**1. Introduction**

The calcineurin inhibitor tacrolimus (Tac) is considered a first-line immunosuppressant in liver transplant (LT) recipients [1–4]. Because of its small therapeutic window, therapy with Tac requires close drug monitoring [5]. In addition, deterioration of renal function induced by acute or chronic calcineurin inhibitor nephrotoxicity (CNIT) is a common side e ffect [6]. Recent studies have reported characteristics of chronic CNIT in up to 70% of LT recipients [7,8]. Furthermore, up to 8.5% of patients develop end-stage renal disease in long-term follow-up [9].

Several studies have revealed that the risk of CNIT is associated with both high Tac trough concentration and high daily Tac dose [10,11], although CNIT may occur even with low-dose regimens [12]. One potential explanation for this association is the correlation between CNIT and a

fast metabolism rate of twice-daily immediate-release Tac (IR-Tac). The Tac blood concentration to daily dose ratio (C/D ratio) has been identified as a simple tool to describe patients' metabolism rate in a steady state, in which a low C/D ratio reflects a high rate of metabolism [13–15]. A low IR-Tac C/D ratio is linked with higher C2 Tac blood concentrations despite comparable trough levels in patients with high C/D ratios [16]. In this regard, a low C/D ratio is strongly associated with an increased risk of CNIT and a faster decline of renal function in both kidney transplant (KT) and LT recipients [13,16–19]. Thus, increasing the C/D ratio by improving Tac bioavailability may result in better nephroprotection. One way of potentially influencing the pharmacokinetics of Tac is to change the formulation of the drug [20].

LCPT is a novel Tac formulation using MeltDose ® technology, in which the particle size of the drug is reduced from 10 μm to the smallest possible units (<0.1 μm), resulting in increased dissolution and thus better absorption [21]. This feature, combined with drug release over the entire intestinal tract, results in LCPT having significantly better bioavailability than other Tac formulations. Tremblay et al. showed that the intraday peak-to-trough fluctuation was approximately 30% lower for LCPT than for standard-release Tac (IR-Tac and once- daily extended-release Tac (ER-Tac)) [20]. A dose reduction of up to 30% has been observed in KT and LT recipients on LCPT [22].

Hence, we hypothesize that conversion from standard-release Tac to LCPT increases C/D ratio and thereby preserves renal function.

#### **2. Materials and Methods**

#### *2.1. Patients and Study Design*

Figure 1 illustrates the enrolment of the subjects in the study. This observational study was performed on patients who had undergone cadaveric liver transplantation at the University Hospital of Münster. LT recipients were included at the time of presentation at our Outpatient Transplant Clinic between March 2017 and August 2018. The study start was defined as the first appointment in this period.

At this time point, the treating physicians made a decision to either leave the patients on their usual immunosuppressive treatment (control group) or to switch them from standard-release Tac (IR- or ER-Tac) to LCPT (intervention group). Data were analysed over a 12-month follow-up. Inclusion criteria were aged over 18 years, intake of standard-release Tac before enrolment, stable graft function and an interval between transplantation and inclusion in the study of at least 1 month. LT recipients were not allowed to receive any medications or agents that could interfere with Tac. The decision about drug conversion was made by treating physicians at their own discretion.

The initial immunosuppressive regimen consisted of Tac (Prograf or Advagraf), mycophenolate mofetil (CellCept, MMF) and prednisolone (Decortin H/Soludecortin H). Tac was given at a dose of 0.1 mg/kg twice daily with a target trough concentration of 8–10 ng/mL during the first month, 6–8 ng/mL from months 2 to 3, and 3–5 ng/mL thereafter. MMF was started at a dose of 1 g twice daily and was adjusted in case of adverse e ffects. Initial prednisolone was given at a dose of 250 mg once daily intravenously before and immediately after LTx and was tapered stepwise. In most cases, prednisolone had been discontinued within 6–12 months after LTx.

Laboratory data were collected at study onset (at the time of conversion to LCPT or the first presentation during the above-mentioned period in the control group (t0)) and after 3 (t3), 6 (t6), 9 (t9) and 12 (t12) months. Serum bilirubin, alanine transaminase (ALT) and international normalized ratio (INR) were measured to assess graft function. General demographic data and information on transplantation and diagnoses were obtained from the patient records.

**Figure 1.** Study design and patient enrolment. A total of 164 liver transplant (LT) recipients were screened for eligibility. Only LT recipients who were started on IR- or ER-Tac (standard-release tacrolimus) and continued taking this drug until the beginning of the study were included. During the enrolment period (March 2017–August 2018), 121 patients met the inclusion criteria and were either switched to LCPT (once-daily MeltDose® tacrolimus (Tac); intervention group) or maintained on standard-release tacrolimus (control group). Clinical data were analysed in a 12-month follow-up. We hypothesized that conversion from standard-release Tac to LCPT increases concentration/dose (C/D) ratio and thereby preserves renal function

The C/D ratio, calculated as the ratio of Tac trough level to the corresponding daily dose, was determined 3 months before study start, at the study start and at subsequent evaluation time points. The t0 C/D ratio in the intervention group was determined the day before first LCPT intake. Renal function was calculated using the estimated glomerular filtration rate (eGFR) in accordance with the Chronic Kidney Disease Epidemiology Collaboration equation at the corresponding time points. The difference from baseline eGFR (t0) was determined at the time points of t3, t6, t9 and t12. A negative value indicates deterioration of eGFR, while a positive value indicates improvement.

The study was conducted in accordance with current medical guidelines and the Declarations of Istanbul and Helsinki. The study was also approved by the local ethics committee (Ethik Kommission der Ärztekammer Westfalen-Lippe und der Medizinischen Fakultät der Westfälischen Wilhelms-Universität Münster, No. 2016-046-f-S). Collected patient data were anonymized and written consent for collection and use of the clinical data was obtained.

#### *2.2. Statistical Analysis*

Statistical analysis was performed with IBM SPSS® Statistics 25 for Windows (IBM Corporation, Somers, NY, USA). Normally distributed data are shown as mean ± standard deviation; non-normally distributed data are shown as median (minimum–maximum). For unrelated groups, normally distributed data were compared with a *t*-test, non-normally distributed data with the Mann–Whitney U-test and categorical variables with Fisher's exact test. Comparison of continuous variables within a connected group was performed with the Wilcoxon signed-rank test. Pearson's test was used to describe normally distributed data, whereas Spearman's test was applied to non-normally distributed data. In all statistical evaluations, two-sided tests were used; a *p*-value of ≤ 0.05 was considered significant for all tests performed.

The study onset was defined as the baseline (t0). In the first approach, eGFR changes (t3-t12) from baseline were compared between the intervention group and the control group. In the next step, eGFR changes from every time point to baseline were compared within each group (eGFR slope). A negative value indicates deterioration in eGFR, whereas a positive value indicates an improvement in renal function.

Multivariable analysis was performed to identify independent predictors of alterations in renal function ( ΔeGFR) after 12 months compared with that at baseline. For this purpose, univariable analysis with factors known to potentially influence renal function was initially performed. Variables that showed a *p*-value < 0.15 in univariable analysis were included in the multivariable analysis. Variables with a significance of <0.05 in multivariable analysis were considered significant.
