamins K and B, proton pump inhibitors, and insulin) throughout the study.
Clinical examination and routine blood tests (albumin, alanine transferase, alkaline phosphatase, bilirubin, prothrombin time, haemoglobin, leucocyte and thrombocyte counts) were performed on the first day and body weight was measured on each day of the study. Patient data are given in Table 1.
Table 1.
Patient data.
| Patient | Age (years) | Liver biopsy | Ultrasound | Encephalopathy | Ascites | Bilirubin (µmol l−1) | Albumin (µmol l−1) | Protrombin time (% activity) | Child-Pugh score |
|---|---|---|---|---|---|---|---|---|---|
| 1 F | 30 | + | + | – | + | 23 | 551 | 70 | A |
| 2 M | 47 | – | + | – | + | 14 | 580 | 64 | A |
| 3 F | 56 | – | + | – | – | 102 | 406 | 43 | B |
| 4 M | 64 | – | + | – | + | 50 | 464 | 43 | C |
| 5 M | 54 | + | + | – | – | 35 | 508 | 60 | A |
| 6 F | 40 | – | + | – | + | 25 | 624 | 84 | A |
| 7 M | 79 | + | + | – | – | 73 | 403 | 32 | C |
| 8 M | 46 | – | + | + | + | 50 | 445 | 40 | C |
| 9 M | 38 | – | + | – | – | 43 | 537 | 56 | A |
| 10 M | 34 | + | + | – | – | 33 | 609 | 76 | A |
+ present; − not present.
Ethics
All patients received verbal and written information and gave their written informed consent. The local Ethics Committee approved the study (no. KA 00008 m). The study was done in accordance with the Declaration of Helsinki.
Study design
An open randomized crossover study was performed. Patients were investigated on 2 days separated by at least 7 days. Citrus products (including juice) were avoided 2 days before the study. The protocol on the 2 days was identical apart from the ingestion of either tap water (control) or grapefruit juice, the order of which was randomized. Standard meals were served 3 and 8 h after midazolam administration.
Following an 8 h fast, the patients were given either 200 ml tap water or 200 ml of normal strength grapefruit juice prepared freshly from frozen concentrate (Minute Maid®, Coca Cola, Glostrup, Denmark) 60 and 15 min before receiving a 15 mg tablet of midazolam (Dormicum®, Roche A/S, Hvidovre, Denmark). Blood samples were taken before the intake of midazolam and after 10, 20, 40 min and 1, 1.5, 2, 2.5, 3.5, 5, 7, 9, and 24 h. The samples were centrifuged at 3000 g for 15 min and plasma stored at −20° C until analysis.
Drug and metabolite analysis
Plasma samples were analysed for midazolam and α-hydroxymidazolam by h.p.l.c. using pure midazolam (Roche A/C, Basel, German) and α-hydroxymidazolam (Lipomed, Arlesheim, Germany) as described by Ha et al.[23]. The lowest detectable concentration was 5 ng ml−1. The interassay coefficients of variation for midazolam and the metabolite were 2.6 and 3.1%, respectively.
Pharmacokinetic analysis
Area under the plasma concentration vs time curve (AUC) was calculated by use of the trapezoidal rule with linear interpolation and extrapolated to infinity using the elimination constant and the last concentration measure. Cmax and tmax were read from the data. t½ was calculated manually from the log concentration vs time curve.
Statistical methods
Data are presented as mean and 95% confidence intervals (CI). A Student’s t-test for paired data was used and a P value of 0.05 was considered statistically significant. Statistical analysis was performed using Statistica for Windows (StatSoft.Inc, Tulca, OK, USA).
Results
Table 1 shows the result of the clinical examination and the blood tests. The degree of cirrhosis is indicated by the Child-Pugh score [24], and six patients were classified as A, one as B and three as C.
Table 2 shows the pharmacokinetic data and Figure 1 the concentration vs time curves for midazolam before and after administration of grapefruit juice in a representative patient. Figure 2 shows the increase in midazolam AUC and decrease in α-hydroxymidazolam AUC following grapefruit juice administration.
Table 2.
The pharmacokinetics of oral midazolam before and after treatment with grapefruit juice. CI confidence interval.
| Water (CI) | GFJ (CI) | P value | |
|---|---|---|---|
| Midazolam | |||
| AUC (ng ml−1 min) | 24 700 (24 400, 25 000) | 57 100 (55 300, 58 900) | P<0.05 |
| Cmax (ng ml−1) | 87 (31–144) | 101 (32, 170) | NS |
| tmax (min) | 85 (39–130) | 105 (57, 153) | NS |
| t½ (min) | 266 (180–351) | 266 (175, 358) | NS |
| α-hydroxymidazolam | |||
| AUC (ng ml−1 min) | 18 000 (17 600, 18 400) | 6900 (6700, 7200) | P<0.05 |
| Cmax (ng ml−1) | 51 (21, 81) | 14 (6, 22) | P<0.05 |
| tmax (min) | 85 (57, 131) | 147 (87, 208) | NS |
| t½ (min) | 231 (160, 302) | 244 (191, 296) | NS |
| Ratio α-hydroxymidazolam/midazolam AUC | 0.77 (0.46, 1.07) | 0.11 (0.05, 0.19) | P<0.05 |
Figure 1.
Concentration-vs-time curve (AUC) for oral midazolam before (•) and after (▪) treatment with grapefruit juice in a representative patient.
Figure 2.
Midazolam (a) and α-hydroxymidazolam (b) AUC before and after treatment with grapefruit juice.
The AUC of midazolam was increased by 106% (16, 197%) (mean (95% confidence interval)) (P<0.05) and that of the metabolite α-hydroxymidazolam decreased to 25% (12, 37%) (P<0.001). The ratio of the AUCs of the metabolite α-hydroxymidazolam to midazolam decreased from 0.77 (0.46, 1.07) to 0.11 (0.05, 0.19) (P<0.05). t½ remained unaltered for both midazolam and the metabolite. Midazolam Cmax, tmax, and α-hydroxymidazolam tmax increased, although not statistically significantly, whereas Cmax of the metabolite decreased to 30% (14, 47%) of control (P<0.05).
Discussion
The results of the present study in 10 patients with liver cirrhosis showed that the systemic availability of oral midazolam was doubled during grapefruit juice intake. Furthermore, the concentration of the CYP3A4 mediated metabolite α-hydroxymidazolam was significantly reduced as was the α-hydroxymidazolam/midazolam ratio. Grapefruit juice did not seem to alter the clearance of midazolam or α-hydroxymidazolam from blood, since no changes in half-lives were observed. Earlier studies have shown that grapefruit juice inhibits intestinal CYP3A4 activity, whereas hepatic activity seemed unaffected [13–15, 19]. Therefore, the results of the present study are consistent with a reduced intestinal CYP3A4-mediated first-pass metabolism of midazolam to α-hydroxymidazolam by grapefruit juice. This should theoretically cause a higher midazolam Cmax. We did found an increase, but it was not statistically significant. This may be due to insufficient blood sampling around Cmax. The patients received routine medication with diuretics and β-adrenoceptor blockers, which may affect the portal blood flow and thereby the bioavailability of midazolam. However, the medication was not changed during the study.
We found a greater increase in the midazolam bioavailability in liver cirrhosis compared with the increase in healthy subjects reported by Kupferschmidt [17]. Thus, the relative contribution of CYP3A4 mediated intestinal drug metabolism to the overall metabolism of midazolam seems to be greater in patients with liver cirrhosis than in healthy subjects. Cirrhotic patients may thus be more susceptible to interactions at the intestinal level.
In conclusion, we have found a marked effect of grapefruit juice on midazolam bioavailability in liver cirrhosis, which was far more pronounced than in healthy subjects. The interaction is likely to occur in the intestine rather than in the liver. The results indicate that patients with liver cirrhosis are relatively more dependent on the intestine for the metabolism of CYP3A4 substrates. The findings support the importance of intestinal midazolam metabolism and the need for investigating drug interactions in patients with liver disease.
Acknowledgments
We are grateful to Coca Cola for the supply of grapefruit juice, Roche A/S for midazolam, The Danish Hospital Foundation for Medical Research, Region of Copenhagen, The Faroe Islands and Greenland for financial support and Lis Nielsen and Tove Laursen for excellent technical assistance.
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