Study design and ethics
This is a prospective, multicentre, open-label, randomised, parallel-group study. It was investigator designed. The study was approved by the Ethics Committee of the Centre Léon Bérard (Lyon, France), declared to the French Agency for the Safety of Health Products (AFSSAPS), registered on ClinicalTrials.gov under number NCT00159159, and conducted in accordance with the European Guidance for Good Clinical Practice and the Declaration of Helsinki.
Randomisation and treatment
Within four days post-surgery, patients undergoing de novo heart transplantation were randomised to receive either a low dose or a standard dose of CsA, in a 1/1 ratio, as part of a triple immunosuppressant regimen, including MMF, and corticosteroids.
Patients were randomised using a centralised procedure. Randomisation was stratified by centre, age, presence of ischemia, and serum creatinine level before transplantation. Patients were followed up for 12 months. During the first three months, the low-dose group received CsA targeting a whole blood pre-dose concentration (C0) of 130 to 200 μmol/L, whereas the standard-dose group received CsA with a C0 between 200 and 300 μmol/L. Both doses reflect current practice . Thereafter, to follow standard practice, the CsA doses of the standard-dose group were tapered to match the C0 of the low-dose group (130 μmol/L to 200 μmol/L).
The immunosuppressive treatment also included MMF (3 g daily) and corticosteroids according to local practice. MMF doses were adjusted according to individual tolerance.
All other concomitant antibacterial, antifungal, antiviral, anticholesterol, or antihypertensive drugs were administered at the discretion of physicians.
Overall, 95 patients were enrolled in 10 French heart transplantation centres. Male and female heart transplant patients, aged 18 to 65, undergoing de novo heart transplantation were eligible. Donors’ cold ischemia time was under six hours. The main recipient exclusion criteria were: unstable haemodynamic status at the time of randomisation, circulatory assistance, serum creatinine level >250 μmol/L, multiorgan transplant, history of malignant disease within the past five years, a human immunodeficiency virus-positive blood test, a positive HB-antigen test, or a positive PCR hepatitis C test. Pregnant or breastfeeding women, patients participating in another trial, drug addicts, and psychiatric patients were also excluded. Each participant gave written informed consent.
Study objectives, primary and secondary endpoints
The primary objective was to compare the renal function between the two groups by assessing changes in serum creatinine levels from inclusion up to 12 months. Creatinine clearance, microalbuminuria, and proteinuria were used as secondary endpoints. Immunosuppressive efficacy endpoints included the incidence of acute rejection episodes, the assessment of cardiac function (ejection and shortening fractions), and treatment failure (defined as death or withdrawal from the study for any reason). Any time during participation, the treatment was to be discontinued in patients of the low-dose group who received less than 1.5 g MMF/day for more than 15 consecutive days.
In compliance with the intention-to-treat (ITT) analysis of the primary endpoint, patients who withdrew early were followed up until the end of the study.
Strategy tolerance was assessed by the incidence of adverse events. Vital signs (weight and clinical manifestations) and cardiovascular risk factors (blood pressure, lipid, and glucose profiles) were closely monitored at each outpatient visit.
Following standard clinical procedures, the patients were monitored every two weeks during the first three months, then at months 4, 5, 6, 9, and 12 post-transplantation to assess the efficacy and safety of the strategy. Serum creatinine level was assessed at each visit. Creatinine clearance, proteinuria, and albuminuria were analysed at months 6 and 12. Cystatin C was assessed at inclusion, then at 1, 2, 3, 6, and 12 months.
Endomyocardial biopsies were performed according to local procedures. Biopsy-proven acute rejection episodes were graded by a local pathologist using the International Society for Heart and Lung Transplantation (ISHLT) scale. All biopsies performed over the study period were considered for analysis . Acute rejection episodes were considered serious adverse events and the sponsor was immediately notified.
Forty-five patients in each group (53 recruited for a drop-out of 15%) would ensure a 80% power to detect an 18% reduction in serum creatinine level of the low-dose group, at 12 months, assuming that the mean in the standard-dose group is 140 μmol/L, the common standard deviation is 43, and the two-sided two-group t test significance level is 0.05 .
According to the intention-to-treat (ITT) principle, analyses were performed on all 95 randomised patients.
Given biological variability and the imprecision of measurements, a mixed model of co-variance was used to take into account all available measures. The main analysis focused on a three- to twelve-month interval. It tested the effects of treatment at three months (low dose vs. standard dose) and whether the expected change lasted until the end of follow-up, when both groups started to receive the same low dose. Missing data were included as random events. The treatment effect was tested both on the intercept (treatment effect at three months) and on the slope (interaction between treatment and time of follow-up). The likelihood ratio test was used for this test comparing the models given below:
Where ηij is the predicted creatinine value (log10-transformed) for patient i and measurement j, X1ij is the time elapsed after transplantation (in days), X2i is the patient group, X1ij X2i is the interaction between the patient group and the time interval, Ui0 is the random intercept for patient i, and Ui1 is the random slope for patient i.
A box plot distribution was used to describe changes in creatinine level over time by treatment group.
A post hoc analysis was conducted on the primary endpoint dichotomised according to a clinically meaningful threshold (>120 μmol/L) in order to check that the low dose was able to decrease the proportion of patient over this threshold after three months and/or up to twelve months.
The threshold of 120 μmol/L was considered as clinically critical on the basis of literature data and clinical observations [18, 19]. Our hypothesis was that the low dose could have a greater impact among the patients with the highest creatinine values. A hierarchical logistic regression model using a penalised quasi-likelihood method (glmmPQL) was used to test this hypothesis. The formula was similar to Model 2, but here, ηij was the probability of having a creatinine level over the threshold (logit transformation).
The statistical analysis used R and SAS™ softwares.