2.1 Study design and population
Adult Chinese patients who underwent lung transplantation between June 2021 and May 2022 at Shanghai Chest Hospital, Shanghai Jiao Tong University, were enrolled in this prospective pharmacokinetic study. The inclusion criteria were as follows: patients who (a) underwent lung transplantation within two weeks, (b) were older than 18 years, and (c) were taking tacrolimus as the primary immunosuppressive agent. Patients were excluded from the analysis if they (a) had intolerable adverse reactions to tacrolimus, (b) received multi-organ transplants, (c) were pregnant or breast-feeding, or (d) had a history of malignant tumours, mental illness, hepatic abnormality, severe gastrointestinal diseases, systemic infection, or any serious condition that might affect tacrolimus pharmacokinetics.
This study was approved by the Ethics Committee of the Shanghai Chest Hospital, and written informed consent was obtained from all patients before enrolment. This study was registered in the Chinese Clinical Trial Registry under the identifier ChiCTR2000036727. All procedures were conducted in accordance with the Helsinki Declaration of 2013 and the Basic & Clinical Pharmacology & Toxicology policy for Experimental and Clinical Studies29.
After lung transplantation, all patients received a triple immunosuppression regimen of tacrolimus (Prograf®; Astellas Ireland Co., Ltd, Killorglin, Ireland), mycophenolate mofetil (MMF; CellCept®; Shanghai Roche Pharmaceutical Co., Ltd, China), and corticosteroids, that included intravenous methylprednisolone (Solu-Medrol®, Pfizer Manufacturing Belgium NV, Belgium) or oral prednisone (Tianjin Jinjin Pharmaceutical Co., Ltd, China). In addition, all patients received induction therapy (Basiliximab, Simulect®; Novartis Pharma Schweiz AG, Switzerland) on the first and fourth days after the surgery.
Tacrolimus treatment was initiated at a dose of 0.5 mg every 12 h (6 am and 6 pm) after surgery and was then adjusted to achieve a target whole blood trough concentration between 8 and 12 ng/mL within 3 months.
The initial mycophenolate mofetil dose was 1000 mg twice daily and was given along with tacrolimus. The dose was modified based on clinical response. A 500 mg (for patients that underwent single lung transplantation) or 750 mg (for patients that underwent bilateral lung transplantation) intravenous methylprednisolone dose was administered during the operation, and then 160 mg was administered every day for the first three days after surgery. The dose was then progressively reduced to 40 mg on the fifth day after surgery, followed by 30 mg of oral prednisone once daily, which was then gradually tapered to a maintenance dose of 5 mg given once daily within two weeks. In addition, each patient was administered 100 mg voriconazole (Pfizer Italia S.R.L., Italy) 1 h after taking tacrolimus for invasive Aspergillus prophylaxis for three months.
During hospitalization, patients received a controlled diet served at 7:00 and 17:00 every day to ensure tacrolimus administration when fasting or 1–2 h after meals. Patient demographics and clinical data were collected from electronic medical records and included: age, weight, height, body mass index, sex, postoperative days (POD), tacrolimus dosage, concomitant medication, haemoglobin, haematocrit, albumin, total protein, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase, blood urea nitrogen, serum creatinine. Glomerular filtration rate was estimated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) Eq. 30.
Within two weeks of transplantation, serial blood samples (5 mL) were collected in tubes containing ethylenediaminetetraacetic acid (EDTA) before the morning tacrolimus dose (0 h) and at 0.5, 1, 2, 4, 6, 8, 10, and 12 h post-dose. Blood was collected from an indwelling catheter placed in the patient's forearm vein and stored at -20°C until analysis.
Tacrolimus whole blood concentration was measured using liquid chromatography/mass spectrometry (LC/MS). The lower quantification limit of the assay was 0.5 ng/mL, with a calibration range of 0.5–100 ng/mL31.
Genomic DNA was extracted from whole blood samples using the Ezup Column Blood Genomic DNA Purification Kit (Sangon Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer’s protocol. Single nucleotide polymorphisms, including CYP3A5*3 (rs776746) and CYP3A4*1G (rs2242480), were genotyped by independent external contractors (Sangon Biotechnology Co., Ltd., Shanghai, China) using a DNA direct sequencing analyser (Applied Biosystems 3730XL, Foster City, CA, USA). The primer and probe sequences used for genotyping are summarized in Table S1.
Hardy-Weinberg equilibrium was tested using the chi-squared test or Fisher’s exact test, and pairwise D’ and r2 were calculated to estimate linkage disequilibrium (LD) between loci within a gene32.
2.2 Pharmacokinetic analysis
Noncompartmental analysis was performed using the WinNonLin (version 8.3, Certara L.P., USA) software to calculate pharmacokinetic parameters. The dosing interval area under the curve (AUC0 − 12h) was estimated using the linear-up log-down trapezoidal method. Apparent clearance (CL/F) was calculated by dividing the tacrolimus dose (mg) by AUC0 − 12h. The maximal (Cmax) and minimal concentration during the dosing interval (Cmin), as well as the time when Cmax was achieved (Tmax), were obtained directly from the concentration-time profiles. Dose-normalized AUC0 − 12, Cmax, and Cmin were calculated, and all pharmacokinetic parameters were log-transformed before statistical analysis.
The association between blood concentration at each sampling time-point and AUC0 − 12 was also evaluated using linear regression. Abbreviated sampling equations were developed to assess the ability of single concentration-time points to predict the tacrolimus AUC0 − 12. The predictive performance was evaluated using the coefficients of determination (R2), mean prediction error (MPE), and mean absolute prediction error (MAE), which were described by Sheiner and Beal33 as follows:
$$\text{M}\text{P}\text{E}=\frac{1}{\text{n}}\sum _{\text{i}=1}^{\text{n}}({\text{A}\text{U}\text{C}}_{\text{p}\text{r}\text{e}\text{d}}-{\text{A}\text{U}\text{C}}_{\text{o}\text{b}\text{s}})$$
1
,
$$\text{M}\text{P}\text{E}=\frac{1}{\text{n}}\sum _{\text{i}=1}^{\text{n}}|{\text{A}\text{U}\text{C}}_{\text{p}\text{r}\text{e}\text{d}}-{\text{A}\text{U}\text{C}}_{\text{o}\text{b}\text{s}}|$$
2
,
where ‘n’ is the number of patients, and ‘AUCpred’ and ‘AUCobs’ refer to the predicted and observed values of AUC0 − 12h in each patient, respectively.
|MPE| percentages within 15% (F15), 20% (F20), and 25% (F25) were calculated. The model is considered clinically acceptable if it yields an MPE ≤ ± 15%, MAE ≤ ± 30%, F15 > 40%, F20 > 45%, and F25 > 50%34.
All continuous variables were summarized using descriptive statistics. Categorical variables were reported as counts and percentages. The correlation between continuous clinical variables and pharmacokinetic parameters was evaluated using Spearman’s correlation test. Differences in pharmacokinetic parameters or demographic characteristics between individuals with different genotypes were compared using analysis of variance (ANOVA). A P-value less than 0.05 was considered statistically significant in the comparison. All statistical analyses were performed using R software (Version 3.6.2, https://cran.r-project.org/).