A Randomized Controlled Trial of Late Conversion from CNI-Based to Sirolimus-Based Immunosuppression Following Renal Transplantation

Authors


*Corresponding author: Christopher JE Watson, cjew2@cam.ac.uk

Abstract

Maintenance immunosuppression with calcineurin inhibitors (CNIs) following renal transplantation is associated with nephrotoxicity and accelerated graft loss. We aimed to assess whether conversion to sirolimus-based immunosuppression would affect the progression of renal impairment.

In this single center, randomized controlled trial, 40 renal transplant recipients between 6 months and 8 years post-transplant were randomly assigned to remain on their CNI (cyclosporin or tacrolimus) or to switch to sirolimus. The primary outcome measure was change in glomerular filtration rate (GFR) measurement at 12 months. Analysis was by intention-to-treat.

Of the 40 patients randomized, 2 patients never took the study drugs and were excluded, leaving 19 patients per group. There was a significant change in GFR at 12 months following conversion to sirolimus (12.9 mL/min, 95% CI 6.1–19.7; p < 0.001). Following conversion, the principal adverse events were the development of rashes (68%), particularly acne, and mouth ulcers (32%). No patient in either group experienced an acute rejection episode.

In renal transplant recipients, a change in maintenance therapy from CNIs to sirolimus is associated with significant improvement in GFR at 12 months.

Introduction

Calcineurin inhibitors (CNIs) have been the main stay of immunosuppressive regimens in renal transplantation since their introduction over two decades ago (1). Although the use of cyclosporin and tacrolimus has markedly improved early graft outcome, both of these CNIs are nephrotoxic and it is well recognized that their long-term use contributes to deteriorating graft function. In the short-term, CNIs produce renal arteriolar vasoconstriction and a decrease in glomerular filtration rate (GFR) that is dose related and reversible (2–4). Long-term exposure to CNIs causes chronic nonreversible changes that are characterized by interstitial fibrosis and obliterative arteriolar changes due to fibrous intimal thickening (5). A recent study of renal transplants undergoing annual protocol biopsies showed histological evidence of CNI toxicity in all grafts by 10 years (6).

To avoid the problems of long-term CNI-nephrotoxicity, a variety of strategies have been explored. These include complete withdrawal of CNIs at some point in the post-transplant period leaving recipients on steroids and azathioprine, substitution of CNI with mycophenolate mofetil (MMF), or simply minimizing the cyclosporin maintenance dose (7–14). Although some success has been achieved with these strategies, withdrawal of CNIs, even as late as 1 year post-transplant, is often associated with acute rejection and the risk of late graft damage.

The availability of the potent immunosuppressant sirolimus offers the potential to substitute CNI therapy with a nonnephrotoxic agent whilst potentially avoiding the risk of acute rejection. Sirolimus has been shown to be effective as de novo therapy after renal transplantation (15,16), and as long-term maintenance therapy with steroids (17–20). It may also have a role as an effective substitute for CNI therapy late after transplantation to avoid further CNI nephrotoxicity(21–23), although its use in this setting has not been previously subjected to evaluation by a randomized controlled study.

We report here the results of an open-label single-center investigator-led study in which renal transplant recipients receiving CNI-based immunosuppression with sub-optimal graft function were randomized to switch to sirolimus-based immunosuppression or to remain on CNI therapy. We demonstrate that compared to maintenance on CNI therapy, switching to sirolimus results in a sustained improvement in graft function as determined by radio-isotope labelled EDTA GFR measurement.

Materials and Methods

Study design

The study was an investigator-led, open-label single-center randomized controlled trial designed to determine whether, in renal transplant recipients with sub-optimal graft function, conversion from CNI-based to sirolimus-based maintenance immunosuppressive therapy resulted in improved graft function.

The primary outcome measure was the change in GFR at 12 months compared to the time of randomization (baseline). Secondary end points were changes in serum creatinine and uric acid concentration, incidence of hypercholesterolemia and hypertriglyceridemia, therapy for hypertension, number of acute rejection episodes, dialysis requirement and mean 24-h blood pressure.

Patients

Adult recipients of kidney transplants performed between 6 months and 8 years previously receiving CNI-based maintenance treatment, were invited to participate. The 6-month time point was chosen to minimize the risk of early acute rejection, while the choice of 8 years was based on the half-life of a cadaver kidney allograft. The principal inclusion criterion was sub-optimal renal function, defined as a serum creatinine between 120 and 400 μmol/L (1.36–4.52 mg/dL). Patients with known allergies to macrolide antibiotics were excluded, as were patients experiencing an acute rejection episode within the preceding 2 months. Other exclusion criteria included histological evidence of recurrent renal disease, presence of a nonrenal transplant, untreated symptomatic hyperuricemia, untreated hypercholesterolemia or hypertriglyceridemia (out with the normal laboratory range for our institution), malignancy within the preceding 5 years (with the exception of adequately treated basal or squamous carcinoma of the skin). Entry of female patients was dependent on a negative pregnancy test, and where relevant, females were asked to use an effective method of contraception for the duration of the study.

Conversion protocol

Consenting patients were randomized to either continue CNI (tacrolimus or cyclosporin) therapy or to undergo an abrupt switch to sirolimus-based immunosuppression. Concomitant immunosuppression, such as prednisolone, azathioprine or mycophenolate was continued, but in those patients receiving mycophenolate, the dose was reduced to a maximum of 1g/day at the time of conversion to sirolimus to avoid mycophenolic acid toxicity. Patients randomized to switch to sirolimus received their last dose of CNI the evening before conversion, 8 mg sirolimus on the following day and 4 mg/day thereafter. The sirolimus dose was then adjusted according to 24-h whole blood trough concentration on days 4, 7 and 14 following conversion. Target range for sirolimus was between 5 and 15 ng/mL (measured by HPLC).

GFR was measured by six-point 51Cr-EDTA clearance, performed on the day of randomization (baseline), and at 3 and 12 months later. In addition, 24-h blood pressure measurement (Tracker NIBP2, Del Mar Reynolds Ltd., Hertford, UK) and full hematological and biochemical screening were undertaken at each of the three time points.

Statistical considerations

Although the principal end point was change in GFR, in the absence of data on which to make a power calculation change in serum creatinine was chosen as a surrogate. Available data on serum creatinine change following conversion to sirolimus were used to derive a sample size. A variety of scenarios were considered, ranging from a mean change in creatinine of 15 μmol/L (SD 20) to a mean change of 25 μmol/L (SD 40). It was felt that the anticipated difference in serum creatinine would be 20 μmol/L, with a standard deviation of 20. To detect such a within patient difference at the 5% level with 90% power would require 94 patients to be randomized. This would also allow detection of a 25 point difference in creatinine with a power of 80% if the SD was 40, and a 15 point difference with a power of 80% if the SD was 20. We acknowledged that such an approach was less precise than calculations based on GFR itself, but the error was likely to over estimate the number of subjects required to show a significant change in GFR. Nevertheless, the lack of good data necessitated a planned interim analysis to validate our assumptions.

When the single planned interim analysis suggested the magnitude of the effect on GFR was much greater than expected, recruitment to the study was stopped prematurely, since it was believed to be ethically unsound to continue randomizing patients to remain on CNIs. At this point, the mean difference in creatinine was −13 μmol/L, standard deviation 30 (ignoring values for patients on dialysis).

An a priori randomization sequence was determined by random numbers generated by a Microsoft Excel Software program, such that odd numbers were allocated to switch to sirolimus. The allocations were then placed in sequentially numbered sealed envelopes by a member of the research team, but concealed from the members who were involved in the enrolment of the participants. The subjects themselves opened the envelopes at the time of randomization. Neither patients nor clinicians were blinded as to therapy. The work was approved by the Local Research Ethics Committee.

For the purposes of the GFR analysis failed grafts, where the participants had returned to dialysis, were arbitrarily assigned a GFR of 10 mL/min, and patients were analyzed on an intention-to-treat basis. Mean values for each group were compared by t tests for independent samples; two-sided significance tests were used throughout.

Results

Entry and participant flow through the study are shown in Figure 1. A total of 40 patients were recruited between May 2002 and January 2004, of whom 38 proceeded beyond randomization. Forty-one patients refused to participate, and 34 were excluded for reasons other than those specified in the inclusion criteria, including receipt of paired paediatric kidneys (3), relocating (2), undergoing investigation for co-morbidity (8), needle-phobia (2), delayed decision until after study closure (8).

Figure 1.

Study flow diagram. The diagram illustrates the study enrolment and disposition of the trial participants.

The two study groups were comparable for age, time since transplant and baseline immunosuppression although there was a predominance of males in the group randomized to continue on CNI therapy (Table 1). The GFR measured at baseline was similar in both sirolimus and CNI groups (37.8 and 36.1 mL/min/1.73 m2, respectively). There was no significant difference in any biochemical or hematological parameter measured (data not shown).

Table 1.  Baseline characteristics of each group
 Sirolimus
n = 19
CNI
n = 19
Years post-transplant (mean, SD)3.2 (SD1.9)3.2 (SD2.3)
Age (mean, SD)46.6 (SD9.9)48.2 (SD10.5)
Gender
 Male1318
 Female61
Initial therapy
 Cyclosporin1612
 Tacrolimus37
Concomitant immunosuppression at baseline
 Prednisolone1818
 Azathioprine1613
 Mycophenolate24
Donor source
 Asystolic donor36
 Heart-beating donor109
 Living donor64
Mean HLA mismatch A : B : DR0.8 : 1.0 : 0.50.7 : 1.2 : 0.8
Creatinine (μmol/L) (mean, SD)190 (SD 73)207 (SD 56)
GFR (mL/min/1.73 m2) (mean, SD)37.8 (SD 11.1)36.1 (SD 9.3)

Two patients returned to dialysis during the course of the study, one in each group at 6 months (sirolimus) and 11 months (CNI) post-randomization. A second patient in the CNI group started dialysis immediately following his 12-month GFR measurement and is considered to have graft function at 12 months. One patient in the CNI group relocated and was lost to follow-up at 6 months; his data are included up to that point. Three patients discontinued sirolimus following randomization at 54, 59 and 104 days due to a painful acneiform rash, a rash and diarrhoea (also on MMF), and lethargy respectively. All three were converted back to CNI-based immunosuppression and continued follow-up; their data are handled in an intention-to-treat basis. One patient in each group declined the 12-month GFR measurement, but other biochemical and hematological data were recorded at that time point.

Renal function (Figures 1,2and3)

Figure 2.

Box and Whisker plot showing 51Cr-EDTA-GFR's. Box and Whisker plot showing 51Cr-EDTA-GFR at baseline and 3 and 12 months following randomization for participants allocated to remain on CNIs (red boxes) and allocated to switch to sirolimus (blue boxes). The boxes enclose the interquartile range, with the median value denoted by the horizontal line. The whiskers enclose the range of values, with the exception of the outliers denoted by small circles. There is a significant difference between values at 3 and 12 months.

Figure 3.

Mean change in GFR from Baseline. Mean (±95% CI) change in GFR from baseline at 3 and 12 months. There is a highly significant difference following conversion to sirolimus.

Baseline GFR's were similar in both groups (37.8 sirolimus, 36.1 CNI); 13 patients (68%) in each group had a GFR below 40 mL/min/1.73 m2. There was a highly significant difference in mean GFR of 7.9 mL/min at 3 months (p < 0.001, 95% CI 4.1– 11.7) and 12.9 mL/min at 12 months (p < 0.001, 95% CI 6.1– 19.7). This was characterized by a mean improvement in GFR of 8.5 mL/min in patients switching to sirolimus, most of which occurred within the first 3 months, and a mean fall of 4.3 mL/min in the patients remaining on CNIs (Figures 2 and 3). There was also a positive correlation between the baseline GFR and the improvement in GFR at 12 months in the sirolimus group (R2= 0.22, p = 0.05 intention-to-treat), suggesting that the better the initial renal function the greater the improvement in GFR following conversion to sirolimus (Figure 4). In spite of the clear difference in GFR, there was no significant change in serum creatinine between groups.

Figure 4.

Relationship between change in GFR at 12 months after randomization compared to baseline GFR. There is a correlation between baseline GFR and subsequent improvement for sirolimus-treated patients (R2= 0.22, p = 0.05), such that the better the initial renal function the greater the improvement in function following conversion.

There was no significant difference in mean changes in 24-h urinary protein between groups (Figure 5), with a median change of 0 and 0.1 g at 3 and 12 months in sirolimus-treated patients and 0 and 0.2 g in CNI-treated patients at the same time points (p = 0.41 and 0.86 for 3 and 12 month differences, respectively).

Figure 5.

Change in 24-h urinary protein from baseline to 12 months. There is no significant difference in the change in proteinuria 12 months after randomization to switch to sirolimus (blue triangles) or stay on CNIs (red circles).

No patient in either group experienced an episode of acute rejection during the study follow-up.

Immunosuppression

Following conversion to sirolimus, the median daily dose of sirolimus at 3 months was 4 mg (range: 1.5–5 mg), and at 12 months 3.5 mg (range: 1–5 mg). Whole blood concentrations were 8.8 ng/mL (range: 6.5–11 ng/mL) at 3 months, and 8.5 ng/mL (range: 4.9–12.5 ng/mL) at 12 months. Patients remaining on tacrolimus had median 12-h trough concentrations of 7.8 and 10.6 ng/mL at 3 and 12 months, respectively, while those on cyclosporin had median concentrations of 176 and 187 ng/mL.

Hematology

Table 2 illustrates the differences at 3 and 12 months in the secondary end points in the study, namely differences in hematological variables between groups. There was a significant fall in hemoglobin in patients on sirolimus at 3 months (mean difference 0.8 g/dL, 95% CI −0.1 to −1.6, p = 0.02), but there was no difference at 12 months. The total leucocyte count fell significantly at 3 and 12 months in patients on sirolimus (p = 0.002 and 0.04, respectively). This was accounted for by significant falls in neutrophils at both time points (p = 0.01 and 0.04, respectively) and a significant fall in lymphocyte count at 3 months (p = 0.04, 95% CI −0.02 to −0.48). There was no change in eosinophil count. Likewise, there was no significant difference in the change of platelet count between groups at either time point.

Table 2.  Changes in hematology variables from baseline
 Months
post-randomization
Change from baselineMean difference
between groups (95% CI)
p-value
CNISirolimus
Hemoglobin (gm/dL)3−0.1−1.0−0.8 (−0.1 to −1.6)0.02
12−0.2−0.60.4 (−0.5 to +1.2)0.47
Leucocyte count (×109/L)3−0.03−1.43−1.4 (+2.25 to −0.55)0.002
12+0.11−1.08−1.19 (−2.30 to −0.09)0.04
Neutrophil count (×109/L)3−0.10−1.18−1.08 (−1.89 to −0.27)0.01
12−0.06−1.02−1.08 (−2.1 to −0.07)0.04
Lymphocyte count (×109/L)30.07−0.18−0.25 (−0.02 to −0.48)0.04
12−0.07−0.11−0.04 (−0.26 to +0.19)0.76
Platelet count (×109/L)3+13−1−14 (−43 to +13)0.29
12−8−17−9 (−32 to +15)0.47

Biochemistry

The significant changes in biochemistry parameters are illustrated in Table 3. There was a significant fall in uric acid, potassium and urea in the sirolimus-treated group. There were also significant changes in bilirubin, ALP and ALT between groups. There were no significant differences in the between group changes in serum creatinine, triglyceride, total, HDL or LDL cholesterol concentrations, although there was a difference in usage of statin therapy. At baseline, 8 CNI patients and 11 sirolimus patients were on statins, and 12 months later, this had changed to 9 CNI patients and 15 sirolimus patients (p = 0.04, Fisher's exact test).

Table 3.  Changes in Biochemistry variables from baseline
 Months
post-randomization
Change from baselineMean difference
between groups (95% CI)
p-value
CNISirolimus
Bilirubin (μmol/L)30−6−6 (−10 to −2)0.003
12−1−5−4 (−8 to 0)0.03
Alanine transaminase (iu/L)3−1+910 (+5 to +15)<0.001
12−1+1819 (+6 to +32)0.005
Alkaline phosphatase (iu/L)3−10+313 (+3 to +24)0.015
12−22−121 (−6 to 50)0.12
Uric acid (mmol/L)3+0.01−0.08−0.09 (−0.13 to −0.05)<0.001
120−0.09−0.09 (−0.17 to −0.04)0.012
Potassium (mmol/L)3+0.1−0.5−0.6 (0.8 to −0.3)<0.001
12+0.2−0.4−0.6 (−1.0 to −0.3)0.001
Urea (mmol/L)3−0.6−3.2−2.6 (−4.4 to −0.8)0.006
12+3.7−2.6−6.3 (−11.3 to −1.3)0.015
Creatinine (μmol/L)3–6−18−12 (−26 to +1)0.075
12+48−19−67 (−148 to 14)0.10

Blood pressure

There was no significant difference in the mean change in systolic or diastolic 24-h blood pressure. There was a nonsignificant change in treatment for hypertension, with three of the sirolimus patients requiring reduction in anti-hypertensive therapy compared to the CNI group where four patients required increase in therapy and three a reduction in therapy.

Adverse events

Adverse events are summarized in Table 4. There was a high incidence of rashes, particularly acneiform rashes in patients following conversion to sirolimus. Most resolved with dose reduction although two patients discontinued sirolimus on their account. Likewise, one third of patients experienced aphthous-type mouth ulcers that resolved over the course of the first 2 weeks with dose adjustment to the lower end of the target range. All three females under 40 who were switched to sirolimus developed amenorrhea for varying duration before resumption of irregular menses. Gynecological investigation revealed concentrations of lutenising hormone (LH), follicle stimulating hormone (FSH) and oestradiol that were within the normal range for the follicular phase.

Table 4.  Principal side effects following randomization
Side
effects
CNI
(n = 19)
Sirolimus
(n = 19)
Acute gout21
Bone pain23
Chest infection24
Coryza17
Diarrhoea46
Dysmenorrhoea03
Epistaxis13
Fatigue58
Gum hypertrophy50
Headache/migraine04
Herpes stomatitis12
Hypercholesterolemia
 Requiring new statin therapy26
 Requiring increased statin therapy44
Hypertension
 Treatment increased40
 Treatment reduced33
Indigestion32
Leg oedema44
Mouth ulcers06
Pulmonary embolism01
Rash
 Acne15
 Other18
Urinary infection46
Vomiting22

Discussion

This randomized controlled study demonstrates that in recipients with sub-optimal graft function conversion to sirolimus within 8 years of renal transplantation results in a significant improvement in graft function, as measured by 51Cr-EDTA GFR. The improvement in GFR after switching to sirolimus was evident at 3 months and maintained at 12 months, and was most evident in those patients with better baseline function, although even patients with poor graft function realized some benefit. Those patients remaining on CNIs suffered deterioration of renal function.

Although the findings of this study are clear cut in terms of the primary endpoint, the number of patients recruited is around half of that originally anticipated. The early closure of the study was justified by the results of a planned interim analysis undertaken after 18 months of recruitment, indicating that further recruitment was unlikely to alter the conclusion significantly given the separation of the 95% confidence limits in measured GFR. Nevertheless, we acknowledge that premature termination of the study, and relatively small numbers may represent a weakness of the study.

The findings from this randomized trial are consistent with previous observations in nonrandomized studies of switching to sirolimus in the presence of impaired function in which improved renal function was noted (24–26). In contrast to previous reports, the present study involved an abrupt switch from CNI-based to sirolimus-based immunosuppression. The abrupt switch was chosen both for simplicity, and to avoid potential over immunosuppression during an overlap period with both sirolimus and CNI therapy, at a time when anti-microbial prophylaxis had been discontinued. Interestingly, the abrupt switch to sirolimus was not associated with acute rejection, in contrast to studies where CNIs were gradually discontinued (9,11,13) or replaced with other agents such as MMF (4,27–29).

Conversion to sirolimus was associated with a high incidence of side effects, particularly skin rash and mouth ulceration. In most cases, these were successfully managed with dose reduction, keeping the sirolimus concentration between 5 and 8 ng/mL. Selection of the initial dose of sirolimus was based on our 10-year experience of the drug, and we were reassured to see that at 3- and 12-month time points, the median dose remained around 4 mg (4 and 3.5 mg, respectively). This is in contrast to our own experience in liver transplantation where a dose of 2 mg achieves concentrations in the same target range. Although an 8 mg loading dose of sirolimus was given to achieve the target range rapidly, this may have contributed to the early side effects observed and in retrospect, the loading dose may be best avoided in this setting, as is our current practice.

Other side effects that were observed after switching included the typical biochemical and hematological changes associated with sirolimus. No patient required supplementary erythropoietin for the treatment of anemia, and reduced leucocyte counts were not associated with any excess in infections save for a high incidence of coryza in the year follow-up period. Raised cholesterol and triglycerides were not significant at 3 or 12 months, but extra therapy was required more often in the sirolimus patients. Reduced potassium and uric acid concentrations were seen as benefits in those patients in whom they occurred, particularly the reduction in uric acid. One of the more notable adverse effects seen was amenorrhea that occurred in all three females under the age of 40 years. Sirolimus inhibits the action of VEGF and inhibits angiogenesis (30), and we speculate that this effect may result in inhibition of endometrial proliferation. An alternative explanation for the observed amenorrhea is that it represents a sirolimus effect on the female hypothalamo-pituitary-gonadal axis. In male heart and renal transplant recipients, sirolimus is associated with lower levels of serum testosterone, with higher levels of FSH and LH than case-controlled patients taking CNIs (31,32).

In the present study, there was no evidence that switching to sirolimus led to a significant increase in urinary protein loss, and no patient developed proteinuria in the nephrotic range. This finding contrasts with previously reported uncontrolled data suggesting that switching to sirolimus markedly increases urinary protein loss (33). The lack of control data illustrating the natural history of proteinuria in these failing grafts may have contributed to this impression. In addition, the target concentration of sirolimus used in our study is lower than in other studies, and we did not overlap CNI and sirolimus during the conversion period, since it is well recognized that combined sirolimus and CNI therapy enhances CNI-nephrotoxicity. An alternative explanation to the lack of significant proteinuria in our study is that the patients that switched to sirolimus all had low levels of proteinuria at baseline, and there is some evidence that heavy proteinuria might be more common in patients who have significant proteinuria at the time of conversion.

Patients in the present study were converted to sirolimus between 6 months and 8 years following renal transplantation. The most appropriate timing of conversion to sirolimus is unknown, but in order to minimize the structural changes and characteristic of CNI toxicity, it seems logical to switch within the first year. On the basis of these findings, we feel there is a good case for converting patients with sub-optimal graft function on CNI-therapy to sirolimus. It is not possible to say with certainty whether patients on CNI-therapy with good graft function after renal transplantation would also benefit from switching to sirolimus. Equally, the long-term outcome following conversion is unknown, but the preservation of renal function following conversion would suggest that graft survival may be extended.

In conclusion, this study has demonstrated that recipients on CNI-based immunosuppression with impaired graft function after renal transplantation show improved GFR after switching to sirolimus. Switching to sirolimus is not associated with acute rejection, and although side effects such as skin rash and mouth ulceration are common, they can usually be managed by careful attention to dosage.

Acknowledgments

The authors would like to acknowledge Linda Cartmel, the Nuclear Medicine Department at Addenbrooke's Hospital, and Addenbrooke's Centre for Clinical Investigation for their help during the conduct of the study. In addition, we are grateful to Mike Allen and George Stanley for their help in setting up the study.

This study was supported by Wyeth Laboratories, Taplow, Maidenhead, U.K.

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