Effect of grapefruit juice on Sandimmun Neoral® absorption among stable renal allograft recipients

Claus Bistrup, Finn Thomsen Nielsen, Unni Elmer Jeppesen and Hans Dieperink

Department of Nephrology Y, Odense University Hospital, Odense, Denmark



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. The oral formulation of cyclosporin A (CsA)—Sandimmun—has a highly variable absorption. The development of a CsA microemulsion—Sandimmun Neoral—resulted in increased bioavailability, and decreased variability of absorption. The first oral formulation (Sandimmun) interacted with numerous other drugs and grapefruit juice. Several of these interactions might be explained by decreased pre-systemic metabolism by a cytochrome-enzyme (e.g. CYP3A4) located in the enteral mucosa, and/or via the P-glycoprotein-mediated decreased transport of CsA back from enterocytes into the gut lumen. The purpose of this pharmacokinetic study was to investigate the interaction between Sandimmun Neoral and grapefruit juice.

Method. Eight stable renal transplant recipients were studied during two 8-h sessions in a randomized cross-over design with 4 weeks interval. Following an overnight fast the patients ingested their habitual morning dose of Neoral either with water or with grapefruit juice. During the 8-h study period 10 blood samples were taken for determination of CsA concentration. These results formed the basis for calculation of area under curve (AUC), and half-life (t1/2). Maximum concentration (Cmax) and time until Cmax (tmax) were obtained from the concentration–time profile.

Results. The median AUC increased by 38% (12–194%) (P<0.05) following co-administration of Neoral with grapefruit juice. There were no significant changes in Cmax, tmax, and t1/2.

Conclusion. Co-administration of Sandimmun Neoral with grapefruit juice resulted in an increased bioavailability of CsA, indicating unchanged pre-systemic enterocyte first-pass metabolism as compared to Sandimmun. There was no impact of an oral grapefruit juice load on systemic clearance of CsA. It seems prudent to advise renal allograft recipients treated with Sandimmun Neoral not to ingest their medication with grapefruit juice.

Keywords: absorption; cyclosporin A; grapefruit juice; interaction; Sandimmun Neoral



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Cyclosporin A (CsA) has had a great impact on renal allograft survival by reducing the rate and severity of rejection episodes [1]. However, the absorption from the first commercially available oral formulation Sandimmun (Sand) (Sandoz, Basel, Switzerland) was markedly variable both interpatiently and intrapatiently [2], which was of particular concern as CsA has a narrow therapeutic window between sufficient immunosuppression and, among other side-effects, nephrotoxicity. The absorption was among others dependent on metabolism in the gut [3], numerous drugs [4], and grapefruit juice (GFJ) [5].

In order to achieve a bioavailability as consistent as possible, a new CsA microemulsion was developed: Sandimmun Neoral (Neoral) (Novartis, Basel, Switzerland) [2]. The absorption of CsA from this microemulsion formulation might be either; (i) paracellular (through tight junctions or temporary openings in the membrane created by shedding of enterocytes within the intestinal tract), and/or (ii) transcellular (with or without intracellular metabolism) [6]. Consequently, using Neoral compared to Sand the influence of enteral mucosa metabolism may be by-passed.

CsA is mainly metabolized through cytochrome P450 (CYP3A4), which is localized both in the liver [7] and in the small bowel enterocytes [8]. Furthermore the enterocyte CYP3A4 was shown to be inhibited by GFJ [8]. In a study of intravenous CsA vs oral Sand it was demonstrated that co-administration with GFJ increased the absorption while the systemic clearance was unaffected, indicating that the effect of GFJ was due to diminished enterocytes metabolism of CsA [9].

Furthermore, the CsA absorption is modulated through the P-glycoprotein [10,11], which is also influenced by GFJ [12]. P-glycoprotein is, among others, located in the epithelial cells lining the gastrointestinal tract, and this protein actively transports xenobiotics (e.g. CsA) back from the epithelial cell into the lumen of the gut, thus decreasing the absorption of the drug.

Despite the almost exclusively use of Neoral nowadays, no study among stable renal transplant recipients has been performed evaluating the impact of GFJ on Neoral absorption. Some authors have called for attention to this problem [13]. To analyse the possible GFJ–Neoral interaction we performed a prospective randomized cross-over study among stable renal allograft recipients.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
Eight stable renal transplant patients participated in the study after written and informed consent approved by the local ethic committee (Den Videnskabsetiske Komite for Vejle og Fyns amter, ref. no. 19980165). The patients were otherwise healthy as judged by medical history, physical examination, and laboratory testing (biochemical and haematological profiles). All patients fulfilled the following inclusion criteria: (i) age between 18 and 75; (ii) stable graft function for 6 months or more; (iii) normal liver biochemistry; and (iv) treatment with a constant Neoral dose for more than 3 months. Excluded from the study were patients who had: (i) antibiotics within 4 weeks prior to the study; (ii) concomitant medications with drugs with known or suspected interaction with CsA; (iii) suffered from gastrointestinal discomfort or illness within 4 weeks; (iv) been eating food containing grapefruit within 7 days prior to the study; or (v) ingested over-the-counter or herbal medications.

All patients were treated with Neoral in combination with mycophenolate mofetil (CellCept, Roche, Switzerland), and did not receive steroids at all. In our centre, the therapeutic interval for whole-blood trough CsA concentration is 100–150 nmol/l (TDxFLx, Abbott, IL, USA) for stable renal transplant patients.

Study design
A cross-over design consisting of two 8-h sessions separated by a washout period of 4 weeks was used. During the first session, and following an overnight fast, the patients ingested their usual dose of Neoral and other medications with either 500 ml GFJ or tap water—during both sessions the first dose of 250 ml fluid was ingested 15 min prior to Neoral intake while the second dose of 250 ml fluid was ingested concomitantly with Neoral. The patients were randomly allocated to ingest either GFJ or water during the first 8-h session. During the second session, the schedule was similar except that the patients received the alternative fluid. The GFJ for the entire study period originated from the same batch.

During both sessions blood samples for determination of CsA concentrations were collected from the patients before and 1/2, 1, 11/2, 2, 21/2, 3, 4, 6, and 8 h after the ingestion of their habitual morning dose of Neoral.

Sample preparation and analysis
Blood samples were collected into EDTA-containing collecting tubes gently inverted several times to allow mixing, and finally frozen (-18°C) at pending. All the samples from the entire study were analysed by means of a monoclonal antibody (TDxFLx, Abbott Laboratories, IL, USA) in one procedure, using reagents from the same batch. According to the inlet with the reagents to TDxFLx, variability in determining CsA-concentrations is less than 4%.

Pharmacokinetic analysis
Pharmacokinetic parameters were calculated by use of non-compartmental methods. Area under the whole-blood concentration curve (AUC) from time zero through 8 h was calculated with use of the linear trapezoidal rule. Cmax (maximum concentration) and tmax (time until Cmax) were obtained directly from the concentration–time profile. Under the assumption of single-pool first-order kinetics t1/2 (half life of whole-blood concentration of CsA) was estimated using the last four points on the concentration–time curve.

Statistical analysis
Non-parametric statistics were used. The level of statistical significance was a priori set at P<0.05.



   Results
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 Abstract
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 Subjects and methods
 Results
 Discussion
 References
 
Eight otherwise healthy patients participated in this study (female/male 4/4). Median age was 41.2 years (range 27.3–72.5) and the median period since last renal transplantation was 3.8 years (range 1.5–12.5). The calculated pharmacokinetic parameters for Neoral when ingested with either GFJ or water are summarized in Table 1Go. The median increase in AUC during the 8-h period was 38%, range 12.3–193.5% (Wilcoxon test, P<0.05). There was no difference in median tmax for CsA whole-blood concentration when ingested with GFJ compared with water; 91 min (interquartile range 67–95) vs 64 min (interquartile range 63–83) respectively (Wilcoxon test, P>0.05). Further, there was no difference in median Cmax for CsA when ingested with GFJ compared with water; 1011 nmol/l (interquartile range 849–1739) vs 935 nmol/l (interquartile range 746–1707) respectively (Wilcoxon test, P>0.05). The median t1/2 for CsA concentration was unaffected by GFJ compared with water, 232 min (interquartile range 179–282) vs 197 min (interquartile range 193–261) respectively (Wilcoxon test, P>0.05).


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Table 1. Patient characteristics and test results

 
Changes in CsA whole-blood concentrations when given with either GFJ or water are individually depicted in Figure 1Go.



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Fig. 1. Cyclosporin A whole blood concentration vs time when Neoral was co-administered with either grapefruit juice (dotted line) or water (solid line). Note different scales on ordinates. Abscissa, Time (min); Ordinate, cyclosporin A concentration (nmol/l).

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this randomized cross-over study we examined the impact of grapefruit juice on the pharmacokinetic of cyclosporin A (Sandimmun Neoral) among stable renal allograft recipients. The calculated pharmacokinetic parameters show that co-administration with GFJ increases AUC (8-h value) significantly. An interaction of comparable magnitude has been reported for healthy volunteers ingesting a single oral load of Neoral [14]. Previous studies using the original formulation of CsA (Sand) have shown similar effect of GFJ on AUC [5,9,15] although one study was unable to demonstrate this effect of GFJ on AUC [16]. This discrepancy might have several explanations: (i) GFJ was given every 3 h, but intake of CsA was separated from GFJ by 11/2 h. (ii) Patients were treated with prednisone, which is know to inhibit CYP3A4 [4], therefore the enzyme was already partially inhibited and additional inhibition by GFJ might not be detectable. (iii) A longer time period was used for collecting blood samples and thereby calculation of AUC (AUC0–24 vs AUC0–8). If in our study a similar longer time period had been used to calculate AUC, the relative increase in AUC—following GFJ and Neoral co-administration—would have been minimized. (iv) A different formulation of CsA—Sand vs Neoral was used. (v) Different juices might contain different amounts/molecules of inhibiting ingredient(s)—see later. To our knowledge, no other study has examined the effect of GFJ on Neoral absorption among stable renal transplant recipients.

We did not see any significant effects of GFJ on Cmax, tmax, or t1/2. This is consistent with a study among healthy volunteers showing only a borderline effect on tmax while both Cmax and t1/2 were unaffected by GFJ [14]. However, conflicting results exists regarding Cmax and tmax [9,15,16].

Two different proteins influence the absorption of CsA: the enzyme CYP3A4 [17] and the transport protein P-glycoprotein [11], both proteins being located in the gastrointestinal mucosa and both being influenced by GFJ.

CYP3A4 is located in the microsomes in the enterocytes [17] and following GFJ administration the enzyme is down-regulated with no effect on the corresponding mRNA [18]. This effect seems detectable within 4 h although daily ingestion of GFJ will increase the effect [8,13,18]. The mechanism of action of GFJ on CYP3A4 is unknown. CsA is converted to its major metabolites by CYP3A4 [17]. Therefore, oral co-administration of GFJ and CsA was expected to diminish first-pass enteral mucosa metabolism of CsA and consequently the amount of absorbed CsA was supposed to increase. This theory is in accordance with Lown et al. [18], who found a significant correlation (for felodipine) between the decrease in enterocytes concentration of CYP3A4 and increase in Cmax as well as in AUC. On the contrary, Edwards et al. [19] was unable to demonstrate that a decreased CYP3A4 concentration in duodenal mucosa was followed by an increased AUC for CsA. Despite the fact that the main reservoir of CYP3A4 is located in the liver cells, only the oral and not the intravenous administration of CsA was affected by an oral load of GFJ [9], indicating that the active ingredient(s) of GFJ do not reach the liver in sufficient concentrations to affect hepatic metabolism of CsA [18].

P-glycoprotein is located in the apical enterocyte membrane and actively pumps xenobiotics (e.g. CsA) back into the intestinal lumen, thereby reducing AUC [11]. Fricker et al. [10] showed that the concentration of P-glycoprotein in the enteral mucosa increases in the anal direction of the gastrointestinal tract and that the amount of absorbed CsA following local application was clearly inversely correlated to the P-glycoprotein concentration. In a series of in vitro experiments using vinblastine, Soldner et al. [12] demonstrated that GFJ activates P-glycoprotein-mediated drug transport, thereby counteracting the inhibitory effects on CYP3A4. The same paper also demonstrated that CsA is a potent P-glycoprotein inhibitor [12], a finding confirmed by others [19]. We are not aware of any investigation evaluating the effect of P-glycoprotein-mediated CsA-transport following stimulation with GFJ and simultaneous inhibition with CsA.

The identity of the inhibitor(s) in GFJ has not been identified. Most studies were using in vitro experiments analysing the inhibitory effect on CYP3A4 from different compounds in GFJ. Fukuda et al. found four different furocoumarins with inhibitory capacity to CYP3A4 [20], and one of these furocoumarins (6,7-dihydroxybergamottin) also was found to have inhibitory capacity in two other in vitro studies [8,19]. However, no effect of 6,7-dihydroxybergamottin on CsA absorption was found among healthy volunteers [19]. Furthermore, two different flavanoids (naringin and nainginin) were thought to account for the increased bioavailability of CsA, while a study in rats could not confirm this [21]. Edwards et al. found no effect of 6,7-dihydroxybergamottin on P-glycoprotein activity [19], when comparing two different juices (GFJ and Seville orange juice) containing the same amount of 6,7-dihydroxybergamottin—only GFJ increased the AUC for CsA.

In summary, following an altered oral formulation of cyclosporin A from Sandimmun to Sandimmun Neoral it was speculated that the impact of GFJ on CsA absorption might be neglected. In this study, however, co-administration of CsA (Sandimmun Neoral) with GFJ resulted in an increased bioavailability of CsA, indicating unchanged pre-systemic enterocyte first-pass metabolism as compared to Sandimmun. Therefore it seems prudent to advise renal allograft recipients treated with Sandimmun Neoral to avoid ingesting their medication with GFJ.



   Acknowledgments
 
Grapefruit juice was a generous gift from Rynkeby Foods, Rynkeby, Denmark. We wish to thank laboratory technician Hannelore Lemming, Tissue Culturing Laboratory, Department of Nephrology Y, Odense University Hospital, Denmark, for preparing the blood cyclosporin A measurements.



   Notes
 
Correspondence and offprint requests to: Claus Bistrup MD, Department of Nephrology Y, Odense University Hospital, DK-5000 Odense C, Denmark.

E-mail: Claus.Bistrup{at}dadlnet.dk Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 29. 3.00
Revision received 29. 8.00.