Everolimus-Based Calcineurin Inhibitor‑Free Immunosuppression Versus Standard CNI Regimen in Liver Transplant Recipients Beyond One Year: A Phase III Randomized Trial

The primary objective is to demonstrate that calcineurin inhibitor‑free immunosuppression provides superior preservation of renal function, assessed by the change from baseline to 14 months in estimated glomerular filtration rate, compared with a standard calcineurin inhibitor‑based regimen in liver allograft recipients without relevant sub‑clinical graft injury. This endpoint addresses the clinical need to reduce nephrotoxicity associated with long‑term calcineurin inhibitor use. The key secondary objective is to establish that the CNI‑free regimen is non‑inferior to standard therapy with respect to the composite of biopsy‑proven acute liver graft rejection or liver graft loss occurring up to month 14, thereby confirming that graft protection is maintained while improving renal safety.

The trial enrolled adult men and women aged 18 to <80 years who had undergone a single‑organ liver transplantation between 12 and 36 months prior to screening and were receiving calcineurin‑inhibitor‑based maintenance immunosuppression. Participants were required to have stable hepatic function (alanine aminotransferase and alkaline phosphatase < 2 × upper limit of normal) and no evidence of relevant subclinical graft injury. Both sexes were eligible, and inclusion required signed informed consent and adherence to contraceptive requirements for individuals of childbearing potential. The sponsor did not provide the total number of participants enrolled in the study.

The ALTERNATION trial is a multicenter, open‑label, phase III study that randomized adult liver transplant recipients who are 12–36 months post‑transplant to receive either an everolimus‑based, calcineurin‑inhibitor‑free regimen (everolimus 2 mg oral daily combined with mycophenolate mofetil, tacrolimus or ciclosporin as required) or to continue standard calcineurin‑inhibitor‑based immunosuppression. Participants are screened for eligibility, provide written informed consent, and undergo a baseline visit during which randomization occurs. Follow‑up visits are scheduled at months 1, 3, 6, 12, 14 (primary endpoint assessment), and subsequently at months 24, 30, and 38 (final end‑of‑study visit). At each visit renal function, liver enzymes, biopsy results, and safety parameters are recorded; quality‑of‑life questionnaires are administered at baseline, month 14, and month 38. The primary efficacy outcome is the change from baseline to month 14 in estimated glomerular filtration rate (eGFR), calculated with the CKD‑EPI equation. Secondary outcomes include biopsy‑proven acute rejection, progression of chronic kidney disease, liver‑related mortality, development of donor‑specific antibodies, and incidence of malignancy or serious infections. Participant involvement extends from the screening visit through the month 38 assessment, totaling approximately 38 months. Early termination may occur if a participant experiences graft loss, severe adverse events, non‑adherence to the protocol, or withdrawal of consent. The study enrolment period is planned from June 2026 to June 2031.

Everolimus is administered orally at a fixed dose of 2 mg per administration. The drug is provided in a tablet formulation and is intended for use as part of the experimental immunosuppressive regimen aimed at CNI‑free maintenance after liver transplantation.

Tacrolimus is given orally at a weight‑based dose of 0.13 mg per kilogram of body weight. It serves as a comparator treatment reflecting standard‑of‑care therapy that includes a calcineurin inhibitor in liver transplant recipients.

Mycophenolate mofetil is delivered orally at a dose of 1500 mg per administration. It is incorporated into both the experimental and control immunosuppressive protocols as an adjunctive antimetabolite.

Ciclosporin is administered orally at a weight‑based dose of 12 mg per kilogram of body weight. This agent represents an alternative standard calcineurin inhibitor regimen used for comparison with the experimental protocol.

Efficacy will be evaluated using both renal and hepatic outcome measures. The primary efficacy parameter is the change in estimated glomerular filtration rate (eGFR) from baseline to 14 months after randomization, calculated with the CKD‑EPI Creatinine equation according to the National Kidney Foundation 2021 guidance. Secondary efficacy parameters include: change in eGFR from baseline to the end of follow‑up (38 months); incidence of biopsy‑proven acute liver graft rejection or graft loss up to month 14 and month 38; progression of chronic kidney disease to stage 4 or 5 or need for renal replacement therapy; liver‑related mortality; progression of subclinical graft injury (fibrosis) and inflammation assessed by repeat liver biopsies at month 14 and at the end of follow‑up; quality of life changes measured with PROMIS and SF‑36 questionnaires; development of de novo donor‑specific HLA antibodies; increase in liver stiffness above 8.4 kPa; incidence of malignancy; occurrence of infections requiring medical intervention or hospitalization; and new onset of hypertension, dyslipoproteinemia, or diabetes mellitus.

Renal function will be assessed at baseline, month 14, and month 38 by obtaining serum creatinine values and applying the CKD‑EPI equation. Hepatic graft status will be monitored through scheduled liver biopsies at baseline, month 14, and month 38; acute rejection is defined by liver enzyme elevation >2 × ULN combined with histological criteria per the most recent BANFF consensus. Fibrosis and inflammation progression are determined by histological scoring, with progression defined as an increase of ≥2 points from baseline. Liver stiffness will be measured using the validated elastography method, applying the 8.4 kPa threshold. Donor‑specific HLA antibodies will be detected by standard immunological assays. Quality of life will be captured using the PROMIS and SF‑36 instruments, administered at the same time points as the renal and hepatic assessments. All data will be collected according to the trial schedule and analyzed using appropriate statistical methods to compare the CNI‑free regimen with standard CNI‑based therapy.