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- Materials and Methods
We performed a randomized trial to compare two regimens of low-risk kidney allograft recipients in the first year after transplantation. Both regimens initially included sirolimus, tacrolimus and steroids; one with long-term maintenance with these drugs vs. tacrolimus withdrawal. Group I: sirolimus levels of 4–8 ng/mL, plus tacrolimus 8–12 ng/mL for 3 months, and 5–10 ng/mL after month 3. Group II: sirolimus concentration of 8–16 ng/mL, plus tacrolimus 3–8 ng/mL with tacrolimus elimination from month 3 onwards. Owing to difficulties in achieving target levels, the protocol was amended to increase the doses. Eighty-seven patients were recruited. In the intention-to-treat analysis, glomerular filtration rate (GFR) at 12 months, adjusted to zero for graft loss, was similar in both groups (58.8 and 59.9 mL/min). Analysis of patients remaining on protocol showed that GFR was higher in group II only in the patients postamendment (58.4 and 72.9 mL/min, p = 0.03). Rates of biopsy-confirmed rejection (BCAR) were 9.3% and 22.7% in groups I and II, respectively (p = NS). After amendment, BCAR rates were 10.3% and 11.1% (p = NS). Diastolic blood pressure was significantly lower in patients who eliminated tacrolimus (80.4 vs. 75.6 mmHg) (p = 0.03). Combining sirolimus and tacrolimus with adequate loading doses was associated with a low incidence of BCAR, and allowed tacrolimus elimination in a high proportion of patients, which may be followed by amelioration in renal function and blood pressure.
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- Materials and Methods
Clinical experience with combined immunosuppression based on both sirolimus and tacrolimus in kidney transplantation is scarce. These two structurally related drugs were once thought to be antagonists based on two phenomena: the blocking effect of sirolimus upon tacrolimus-mediated inhibition of interleukin-2 messenger ribonucleic acid (mRNA) production in T-cell cultures (1) and the interaction of both drugs with the same immunophilin inside the T cells, FK-binding protein (FKBP-12; 2). Nevertheless, it seems that only a small fraction of the abundant FKBP-12 needs to be occupied by the drugs to reach maximal immunosuppression (3). The concomitant use of these two macrolides has also been supported by a model of vascularized heart allograft in rats, in which the combination of sirolimus and tacrolimus was shown to produce longer graft survival in the short term than each agent alone (4). Likewise, low doses of tacrolimus combined with sirolimus prolonged kidney allograft survival in monkeys without additive or synergistic side-effects (5).
Based on these findings, early experiences of this combination in solid organ transplantation were reported in Halifax (6). Unlike combinations with cyclosporine, pharmacokinetic studies of sirolimus plus tacrolimus show that neither drug influences the other, and therefore simultaneous administration seems advisable (7).
The combination of sirolimus, cyclosporine and steroids after transplantation, followed by cyclosporine withdrawal in month 3 onwards, seems to be a promising schedule to prevent early acute rejection while preserving allografts from the deleterious long-term nephrotoxicity of calcineurin inhibitors (8,9). Therefore, we performed a pilot randomized open-label trial to evaluate the feasibility of two combinations in the early postoperative period, one with low doses of sirolimus plus conventional doses of tacrolimus and the other with high doses of sirolimus plus low doses of tacrolimus. In addition, we tested whether tacrolimus can be withdrawn from the latter regimen to avoid long-term calcineurin inhibitor toxicity.
Materials and Methods
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- Materials and Methods
A multicentered open-label pilot randomized trial was conducted in seven Spanish transplant units to investigate two different schedules of combinations of sirolimus and tacrolimus in low-risk adult kidney cadaveric allograft recipients. The trial was approved by the Institutional Review Board of each center and written informed consent was obtained from all participants. Exclusion criteria comprised infection with human immunodeficiency virus, panel reactive antibodies with greater than 50% positive cells, donors younger than 9 or older than 65 years old, cold ischemic time longer than 36 h or nonheart-beating donors, infection with either hepatitis B or C virus with impairment in liver function tests (greater than 2.5-fold above the normal upper limit of aspartate aminotransferase or alanine aminotransferase) and a history of malignancy in the previous 10 years (except for successfully excised nonmelanocytic skin cancers). Second transplantations were allowed only if the first graft was maintained for at least 6 months or if the graft was lost owing to technical surgical causes. Women of childbearing age with a negative pregnancy test who agreed to use a medically acceptable contraceptive throughout the study were eligible. Patients were also required to have a white blood cell count ≥ 3 × 109/L, platelets ≥ 100 × 109/L, triglycerides ≤ 400 mg/dL (4.5 mmol/L) and fasting cholesterol ≤ 300 mg/dL (7.8 mmol/L).
Patients were randomly allocated in a 1: 1 proportion to one of two groups using computer-generated randomization envelopes prepared by Wyeth without stratification. The block size was not revealed to the investigator and the assigned therapy was not blinded. Group I patients received a loading dose of 6 mg of sirolimus on day 1, followed by a maintenance dose of 2 mg/day, which was subsequently adjusted to reach trough levels between 4 and 8 ng/mL measured by high-performance liquid chromatography (HPLC)/UV. Tacrolimus was administered at a dose of 0.1 mg/kDa/day, was adjusted to achieve trough levels of 8–12 ng/mL (immunoassay) for the first 3 months, and was then decreased to 5–10 ng/mL thereafter. Group II received a loading dose of 15 mg of sirolimus on day 1 followed by a maintenance dose of 5 mg/day to target HPLC levels of 8–16 ng/mL. In this group, tacrolimus was begun at 0.05 mg/kg/day and fitted to reduced levels of 3–8 ng/mL. Patients in group II showing stable renal function, and sirolimus trough levels of between 12 and 20 ng/mL that had not required treatment for acute rejection in the previous 3 weeks were withdrawn from tacrolimus from month 4 onwards. Thereafter, these patients received maintenance immunosuppression based on sirolimus levels of 12–20 ng/mL and steroids. Patients experiencing acute rejection were treated according to local practice, but were withdrawn from the study if they required immunosuppressants other than the steroids, antibodies or increased doses of drugs studied in the protocol. Owing to difficulties in achieving target sirolimus and tacrolimus concentrations in both groups, the protocol was amended to increase the drug doses. In group I, sirolimus was increased to 6 mg on days 1–3 and was then followed by 3 mg/day and tacrolimus at a dose of 0.2 mg/kg/day; in group II, sirolimus was increased to 15 mg on days 1–3, and to 6 mg/day thereafter combined with tacrolimus at a dose of 0.1 mg/kg/day. The use of antibodies was not allowed as induction therapy. In both arms, prednisone was administered at a dose of 0.25 mg/kg/day, tapering to 10 mg/day at the end of the first month and 5 mg/day from month 3 onwards. Tacrolimus was administered in two doses daily. Sirolimus was given as a single morning daily dose.
The primary end point of the study was renal function measured as serum creatinine and creatinine clearance calculated with the Cockcroft-Gault algorithm at 12 months. Secondary end points included the rate of biopsy-confirmed acute rejection (BCAR), graft and patient survival, and the toxicity of the combination. Acute rejection episodes were histologically graded according to the Banff 1997 criteria.
In this pilot study, a sample size of 45 patients in each group was estimated to have approximately 50% power to detect a difference in mean creatinine levels as small as 0.2 mg/dL at 12 months assuming a common standard deviation of 0.6 mg/dL (α= 0.05 two-sided significance level).
Statistical analyses were calculated in two populations: intention-to-treat analysis (ITT) was performed to evaluate renal function, blood pressure, graft and patient survival and cumulative BCAR and included all the patients who had received at least one dose of sirolimus, regardless of the drugs they were currently receiving in month 12. Therefore, graft losses, acute rejections and deaths occurring after discontinuation of the study were included in the analyses. No patient was lost to follow up for acute rejection, glomerular filtration rate and graft and patient survival. Graft survival was calculated assuming as events the death, return to dialysis or nephrectomy (whichever occurred first), and censoring patients at last contact. For the ITT analysis of patients with graft loss, calculated creatinine clearance was assigned a value of zero, while the value of serum creatinine was considered lost. Analysis of patients on protocol included all the patients who remained on the assigned drugs as per protocol at 12 months. Differences in adverse events and other categorical variables between the two arms were calculated with Fisher's exact test. Differences in quantitative values at all evolutionary time points were calculated with the Student's t-test, and differences in rates of graft and patient survival were measured with the Mann–Whitney test.
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- Materials and Methods
Patients were consecutively recruited from December 7, 2000 to January 21, 2002. Eighty-seven patients were enrolled in the trial (43 in group I and 44 in group II). The protocol amendment was applied to 64% of these patients (29 in group I and 27 in group II). Patient demographics and baseline characteristics including sex, age, etiology of end-stage renal disease, donor age, cold ischemia time, number of human leukocyte antigens mismatches, percentage of reactive antibodies, and number of second transplantations were similar between both groups (Table 1). Only one patient in each group was serologically positive for hepatitis C virus.
Table 1. Patient demographics and donor characteristics
| ||Group I (n = 43)||Group II (n = 44)|| P-value|
|Age (years)||47.4 (11.2)||45.2 (13.5)||NS|
|Race (Caucasian/non-Caucasian) (%)||97.7/2.3||93.2/6.8||NS|
|Sex (male/female) (%)||69.8/30.2||70.5/29.5||NS|
|Cause of end-stage renal disease (%)|| || ||NS|
| Hypertension|| 7.0||13.6|| |
| Diabetes mellitus|| 4.7|| 6.8|| |
| Polycystic kidney disease||18.6||20.5|| |
| Interstitial nephropathy|| 4.7||13.6|| |
| Glomerulonephritis||30.2||25.0|| |
| Other||13.9|| 6.8|| |
| Not defined/unknown||20.9||18.1|| |
|Second transplantations (%)|| 7||11.4||NS|
|Time on dialysis (months)||24.4 (18.8)||28.1 (27.6)||NS|
|Donor age (years)||42.9 (16.3)||40.7 (15.2)||NS|
|Cold ischemia time (h)||18.7(4.0)||17.9 (7.4)||NS|
|Number of HLA mismatches|| 3.6(0.7)|| 3.8 (0.7)||NS|
|Patient with PRA > 20 (%)|| 2.3|| 0.0||NS|
In 33 patients in group II (75%) tacrolimus was withdrawn at a median of 5 months after transplantation (95% CI: 4.1–6 months). Seven patients in group I and 18 patients in group II (28% of the whole group) did not complete the assigned treatment as described in the protocol at the end of month 12. The number of patients who dropped out was slightly higher after the amendment (32% vs. 22%). The causes and timing (pre or post amendment) are shown in Table 2. In group I there were no differences in sirolimus and tacrolimus trough levels at hospital discharge between preamendment and postamendment patients. In group II, only tacrolimus levels at discharge were higher in postamendment than in preamendment patients (Student's t-test, p = 0.017) (Table 3). At 12 months median sirolimus trough levels were 6 (SD 2.4) and 14 (SD 4.4) ng/mL in groups I and II, respectively, with median doses of 2 (SD 1) and 7 (SD 2.6) mg per day. In group I, median tacrolimus trough levels and doses at month 12 were 7.3 (SD 2.4) ng/mL and 4 (SD 1.9) mg/day, respectively.
Table 2. Patients who dropped out of the study protocol within 12 months
| Patient number|| Group pre/post|| amendment||Time of drop- out (days post- transplantation)|| Cause of drop-out|| Treatment at 12 months|
|3/1651||1||Pre||46||Lung hemorrhage and cardiac arrest. Died.||NA|
|6/1977||1||Post||4||Renal arterial thrombosis. Nephrectomy.||Dialysis|
|9/1977||1||Post||64||Died owing to pulmonary embolism (prior|| |
| ||antecedents of mitral mechanic prosthesis,|| |
| ||lupus, hypertension, and coronary bypass).||NA|
|6/1975||1||Post||140||FK withdrawn owing to nephrotoxicity.||SRL + STE|
|2/1973||1||Post||163||Polyarthralgias in knees and ankles owing|| |
| ||to osteonecrosis.||FK + MMF + STE|
|13/1977||1||Post||33||FK-related HUS. BCAR.||SRL + STE|
|15/1651||1||Post||285||Microcytic anemia.||FK + STE|
|1/1651||2||Pre||43||Thrombocytopenia and anemia.||MMF + FK + STE|
|2/1651||2||Pre||125||Died owing to intestinal PTLD.||NA|
|9/1651||2||Pre||2||Vascular BCAR grade III, nephrectomy.||Dialysis|
|10/1665||2||Pre||6||Patient's request.||MMF + FK + STE|
|8/1665||2||Pre||130||Died owing to mitral valvulopathy and heart failure.||NA|
|11/1651||2||Pre||365||No FK withdrawal owing to depression with|| |
| ||noncompliance treatment and prior BCAR.||SRL + FK + STE|
|9/1976||2||Post||88||CMV and leukopenia.||FK + STE|
|6/1973||2||Post||41||Thrombocytopenia.||MMF + FK + STE|
|16/1651||2||Post||37||Switch to CsA owing to PTDM.||SRL|
|6/1974||2||Post||6||Thrombosis upon an antiphospholipid syndrome.|| |
|5/1975||2||Post||173||Reintroduction of FK after BCAR.||SRL + FK + STE|
|7/1977||2||Post||173||Reintroduction of FK after BCAR and||MMF + FK + STE|
| ||thrombocytopenia.|| |
|2/1974||2||Post||193||Reintroduction of FK after BCAR.||MMF + FK + STE|
|15/1977||2||Post||112||Systemic CMV infection, HUS.||SRL + MMF + STE|
|14/1977||2||Post||167||Introduction of MMF owing to clinically diagnosed|| |
| ||AR after FK withdrawal.||MMF + FK + STE|
|10/1973||2||Post||165||Polyarthralgias in knees and ankles owing||MMF + FK + STE|
| ||to osteonecrosis|| |
|14/1065||2||Post||365||No FK withdrawal owing to sirolimus|| |
| ||related anemia and prior BCAR.||SRL + FK + STE|
|11/1976||2||Post||365||No FK withdrawal owing to sirolimus related leukopenia.||SRL + FK + STE|
Table 3. Immunosuppressive drug trough levels at hospital discharge
|Levels (ng/mL) at discharge||Group I||Group II|
|Pre-amendment||Post-amendment||p-value||Pre amendment||Post amendment||p-value|
|SRL||3.7 (1)||4.3 (2)||NS||6.8 (3.6)||8.7 (4.8)||NS|
|FK||103 (48)||9.7 (2.8)||NS||6.0 (1.9)||8.1 (2.9)||0.017|
In the ITT analysis, neither serum creatinine nor creatinine clearance differed between the two groups, either before or after the amendment, although glomerular filtration rate tended to be higher in group II in all comparisons. In the analysis of patients on protocol, serum creatinine value at 12 months was statistically significantly higher in the group that remained on tacrolimus therapy [1.6 (SD 0.4) mg/dL vs. 1.3 mg/dL (SD 0.3), p = 0.03]. Creatinine clearance in the overall group tended to be better in the group with tacrolimus withdrawal but this difference did not reach statistical significance. However, in the patients grafted after the amendment, creatinine clearance was significantly higher in group II than in group I [72.9 (SD 20.1) and 58.4 (SD 18.9) ml/min, p = 0.03, respectively,]. Overall, in the ITT analysis, four out of 43 patients (9.3%) in group I and 10 out of 44 patients (22.7%) in group II developed BCAR (p = 0.14). The rate of BCAR in group II decreased from 41% before the amendment to 11.1% after increasing the loading doses. The median time of first rejection was 6.5 days in group I and 9 days in group II. Of the 10 patients who experienced an acute rejection in group II before tacrolimus withdrawal, four finally showed successful tacrolimus elimination with an uneventful clinical course. After tacrolimus withdrawal, two patients suffered acute rejection, both on day 36 after elimination with concurrent sirolimus trough levels of 7.9 and 10.2 ng/mL, respectively. In group I, the severity of acute rejection episodes was graded according to Banff ‘97 histological classification as grade II in three patients, and grade III in one patient. In group II, rejection events were scored as grade I in five patients, grade II in four patients and grade III in one patient.
When analyzing the ITT population, diastolic blood pressure was significantly higher in group I [80.4 (SD 10.2) mm-Hg] than in group II [75.6 (SD 10.6) mm-Hg, p = 0.03]. Systolic blood pressure also tended to be higher in group I [141 (SD 24.2) mm-Hg] than in group II [131.8 (SD 19.5) mm-Hg], but this difference was not significantly different (p = 0.06). In the analysis of patients on protocol, blood pressure was found to be statistically significantly higher in those remaining on tacrolimus. Thus, systolic blood pressure was 140.7 (SD 21.7) and 129.1 (SD 17.7) mmHg in groups I and II (p = 0.03) and diastolic blood pressure was 80.9 (SD 10.8) and 74.2 (SD 8.8) mm of Hg in groups I and II (p = 0.01).
There were no differences in survival between groups: graft survival was 93% and 90.9% and patient survival was 95.3% and 95.4%, in groups I and II, respectively.
The main laboratory parameters at 12 months are depicted in Table 4. Levels of total cholesterol, low-density lipoprotein cholesterol and magnesium were higher in group II than in group I. Potassium levels showed no statistically significant differences at 12 months but were statistically lower in group II several times during the study. There were no significant differences between groups I and II in reported events except for thrombocytopenia (16.3% vs. 40.9%, p < 0.05), headache (20.9% vs. 4.5%, p < 0.05), or anxiety (0 vs. 11.4%, p < 0.05). Delayed graft function, defined as the need for dialysis within 1 week of transplantation, was similar in both groups (29.3% in group I vs. 23.8% in group II, p = NS). Edemas were reported in 42% of group I and 29% of group II (p = NS). Post-transplantation diabetes mellitus developed in 7% of group I and 2.3% of group II. The percentage of patients on hypolipemiant therapy was similar in both arms (65% in group I vs. 75% in group II, p = NS). Potassium supplements were administered to 7% of patients. Anemia was reported in 60.5% of the patients in group I and in 54.5% of those in group II (p = NS). Erythropoietin was temporarily administered to 38% of all the patients. The mean hemoglobin value was 9.5 g/dL at initiation of erythropoietin. One month later, the mean increment observed was 1.5 g/dL (Wilcoxon test, p < 0.05). Two patients in each group died. Causes of death included lung hemorrhage and lung embolism in group I and, post-transplantation lymphoproliferative disorder and acute lung edema in group II.
Table 4. Main laboratory parameters measured in the population on protocol at 12 months
| ||Group I||Group II||P|
| Hemoglobin (g/dL)||13.3 (2.5)||13.3 (1.6)||NS|
| Leukocyte × 109/L||6.7 (2.2)||7.1 (1.7)||NS|
| Platelet × 109/L||195 (49)||192 (57)||NS|
| Creatinine (mg/dL)||1.6 (0.4)||1.3 (0.3)||<0.05|
| Creatinine clearance (ml/min)||64.3 (22.8)||71.4 (18.8)||NS|
| Glucose (mg/dL)||102 (20.5)||111 (53)||NS|
| Sodium (mmol/L)||141 (2.2)||141 (2.2)||NS|
| Potassium (mmol/L)1||4.3 (0.5)||4.1 (0.4)||NS|
| Chloride (mmol/L)||108 (3.5)||107 (2.9)||NS|
| Calcium (mg/dL)||9.8 (0.7)||9.8 (0.6)||NS|
| Magnesium (mg/dL)||1.8 (0.2)||2 (0.2)||<0.05|
| Phosphorous (mg/dL)||2.9 (0.6)||2.9 (0.6)||NS|
| ALT (UI/L)||35 (31)||33 (17)||NS|
| AST (UI/L)||27 (11)||26 (9)||NS|
| LDH (UI/L)||365 (105)||394 (147)||NS|
| Alkaline phosphatase (UI/L)||172 (136)||162 (90)||NS|
| Cholesterol (mg/dL)||208 (33)||241 (36)||<0.05|
| LDL-cholesterol (mg/dL)||119 (28)||146 (24)||<0.05|
| HDL-cholesterol (mg/dL)||57 (17)||56 (13)||NS|
| Triglycerides (mg/dL)||167 (87)||187 (75)||NS|
| Lipoprotein A (mg/dL)||48 (67)||66 (76)||NS|
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- Materials and Methods
To the best of our knowledge, this is the first prospective trial in kidney transplantation that has tested the feasibility of tacrolimus withdrawal from an immunosuppressive regimen with tacrolimus and sirolimus in the postoperative period. We found that this approach provides excellent renal function after tacrolimus elimination with a conventional rate of acute rejection.
This trial included 87 low-risk kidney cadaveric allograft recipients. An unexpectedly high rate of acute rejection in the group with reduced tacrolimus exposure together with concomitant low sirolimus and tacrolimus trough levels prompted us to amend the protocol to increase the doses (10). Discharge levels in group II were higher after the amendment and the rate of acute rejection decreased. However, higher sirolimus levels and possible related toxicities may have led to a greater number of patients who dropped out postamendment. We have not found the increasing requirements in sirolimus doses to maintain levels throughout the study period, as has been reported by Kuypers et al. (11).
The results of this study emphasize the importance of reaching adequate trough levels of both sirolimus and tacrolimus in the early post transplant period. Moreover, El-Sabrout et al. have retrospectively addressed the value of using a loading dose of at least 10 mg of sirolimus on the first day post-transplantation to increase rejection-free graft survival (12). Similarly, a recently reported pooled analysis of an international trial of 361 de novo kidney recipients stressed the need for an amendment similar to our own to minimize the risk of early acute rejection (13). Another alternative might be the use of full therapeutic doses of tacrolimus with sirolimus, but this strategy resulted in a high incidence of biopsy-proven tacrolimus nephrotoxicity (14). The use of induction strategies with antibodies followed by a scheme of low-dose tacrolimus and sirolimus could provide an acceptable rate of rejection (15–17). Moreover, the combination of sirolimus and tacrolimus seems to offer excellent protection against rejection even in high-risk populations (14,18), and has allowed steroid withdrawal in patients at high risk of developing long-term steroid toxicity (19).
A recent approach that tested the feasibility of withdrawing sirolimus from a regimen of standard tacrolimus doses plus extremely low doses of sirolimus attained excellent rejection rates, but the suspension of sirolimus was not followed by an improvement in renal function (20). Interestingly, data from the present study suggests that the transient combination of these two macrolides may provide a low incidence of rejection as well as a trend towards better renal function in the patients who are withdrawn from tacrolimus. Large retrospective studies have suggested that the lower the serum creatinine in the first year, the longer the graft survival (21). Short-term improvement in serum creatinine may reflect the cessation of the deleterious hemodynamic effect of calcineurin inhibitors. Longer follow up could provide information on the potential beneficial effect of tacrolimus elimination, although the number of patients in the present exploratory study will be probably too small to address this issue. Nevertheless, long-term follow up in a larger trial of cyclosporine elimination from an initial regimen with sirolimus and steroids reported sustained improvement in renal function (22,23). Similarly, randomized trials with grafted patients on regimens based on sirolimus with avoidance of calcineurin inhibitors show better long-term renal function when compared with control groups administered cyclosporine (24,25) and better scores of biopsy-proved chronic allograft nephropathy when compared with tacrolimus (26) or cyclosporine (27).
Our results suggest more effective blood pressure control in patients who were withdrawn from tacrolimus, which may be beneficial considering that systolic and diastolic blood pressure at 1 year is highly predictive of long-term graft survival (28). The avoidance of calcineurin inhibitors with the use of sirolimus or mycophenolate mofetil may allow more effective long-term blood pressure control (22,29).
Two major concerns regarding the combination of sirolimus and tacrolimus remain. Cholesterol levels tend to increase proportionally with higher doses of sirolimus, as shown in the present trial, although this increase does not seem to cause large increments in the Framingham risk model score (30) or to increase the risk of cardiovascular events after transplantation at least in the short term (31). Hyperglycemia has been reported in approximately a quarter of patients on sirolimus and tacrolimus, especially in the African American populations (14,18). Despite this finding, sirolimus and tacrolimus have been successfully used in pancreas transplantation in diabetic patients (17,32).
The major limitation of this pilot study is probably related to the small number of patients. The trial was planned as a pilot investigation with a calculated beta error of 50%. The rate of discontinuations, coming near to the third part of recruited patients and slightly higher after amendment, albeit similar to other reported trials of tacrolimus with sirolimus (13,33), has also probably accounted for this issue. Interestingly, eight of the 25 patients who dropped out remained on sirolimus therapy at 1 year.
In conclusion, despite the limitations owing to the inclusion of relatively low-risk patients, the protocol amendment introduced during the trial, and the small sample size, the findings of this exploratory study suggest that an acceptable rejection rate may be achieved once adequate early exposure to sirolimus and tacrolimus is attained. Early tacrolimus elimination from a triple regimen of sirolimus, tacrolimus and steroids may result in better renal function and blood pressure control, which might contribute to better long-term graft survival. The knowledge drawn from this exploratory trial may help to design future larger prospective studies to determine the efficacy and safety profile of this immunosuppressive strategy.