The effect of very low dose diltiazem on tacrolimus exposure very early after kidney transplantation: a randomized controlled trial
This prospective, randomized, double-blind, placebo-controlled study was conducted in four parallel KTR groups. Eligible KTRs were randomly assigned to receive either immediate-release oral diltiazem (Ditizem®; Siam Pharmaceutical, Bangkok, Thailand) 30 mg q12h or physically identical placebo, which started on the day of the KT (day 0), in a 1:1 ratio according to a sequence of computer-generated random schedules in permuted blocks of 4 with stratification on the CYP3A5 genotypes (expressors and non-expressors of CYP3A5) by the program nQuery version 4.0 (Statistical Solutions Ltd), Cork, Ireland. The randomization schedule was concealed in sequentially numbered sealed opaque envelopes and kept in a locked cabinet in the central pharmacy of Siriraj Hospital. Envelopes were opened sequentially only after each participant had registered. The containers of diltiazem and placebo, which were given a drug number and assigned to consecutive patients in sequential order, were tamper evident, of equal weight and similar in appearance. A pharmacist not involved in the care of study patients performed the randomization, distributed the study agents, and held the trial codes, which were disclosed after the study. Physicians, nurses, other healthcare providers, and study patients were unaware of group allocations.
All adult KTRs (>18 years old) who received their life-related KTs at Siriraj Hospital, a teaching hospital in Thailand, between March 2017 and December 2020 and were prescribed tacrolimus as their primary immunosuppressive therapy after the KT were eligible to participate. Exclusion criteria included multiple organ transplantation, KT incompatible with ABO blood group, cirrhosis, aspartate aminotransferase > 100 U/L, alanine aminotransferase > 120 U/L, infection with hepatitis C virus, an active gastrointestinal disorder, signs of infection and blood pressure
Relevant demographic and clinical data were obtained from medical records and the electronic laboratory reporting program (Éclair program). All transferred data has been verified and validated.
DNA extraction and genotyping
After recruitment, venous blood samples were collected in tubes containing EDTA. Genomic DNA was extracted from peripheral blood leukocytes using a Puregene blood kit (Qiagen®). The concentration of extracted DNA was measured using a Nanodrop2000 spectrophotometer (Thermo Scientific, Waltham, MA) and diluted to 20 ng/μL for genotyping analysis. Genotyping of CYP3A5 (6986A>G, rs776746, assay ID: C_26201809_30) was evaluated using 2 μL genomic DNA samples (20 ng/μL), TaqMan® Genotyping assay (20×) 1 μL, TaqMan ® Universal PCR master mix (2×) 10 μL, distilled water 7 μL (total 20 μL). TaqMan Drug Metabolism Genotyping Assay reactions were performed with the following thermal cycling profile: 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 90 s. The analysis of the cycles and of the allelic discrimination was carried out using the QuantStudioTM 3 Real-Time (Applied Biosystems®).
KTR heterozygous or homozygous carriers of the CYP3A5*1 allele were classified as expressing CYP3A5 and those that are homozygous carriers of CYP3A5*3 alleles were classified as non-expressors of CYP3A5.
Immunosuppressive drug regimen and use of other drugs
All participants received oral immediate-release tacrolimus (Prograf®; Astellas, Kerry, Ireland). According to the protocol of Siriraj Hospital, the initial dose (0.05 mg/kg every 12 h) of tacrolimus was started 2 days before KT (day – 2) and then adjusted after KT according to the efficacy and toxicity and to maintain morning trough concentrations of 7-10 ng/mL in standard-risk patients and 10-12 ng/mL in high-risk patients during the first week after transplantation. Participants also received either mycophenolate mofetil (Cellcept®; F Hoffmann-La Roche Ltd, Basel, Switzerland or Immucept®; Intas Pharmaceuticals Ltd., Gujarat, India) or mycophenolate sodium (Myfortic®; Novartis Pharma Stein, Switzerland for Novartis AG, Basel, Switzerland) at a dosage equivalent to 2 g/day of mycophenolate mofetil. A tapered corticosteroid regimen was administered consisting of an intravenous bolus of 500 mg methylprednisolone (Solu-medrol®; Novartis Pharma Stein, Switzerland for Novartis AG, Basel, Switzerland) on day 0, 250 mg on day 1, 125 mg on day 2 followed by 40 mg/day of prednisolone on day 4 after KT. Subsequent doses of prednisolone were gradually reduced thereafter. Induction treatment with basiliximab (Simulect®; Novartis Pharma AG, Basel, Switzerland) was authorized.
Either 30 mg of diltiazem or a matched placebo capsule was administered every 12 h at the same time as the tacrolimus dose. All patients received a fixed dose of omeprazole 40 mg/day. Other antihypertensive drugs, except those significantly affected by the pharmacokinetics of tacrolimus, were permitted at physician discretion as per standard of care.
Tacrolimus Concentration Analysis
On day 7 post-KT, after the tacrolimus dose had been stable for at least 48 h, venous blood samples (5 mL) were collected pre-dose and at 1, 2, 4, 6, 8, and 12 h after the morning dose of tacrolimus. Samples were collected in EDTA tubes and stored at 4 degrees Celsius until analysis. Concentrations of tacrolimus in whole blood were determined on the same day of blood collection using a chemiluminescent microparticle immunoassay method with the ARCHITECT® i2000 system (Abbott, Abbott Diagnostic, USA). The method was linear in the concentration range up to 30 ng/mL, the limits of quantification were 0.8 ng/mL, and the coefficient of variation of within-run and between-run precision was less than 10% . The average recovery was 100 ± 10% according to manufacturer’s information20.
The primary endpoint was AUC/D on day 7 post-KT. Secondary outcomes were the proportions of patients with significantly undertherapeutic tacrolimus C0 ( 15 ng/mL) and systolic and diastolic blood pressures measured on days 3 and 7 after KT.
The area under the 12 h curve of tacrolimus concentration (AUC) was calculated by the linear logarithmic trapezoidal method. For the calculation of the AUC/D and the C0/D, the AUC and C0 values were adjusted to the dose by dividing the values observed by the dose recorded on the day of the blood sample. Early morning blood pressures were measured the same day of blood collection for pharmacokinetic analysis by a ward nurse as part of a routine examination, at rest in a seated position with a calibrated and well-maintained automatic sphygmomanometer. Delayed graft function (DGF) was defined as the inability of the kidney graft to function immediately, with the need for dialysis within the first 7 days after KT. A biopsy-confirmed acute rejection (BPAR) episode within 3 months of KT was diagnosed based on histological findings in the indication renal graft biopsy according to the 2015 Banff criteria.
The distribution of continuous data was assessed by the Shapiro-Wilk test, and thereafter parametric tests or non-parametric tests were applied as appropriate. Normally distributed continuous data are presented as mean ± standard deviation (SD) and non-normally distributed continuous data as medians with interquartile ranges (IQR), unless otherwise specified. Numbers and percentages are expressed for categorical data.
Genotypic frequencies of polymorphisms were tested for deviations from Hardy-Weinberg equilibrium using appropriate chi-square tests. Analysis of variance (ANOVA) or Kruskal-Wallis test was used to compare continuous data. For comparison of AUC/D between the four study groups, eta squared, a measure of effect size for ANOVA was calculated if necessary; otherwise, squared epsilon, a measure of effect size for Kruskal-Wallis, was calculated based on the H statistic, which was adjusted for links. For ANOVA, Bonferroni’s correction was performed for multiple pairwise tests when variances are homogeneous and Dunnett’s T3 test was applied when variances are not homogeneous. For the Kruskal-Wallis test, the Bonferroni procedure was performed for pairwise multiple comparison. Fisher’s exact test was used to compare proportions between groups. Bivariate correlations between continuous variables were assessed by either Pearson’s correlation test or Spearman’s rank correlation test.
The calculation of the power has been carried out. A required total sample size of at least 40 was estimated by G*Power 220.127.116.11 software, Heinrich-Heine-Universität, Düsseldorf, Germany (with an effect size of 0.50, a power of 0 .9, an estimated dropout rate of 50%, and a significance level of 0.05).
All statistical tests were performed against a two-sided alternative hypothesis using a significance level of 0.05. All analyzes were performed using IBM SPSS 28 statistics (IBM, Bangkok, Thailand).
This study was conducted in accordance with the provisions of the Declaration of Helsinki, Declaration of Istanbul and Good Clinical Practice, approved by the Siriraj Institutional Review Board, Faculty of Medicine, Siriraj Hospital, Mahidol University (approval number Si 721/2016), and registered in the Thai Clinical Trials Registry (TCTR20170206005) with full date of first registration as 2017-02-06. All patients provided written informed consent prior to enrollment.