Influence of Co-Medication with Sirolimus or Cyclosporine on Mycophenolic Acid Pharmacokinetics in Kidney Transplantation

Authors

  • D. Cattaneo,

    Corresponding author
    1. Department of Immunology and Transplantation, Ospedali Riuniti–Mario Negri Institute for Pharmacological Research, Bergamo, Italy
    2. Center for Research on Organ Transplantation ‘Chiara Cucchi De Alessandri e Gilberto Crespi’, Bergamo, Italy
      Corresponding author: Dario Cattaneo, dcattaneo@marionegri.it
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  • S. Merlini,

    1. Department of Immunology and Transplantation, Ospedali Riuniti–Mario Negri Institute for Pharmacological Research, Bergamo, Italy
    2. Center for Research on Organ Transplantation ‘Chiara Cucchi De Alessandri e Gilberto Crespi’, Bergamo, Italy
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  • S. Zenoni,

    1. Department of Immunology and Transplantation, Ospedali Riuniti–Mario Negri Institute for Pharmacological Research, Bergamo, Italy
    2. Center for Research on Organ Transplantation ‘Chiara Cucchi De Alessandri e Gilberto Crespi’, Bergamo, Italy
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  • S. Baldelli,

    1. Department of Immunology and Transplantation, Ospedali Riuniti–Mario Negri Institute for Pharmacological Research, Bergamo, Italy
    2. Center for Research on Organ Transplantation ‘Chiara Cucchi De Alessandri e Gilberto Crespi’, Bergamo, Italy
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  • E. Gotti,

    1. Department of Immunology and Transplantation, Ospedali Riuniti–Mario Negri Institute for Pharmacological Research, Bergamo, Italy
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  • G. Remuzzi,

    1. Department of Immunology and Transplantation, Ospedali Riuniti–Mario Negri Institute for Pharmacological Research, Bergamo, Italy
    2. Center for Research on Organ Transplantation ‘Chiara Cucchi De Alessandri e Gilberto Crespi’, Bergamo, Italy
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  • N. Perico

    1. Department of Immunology and Transplantation, Ospedali Riuniti–Mario Negri Institute for Pharmacological Research, Bergamo, Italy
    2. Center for Research on Organ Transplantation ‘Chiara Cucchi De Alessandri e Gilberto Crespi’, Bergamo, Italy
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Corresponding author: Dario Cattaneo, dcattaneo@marionegri.it

Abstract

The pharmacokinetics of mycophenolic acid (MPA)—the active metabolite of mycophenolate mofetil (MMF)—is significantly influenced by co-medications. The impact of sirolimus on daily MPA exposure, however, has not been investigated so far. As a part of the study aimed at investigating the efficacy of Campath-1H induction therapy in a steroid-free regimen in kidney transplantation, MPA plasma levels were serially measured in 21 patients treated with low-dose sirolimus (SRL) or low-dose CsA both in addition to low-dose MMF over 12 months post-operatively. Full pharmacokinetic profiles were compared at month 6 and 12 post-surgery. Mean dose-adjusted MPA trough levels were 4.4-fold higher in patients on combined SRL and MMF than in those given CsA and MMF. Pharmacokinetic studies demonstrated that mean MPA Cmax and Tmax were comparable in the two groups, while mean MPA AUC0-12 was higher in SRL than CsA treated patients. The pharmacokinetic profile of SRL- but not of CsA-group showed a second peak consistent with the enterohepatic recirculation of MPA. These findings suggest that SRL and CsA have different effects on MPA metabolism and/or excretion eventually affecting its immunosuppressive property and/or toxicity. CsA, but not SRL, inhibits MPA enterohepatic recirculation, reducing MPA daily exposure.

Introduction

Mycophenolic acid (MPA), the active form of the pro-drug mycophenolate mofetil (MMF) and the new mycophenolate sodium, is part of standard immunosuppressive regimens in organ transplantation (1). MMF is commonly administered in a fixed daily dose as adjunctive therapy in combination with a calcineurin inhibitor and corticosteroids. Recent evidence, however, suggests that a fixed dose regimen—adopted in the majority of transplant units worldwide—no longer might be the best approach for the management of transplant patients, since wide inter-patient variability in drug exposure has been observed in kidney transplant recipients given the fixed daily MMF dose (2). Therefore, therapeutic drug pharmacokinetic monitoring is advised (3–6). A significant predictive value for acute rejection (3,5,6), renal function (2) and drug-related side effects (7,8) has been found for the 12-h dose interval MPA area under the concentration-time curve (AUC0-12) and for the predose trough MPA concentration (C0).

Following oral administration, MPA is converted to inactive metabolites by glucuronidation mediated by the uridine diphosphate glucuronosyltransferase (UDP-GT) enzyme family (1,9). The main metabolite, 7-hydroxy-glucuronide (MPAG), is excreted in the urine but may also contribute to the enterohepatic recirculation of MPA after excretion into the bile and hydrolysis in the gastrointestinal tract (10). Recently, three carboxyl-linked additional glucuronides have also been detected in vitro and in vivo (11).

Concomitant immunosuppressive therapy significantly influences MPA exposure. In particular glucocorticoids, by inducing UDP-GT expression, interfere with MMF metabolism (12). Others reported a significant increase in plasma MPA concentrations in patients treated with MMF plus tacrolimus (13,14), attributed to the inhibition of MPA glucuronidation by tacrolimus (13,15). At variance, combining MMF with the other calcineurin inhibitor cyclosporine (CsA) reduced MPA exposure (16). Recently, experimental animal studies have shown that CsA inhibits the multidrug resistance-associated protein 2 (MRP2)—a protein expressed at the apical surface of hepatocytes—which excretes conjugation products of drug metabolites, as MPA glucuronides, into bile (17,18). Therefore CsA, by blocking the transport of MPA metabolites into bile from hepatocytes, reduces enterohepatic recirculation of MPA, ultimately leading to a decrease in MPA concentrations in the 4 to 12-h window of the AUC profile (AUC4-12) (17).

Sirolimus (SRL) is a new immunosuppressant characterized by a unique mechanism of action and a potential capacity to synergize with other antirejection drugs (19). In the clinical practice, SRL is usually given in association with calcineurin inhibitors and steroids or antimetabolites such as MMF and azathioprine (20).

Preliminary retrospective evidence indicates that concomitant SRL therapy enhances MPA trough level as compared with CsA (21,22). However, there are no prospective studies, with strict drug monitoring and full pharmacokinetic evaluations aimed at formally assessing the effects of SRL and CsA on MPA exposure.

The current study—which is part of a protocol aimed at investigating the efficacy of Campath-1H induction therapy in a steroid-free regimen in kidney transplant patients—was designed to compare prospectively the effects of SRL- or CsA-coadministration with MMF on MPA exposure. Given its potent effect of depleting both T and B lymphocytes, Campath-1H should allow the minimization of maintenance anti-rejection therapy. We have, therefore, planned to use very low doses of CsA or SRL, in combination with low-doses of MMF. In the present study, we sought to : (1) serially monitor MPA trough levels in patients given low-dose SRL or low-dose CsA both in addition to low-dose MMF as maintenance immunosuppression over 12 months post-operatively; (2) compare 12-h MPA pharmacokinetic profiles in both groups at month 6 and 12 after surgery and (3) examine the effects of SRL and CsA on MPA enterohepatic recirculation, expressed as MPA AUC4-12.

Methods

Patients

Twenty-one patients (13 men; 8 women) with end-stage renal disease who underwent primary kidney transplant were enrolled under an Ethics Committee-approved protocol at the Ospedali Riuniti Bergamo, Italy, following written informed consent. They were allocated to one of the following two study groups according to a randomization design: Group 1 (n = 11) was assigned to Campath-1H, low-dose SRL and low-dose MMF; group 2 (n = 10) entered a regimen with Campath-1H, low-dose CsA and low-dose MMF.

Campath-1 (Alemtuzumab, Schering Plough, Milano, Italy) was given as a single intravenous infusion (30 mg, over 2 hours) intraoperatively on the day of transplant (day 0). Corticosteroids were administered intraoperatively and for the first 2 days after transplantation. Thereafter, patients were free of steroids. Patients randomized to SRL received the drug (Wyeth, Rome, Italy) at the oral dose of 4 mg/day in a single morning administration starting on the day 1 after transplant. SRL dosing was adjusted to maintain whole blood levels within the 5–10 ng/mL range. In the CsA-based group, the drug was started just after surgery (1–2 mg/kg/day) and CsA doses were adjusted to achieve trough blood concentration of 120–200 ng/mL in the first month post-surgery and of 70–120 ng/mL thereafter. Both groups were given MMF at the oral low-dose of 500 mg twice a day starting on day 1 post-operatively, with no specific MPA targets. During the study, the MMF dose was adjusted by the attending physicians, which were unaware of the MPA concentrations. Modifications of MMF doses were mainly performed taking into account blood leukocyte count as well or other clinical conditions related to drug tolerability and adverse effects.

The administration of prokinetic drugs, resins or any agent known to interfere with MPA absorption, distribution, metabolism and/or elimination was not allowed during all the study period.

Study design and pharmacokinetic measurements

This prospective study examined the effects of SRL and CsA on dose-adjusted MPA trough levels measured every 5 days starting from day 5 post-Tx—when patients reached steady-state of drug distribution—up to day 90 and then at month 4, 6 and 12 post-surgery.

Moreover, at month 6 and 12 post-transplant, all patients underwent a 12-h MPA pharmacokinetic profile. On the morning of the pharmacokinetic studies, blood samples were collected for routine hematological, biochemical analysis as well as estimation of glomerular filtration rate by iohexol clearance (23) and for the determination of trough levels of plasma MPA and blood SRL or CsA. Then patients were given the morning dose of MMF and SRL or CsA. MPA pharmacokinetic analysis was based on EDTA-collected blood samples from antecubital vein at 20, 40, 75, 120 minutes and 3, 4, 5, 6, 7, 8, 10 and 12 h after drug administration. Samples were centrifuged at 3000 g, plasma separated and stored at −20°C until analysis. For SRL and CsA pharmacokinetics, blood samples were collected in heparinized tubes at 0.5, 1, 2, 3, 4, 5, 6, 8, 10 and 12 h after dosing and stored at −20°C until analysis. All drug measurements were performed by high-performance liquid-chromatography (HPLC) as previously described (2,24,25). MPA, SRL and CsA concentration-time profile was recorder for all patients, together with the time to reach the maximum concentration (Tmax) and the maximum drug concentration (Cmax). The AUC from time equal to 0 to the last sampling point (12 h) was calculated by the trapezoidal rule.

To study the different effect, if any, of SRL and CsA on MPA bioavailability, we measure MPA AUC0-2, as a surrogate marker of MPA absorption. Additionally, to test the hypothesis that CsA may decrease the MPA enterohepatic recirculation (17), we also estimated the MPA AUC from 4 h to 12 h after drug administration (AUC4-2), a time interval corresponding to the appearance of a secondary MPA peak due to enterohepatic recirculation, related to the reabsorption of MPA glucuronidated metabolites as MPA. Both MPA AUC0-2 and AUC4-12 were estimated using the trapezoidal rule.

Statistical analysis

Results are reported as means ±SD. Correlation between MPA trough levels and the corresponding AUC0-12 was performed in both groups (SRL and CsA) by linear regression analysis. To take into account the confounding factor of MMF drug dose changes, MPA trough levels and pharmacokinetic parameters were adjusted for the daily MMF dose. Moreover, to prove the linearity between daily MMF doses and MPA exposure, analyses were also performed using non-dose adjusted MPA pharmacokinetic parameters in subgroups of patients given MMF at the daily dose of 500 or 1000 mg.

Unpaired t-test was used to compare MPA levels between group 1 (given MMF with SRL) and group 2 (given MMF and CsA). The statistical significance was defined as p < 0.05. The ratio between each MPA value measured in the SRL group and the corresponding value measured in the CsA group was used to estimate the degree of the differences between the two groups as follows:

image

Results

Recipients demographics

All patients enrolled in the study were Caucasians. Mean age of recipients was 49 years (range: 24–71 years). Patients randomized to SRL- or CsA-based maintenance immunosuppression were comparable as for the distribution of baseline demographics, including age, sex and HLA matching among the donors and the recipients (data not shown). As reported in Table 1, no differences in the main biochemical and hematological parameters that could potentially affect MPA pharmacokinetics were found between the SRL- and CsA-arm. Of note, 3 patients in the SRL arm and 1 patient in the CsA group had delayed graft function.

Table 1.  Main hematological and biochemical parameters
Months post-TxSRL groupCsA group
1361213612
WBC (×106/μL)4.1 ± 1.34.4 ± 1.44.6 ± 1.25.2 ± 1.06.4 ± 6.14.9 ± 3.14.8 ± 1.76.1 ± 2.0
RBC (×106/μL)3.9 ± 0.34.6 ± 0.74.7 ± 0.94.8 ± 1.14.1 ± 0.24.3 ± 0.44.1 ± 0.34.4 ± 0.6
S. creat (mg/dL)2.0 ± 1.41.4 ± 0.51.4 ± 0.41.8 ± 1.11.8 ± 0.81.9 ± 0.61.7 ± 0.51.6 ± 0.4
S. albumin (mg/dL)3.7 ± 0.43.6 ± 0.33.4 ± 0.43.3 ± 0.53.8 ± 0.73.6 ± 0.43.5 ± 0.33.3 ± 0.5
GFR (mL/min)61 ± 1261 ± 2051 ± 1554 ± 13
GOT (IU/L)28 ± 1127 ± 1019 ± 1217 ± 524 ± 1327 ± 1022 ± 820 ± 6
GTP (IU/L)35 ± 3339 ± 2126 ± 1122 ± 642 ± 2639 ± 2127 ± 2217 ± 11

Immunosuppressive drug monitoring

Mean immunosuppressive drug dose, whole blood trough SRL and CsA and plasma trough MPA concentrations are shown in Table 2. SRL AUC0-24 were comparable at 6 and 12 months post-transplant (month 6: 283 ng* h/mL, month 12: 334 ng h/mL, p = 0.40). The mean SRL trough levels fell within the planned range of 5–10 ng/mL with not significant difference during the 12-month study period. The mean dose to achieve these levels was within 3.6 and 4.3 mg/day. Similarly, mean CsA trough levels were maintained within the anticipated low targets during the 12-month follow-up (Table 2). CsA AUC0-12 at 6 and 12 month post-surgery did not differ at significant extent (month 6: 3246 ng* h/mL, month 12: 3080 ng* h/mL, p = 0.63).

Table 2.  Immunosuppressive drug dosing and trough levels
Sirolimus group (n = 11)

Time post-Tx
Mean SRL dose
(mg/day)
Mean SRL trough
(ng/mL)
Mean MMF dose
(mg/day)
Mean MPA trough
(μg/mL)
  1. *p < 0.05 versus CsA group at same time point; °p < 0.01 versus CsA group at same time point.

 2 weeks4.3 ± 0.89.1 ± 2.2484 ± 630.88 ± 0.63*
 1 month4.3 ± 1.79.4 ± 3.2571 ± 1821.73 ± 0.94°
 3 months4.3 ± 1.88.3 ± 2.7621 ± 4301.54 ± 1.05°
 6 months4.0 ± 1.57.9 ± 2.3591 ± 2021.67 ± 1.12*
 12 months4.0 ± 1.010.1 ± 4.51200 ± 4472.75 ± 1.63°
 
Cyclosporine group (n = 10)
 

Time post-Tx
Mean CsA dose
(mg/day)
Mean CsA trough
(ng/mL)
Mean MMF dose
(mg/day)
Mean MPA trough
(μg/mL)
 
 2 weeks243 ± 106134 ± 59550 ± 1580.33 ± 0.32
 1 month279 ± 109133 ± 61559 ± 1660.26 ± 0.14
 3 months263 ± 74124 ± 52608 ± 2040.41 ± 0.34
 6 months269 ± 78119 ± 34750 ± 2670.49 ± 0.38
 12 months207 ± 57101 ± 31813 ± 2590.66 ± 0.24

Overall, mean MPA trough levels ranged from 0.22 to 2.75 μg/mL. However, MPA trough concentrations were close to the high threshold for patients belonging to the SRL group and to the low threshold for those of CsA group. To exclude the confounding factor of different MMF doses, MPA trough levels were adjusted for the daily drug dosing given to each patient. As shown in Figure 1, starting from day 10 post-surgery, mean dose-adjusted MPA trough levels were significantly higher in patients given SRL as compared to those receiving CsA. The average ratio of dose-adjusted MPA trough levels between SRL and CsA groups was 4.4.

Figure 1.

Mean dose-adjusted MPA trough levels from day 5 to year 1 after transplantation in kidney transplant recipients treated with SRL or CsA, both in addition to MMF.°p < 0.01 vs CsA group; *p < 0.05 vs CsA group.

MPA pharmacokinetic studies

Results of the 12-h MPA pharmacokinetic studies, performed at month 6 and 12 post-transplant, are given in Tables 3 and 4. Overall, linear regression analysis between MPA trough levels and MPA AUC evidenced a significant (p < 0.01) correlation in the SRL (r = 0.70) and CsA (r = 0.75) groups.

Table 3.  Mycophenolic acid (MPA) pharmacokinetic parameters at month 6 post-transplantation in patients treated with sirolimus (SRL) or cyclosporine (CsA) both in combination with mycophenolate mofetil (MMF)
MPA pharmacokinetic
parameters
SRL group
(n = 11)
CsA group
(n = 10)

p-value
MMF dose (mg/day)591 ± 202750 ± 2670.1570
MPA C0 (μg/mL)1.67 ± 1.120.49 ± 0.380.0117
C0/dose (μg/mL/g MMF)2.87 ± 2.080.63 ± 0.400.0082
Cmax (μg/mL)10.66 ± 9.2811.74 ± 6.560.7829
Cmax/dose (μg/mL/g MMF)17.09 ± 8.9715.28 ± 5.600.6221
Tmax (min)63 ± 10046 ± 190.6444
AUC0–12 (μg*h/mL)24.63 ± 13.8818.78 ± 11.470.3441
AUC0–12/dose (μg*h/mL/g MMF)41.52 ± 14.7623.66 ± 7.770.0006
AUC0–2/dose (μg*h/mL/g MMF)16.21 ± 6.0315.95 ± 9.150.9543
AUC4–12/dose (μg*h/mL/g MMF)19.93 ± 9.206.52 ± 3.310.0012
Table 4.  Mycophenolic acid (MPA) pharmacokinetic parameters at month 12 post-transplantation in patients treated with sirolimus (SRL) or cyclosporine (CsA), both in combination with mycophenolate mofetil (MMF)
MPA pharmacokinetic
parameters
SRL group
(n = 11)
CsA group
(n = 10)

p-value
MMF dose (mg/day)1200 ± 447813 ± 2590.0706
MPA C0 (μg/mL)2.75 ± 1.630.66 ± 0.240.0037
C0/dose (μg/mL/g MMF)2.30 ± 1.300.89 ± 0.440.0151
Cmax (μg/mL)13.46 ± 6.3212.53 ± 3.910.7471
Cmax/dose (μg/mL/g MMF)11.11 ± 2.9615.89 ± 4.300.0528
Tmax (min)24 ± 930 ± 110.3193
AUC0–12 (μg*h/mL)43.92 ± 24.6020.37 ± 6.570.0236
AUC0–12/dose (μg*h/mL/g MMF)35.45 ± 10.0226.00 ± 8.610.0974
AUC0–2/dose (μg*h/mL/g MMF)17.16 ± 9.9015.30 ± 5.080.6458
AUC4–12/dose (μg*h/mL/g MMF)17.22 ± 6.496.78 ± 2.530.0016

At month 6, unadjusted or dose-adjusted MPA trough levels were significantly higher in the SRL group than in CsA-treated patients. The ratio between the two series of MPA trough levels was 4.5. Although less pronounced, the same trend was confirmed at month 12 (2.3 ± 1.3 vs 0.9 ± 0.4 μg/mL/g MMF, p = 0.0151). By contrast, only slight but not significant difference for MPA Tmax, Cmax and AUC0-2—all surrogate markers of MPA absorption—between the two groups of treatment was found. Dose-adjusted MPA AUC0-12 was significantly higher in the SRL group than in patients given CsA, leading to an AUC0-12 ratio between the two series of 1.8. Difference in MPA pharmacokinetic parameters among the SRL- and CsA-treated patients was confirmed at 12 months post-transplant (Table 4), with the exception of dose-adjusted MPA AUC, that was numerically but not significantly higher in SRL- than in CsA-group.

We then repeated the above mentioned analysis without the adjustment of MPA pharmacokinetic parameters for the daily MMF dose. As shown in Table 5, again MPA trough levels and AUC were significantly higher in patients given SRL than those on CsA, both in patients receiving MMF at 500 or 1000 mg/day.

Table 5.  Mycophenolic acid (MPA) pharmacokinetic parameters in patients treated with sirolimus (SRL) or cyclosporine (CsA), divided according to daily mycophenolate mofetil (MMF) dose given to kidney transplant recipients
MPA pharmacokinetic
parameters

SRL group

CsA group

p-value

Ratio
MMF: 500 mg/day
 MPA C0 (μg/mL)1.47 ± 1.160.42 ± 0.280.00313.5
 Cmax (μg/mL)7.85 ± 3.167.89 ± 3.020.98751.0
 AUC0–12 (μg*h/mL)20.64 ± 6.5812.01 ± 4.920.01181.6
 AUC0–2 (μg*h/mL)9.18 ± 3.938.87 ± 4.940.79851.0
 AUC4–12 (μg*h/mL)10.35 ± 4.663.19 ± 1.520.00173.2
MMF: 1000 mg/day
 MPA C0 (μg/mL)2.39 ± 1.170.69 ± 0.310.00103.4
 Cmax (μg/mL)15.07 ± 11.2215.44+4.070.93241.0
 AUC0–12 (μg*h/mL)36.68 ± 15.7425.46 ± 6.840.04121.5
 AUC0–2 (μg*h/mL)14.68 ± 8.2914.14 ± 4.160.82381.0
 AUC4–12 (μg*h/mL)18.62 ± 4.586.83 ± 2.860.00012.8

Effects of SRL and CsA on MPA enterohepatic cycle

As shown in Figure 2, the main difference in the MPA pharmacokinetic profiles between SRL and CsA groups was found in the late phase, starting 4 h after MMF administration. Four to 12 hours after MMF administration, MPA metabolites undergo enterohepatic recirculation, and are reabsorbed as MPA, as documented by the appearance of a secondary MPA peak in the kinetic profile (10).

Figure 2.

Dose-adjusted MPA concentration-time curves from 0 to 12 hours after MMF administration in patients at month 6 post-surgery and treated with SRL or CsA.°p < 0.01 vs CsA group; *p < 0.05 vs CsA group.

To assess whether SRL and CsA might indeed affect the MPA enterohepatic recirculation, MPA AUC from 4 h to 12 h after drug dosing (AUC4-12) was measured. Dose-adjusted MPA AUC4-12 was significantly higher in patients on SRL than those treated with CsA at month 6 (19.9 ± 9.2 vs 6.5 ± 3.3 μg* h/mL) and 12 post-surgery (17.2 ± 6.5 vs 6.8 ± 2.5, μg* h/mL). The AUC4-12 ratio between SRL and CsA groups was higher as compared with that estimated for the corresponding full AUC0-12 (AUC4-12, month 6: ratio = 3.1; month 12: ratio = 2.5. Ratio AUC0-12, month 6: ratio = 1.8; month 12: ratio = 1.4).

Discussion

Potential pharmacokinetic interactions between MMF and other immunosuppressive agents may induce significant changes in patient exposure to the biologically active MMF metabolite, MPA, with relevant clinical consequences in terms of efficacy and side effects, especially when the drug is administered in a fixed dose regimen.

In this study, we found that in kidney transplant recipients on maintenance immunosuppression with MMF, MPA plasma levels were markedly influenced by the concomitant immunosuppressive regimen.

So far, only two studies have previously investigated the impact of SRL co-administration on MPA plasma levels in transplant patients (21,22). However, shortcomings of these observations were the retrospective nature of the studies, the evaluation of a single pharmacokinetic parameter such as MPA trough levels as surrogate marker of daily drug exposure and short-term (3 months) drug monitoring post-transplantation. Moreover, in these studies MPA concentrations were assessed by immunoassay, a method biased by cross-reaction of the test antibody with MPA metabolites, eventually leading to MPA value overestimation (26,27). At variance with these studies, we have prospectively compared two cohorts of kidney transplant recipients randomized to receive SRL or CsA together with MMF. In addition, patients were monitored for MPA trough levels and AUC0-12 pharmacokinetic parameters over 1 year follow-up using the gold standard HPLC method to assess MPA plasma concentrations (28,29). A possible shortcoming of the present study, however, is the fact that we have monitored the different effects of SRL or CsA on MPA exposure, in patients given MMF at doses lower than conventional. With this approach, we found that co-administration of SRL and MMF was consistently associated with 4.4-fold higher dose-adjusted MPA trough levels than those measured under CsA-based regimen during the 1 year study period.

The effects of SRL and CsA on MPA trough levels could be explained by their interference with MPA metabolic pathway. However, since SRL and CsA are metabolized by the cytochromial system (19) with phase I reactions, whereas MPA is mainly detoxificated by phase II metabolic reaction, not involving the cytochromial system (1,10), a potential influence of SRL or CsA on MPA metabolism would be negligible, if any.

As alternative hypothesis, SRL and CsA might differentially affect MPA absorption, distribution and/or elimination. However, since in the pharmacokinetic profile no significant difference in mean drug-adjusted MPA Cmax, Tmax and AUC0-2—as surrogate markers which mainly reflect drug absorption—was observed between the two groups, our findings would also exclude different effect of SRL and CsA on MPA gastrointestinal absorption and thus on drug bioavailability.

The plasma concentration-time profile of MPA after oral MMF administration is usually characterized by a sharp initial peak around 1 h and the occurrence of a secondary peak approximately 6–12 h postdose (10). This late peak has been attributed to glucuronidated MPA metabolites that undergo enterohepatic recirculation (EHC), a process by which a given drug or its metabolites, are excreted by the liver into the bile and then reabsorbed back into the portal circulation (4). In the case of conjugated drug metabolites, such as MPA metabolites, deconiugation mediated by the colonic bacteria in the gut flora precedes the uptake process. After deglucuronidation of the metabolites, MPA is reabsorbed in systemic circulation leading to the appearance of the secondary peak on the pharmacokinetic profiles (10). Drugs capable to influence EHC of MPA metabolites may, therefore, affect daily MPA exposure.

Despite comparable drug-adjusted MPA AUC in the first few hours after MMF administration, here we found a second late MPA peak in the pharmacokinetic profile in patients on SRL but not on CsA therapy. This translated in a significantly higher dose-adjusted MPA AUC4-12 in SRL than CsA group. These findings indicate that the enterohepatic recirculation of MPA is markedly and selectively suppressed by CsA. This observation confirms for the first time in humans previous experimental evidence in rats that CsA decreased the enterohepatic recirculation of MPA (17). As documented in this animal model (17), we speculated that also in patients the suppressive effect on MPA recirculation could be attributed to CsA-induced inhibition of MPA metabolites transport into bile from hepatocytes. Moreover, in our study the full AUC curves were drawn at month 6 and 12 after transplantation, when target CsA concentrations were low. Therefore, it can be reasonably speculated that the differences found in the present study may be more pronounced shortly after transplantation, when patients are exposed to higher CsA concentrations.

To take into account the confounding factor of MMF drug dose changes, MPA pharmacokinetic parameters were adjusted for the daily MMF dose. This approach presumes linearity between MMF doses and MPA levels, an unproven conditions that might have biased the results. To address this issue, we have compared the main non-dose adjusted MPA pharmacokinetic parameters between CsA and SRL groups, in subgroups of patients given MMF at 500 or 1000 mg/day. Also using this approach, we confirmed previous findings with dose-adjusted parameters, suggesting that the assumption of dose-linearity was valid.

The present study confirmed that CsA significantly interfere with MPA exposure and suggest that SRL does not share this effect on MPA pharmacokinetics. The peculiar effect of CsA is also supported by studies showing that patients given tacrolimus in combination with MMF had similar mean MPA trough levels, Cmax and AUC to those found in our patients who received SRL and MMF (13, 30–33). This indicates that in tacrolimus and SRL-treated patients, the MPA exposure is comparable and significantly higher than in patients given CsA and MMF.

The different effect of SRL and CsA on the secondary MPA peak may have also significant impact on plasma MPA trough levels, since the drug MPA enterohepatic recirculation usually occurs 6–12 (trough) h after MMF dosing. Indeed, this explains why dose-adjusted MPA trough concentrations were significantly higher in patients receiving combined SRL and MMF than those given CsA and MMF. Thus, monitoring MPA trough levels could be a reasonable surrogate marker for patient exposure to MPA, as documented by significant correlations between these values and the corresponding MPA AUC in both groups of patients. Moreover, it should be pointed that the published guidelines for therapeutic drug monitoring of MPA are based on studies where MMF was combined with calcineurin inhibitors. Therefore, concentration-controlled studies in patients given MMF in combination with SRL, as we did, are needed to identify MPA therapeutic window also in this setting.

In conclusion, we have documented that combined administration of SRL or CsA with MMF differently influence MPA exposure in kidney transplant recipients. This is the first clinical documentation that CsA, but not SRL, inhibits MPA enterohepatic recirculation, ultimately resulting in 50% lower MPA daily exposure as compared with SRL. These results should be carefully taken into account when MMF-based immunosuppressive regimens are implemented in transplantation, especially when the drug is administered at fixed daily dose, as routinely occurs, and patients are switched from CsA to SRL or viceversa to minimize their toxicity or maximize their inhibitory effect on the immune response.

Acknowledgments

Dario Cattaneo is a recipient of ‘Fondazione Monzino’ fellowship, Simona Merlini is a recipient of a fellowship from Associazione Ricerca Trapianti (ART) Milan, Italy and Sara Baldelli is a recipient of a fellowship from ARMR (Association for Research on Rare Disease). This work was partially supported by the ART.

Part of this study was presented at the American Transplant Congress, Seattle, Washington, USA, May 21–25, 2005.

Ancillary