Background: Endotoxemia, characterized by elevated plasma lipopolysaccharide (LPS), is associated with gut microbiota dysbiosis and increased intestinal permeability. In transplant patients, immunosuppressive drugs may contribute to or exacerbate endotoxemia. LPS can modulate hepatic metabolic enzymes and drug transporters, potentially altering the pharmacokinetics of immunosuppressants.

Aims: Understanding these interactions is crucial for optimizing therapy and improving transplant outcomes.

Methods: We analyzed plasma samples from 48 transplant recipients at one-year post-transplantation, sourced from the CRBIOLIM biobank (Limoges University Hospital) through the PIGREC (heart), STIMMUGREP (lung), and BIOMARGIN (kidney) studies in organ transplant recipients. In addition, samples from 24 healthy volunteers were obtained with informed consent through the Clinical Investigation Center (Limoges University Hospital). Patients included were on a standard immunosuppressive regimen with mycophenolate mofetil (MMF), known to disrupt gut microbiota and promote endotoxemia. Plasma LPS levels were quantified using ELISA and compared between groups (Mann-Whitney test). Patient area under the concentration-time curve (AUC) of MMF, tacrolimus, and cyclosporine was extracted from databases, and correlations with LPS levels were assessed using Spearman’s rank correlation coefficient.

Results: Transplant recipients had significantly higher plasma LPS concentrations than healthy volunteers (99 ± 22 pg/mL vs. 257 ± 32 pg/mL; mean ± SEM; p < 0.01), confirming endotoxemia. Among immunosuppressants, only cyclosporine AUC correlated with LPS levels (Spearman’s ρ=0.818, p=0.002).

Conclusion: The strong correlation between cyclosporine exposure and LPS plasma levels suggests that endotoxemia may influence cyclosporine metabolism by modulating hepatic enzymes and transporters. Further research is needed to elucidate the mechanisms.

Keywords: endotoxemia, lipopolysaccharide, cyclosporine A

Background: Due to its narrow therapeutic range, tacrolimus requires therapeutic drug monitoring (TDM) to minimize risks of over- or under-exposure. While the AUC is the most reliable measure of drug exposure, trough concentration (Cmin) is commonly used in clinical practice. However, Cmin may not always accurately reflect true exposure, leading to potential unexpected effects.

Aims: This study aims to identify risk factors associated with AUC-Cmin discrepancies in transplant recipients treated with tacrolimus.

Methods:This retrospective study included patients who benefited from full microsampling tacrolimus PK profiles between one and six months post-transplantation. Age, weight, transplantation date, dose, creatinine, liver enzymes, hematocrit, proteins and co-treatments were collected. AUC-Cmin discrepancy was defined as a difference >20% between AUC calculated using the trapezoidal method and AUC estimated from Cmin using a linear regression equation. A logistic regression was conducted to identify risk factors for discrepancy.

Results: Ninety-eight patients were included in the study (68 kidney, 29 liver and 1 kidney-liver transplant recipients). Discrepancies were observed in 28.6% of patients with a lower Cmin/dose ratio (p=0.03), hematocrit (p=0.007) and total protein (p=0.01) and higher Alanine Aminotransferase (p=0.007) in the group presenting a discrepancy. No covariate remained statistically significant in the multivariate analysis.

Conclusions: No risk factor for AUC-Cmin discrepancy has been evidenced underlining the need for systematic AUC monitoring to detect patients with discrepancies and exposure-targeted individualization of tacrolimus treatment. Significant discrepancies between AUC and Cmin was experienced in 28.6% of patients, highlighting that Cmin may not reliably reflect tacrolimus exposure.

Keywords: Immunosuppressants; pharmacokinetics

Background: Tacrolimus, essential for allogeneic stem cell transplant patients, requires therapeutic drug monitoring (TDM) due to high variability and a narrow therapeutic window. Finger-prick dried blood spots (DBS) could replace venous blood draws, enabling home monitoring and improving patients’ quality of life.

Aims: This study aimed at evaluating the clinical applicability of capillary finger-prick sampling for tacrolimus TDM and creatinine determination in the hospital (part 1), and in a homesampling context (part 2), using DBS and Capitainer® qDBS devices.

Methods: Patients collected qDBS samples at home at trough levels over three days. LC-MS/MS analysis methods were used. Analytical and intra-patient variation based on home-sampled duplicates were evaluated as well as therapy adherence. For each homesampling participant, tacrolimus levels were compared to their individual target ranges. Patients’ experience was assessed via questionnaires.

Results: Assisted sampling confirmed the clinical applicability of capillary (q)DBS for tacrolimus TDM and creatinine determination, with qDBS showing better clinical agreement (98% of tacrolimus and 93% of creatinine results within 20% of reference values). Homesampling using qDBS is feasible, with the CV below 8%. In stable-dose patients, within-subject variation for tacrolimus ranged from 4.1% to 24.7%, suggesting strong adherence. Homesampling improved tacrolimus trough level resolution and enabled closer monitoring after dose changes, with positive patient feedback.

Conclusions: Capillary qDBS show great potential as a complement to venous blood draws for tacrolimus TDM and creatinine determination. Homesampling is promising for this patient population, addressing the need for individualized TDM.

Key Words: Therapeutic Drug Monitoring, Tacrolimus, Creatinine, Dried Blood Microsampling

Background: Donor derived cell free DNA (dd-cfDNA) is a non-invasive biomarker for allograft monitoring, useful for transplant surveillance. However, current quantification methods are expensive and time-consuming. Droplet digital PCR (ddPCR) offers a more affordable alternative to next-generation sequencing (NGS) in resource-limited settings.

Aims: To develop and validate a ddPCR-based method for quantifying dd-cfDNA in pediatric liver transplant recipients (PLTR), and compare its performance with an NGS-based methodology.

Methods: Donor and recipient genomic DNA were typed using biallelic deletion/insertion polymorphisms (DIPs) to identify informative alleles. Genomic DNA from healthy volunteers was sonicated and mixed at different proportions to simulate patient- and donor-derived cfDNA. DNA input, primer concentrations, and annealing temperatures were optimized. Precision, sensitivity, and linearity were assessed, considering a coefficient of variation (CV) < 30% as acceptable. The relation between dd-cfDNA% and donor DNA quantity (ng) was evaluated. The methodology was validated by comparing dd-cfDNA% from PLTR samples quantified using NGS.

Results: The limit of quantification was 0.084 ng (0.47% dd-cfDNA) with an optimal input of 18 ng per PCR reaction. Precision was acceptable across all dd-cfDNA% levels. dd-cfDNA% showed an acceptable linear relation with donor gDNA (ng) (R² > 0.99). A strong correlation was observed between ddPCR and NGS (Pearson r = 0.96, p < 0.001).

Conclusions: The validated ddPCR methodology offers a reliable, cost-effective, and faster alternative to NGS for dd-cfDNA quantification, potentially improving early detection of graft injury in PLTR with a prompt intervention aiming to improve patient outcomes.

Keywords: ddPCR, dd-cfDNA, pediatric liver transplant, non-invasive monitoring

Background: Mycophenolic Acid (MPA), an ester prodrug of Mycophenolate Mofetil i.e. is an immunosuppressive agent, used with tacrolimus to prevent organ rejection in transplant patients. Traditionally, Therapeutic Drug Monitoring (TDM) relies on plasma measurements, but alternative matrices like saliva offers a non-invasive approach for assessing drug exposure. This study investigates the feasibility of estimating MPA from saliva and explores its potential as a surrogate for plasma measurements in renal transplant recipients.

Aims: The study aims to evaluate the feasibility of measuring MPA in saliva, assessing salivary and plasma MPA exposure through area under curve (AUC) and finding their concentration correlation for further clinical outcomes.

Methods: Saliva and plasma samples were collected from 10 adult renal transplant patients with stable allograft ≥4 weeks. Salivary MPA (sMPA) was quantified using High Performance Liquid Chromatography-Fluorescence Detector (HPLC-FLD) and plasma MPA by established HPLC-UV method. The correlation between salivary and plasma MPA concentrations and mean AUC0-12h was calculated.

Results: The mean plasma MPA AUC0-12h was 88.21 ± 35.03 µg*h/mL, while the mean sMPA AUC0-12h was 195.82 ± 126.81 ng*h/mL. In our study, the correlation between plasma and sMPA concentrations was 0.903±0.133,concluding sMPA concentrations may reflects its plasma concentrations.

Conclusions: Out of the 10 patients, 7 exhibited a strong correlation between plasma and sMPA concentrations (>0.900). These findings suggest that saliva may serve as a potential alternative matrix for TDM of MPA, which could aid in non-invasive assessment and optimization for further clinical outcomes.

Keywords: Mycophenolate, Saliva, Therapeutic Drug Monitoring, Area under the Curve, HPLC-FLD

Introduction: Tacrolimus (Tac) is a key immunosuppressant in renal transplantation, often combined with anti-thymocyte globulin (ATG) for induction therapy. However, ATG may be obviated in low-risk patients. Tac inhibits nuclear factor of activated T cells (NFAT), reducing gene expression (IL-2, TNF-α, GM-CSF, IFN-γ). Study examines the in-vitro and in-vivo pharmacodynamic (PD) effects of Tac using NFAT residual gene expression (NFAT-RGE) in renal-transplant recipients with/without ATG.

Methods: Sixty patients (≥18 years), planned for de-novo renal-transplant. Blood samples were collected pre-transplant (10ml) and on post-transplantation day 7 (POD 7) (2ml at Tac-C0 and C1.5). Samples were spiked with varying concentrations of Tac (0, 2.5, 5, 10, or 20 ng/ml) and ex-vivo immune activation was done with RPMI 1640 medium, phorbol myristate acetate (PMA), and ionomycin. Total RNA was extracted, cDNA was synthesized. Real-time PCR was performed for gene expression quantification. Fold change was calculated using ∆Ct and ∆∆Ct. Tac concentration was estimated in whole blood samples using LC-MS/MS system with TAC-13C-d2 as the internal standard.

Results: Recipients (n=60) had an average NFAT-RGE of 53.7±21.8% (2.5ng/ml), 36.6±18.7% (5ng/ml), 24.8±16.2% 10ng/ml) and 13.5±11.6% (20ng/ml) in-vitro. In comparison, the POD7 NFAT-RGE was 46.9±22.3% at C0 (11.15±2.7ng/ml) and 19.5±13.1% at C1.5 (16.6±4.3ng/ml). The NFAT-RGE in ATG-induced patients (n=34) at POD7 was 25.13±30.83% compared to 37.68±34.36% in the non-ATG (n=26) at C0. The corresponding values were 11.92±13.11% and 14.29±15.76% for C1.5.

Conclusions: NFAT-RGE could be used as potential biomarker in individualizing immunosuppression in non-ATG triple drug-induced patients.

Keywords: Renal transplantation, Pharmacodynamic biomarkers, tacrolimus, residual gene expression, NFAT