Pharmacokinetics of Envarsus in pediatric kidney transplant recipients – phase 1 pilot conversion study

Tacrolimus is the standard immunosuppressant for pediatric kidney transplants and is routinely administered twice daily (BD‐tac). Envarsus (LCP‐tac), an extended‐release formulation, is approved for adults but not for pediatric patients.

Patients with kidney transplants are required to take immunosuppression to prevent rejection.The most commonly prescribed immunosuppression is tacrolimus.2][3] Tacrolimus functions by blocking calcineurin and inhibits activation of T cells.
Nonetheless, calcineurin is also present in other organs in the body including kidney, heart, blood vessels, and brain. 4The side effects of tacrolimus are now well recognized including increased risk of hypertension, diabetes, and neurological side effects, particularly tremor. 5Transplant patients often experience headaches, difficulties sleeping, and poor mood, which have been attributed to calcineurin inhibitors, but often these side effects are multi-factorial.
Importantly, the class of calcineurin inhibitors is associated with kidney problems including electrolyte disturbances and kidney scarring with long-term use. 6,7This was demonstrated in animal models which showed constriction of the blood vessels supplying the kidney in rats treated with cyclosporine. 8In addition, other patients who receive calcineurin inhibitors (e.g., non-renal transplants and patients with nephrotic syndrome) have a clear link to worsening renal function. 6Clinicians and families therefore need to balance the benefits of immunosuppression against the potential side effects, both of which can be related to measurements of tacrolimus levels, usually done through whole blood assays.
Tacrolimus is absorbed throughout the gastrointestinal tract and undergoes significant first-pass metabolism in the liver. 9The levels of tacrolimus are therefore affected by the concomitant presence of food.Patients are advised to take tacrolimus on an empty stomach (fasting for 1 h before and 2 h after the medication).As tacrolimus is taken twice a day 12 h apart, this regimen has a significant impact on patients' lifestyle.The variation with food intake will affect trough levels of tacrolimus which potentially will affect graft function (if levels are low when taken with food) or increase risk of side effects (if taken on an empty stomach, leading to high levels). 9With standard twice-daily tacrolimus, the peak blood level (C max ) is usually achieved 2 h post-dose (T max ). 10 There are a rapid decline and a subsequent smaller peak after the evening dose. 11P-tac (Envarsus) is an extended-release formulation of tacrolimus designed for once-daily administration. 12Phase I and II adult LCP-tac studies confirmed broader absorption in the intestinal tract and more sustained, consistent tacrolimus levels and is distinct from other once-daily tacrolimus preparations which maintain a high C max 2 h post-dose. 12,13Clinically, LCP-tac is licensed in the European Union, the United Kingdom, and the United States of America for the prevention of rejection in adult kidney transplant recipients.This was based on the phase III double-blind randomized control study comparing LCP-tac to twice-daily tacrolimus. 12This trial was powered to show non-inferiority of LCP-tac in preventing acute rejection, graft loss, and mortality at 12 months post-transplant.The trial met this primary outcome and had similar serious adverse events between the two groups.The results persisted at 2 years. 14Secondary outcome of eGFR was similar between the two groups.There was a high incidence of new-onset diabetes after transplantation (NODAT) in the LCP-tac group though this did not reach statistical significance.
The LCP-tac group required small doses of medication to achieve the same tacrolimus levels which endorses the better absorption profile of LCP-tac.LCP-tac is not licensed for use in pediatric patients.
We therefore set up a phase 1, Pk study to establish whether the smoother Pk profile is consistent in children.

| Study design and population
We performed a pilot, open-label, dose conversion, and pharmacokinetic study in a group of pediatric kidney transplant recipients.Patients were required to have stable graft function (eGFR >30 mL/min/1.73m 2 and no decline of more than 20% in the preceding 6 months) and no treatment for rejection in the preceding 6 months.Immunosuppression needed to include any form of twice-daily (immediate-release) tacrolimus (BD-tac).As the tablet form of LCP-tac needed to be swallowed whole, we pragmatically recruited patients between the ages of 6 and 17 years inclusive.We were funded to recruit six patients, with a plan to recruit at least one patient between 6 and 10 years old.Patients were recruited from a single center (Nottingham University Hospitals NHS Trust).

| Study objectives
The primary objective was to study the pharmacokinetics of LCPtac compared to standard BD-tac, including maximum concentration (C max ), time to maximum concentration (T max ), and 24-h tacrolimuslevel area under the curve (AUC).Secondary objectives were related to safety and adverse events.Pre-specified secondary clinical objectives included difference in eGFR 6 months after conversion, episodes of biopsy-proven acute rejection, patient/graft survival, and ratio of tacrolimus level to total daily dose (C/D).
We also performed the following exploratory analyses at baseline (first Pk visit on BD-tac) and at the end of the study

| Tacrolimus conversion and pharmacokinetic study
We performed two 24-h Pk studies for each patient (Figure 1).Following consent, the first Pk study was performed while taking BD-tac.One week later, patients were converted to LCP-tac with a conversion ratio of 1:0.7 (total daily BD-tac to LCP-tac).Tacrolimus levels were monitored weekly, and LCP-tac dose was adjusted to maintain equivalent levels.Target trough levels are individualized for each patient.Our center policy is to aim for trough levels between 4 and 6 ng/mL after 6 months post-transplant.The second Pk study was performed at a minimum of 4 weeks after conversion, or longer to achieve stable levels without any change in LCP-tac dose for 2 weeks.Over the 24 h, tacrolimus levels were obtained at the following time points: pre-dose, 1, 2, 3, 4, 5, 6, 8, 10, 12, 13, 14, 16, and 24 h.Venous samples were initially obtained through a cannula following appropriate blood volume discard and applied to a 10 μL volumetric absorptive microsampling device (Mitra).At later time points, samples could be obtained through a finger-prick capillary sample directly onto the microsampling device.For the Pk study, tacrolimus levels were performed at the central laboratory (using mass spectrometry) with our previously validated protocol. 15Patients were followed up for 6 months after conversion and were eligible to continue on LCP-tac after the end of the study.

| Statistics
To assess the Pk profile for tacrolimus, we performed a noncompartmental analysis using R package PKNCA (version v.10.1). 16 studies were performed after stable tacrolimus levels were obtained.AUC (0-24 h) and C max were summarized using the geometric mean and geometric coefficient of variation.T max was defined as the first peak after the morning dose and summarized as the median and range.As this was a pilot study, no formal power calculations were performed to determine effective sample size for Pk analysis.The study was designed as a descriptive study, and with the small sample size, no comparative statistics were performed.

| Patient characteristics
We started recruitment in January 2020 but had to pause the study during the COVID-19 pandemic.Thirteen patients were screened, of whom 7 patients consented for participation.Two patients were withdrawn as they no longer met the study criteria after consenting: The first consented before COVID-19 but was over 18 years of age when the study recommenced; the second developed graft dysfunction after consenting.The latter patient was 9 years old.Therefore, five patients underwent conversion to LCP-tac with no drop-outs or reconversions.Patient characteristics are provided in Table 1.

| Pk results
The Pk results for BD-tac and LCP-tac are summarized in Figure 2 and Table 2. Patients on BD-tac had a higher C max (14.4  9.9 ng/mL) and earlier Pk peak max 1 h versus 5 h post-dose) compared to LCP-tac.Therefore, the Pk curve of LCP-tac was consistent with a slow-release profile with a gradual curve and lower peak, compared to BD-tac with a bimodal peak shortly after each dose.
The 24-h AUC was comparable for both medications: 164 (SD 27.8) ng/mL BD-tac and 141 (SD 46.5) ng/mL LCP-tac.Of note, the mean trough level for LCP-tac at the end of conversion was lower and AUC results were not normalized to the trough level.The conversion ratio after stable tacrolimus levels was 0.6 which is lower than the current recommendation for dose conversion of 0.8 in adults.

| Secondary outcomes
There were no episodes of rejection (biopsy proven or presumed).
There was one serious adverse event during the whole study period which was a hospital admission unrelated to the study medication.No new safety concerns or side effects arose from the study.
One patient had subjective improvement in their hand tremor.

| Exploratory analyses
Patients responded to the BAASIS questionnaire by saying they missed their medication at a median of 2 times (range 0-2) within the last 4 weeks, while taking BD-tac.After conversion to LCP-tac, the median missed dose was 0 (range 0-1).Nonetheless, one patient had adherence concerns raised retrospectively by the direct care team after the end of the follow-up, as subsequent tacrolimus levels were low.Paired EQ-5D-Y results at baseline and end of study were available for four patients on BD-tac and all five patients on LCP-tac.
Two young adults expressed feeling "a bit worried, sad, or unhappy" at both time points, which were also reported by their parents/carers.Young adults reported a decrease in the visual analog scale (EQ VAS, median −3.5%, range −6% to 0%, Figure S1a).Parents/carers reported an increase in the EQ VAS (median 6%, range −9% to 12%, Figure S1b).

| DISCUSS ION
In this pilot study, we converted stable pediatric kidney transplant recipients who were taking twice-daily immediate-release tacrolimus to once-daily, extended-release tacrolimus, LCP-tac.Our Pk results confirmed that LCP-tac had a more gradual Pk curve with lower and later C max and equivalent 24-h AUC.LCP-tac was better absorbed, and C/D ratio was nearly double that of BD-tac.The final TA B L E 2 Pharmacokinetics of BD-tac and LCP-tac.

BD-tac LCP-tac
T max , ng/mL a 1 (1-4) 5 (3-8)   C max , ng/mL b 14.4 (46.9) 9.9 (49.3)F I G U R E 2 24-h pharmacokinetic profile of BD-tac and LCP-tac (during fasting conditions) analyzed using non-compartmental analysis.Dots and bars represent the mean and standard deviation.Comparable to other studies, the dose of LCP-tac after conversion was 40% less than BD-tac highlighting the better absorption.
The result is comparable to the adult ASTCOFF Pk study, which showed that LAC-tac dose was 30% less compared to BD-tac and 36% less compared to Advagraf, the first available once-daily tacrolimus preparation. 10Similarly, ElChaki converted three adolescent patients to LCP-tac and showed prolonged T max of 9 h. 17Of note, direct Pk comparisons between BD-tac and LCP-tac are not feasible because dose adjustments were made after conversion.Tacrolimus is extensively metabolized by first-pass metabolism in the small bowel and liver, mainly through CYP3A4 and CYP3A5.Patients with CYP3A5*1 polymorphisms (CYP3A5 expressers) exhibit higher clearance of tacrolimus and require larger doses, resulting in higher C max and larger C max -C0 differences. 9The high C max is postulated to result in higher toxicity including renal fibrosis. 18An initial study showed that CYP3A5 expressers had lower C max with Envarsus. 19 did not have CYP3A5 details in our study, though our patients were all Caucasians in whom the CYP3A5 expression is rare.Our C/D for BD-tac is considerably low (mean 0.8), with a C/D ratio <1.05 associated with high metabolizers. 20Further studies are needed to elucidate whether LCP-tac has better efficacy in patients who are high metabolizers.Additionally, patients fasted around the time of tacrolimus dosing during the Pk study, while they potentially took tacrolimus around the time of food at home.Pk studies for nonfasting doses showed a flat Pk profile. 21ere were no significant serious adverse events in this study.
The side effect profile was similar.One patient reported a subjective improvement in tremors and headaches, as reported in the literature. 22There was no statistical difference in eGFR though the follow-up time was short.In the one patient who had a decrease in eGFR, tacrolimus trough level was higher after LCP-tac.Young adults reported a lower quality-of-life score after the end of the study though the overall score was high in our cohort of patients.
Of note, two patients reported feeling "worried, sad, and unhappy" which is under-recognized in our transplant patients. 23,24The number of patients was small and precludes firm conclusions but raises areas for attention in future studies.
6][27][28] The effects of missed doses for BD-tac have been simulated using population Pk characteristics. 29Missing a morning dose will result in a significant drop in subsequent AUC with a relatively lower drop in C 0 level.Many days are required to re-establish stable AUC levels.The effect of missed morning doses is more pronounced in patients who are high metabolizers for tacrolimus.The authors suggest a compensatory increase in the next dose by 150% though this requires self-reporting by the patient. 29The authors also show a significant effect of delayed doses. 29Patients describe significant stress over needing to take their medications as close to 12 hourly as possible.Therefore, once-daily preparations are more convenient and preferable to patients. 26,27The effect of missed or delayed doses on Pk characteristics for LCP-tac is not known.The current advice is to take the same dose within 15 h of prescribed dose (14 h for Advagraf).Nonetheless, "pill burden" is only one aspect of adherence. 30In this study, one patient was retrospectively suspected to be non-adherent after the end of the study as the tacrolimus levels were subsequently sub-therapeutic.
Tacrolimus exposure is most commonly measured with trough levels, due to its practicality.Though there is good correlation between trough levels and AUC, for some patients the AUC may be lower (or higher) which might increase the risk of rejection. 9The target AUC is not well established, and the suggested target is >150 ng/ mL for standard-risk patients. 9In the ASTCOFF study, AUC for LCPtac was higher (117%), but after adjusting for the equivalent trough level, the AUC was similar (100.6%).Correlation for trough and AUC better for LCP-tac (Pearson's correlation coefficient, r = 0.92) versus BD-tac (0.81). 10 Limited sampling strategies are now available for LCP-tac, and for all tacrolimus preparations, AUC/C trough calculations can be personalized for at patient, done after a steady period. 31,32Innovations in microsampling and tacrolimus measurement assays might make it easier for limited sampling to be done, even by patients themselves at home. 15 are aware of the high variability in tacrolimus levels in the Pk study which likely relates both to variability between patients, small sample size, as well as variation due to the tacrolimus measurement method.The latter can be ameliorated through repeat sampling at each time point or through using alternative microsampling devices. 33larger study is needed to further evaluate the population Pk characteristics in the children though our Pk results were consistent with adult studies.We did not pre-specify an analysis of tacrolimus IPV due to the short-term follow-up.Though we showed that LCP-tac was well absorbed, further studies are required to assess tacrolimus IPV and the effect of missed doses on overall tacrolimus levels over time.We did not include assessment of puberty, and majority of participants were close to 18 years of age.Extended-release tablets need to be swallowed whole which can be difficult for younger patients.
Nonetheless, facilitative educational tools are available which have been successful in converting children from liquid to tablets. 34crolimus is the main anti-rejection medication for kidney trans- Written consent was provided by the patients or parent/guardian for patients below 16 years of age.The study was approved by the East of England-Essex Research Ethics Committee (19/EE/0126) and was registered on EudraCT (European Union Drug Regulating Authorities Clinical Trials Database: 2018-003593-13).
(6 months after conversion to LCTP-tac): a) Adherence through the Basel Assessment of Adherence to Immunosuppressive Medications Scale (BAASIS), administered by a research nurse independent from the clinical care team.b) Quality of life using Generic EQ-5D-Y (3-level version) completed by the patient and parent/carer.
ng/mL versus F I G U R E 1 Study design and pharmacokinetic time points.
conversion ratio was 0.6 which is lower current adult recommendations.No new safety signals arose, and there was no difference in eGFR at the end of follow-up of 6 months.

F I G U R E 3
Results of secondary analysis: (A) eGFR and (B) concentration/dose (C/D) ratio.
plantation despite its narrow therapeutic window.With advances in measurement techniques and better understanding of Pk profiles, patient care can be improved through personalized tacrolimus management.In this pilot study in young adults, we showed that the Pk profile of LCP-tac is consistent with an extended-release profile and comparable to their adult counterparts.LCP-tac can therefore be considered for use in this age group for the same indications.AUTH O R CO NTR I B UTI O N SJJK, LL, and DM designed the study.JJK and LL obtained ethical approval and sponsorship approval.DM performed biochemical measurements.JJK, AM, AL, and MM recruited patients.JJK, LL, AM, AL, MM, and AW performed study procedures.JJK performed statistical analysis.JJK and AW wrote the draft.All authors contributed to the article and approved the submitted version.