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Influence of Everolimus on Steady-State Pharmacokinetics of Cyclosporine in Maintenance Renal

Transplant Patients
Klemens Budde, Gustav Lehne, Michael Winkler, Lutz Renders, Arno Lison, Lutz Fritsche, Jean-Paul Soulillou, Per
Fauchald, Hans-Hellmut Neumayer, Jaques Dantal and RADW 102 Renal Transplant Study Group
J. Clin. Pharmacol. 2005; 45; 781
DOI: 10.1177/0091270005277196

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BUDDEINTERACTIONS
10.1177/0091270005277196
EVEROLIMUS
DRUG ET AL AND CYCLOSPORINE IN RENAL TRANSPLANT PATIENTS
Influence of Everolimus on Steady-State
Pharmacokinetics of Cyclosporine
in Maintenance Renal Transplant Patients
Klemens Budde, Gustav Lehne, Michael Winkler, Lutz Renders, Arno Lison, Lutz Fritsche,
Jean-Paul Soulillou, Per Fauchald, Hans-Hellmut Neumayer, and Jaques Dantal,
on Behalf of the RADW 102 Renal Transplant Study Group
To investigate possible interactions of the novel
immunosuppressant everolimus with cyclosporine, a
multicenter, randomized, double-blind, placebo-controlled,
dose-escalating phase I study was performed. Everolimus
regimens (0.75-10 mg/d) were administered for 28 days to sta-
ble renal allograft recipients receiving the microemulsion
form of cyclosporine. Steady-state cyclosporine profiles were
assessed at baseline on day 0 (cyclosporine alone) and on day
21 with everolimus on steady state. By day 21, mean dose-
normalized cyclosporine AUC0-12 increased by 15% in pa-
tients receiving placebo. In everolimus-treated patients,
mean increases in cyclosporine AUC0-12 ranged from 7% to
T he novel macrocyclic immunosuppressant
everolimus (40-O-(hydroxy)ethyl-rapamycin) is a
promising candidate for potent adjunctive
immunosuppressive therapy in solid organ transplan-
From the University Clinic Charité, Berlin, Germany (Dr Budde, Dr Fritsche,
Dr Neumayer); Nephrology Service, CHU Hotel Dieu, Nantes, France (Dr
Soulillou, Dr Dantal); Rikshospitalet, Oslo, Norway (Dr Lehne, Dr
Fauchald); Medizinische Hochschule, Hannover, Germany (Dr Winkler);
Universitätsklinikum Kiel, Germany (Dr Renders); and Zentral-
krankenhaus, Bremen, Germany (Dr Lison). The study group contains the
following additional members: L. Lerat (Nantes, France), K. Nordal (Oslo,
Norway), L. Müller, R. Brunkhost (Hannover, Germany), I. A. Hauser
(Frankfurt, Germany), B. Charpentier (Safety Board member, Le Kremlin
Bicêntre, France), P. McMaster (Safety Board member, Birmingham, United
Kingdom), Albrecht-Georg Schmidt, Annette Jappe, Nathalie Cambon,
Janet Von Fellenberg, Khazal Paradis, Silke Appel-Dingemanse, Francois
Legay (Basel, Switzerland), and Christophe Gerbeau (Paris, France). Ad-
dress for reprints: Klemens Budde, University Clinic Charité, Schumannstr.
20/21, Berlin 10098, Germany.
DOI: 10.1177/0091270005277196
J Clin Pharmacol 2005;45:781-791
43%, which were not significantly different across all dosing
cohorts including placebo. Linear regression of everolimus
AUC on day 21 versus the increase in cyclosporine AUC0-12
yielded a slope not significantly different from a horizontal
line (P = ns). In conclusion, these results suggest that steady-
state everolimus exposure over the wide range assessed
in this study did not affect steady-state cyclosporine
pharmacokinetics.
Keywords: Cyclosporine; everolimus; pharmacokinetics; re-
nal transplantation
Journal of Clinical Pharmacology, 2005;45:781-791
©2005 the American College of Clinical Pharmacology
tation.1,2 In experimental models, everolimus was syn-
ergistic with cyclosporine (CsA) in the prevention of
acute and chronic rejection and has been approved in
several countries in combination therapy with CsA af-
ter kidney and heart transplantation.1-3 Everolimus be-
longs to the same drug class as sirolimus, but relatively
minor structural differences between the 2 agents con-
fer different pharmacokinetic characteristics. 4-8
Everolimus has a shorter half-life (approximately 24
hours vs 60 hours), exhibits more stable/consistent ab-
sorption characteristics than oral sirolimus does,4-8 and
may exert different effects on microsomal and mito-
chondrial metabolism.9,10
Sirolimus and everolimus have potent immunosup-
pressive activity and have a mode of action different
from that of CsA and other classes of immuno-
suppressants.1,11 They target the signal transduction
pathway involved in cell-cycle progression by inhibit-
ing the mammalian target of rapamycin (mTOR), thus
leading to subsequent inhibition of interleukin-2 (IL-
781
 BUDDE ET AL
2)–induced T-cell proliferation as well as inhibition of
general growth-factor–dependent proliferation of
hematopoietic and nonhematopoietic cells, including
vascular smooth muscle cells.11,12 Chronic rejection has
been attributed to proliferation of vascular smooth
muscle cells.12 Therefore, improved immunosup-
pression and a direct effect of these drugs on smooth
muscle cell proliferation may have an impact on long-
term outcomes.12
Sirolimus, the first drug of the class of mTOR inhibi-
tors, is primarily metabolized through the hepatic
cytochrome P450 (CYP) 3A4 enzyme system, which is
shared with CsA mutual microsomal metabolism.7-10 In
addition, both drugs are substrates for p-glycoprotein
countertransport.7-10 Not surprising, several studies
have demonstrated a strong pharmacokinetic interac-
tion between sirolimus and CsA.13-20 Interestingly, CsA
pharmacokinetics is unaffected by sirolimus in renal
transplant recipients.13,14 In contrast, CsA administra-
tion resulted in markedly (1.8- to 2.3-fold) elevated
sirolimus exposure20; even the timing of CsA dosing af-
fected sirolimus pharmacokinetics. Whole-blood
trough levels increased by about 30% with concomi-
tant dosing; the time to peak levels was also shorter (1.8
vs 2.5 hours) compared to timely separated administra-
tion.20 As a consequence, sirolimus should be given 4
hours after CsA according to the dosing recommenda-
tions. No such effect of timing of drug administration,
however, could be found on CsA pharmacokinetic
parameters.20
Since everolimus is intended to be used with CsA
and both compounds are metabolized in part by
CYP3A4,9,10 the potential influence of everolimus on
CsA pharmacokinetics needs to be investigated. This is
the first study to investigate the tablet formulation of
everolimus across a wide range of doses, unlike the
previously reported phase I single-dose study5 and a
multiple-dose study that used in development a ser-
vice capsule formulation6 that will not be commer-
cially available. This is of particular importance as the
tablet has a 2.6-fold higher bioavailability compared to
the service capsule.21 The aim of the present study was
to assess the effect of steady-state everolimus
coadministration over a wide dose range on detailed
steady-state pharmacokinetics of CsA in stable re-
nal allograft recipients. Unlike previously reported
studies, 22-26 complete 12-hour CsA pharmaco-
kinetics in stable renal allograft recipients receiv-
ing the microemulsion formulation of CsA was as-
sessed, as it is well known that CsA exposure varies
over time22 and trough levels are a poor indicator of
CsA exposure.27
782 • J Clin Pharmacol 2005;45:781-791
MATERIAL AND METHODS
Patients
Stable recipients of a primary renal transplant
(cadaveric or living donor) were eligible for inclusion
in the study. All subjects gave written informed con-
sent prior to inclusion. The study was conducted in ac-
cordance with good clinical practice guidelines and in
line with the Declaration of Helsinki. The protocol was
approved by the Ethics Committee at each center (see
the appendix). For at least 3 months before enrollment
into the study, all patients were receiving the
microemulsion formulation of CsA (Neoral; Novartis
Pharma, Basel, Switzerland) sufficient to produce
morning trough levels of 80 to 200 ng/mL. For inclu-
sion in the study, women had to be practicing an ac-
cepted method of birth control. Individuals were ex-
cluded from the study if they had received any other
immunosuppressive medication within 4 months of
baseline. Any treatment interfering with CsA
pharmacokinetics was not allowed for 4 weeks prior to
enrollment. The detailed study protocol and patient
characteristics were published recently.21 In brief, a to-
tal of 54 patients were randomized into 1 of the 7 differ-
ent treatment groups, 44 subjects received everolimus
(Novartis Pharma, Basel, Switzerland), and 10 patients
received placebo.
Study Design
This was a randomized, double-blind, placebo-
controlled, dose-escalation study conducted at 6 cen-
ters. Subjects fulfilling the entry criteria were ran-
domly assigned in a 3:1 ratio to either 1 of 4 everolimus
dose groups or placebo. This comprised 7 treatment
groups to examine the effect of dose level, dosing fre-
quency (once daily [QD] or twice daily [BID]), and for-
mulation (capsule and tablet at 0.75 mg QD). Dose
groups (0.75, 5, and 10 mg/d) were initiated sequen-
tially at weekly intervals so that a preliminary assess-
ment of safety and tolerability could be made at each
dose level prior to escalation to the next dose level.
Safety and tolerability were monitored in an ongoing
manner throughout the study by an independent, un-
blinded Safety Monitoring Board constituted specifi-
cally for this study. An additional dose group (2.5 mg
QD, open label) was added toward the end of the study.
In addition, 4 patients were added in an open-label
fashion to the 2.5-mg-BID group to further assess the
safety and tolerability of the 5-mg daily dose, given
concomitantly with cyclosporine in a BID dosing regi-
 EVEROLIMUS AND CYCLOSPORINE IN RENAL TRANSPLANT PATIENTS
men. Treatment at each dose level was administered for
4 weeks. At the baseline visit (before administration of
study drug, while the patients were hospitalized for the
baseline CsA pharmacokinetic profile), creatinine
clearance was determined from a 24-hour urine sam-
pling period using the local laboratory at each center.
Pneumocystis carinii prophylaxis with cotrimoxazole/
sulfamethoxazole was mandated during the course of
the study. This study was of an exploratory design; that
is, the study was not powered to address a specific sta-
tistical hypothesis. The results of the safety and
pharmacokinetic exploration of everolimus were
published separately.21
Pharmacokinetic Assessment
Morning trough whole-blood levels (Cmin) of CsA were
measured at 1- to 3-day intervals throughout the study.
Steady-state CsA pharmacokinetic profiles over the 12-
hour dosing interval were obtained at baseline in the
absence of everolimus and on day 21 during
everolimus coadministration when steady-state
everolimus blood concentrations would have been at-
tained. For CsA profiling, blood samples were drawn
predose and after 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8,
10, and 12 hours. Full pharmacokinetic profiles of
everolimus over a 12- or 24-hour dosing interval were
obtained on days 1 and 21. For everolimus profiles,
blood samples were obtained from a forearm vein via
an indwelling cannula predose and then 0.25, 0.5, 0.75,
1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 8, 10, 12, 16, and 24
hours thereafter. On profiling occasions, patients had
an overnight fasting period of at least 12 hours, and af-
ter administration of the medication, they remained
fasting for an additional 4 hours; only water was al-
lowed in this period. All blood samples were drawn
into EDTA-coated collection tubes, gently inverted sev-
eral times, frozen at –20°C or below, and analyzed in a
central laboratory.
Whole-blood everolimus concentrations were deter-
mined in duplicate by a validated enzyme-linked
immunosorbent assay method.5,6 Assay performance
was based on a 7-point calibration curve (1.6 to 100 ng/
mL) and 5 quality control concentrations (2 to 80 ng/
mL) determined with each assay run. Assay precision
(coefficients of variation) ranged from 11.2% to 26.3%,
and accuracy (deviation from nominal value) ranged
from –1.6% to –8.8%. The limit of quantification was 2
ng/mL. CsA was quantified in whole blood by a com-
mercially available radioimmunoassay using a
monoclonal antibody specific for the parent com-
pound (INCSTAR Cyclo-Trac SP, Stillwater, Minn). As-
say performance was based on a 7-point calibration
DRUG INTERACTIONS
curve (25-2500 ng/mL) and 5 quality control concen-
trations (30-1600 ng/mL). The precision of quality
control samples (coefficients of variation) ranged from
5.1% to 13.5%, and the assay quantification limit was
25 ng/mL.
Statistical Evaluation
Standard noncompartmental pharmacokinetic param-
eters were derived including the peak concentration
(Cmax) and the time of its occurrence (tmax), the area un-
der the concentration-time curve over the dosing inter-
val (AUC), and the percentage peak-trough fluctuation
(PTF). Attainment of steady state for everolimus and
CsA was assessed by linear regression analysis of the
serial trough concentrations over time. A slope not sig-
nificantly different from zero (a horizontal line) was
taken as evidence for steady-state conditions. Serial
CsA trough concentrations were plotted for each
everolimus dosing group and inspected for trends over
the study duration. Linear regression analysis was per-
formed for each patient’s troughs over time. A signifi-
cant positive slope was interpreted as a rise in CsA
exposure over the course of the study.
The influence of everolimus on CsA pharmaco-
kinetics was explored by an analysis of variance
(ANOVA) on dose-normalized CsA parameters from
baseline (without everolimus) and day 21 (under
steady-state everolimus). The ANOVA included terms
for everolimus dose level (including placebo), patient
nested within everolimus dose level, study day (days 0
and 21), and day by everolimus dose-level interaction.
This model explored whether differences in CsA
pharmacokinetic parameters between baseline in the
absence of everolimus and day 21 during everolimus
coadministration were different among the everolimus
dose levels including placebo. Pharmacokinetic pa-
rameter ratios (coadministration on day 21/baseline
on day 0) were also derived and compared among
everolimus dosing levels in a 1-way ANOVA. Linear
regression analysis was performed on everolimus
AUC on day 21 versus the CsA AUC ratio. A slope
not significantly different from zero was taken as evi-
dence that everolimus had no influence on CsA
pharmacokinetics.
RESULTS
Subjects
A total of 54 subjects were randomized for treatment
with everolimus (n = 44) or placebo (n = 10). There
were no significant demographic differences between
783
 BUDDE ET AL

different treatment groups and placebo (Table I). The Table I Baseline Demographics and Medical
median age was 47 years (range, 25-66), the majority of Characteristics
patients were Caucasian (93%) and male (69%), and no
black patients participated. They were at least 6 Everolimus Placebo
months posttransplant (mean = 5.3 years) and had sta- Variable (n = 44) (n = 10)
ble serum creatinine levels (mean = 147 ± 46 µmol/L;
Age, y
everolimus 149 ± 49 µmol/L; placebo 139 ± 42 µmol/L).
Median (range) 47 (25-66) 46 (25-57)
The average glomerular filtration rate (using 24-hour
Gender, %
urine collection) was 68 ± 22 mL/min (range, 28-128) at
Male:female 68:32 70:30
baseline. As shown in Table I, the immunosuppressive
Race, % Caucasian 93 90
therapy mainly consisted of CsA and prednisone; the
Weight, kg 69 ±14 75 ± 6
mean prednisone dose was 6.7 mg/d (everolimus 6.9 ±
Height, cm 170 ± 10 172 ± 9
1.7 mg/d; placebo 6.0 ± 1.8 mg/d). Eighty percent of the
Years since transplantation 5.3 ± 4.5 5.1 ± 4.9
patients were hypertensive, and only 2 diabetic pa- Baseline creatinine clearance, 66 ± 20 80 ± 28
tients (4%) were included. Thus, the most frequent mL/min
concomitant medications were antihypertensive drugs Medical disorders, %
(Table I). In general, the patients’ usual medication re- Hypertension 79.5 80
mained unchanged over the study period. Pre- Diabetes 4.5 0
transplant and transplant history as well as past or co- Concomitant medication, %
existing medical conditions revealed no specific Glucocorticoids 93.2 100
clustering in certain treatment groups and reflected the Calcium channel blockers 47.7 30
intended target population. Treatment groups were (dihydropyridines)
matched for demographic data, prior nonimmunosup- β-Blockers 45.5 50
pressant medications, kidney transplant history, and Diuretics 22.7 20
background medical characteristics, as reported Angiotensin-converting 18.2 10
elsewhere in greater detail.21 enzyme inhibitors
Eight (80%) of the 10 patients in the placebo group α-Blockers 13.6 10
and 29 (80.5%) of the 36 patients in the everolimus Statins 27.3 10
dose group up to 5 mg/d received study drug for the full
duration of treatment, while only 1 patient (12.5%) of
the 8 enrolled in the 10-mg dose group completed the treatment discontinuations nor serious AEs were
study. If samples for pharmacokinetic analysis were reported for the placebo group. One (10%) subject in
obtained, these were included in the results, even if the the placebo group withdrew because of a protocol
patients discontinued the study. A total of 9 serious ad- violation.
verse events (AEs) were reported in 8 everolimus-
treated patients, consisting of 2 viral infections and 1 Cyclosporine Dosing
case each of gastroenteritis, pneumonia, intestinal ob-
struction, myocardial infarction, stomatitis, increased Initial CsA doses were comparable in each of the
creatinine, and thrombopenia, leading to 4 treatment everolimus dosing cohorts: in the placebo group, the
discontinuations. Treatment discontinuations in the mean dose was 107 ± 36 mg BID and among the patients
everolimus group were due to AEs in 11 subjects, and receiving everolimus, the mean cohort doses ranged
in a further 3 subjects, discontinuation was due to the from 100 ± 50 (0.75 mg QD), 133 ± 44 (2.5 mg QD), 133 ±
decision of the Safety Monitoring Board to discontinue 17 (5 mg QD), 135 ± 45 (2.5 mg BID), to 137 ± 40 (0.75
treatment at the 10-mg/d dose level. AEs leading to dis- mg QD capsule mg BID). At the end of the study, the
continuation were thrombocytopenia (n = 4), infection mean CsA dose differed not significantly from starting
(n = 2), stomatitis and thrombocytopenia, hypertension doses (placebo: 107 ± 36; 0.75-mg capsule: 133 ± 33;
and headache, increased serum creatinine, intestinal 0.75 mg QD: 95 ± 48; 2.5 mg QD: 129 ± 51; 5 mg QD: 133
obstruction (each n = 1), and a patient with gastritis, ± 17; 2.5 mg BID: 129 ± 47; 10 mg QD (n = 1): unchanged
atrial fibrillation, edema, and elevated lipids. Most AEs 150 mg BID).
leading to discontinuation (especially thrombo- Of the 52 patients providing CsA concentration-
cytopenia) occurred in the highest dosage groups.21 time data (profiles and/or troughs), 38 (73%) main-
The highest dosage group was therefore prematurely tained the same dosage schedule throughout the trial.
terminated by the Safety Monitoring Board. Neither The remaining 14 patients had dose reductions of 20

784 • J Clin Pharmacol 2005;45:781-791

 EVEROLIMUS AND CYCLOSPORINE IN RENAL TRANSPLANT PATIENTS
250
ng/ml
200
CsA trough level
150
100
0.75mg capsule
0.75mg tablet
2.5mg QD
50
0 7
Figure 1. Synoptic view of mean cyclosporine trough concentrations in patients receiving different doses of everolimus or placebo.
mg (n = 1), 25 mg (n = 4), 50 mg (n = 8), and 75 mg (n =
1). These dose reductions were started roughly equally
in each study week: 3 in week 1, 5 in week 2, 2 in week
3, and 4 in week 4. While no patient randomized to re-
ceive placebo had a CsA dose alteration, the 14 dose re-
ductions were equally spread among the rising
everolimus dose levels.
Serial Trough Concentrations
Serial trough concentrations were available from 52
patients. Not surprisingly, CsA trough levels exhibited
inter- and intrapatient variability. For patients without
a dose change, the intrapatient coefficient of variation
was calculated. Placebo-treated patients had a coeffi-
cient of variation of CsA trough levels of 16.8% ± 6.1%,
similar to everolimus-treated patients (16.0% ± 7.5%).
Synoptic views of mean CsA trough levels are shown in
Figure 1. For patients who withdrew from the study
DRUG INTERACTIONS
5.0mg QD placebo
10mg QD
2.5mg BID
14 21 28
day
prior to day 28 or who had a CsA dose reduction, con-
centrations up to the day of these events were used.
Linear regression was performed on through trough
levels over time to test for a significantly positive slope.
This was taken as an indication for a rise in CsA expo-
sure over the course of the trial in the absence of CsA
dose changes. Interestingly, there was a general rise in
troughs over time; indeed, the majority of patients re-
ceiving placebo (6 of 9, or 67%) had a significantly pos-
itive regression slope. The proportion of patients in
everolimus dosing cohorts for which regression slopes
were significantly positive was similar or lower com-
pared with the placebo group: 0/6 (0.75-mg QD cap-
sule), 3/6 (0.75 mg QD), 2/6 (2.5 mg QD), 5/6 (5 mg QD),
1/2 (10 mg QD), 7/11 (2.5 mg BID), and 2/6 (5 mg BID).
Across all dose levels including placebo, ANOVA did
not detect any differences in the magnitude of the
trough increases among everolimus dose levels or
placebo (P = .11).
785
 BUDDE ET AL

Table II Cyclosporine Pharmacokinetics

RAD-Placebo (n = 9) 0.75 mg QD (n = 5) 2.5 mg QD (n = 6) 5 mg QD (n = 5)
Parameter Day 0 Day 21 Day 0 Day 21 Day 0 Day 21 Day 0 Day 21

tmax, h 1.0 1.5 1.0 1.0 1.5 1.5 1.0 1.5

(1.0-2.0) (1.0-1.5) (1.0-1.5) (1.0-1.5) (1.0-1.5) (1.5-2.5) (1.0-1.5) (1.0-2.0)
Cmin, ng/mL 119 ± 62 133 ± 46 149 ± 56 163 ± 93 112 ± 26 117 ± 39 89 ± 26 140 ± 38
Cmin/dosea 1.12 ± 0.47 1.29 ± 0.37 1.56 ± 0.30 1.70 ± 0.62 0.95 ± 0.54 1.05 ± 0.54 0.67 ± 0.15 1.06 ± 0.28
Cmax, ng/mL 978 ± 284 1061 ± 267 1162 ± 560 1278 ± 725 1106 ± 444 843 ± 451 1176 ± 187 1621 ± 366
Cmax/dosea 9.9 ± 3.4 10.6 ± 3.4 11.8 ± 1.5 13.0 ± 2.1 8.5 ± 3.2 7.2 ± 3.8 8.9 ± 0.9 12.3 ± 2.9
C2h, ng/mL 681 ± 146 834 ± 339 829 ± 397 945 ± 599 794 ± 339 730 ± 328 717 ± 194 1080 ± 275
AUC12, 3470 ± 974 3942 ± 1064 4078 ± 1554 4774 ± 2448 3917 ± 1148 3664 ± 1102 3684 ± 775 5215 ± 824
ng•h/mL
AUC12/dosea 34.0 ± 8.8 38.9 ± 10.8 42.5 ± 5.2 49.9 ± 12.6 31.0 ± 11.5 32.1 ± 14.2 27.7 ± 4.0 39.5 ± 6.4
Peak-trough 309 ± 87 287 ± 43 293 ± 62 281 ± 62 290 ± 81 226 ± 74 362 ± 70 344 ± 88
fluctuation, %
Values are mean ± SD except for tmax, which is median (range).
a. Dose-normalized parameters are per milligram cyclosporine.

Cyclosporine Profiles average in patients receiving placebo and between

Paired CsA profiles from day 0 (CsA alone) and day 21
(CsA + everolimus) were evaluable from 9 patients re-
ceiving placebo and 32 patients receiving everolimus.
None of the patients randomized to receive 5 mg
everolimus BID remained in the study to day 21; hence,
paired CsA profiles were not available at this dose
level. Pharmacokinetic parameters are summarized in
Table II for 3 representative everolimus dose levels us-
ing the tablet formulation. The administration of the
capsule formulation yielded similar results (not
shown). The global ANOVA did not detect any group-
by-day interactions; hence, the main effects compari-
son was used to assess differences in parameters be-
tween days. No differences in tmax or PTF were noted
when CsA was coadministered with everolimus com-
pared with administration alone (P = .60 and .17, re-
spectively). Dose-normalized Cmin and AUC were sig-
nificantly increased (P < .01 for both) across all dose
groups (including placebo), while dose-normalized
Cmax showed a similar trend (P = .06). Representative
plots to examine the mean dose-normalized parame-
ters across 3 dosing groups and the placebo group on
the 2 profiling occasions are shown in Figure 2.
Cyclosporine Parameter Ratios
10% and 19% for the lower dose everolimus regimens
(0.75-2.5 mg QD). Of the 2 regimens delivering the
same total daily dose of 5 mg, there was a 58% increase
for 5 mg QD but a 28% increase for 2.5 mg BID; this lack
of an apparent dose relationship was due to individual
variability. At the highest dose level of 10 mg QD (n =
1), no change in CsA trough was noted. Similar pat-
terns were noted for dose-normalized Cmax and AUC. In
the placebo arm, these parameters rose on average by
11% and 15%, respectively, with similar increases at
all other everolimus dose levels with the apparent ex-
ception of 5 mg QD (38% and 43%). Nonetheless,
ANOVA did not detect any differences in the magni-
tude of the Cmax or AUC increases (P = .17 and .13, re-
spectively) across all dose levels including placebo.
The plot of dose-normalized AUC ratios is reproduced
in Figure 3. In agreement with the global ANOVA, CsA
peak-trough fluctuation was not altered upon
coadministration with everolimus (P = .17).
An additional analysis of the impact of everolimus
exposure on CsA disposition was based on linear re-
gression of everolimus AUC versus the
coadministration/baseline CsA AUC ratios (day 21/0).
As shown in Figure 4, the slope of the relationship was
not significantly different from that of a horizontal line
(P = .24).

An alternative approach to examine CsA changes DISCUSSION

across everolimus dose groups was based on the CsA
parameter ratios (ratio coadministration/baseline). This article reports the results of the first multiple oral
These data are summarized in Table III. Over the course dose clinical study of everolimus tablets. Everolimus
of the study, CsA trough concentrations rose by 24% on or placebo was administered as add-on therapy to

786 • J Clin Pharmacol 2005;45:781-791

 EVEROLIMUS AND CYCLOSPORINE IN RENAL TRANSPLANT PATIENTS

2000

RAD-placebo
CsA Conc (ng/mL)

1500

1000

500

0
0 2 4 6 8 10 12
Time (h)

2000 2000
CsA Conc (ng/mL)

CsA Conc (ng/mL)

0.75mg qd 2.5mg qd
1500 1500

1000 1000

500 500

0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
Time (h) Time (h)

2000
CsA Conc (ng/mL)

5mg qd
1500

1000

500

0
0 2 4 6 8 10 12
Time (h)

Figure 2. Mean (SD) cyclosporine plots comparing administration alone ( ) and with various everolimus regimens ( ).

Table III Cyclosporine Parameter Ratios

(Ratio Coadministration/Baseline)
RAD Placebo 0.75 mg QD Tablet 2.5 mg QD Tablet 5 mg QD Tablet
Parameter (n = 9) (n = 5) (n = 6) (n = 5)

Cmin/dose, ng/mL/mg 1.24 ± 0.30 1.10 ± 0.34 1.12 ± 0.32 1.58 ± 0.18
Cmax/dose, ng/mL/mg 1.11 ± 0.24 1.12 ± 0.22 0.93 ± 0.47 1.38 ± 0.26
AUC/dose, ng•h/mL/mg 1.15 ± 0.16 1.18 ± 0.28 1.07 ± 0.38 1.43 ± 0.16
Peak-trough fluctuation, % 0.98 ± 0.24 0.96 ± 0.10 0.80 ± 0.21 0.95 ± 0.13
A ratio of 1.00 implies no change in the respective cyclosporine parameter with everolimus or placebo coadministration. Values are mean ± SD.

maintenance renal transplant patients on a double teristics of subjects enrolled in the study matched those
immunosuppressive regimen consisting of CsA of the intended target population; however, it is impor-
microemulsion formulation and steroids. The charac- tant to note that no black patients were included in this

DRUG INTERACTIONS 787


2.00
1.75
AUC/Dose Ratio (Day 21/Day 0)
1.50
1.25
1.00
0.75
0.50
0.25
0.00
Placebo 0.75qd* 0.75qd 2.5qd 5qd
RAD regimen (mg)
Figure 3. Cyclosporine AUC ratios compared across everolimus
dosage levels and placebo cohorts. Shown are individual values ( )
and group means ( ). A ratio of 1.00 implies no change in
cyclosporine exposure with everolimus or placebo coadministration.
All everolimus regimens are with the tablet formulation except 0.75
mg QD (capsule).
trial. It is noteworthy that patients randomized to re-
ceive placebo had a significant increase in CsA expo-
sure over the course of the study on average by 15%. It
is likely that this reflects improved compliance while
participating in a controlled clinical trial. A similar
15% to 20% increase of CsA pharmacokinetic parame-
ters was noted in placebo-treated patients of a parallel
North American trial.6 As a consequence, changes in
CsA pharmacokinetics in patients receiving
everolimus needed to be assessed in comparison to the
placebo arm of the study. This was addressed at 3 levels
of increasing refinement: (1) CsA dose histories, (2)
comparison of CsA pharmacokinetic parameters across
everolimus dose groups, and (3) regression of
everolimus exposure versus the change in CsA
exposure.
No CsA dose reductions were performed in the pla-
cebo arm, and 28 of 44 patients (64%) randomized to
receive everolimus maintained the same CsA dose
throughout the study. The remaining 14 patients had
generally small dose reductions of ≤50 mg, probably re-
flecting the improved compliance during the course of
the study. Only 1 patient had a reduction of 75 mg.
Since these patients were evenly distributed across all
everolimus dose levels, increasing everolimus expo-
sure did not appear to have a strong influence on CsA
exposure based on CsA-dosing histories. This observa-
tion extends the previous report on 24 patients receiv-
ing the service capsule formulation of everolimus.6 No
dose changes under everolimus treatment were re-
ported in this smaller study, suggesting no major im-
788 • J Clin Pharmacol 2005;45:781-791
BUDDE ET AL
2.00
CsA AUC/Dose Ratio (Day 21/Day 0)
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
0 200 400 600 800 1000 1200
2.5bid 10qd RAD AUC(0-24) (ng.h/mL)
Figure 4. Linear regression to explore the relationship of
everolimus exposure and the change in cyclosporine AUC during QD
steady-state coadministration. Regression equation: Y = 0.0001 • X +
1.13; slope not significantly different from zero (P = .24).
pact on CsA pharmacokinetics under steady-state con-
ditions. However, the fact that all patients who re-
ceived CsA dosage adjustments were on active
everolimus and none on placebo and that all were CsA
dosage reductions might suggest that there was indeed
a minor interaction occurring. Unfortunately, even the
analysis of 2 large prospective trials with more than
580 patients each could not solve this question as the
mean CsA dose was about 9% lower in everolimus-
treated patients, but this reached statistical signifi-
cance only in the global trial, not in the American
trial.22 In this regard, it is important to note that smaller
drug interactions cannot be detected based on CsA-
dosing histories alone, mainly because CsA dose
changes depend on the fluctuating CsA trough levels
(with a coefficient of variation in this study of 14%-
20%), which precludes the detection of smaller
pharmacokinetic interactions.
We next analyzed CsA trough concentrations in
greater detail. To avoid any additional interassay vari-
ability, all CsA concentrations were determined in a
central laboratory. CsA trough concentration trajecto-
ries demonstrated a general rise in troughs over the
study duration in the majority of patients receiving pla-
cebo, suggesting better patient compliance during the
course of the clinical trial. In cohorts of patients receiv-
ing everolimus, a similar or even lower proportion of
patients had significant increases in trough levels.
Again, this observation is in line with the previously
reported North American study, in which 29% of pa-
tients had increasing CsA trough levels over the course
of the study.6 Furthermore, even large studies in de
novo renal allograft recipients failed to demonstrate a
clear effect of low everolimus doses (1.5-3 mg/d) on
 EVEROLIMUS AND CYCLOSPORINE IN RENAL TRANSPLANT PATIENTS
CsA trough level.22 Although dose-normalized Cmin was
approximately 10% higher in everolimus-treated pa-
tients, this difference reached statistical significance
only in the global trial and failed to reach statistical sig-
nificance in the American trial. Using a population
pharmacokinetic approach, it was found that
everolimus-treated patients had similar CsA trough
levels compared to mycophenolate mofetil–treated pa-
tients, and there was only a weak correlation between
everolimus exposure and CsA trough levels, suggesting
congregation of high absorber status and/or poor clear-
ance for both drugs.25 In addition, 3 de novo trials could
not demonstrate a significant impact of everolimus
dosing (0.5-4 mg) on CsA levels24,28,29; however, these
trials had no control group of patients not receiving
everolimus. The detailed analysis in our European co-
hort of stable maintenance patients provided further
evidence that everolimus, assessed over a wide dosing
range, has no major impact on CsA trough levels,
which would be expected to have a clinical meaning-
ful effect. Important to note is the rather large coeffi-
cient of variation (14%-20%) in consecutive CsA
trough level determinations, even in this closely moni-
tored, selected, and compliant study population. This
probably reflects the highly variable CsA pharma-
cokinetics and makes the detection of smaller changes
of CsA exposure based on trough level monitoring
difficult.27
Because CsA trough levels do not adequately reflect
CsA drug exposure,27 individual CsA pharmacokinetic
parameters from the full profiles were compared be-
tween baseline and coadministration with everolimus
across different everolimus dose groups. ANOVA de-
tected higher CsA exposure during coadministration
with everolimus; however, this was also detected in the
placebo arm. To more specifically address potential
differences among everolimus dose levels, the CsA
AUC ratios (coadministration/baseline) were com-
pared across groups. The mean ratio was 1.15 for pla-
cebo and did not significantly differ in the everolimus
dose groups in which the ratios ranged from 1.08 to
1.43. Finally, the above approaches categorize each pa-
tient’s everolimus exposure into the nominal
everolimus regimen they received, which may down-
play interindividual variation in the systemic
everolimus exposure. By contrast, linear regression
analysis incorporates everolimus exposure (AUC on
day 21) as a continuous variable. When regressed
against the change in CsA exposure, the relationship
was not different from a horizontal line over the full
range of everolimus AUCs achieved in this study. Us-
ing abbreviated pharmacokinetic profiles from 256 de
novo patients, Kovarik et al22 could not find differences
DRUG INTERACTIONS
between everolimus-treated patients and patients re-
ceiving mycophenolate mofetil with regard to CsA
AUC and Cmax. In 953 paired observations, they ob-
served only a weak correlation (r = 0.38) between
everolimus exposure and CsA exposure and only a
modest rise in dose-adjusted Cmin. The effect of
everolimus on CsA exposure was clearly much less
pronounced than the intra- and interindividual
variability of CsA.22
Within the limitations of the study, our comprehen-
sive and thorough evaluations over a broad range of
everolimus exposures suggest that steady-state CsA
pharmacokinetics was not influenced by steady-state
coadministration of everolimus to a significant degree
that would be expected to have a clinical effect, based
on what is known about CsA pharmacokinetics. This
conclusion is in agreement with preclinical pharma-
cokinetic studies in rats and monkeys9,30 and with pre-
vious clinical studies6,22,24,26 in which steady-state
coadministration of 0.5 to 7.5 mg everolimus with CsA
did not affect the pharmacokinetics of the latter. In
addition, single-dose administration of everolimus to
renal transplant patients5 or healthy volunteers23 re-
sulted in no interaction with CsA pharmacokin-
etics. Kirchner and colleagues31,32 performed a detailed
analysis of CsA metabolites in a small number of pa-
tients. They could not detect an impact of everolimus
on CsA metabolites after single-dose and multiple-dose
administration,31,32 which excluded a significant influ-
ence of everolimus on the time-concentration relation-
ship and the metabolism of CsA under steady-state
conditions. Finally, the single and multiple oral ad-
ministration of sirolimus did not reveal any pharmaco-
kinetic interaction on CsA blood concentrations13-15,20
in renal transplant recipients. Because both drugs are
structurally closely related and share most metabolic
pathways, this observation provides further evidence
that there is no significant drug interaction of evero-
limus on CsA pharmacokinetics, but this cannot rule
out pharmacodynamic interactions of both drugs.
Similar to tacrolimus and CsA, sirolimus and
everolimus are metabolized to some extent in the small
intestine and extensively in the liver via cytochrome
P450 3A4 enzymes and are countertransported in
the gut lumen by the multidrug efflux pump, p-
glycoprotein; these processes account for low
bioavailability and high pharmacokinetic variabil-
ity.2,4,13 As a consequence of this wide interpatient vari-
ability, it is not possible to rule out that some patients
have an interaction to everolimus. This has been de-
scribed for other drugs interacting with CsA, in which
some patients are more sensitive to the interaction than
are others (eg, serial metabolic inhibition studies with
789
 BUDDE ET AL
diltiazem33). Larger, specifically designed studies are
needed to further explore this issue.
As immunosuppressive doses and concentrations of
CsA in humans are usually 100-fold higher than those
of everolimus, the competition between both drugs for
elimination pathways would favor an influence of CsA
on everolimus compared with the reverse situation.
This indeed has been shown in animal models, in
which CsA increased the systemic exposure to
everolimus. Similarly, CsA seems to exert rather strong
effects on everolimus exposure in humans, as demon-
strated in a single-dose study.23 The effect of CsA seems
to be dependent on the CsA formulation, as Neoral ex-
hibited much stronger effects on everolimus exposure
(168% increase) compared with the previous CsA for-
mulation (Sandimmun; 74% increase). Because
everolimus was added to patients already receiving
CsA, there was no opportunity to examine the possible
effect of CsA on everolimus pharmacokinetics in this
study. Similar to everolimus, in preclinical models,
sirolimus increased the bioavailability of CsA by 2- to
3-fold,19 with an approximately 2-fold increase in
the CsA concentrations in rat tissues.9,16,18 From these
studies, it was suggested that the pharmacokinetic in-
teraction between both drugs might contribute to the
observed in vivo synergism. However, this pharmaco-
kinetic interaction with increased drug concentrations
in renal tissue might be responsible for the aggravation
of CsA-induced renal dysfunction, as has been sug-
gested by Podder et al.16 Whether these data derived
from experimental models transfer to humans is yet
unclear.
Despite their structural similarity and identical
mode of action, sirolimus and everolimus clearly differ
in their pharmacological metabolism.17 Although CYP
3A4 is mainly responsible for the metabolism of both
sirolimus and everolimus, the total intrinsic clearance
of the CYP-dependent formation of everolimus metab-
olites is 3-fold lower than for sirolimus, and some of the
main metabolic rate constants are drastically (up to 15-
fold) reduced.17 In contrast to sirolimus, everolimus de-
creased CsA concentration in rat brain mitochondria.9
Furthermore, sirolimus enhances CsA toxicity on rat
brain; everolimus, however, had no effect on CsA toxic-
ity and even improved some deleterious effects of CsA
on high-energy phosphate metabolism.9
Taken together, our data provide further evidence
that everolimus coadministration exhibits no relevant
effect on pharmacokinetics and CsA exposure in Cau-
casians that would be expected to have a clinical effect,
based on what we know today about CsA pharmaco-
kinetics. This observation clearly extends our knowl-
790 • J Clin Pharmacol 2005;45:781-791
edge on this interesting new immunosuppressive com-
pound, suggesting that everolimus could be used con-
comitantly with the microemulsion formulation of
CsA using either QD or the standard BID dosage. How-
ever, the present study did not address the question of
pharmacodynamic interactions, which might be re-
sponsible for the increased incidence of calcineurin-
related toxicity, seen in clinical studies, when
everolimus or sirolimus were coadministered with
CsA.1,3,10,16,28,29 Because of limitations of the present
study, which do not allow us to detect smaller changes
or changes in subpopulations (eg, black patients; pa-
tients who differ in their expression of CYP 3A or p-
glycoprotein), this finding will, however, need to be
confirmed over longer periods of coadministration in
broader patient populations.
This study was supported by Novartis Pharma AG, Basel, Swit-
zerland. The authors are grateful for the help of J. Kovarik in prepar-
ing the article.
APPENDIX
List of Institutional Review Boards
Ethik-Komitee der Medizinischen Hochschule
Hannover; 30623 Hannover, Germany
Ethikkommission, Universitätsklinikum Charité;
Schumannstrasse 20/21, 10177 Berlin, Germany
Der Dekan der Medizinischen Fakultät (Ethik-
kommission); Universitätsstrasse 40; 91054 Erlangen,
Germany
Ethikkommision des Landes Bremen, ZKH St.-
Jürgenstr.; 28205 Bremen, Germany
Comite consultatif de protection des persones; CHU
de Nantes, Immeuble Deurbroucq, 44093 Nantes,
France
Regional Komite for Medicinsk Forskningsetikk
Forskningsparken, Gaustadalleen 21 0371 Oslo,
Norway
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